CN101657468B - Human monoclonal antibodies to the thyrotropin receptor which act as antagonists - Google Patents
Human monoclonal antibodies to the thyrotropin receptor which act as antagonists Download PDFInfo
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Landscapes
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention provides an isolated antibody for the TSHR which is an antagonist of TSH. The invention also relates to methods of using antibodies of the invention.
Description
Technical field
The present invention relates to the antibody reacting with thyrotropic hormone (TSH) acceptor (TSHR), and particularly, but relate to nonexclusively such antibody, this antibody can be combined with TSHR and can be by the activation of TSH-pungency antibody or TSHR-pungency antibody blocking TSHR.
Background technology
Thyrotropic hormone (Thyrotropin) or thyrotropic hormone (thyroid stimulatinghormone) are (TSH) a kind of pituitrin (Szkudl inski MW that regulates thyroid function by TSHR, Fremont V, Ronin C, Weintraub BD 2002 Thyroid-stimulating hormone andTSHR structure-function relationships.Physiological Reviews 82:473-502).TSHR is a kind of g protein coupled receptor, formed by 3 structural domains: be rich in leucic structural domain (LRD), cutting structure territory (CD) and membrane spaning domain (TMD) (Nunez Miguel R, Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Blundell TL, Rees SmithB, Furmaniak J 2004 Analysis of the thyrotropin receptor-thyrotropininteraction by comparative modelling.Thyroid 14:991-1011).TSH is attached to the upper receptor signal that causes of TSHR and conducts, and it promotes Triiodothyronine: formation and the release of thyroxine (T4) and triiodothyronine (T3).Have a kind of Feedback mechanism to relate to the level of T4 and T3 in circulation, this mechanism control discharges TSH (with the throtropin releasing hormone of hypothalamus secretion) from pituitary body, and TSH controls again that Tiroidina activates and the level of Thyroid Hormones In Serum.
A lot of data explanations of this area, the patient of some autoimmune thyroid disease (AITD) has autoantibody (the Rees Smith B reacting with TSHR, McLachlan SM, Furmaniak J 1988Autoant ibodies to the thyrotropin receptor.Endocr ine Reviews 9:106-121).In most of the cases, these autoantibodies are combined with TSHR and are imitated the effect of TSH, thereby stimulate Tiroidina to produce high-caliber T4 and T3.These autoantibodies are called as Tiroidina pungency autoantibody or TSHR autoantibody (TRAb), have pungency activity or TSH agonist activity.The physiology Feedback mechanism of thyroid function control mentioned above is in the situation that this Tiroidina pungency autoantibody exists and occur Thyroid Activity excessively or in the patient of thyrotoxicosis (Thyroid Hormones In Serum is excessive) without effect.This illness is called as Graves disease (Graves ' disease).It is believed that, in some patients, during the TRAb with stimulating activity organizes after socket of the eye, interact with TSHR, and caused the eye symptom of Graves disease.Human monoclonal antibodies (hMAb TSHR1) as powerful thyroid-stimulating factor is recorded in patent application WO2004/050708A2 in detail.
From different in the patient of some trouble AITD, autoantibody can be incorporated into described acceptor, but not stimulate the ability of TSHR in conjunction with TSHR, prevention TSH.The autoantibody of these types is called as the TRAb with blocking-up activity or TSH antagonistic activity, and may there is active not enough symptom (hypothyroidism) (the Rees Smith B of Tiroidina in the patient who has barrier TRAb in its serum, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor.EndocrineReviews 9:106-121).Particularly, if be present in conceived women's serum, having the active TRAb of blocking-up can and can block the Tiroidina TSHR of fetus through placenta, causes congenital hypothroidism and serious growth consequence.Moreover, thering is the active TRAb of blocking-up and can come across in infected mother's breast milk, this may further cause clinical hypothyroidism in baby.Up to now, also not yet obtain the human monoclonal autoantibody of the anti-TSHR with TSH antagonistic activity.Therefore, for such autoantibody how with the interactional scrutiny of TSHR, and for their and interactions of TSHR and the autoantibody of stimulus type (for example M22) and compared with TSH how study in great detail all and be restricted.
Human chorionic gonadotrophin is a kind of hormone with gentle Tiroidina stimulatory effect producing in During Pregnancy.
Be definitely important to being characterized in following research of character that has stimulating activity or block active TRAb, described research is that its target is diagnosis and the treatment improving the disease relevant to the autoimmune response of TSHR.The invention of describing in patent application WO2004/050708A2 provide about have powerful stimulating activity human monoclonal autoantibody character and with the interactional details of TSHR.Moreover, patent application WO2006/016121A discloses a kind of TSHR goods of the sudden change that comprises at least one point mutation, it is used in from treating in patient's the humoral sample of examination, and patients serum's pungency TSHR autoantibody, patients serum's barrier TSHR autoantibody and TSH are carried out to difference examination and qualification.Patent application WO2004/050708A2 has also described to be had TSHR and blocks active mouse monoclonal antibody (9D33).9D33 is with high-affinity (2 × 10
10l/mol) be combined with TSHR, and be a kind of TSH, hMAb TSHR1 (M22) and there is stimulating activity or block effective antagonist (patent application WO2004/050708A2 and the SandersJ of active patients serum TRAb, Allen F, Jeffreys J, Bolton J, Richards T, Depraetere H, Nakatake N, Evans M, Kiddie A, Premawardhana LD, Chirgadze DY, Miguel RN, BlundellTL, Furmaniak J, Rees Smith B 2005 Characteristics of a monoclonalantibody to the thyrotropin receptor that acts as a powerfulthyroid-stimulating autoantibody antagonist.Thyroid 15:672-682).Although mouse monoclonal antibody 9D33 at least can show some features with the active patients serum TRAb of blocking-up, but it is a kind of by the mouse antibodies producing with TSHR immunization experiment animal, and therefore can not the interior autoantibody to the TSHR producing in TSHR autoimmune response process of authentic representative human body.As a kind of mouse monoclonal antibody, in order to be applied in human body, need to be by 9D33 humanization.Consider the cost and the complex operations that in humanization process, relate to, this may be disadvantageous.
The present invention results from generation and the character of human monoclonal autoantibody (5C9) of anti-TSHR, and this antibody is effective antagonist of a kind of TSH and pungency TRAb in patients serum.Be separated to 5C9 from the periphery lymphocyte of the patient with hypothyroidism and high-level TSHR autoantibody.In the following manner by this lymphocyte immortalization: infect and positive colony and mouse/human cell line are merged with Epstein epstein-Barr virus (EBV), to form stable clone.From the supernatant liquor IgG purification of cloned culture, assessment 5C9IgG is incorporated into TSHR and affects the ability of TSHR activity.Particularly, studied 5C9 and suppressed the ability that TSH is combined with TSHR, and suppressed the ability of the ring AMP stimulating activity of TSH.Moreover, also assess 5C9 and suppressed the ability of pungency or the patients serum TRAb of barrier and the combination of TSHR, and suppressed their bioactive ability.In addition, studied 5C9 in the purposes in the mensuration of TSHR antibody, TSH and related compound.Variable region (V district) gene of the heavy chain (HC) to 5C9 and light chain (LC) checks order, and specifies complementary determining region (CDR).
Summary of the invention
According to a first aspect of the invention, provide people's antibody of a kind of TSHR of separation, this antibody is the antagonist of a kind of TSH.
According to a second aspect of the invention, provide the humanized antibody of a kind of TSHR of separation, this antibody is the antagonist of a kind of TSH.
Be " a kind of according to antibody of the present invention " according to the antibody of first aspect present invention or second aspect.
Antibody according to the present invention can be a kind of antagonist of thyroid stimulating antibody.
Can there is patients serum TSHR autoantibody according to the antibody of the present invention TSH antagonist feature of---a kind of TSH antagonist---.
Antibody according to the present invention can be the antagonist of TSH and the antagonist of thyroid stimulating antibody.
Can there is patients serum TSHR autoantibody according to the antibody of the present invention antagonist feature of---antagonist of thyroid stimulating antibody---.
Can be a kind of TSH, M22 or TSHR is had the antibody of stimulating activity or has the active antibody of blocking-up and the inhibitor of TSHR or its a part of combination according to antibody of the present invention.TSHR part can comprise LRD or its sizable part.Preferably, a kind of antibody of this combination capable of blocking.
Antibody according to the present invention can be monoclonal antibody or recombinant antibody, or comprises its fragment or be made up of its fragment, is the antagonist of a kind of TSH.Can comprise such V according to antibody of the present invention
hdistrict, i.e. this V
hdistrict comprises one or more and is selected from the CDR 1, the CDR 2 that in Fig. 2, show or the CDR of CDR 3, or one or more aminoacid sequences basic and these CDR homologies.In addition or in addition, can comprise such V according to antibody of the present invention
ldistrict, i.e. this V
ldistrict comprises one or more and is selected from the CDR 1, the CDR 2 that in Fig. 3, show and the CDR of CDR 3, or one or more aminoacid sequences basic and these CDR homologies.
Can have approximately 10 according to antibody of the present invention and people's total length TSHR
10the binding affinity of L/mol.Preferably, can have approximately 10 according to antibody of the present invention and people's total length TSHR
9the binding affinity of L/mol.
The present invention can help skilled in the art to understand the amynologic mechanism that orders about pungency and the development of barrier TSHR autoantibody and generation.In addition, the present invention can help skilled in the art to understand and have the TSHR autoantibody of Tiroidina stimulating activity and have the molecular difference between the active TSHR autoantibody of blocking-up.In addition, treatment treatment process of the present invention and pharmaceutical composition provide new Tiroidina associated conditions therapy.
A kind of preferred antibody of the present invention is 5C9.Be surprised to find that 5C9 can suppress the constitutive activity of thyrotropin receptor, that is to say in the situation that not there is not thyrotropic hormone or M22, in test system, produced ring AMP.This may be particularly conducive to treatment remaining in Tiroidina or thyroid carcinoma cell that shift, and especially, aspect stoping or postponing regrowth, this is because the constitutive activity of thyrotropin receptor can cause these cells to grow more quickly.
Term used herein " antibody " and homology term for example " Multiple Antibodies " based on context comprise the bound fraction based on immunoglobulin (Ig), for example mono-clonal and polyclonal antibody, single-chain antibody and multi-specificity antibody, and comprise that those skilled in the art can be used for replacing the bound fraction of this bound fraction based on immunoglobulin (Ig), for example domain antibodies, double-chain antibody, IgG Δ C
h2, F (ab ')
2, Fab, scFv, V
l, V
h, dsFv, miniantibody, three chain antibodies, four chain antibodies, (scFv)
2, scFv-Fc and F (ab ')
3(Holliger P, Prospero T, Winter G 1993 " Diabodies:small bivalent and bispecificantibody fragments " Proc Natl Acad Sci USA 90:6444-6448.), (Carter PJ2006 " Potent antibody therapeutics by design " Nat Rev Immunol 6:343-357).
Term " TSHR " refers to the total length thyroid stimulating hormone receptor with the aminoacid sequence being shown in Fig. 4, or it with variant or the fragment of thyrotropin receptor height homology.Preferably, this variant and fragment and be shown in aminoacid sequence in Fig. 4 and have the homology of 70-99.9%.
According to a further aspect in the invention, provide a kind of Nucleotide, described Nucleotide comprises:
A) coding is according to the nucleotide sequence of the antibody of first aspect present invention;
B) the antibody V of coding as shown in Fig. 2 or 3
hstructural domain, antibody V
lthe nucleotide sequence as shown in Fig. 2 or 3 of the aminoacid sequence of structural domain or CDR; Or
C) nucleotide sequence height homology and a) or b) coding are with at least about 10
9the nucleotide sequence of the antibody that the avidity of L/mol is combined with TSHR.
According to a further aspect in the invention, providing a kind of comprises according to the carrier of the Nucleotide of above aspect of the present invention.
Described carrier can be a kind of plasmid, virus or their fragment.The carrier of the known number of different types of those skilled in the art.
According to a further aspect in the invention, providing a kind of comprises according to the cell of the separation of antibody of the present invention, Nucleotide and/or carrier; The cell of described separation can be expressed a kind of according to antibody of the present invention.Preferably, the emiocytosis one of described separation is according to antibody of the present invention.Preferably, according to the cell of separation of the present invention from a stable hetero hybridoma cells be.
According to another aspect of the invention, provide determine the TSHR autoantibody of concentration and comprise the composition according to antibody of the present invention a kind of comprising.This composition can comprise a kind of TSHR autoantibody with TSH antagonistic activity of definite concentration, and comprises a kind of according to antibody of the present invention.
Or, can comprise a kind of TSHR autoantibody---thyroid stimulating antibody antagonist of definite concentration according to the composition of this aspect of the present invention, and comprise a kind of according to antibody of the present invention.Composition can comprise a kind of TSHR autoantibody with TSH antagonistic activity---a thyroid stimulating antibody antagonist of definite concentration, and comprises a kind of according to antibody of the present invention.
According to a further aspect in the invention, provide a kind of thin compound of medicine that carries out the treatment of Tiroidina associated conditions for giving mammalian subject, comprised a kind of according to antibody of the present invention and a kind of pharmaceutically useful carrier.Described Tiroidina associated conditions can be selected from Thyroid Activity excessively, hyperthyroidism, circumscribed myxedema, thyroid carcinoma and the thyroiditis of graves' ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotrophin induction.
Pharmaceutical composition according to the present invention can be used for the administration to people.Preferably, pharmaceutical composition according to the present invention to experimenter's immunity system without significant adverse effect.
Can comprise other thyrotropin receptor antagonist according to pharmaceutical composition of the present invention.A kind of suitable other thyrotropin receptor antagonist is disclosed 9D33 in WO2004/050708.
Consider that pharmaceutical composition of the present invention can have various ways.What be used for the treatment of Tiroidina associated conditions can be a kind of injectable form according to pharmaceutical composition of the present invention.The pharmaceutical composition according to the present invention that is used for the treatment of circumscribed myxedema is preferably a kind of topical form.The pharmaceutical composition according to the present invention that is used for the treatment of graves' ophthalmopathy is preferably the form of eye drops.
Pharmaceutical composition of the present invention comprises according to any antibody of the present invention and pharmaceutically useful any carrier, adjuvant or vehicle.Can be used for the pharmaceutical carrier of pharmaceutical composition of the present invention, adjuvant and vehicle include but not limited to ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein (as human serum albumin), buffer substance (as phosphoric acid salt), glycine, Sorbic Acid, potassium sorbate, the partial glycerol ester mixture of saturated vegetable fatty acid, water, salt or ionogen (as protamine sulfate), Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, silicon sol, Magnesium Trisilicate, polyvinylpyrrolidone, cellulose base material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, wax, polyethylene polyoxypropylene block polymer, polyoxyethylene glycol and lanolin.
Pharmaceutical composition of the present invention can pass through oral administration, non-through enteral administration, by spraying inhalation, topical, by eye drop administration or eye ointment administration, rectal administration, intranasal administration, orally administering, vagina administration or via implanting holder administration.Our preferred oral administration or drug administration by injection.That term used herein " non-through intestines " comprises is subcutaneous, in intracutaneous, intravenously, intramuscular, intraarticular, synovial membrane, in breastbone, in sheath, intralesional and intracranial injection or infusion techn.
Described pharmaceutical composition can be the form of sterile injectable preparation, as the aqeous suspension of sterile injectable or oil suspension.This suspension can use suitable dispersion agent or wetting agent (as Tween 80) and suspending agent to make according to techniques known in the art.Sterile injectable solution or suspension that sterile injectable reagent is also made up of the outer acceptable thinner of nontoxic intestines or solvent, as be dissolved in the solution form of 1,3 butylene glycol.Spendable acceptable vehicle and solvent are N.F,USP MANNITOL, water, ringer's solution and isotonic sodium chlorrde solution.In addition, aseptic fixed oil is often used as solvent or suspending medium.For this purpose, the fixed oil of any gentleness be can use, synthetic direactive glyceride or double glyceride comprised.Lipid acid (as oleic acid and glyceride derivative thereof) can be used for preparing injectable agent, as the acceptable oils of natural pharmacy (as sweet oil or Viscotrol C) especially its polyoxy ethylization form.These oil solutions or suspension also can contain long-chain alcohol thinner or dispersion agent as Ph.Helv or similar alcohol.
Pharmaceutical composition of the present invention can any oral acceptable medicine type oral administration, includes but not limited to capsule, tablet, aqeous suspension and solution.For tablet for oral use, conventional carrier comprises lactose and W-Gum.Conventionally also add lubricant as Magnesium Stearate.For capsule form for oral use, useful thinner comprises lactose and dry W-Gum.In the time that aqeous suspension is oral administration, its activeconstituents is combined with emulsifying agent and suspension agent.If need, also can add some sweeting agent and/or seasonings and/or tinting material.
Pharmaceutical composition of the present invention also can rectal administration suppository form administration.Can be by compound of the present invention is mixed to prepare these compositions with suitable non-irritating excipient, described vehicle is at room temperature solid-state but is liquid under rectal temperature, thereby thereby can in rectum, dissolve and discharge active ingredient.This material includes but not limited to theobroma oil, beeswax and polyoxyethylene glycol.
In the time that required treatment comprises the easy region arriving of local dispenser or organ, the topical of pharmaceutical composition of the present invention is particularly useful.For local skin administration, described pharmaceutical composition should be prepared with containing the suitable ointment suspending or be dissolved in the active ingredient in carrier.Include but not limited to mineral oil, liquid petroleum, white oil, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water for the carrier of the compounds of this invention local application.Or described pharmaceutical composition can be prepared with containing the suitable lotion or the creme that suspend or be dissolved in the active compound in carrier.Suitable carrier includes but not limited to mineral oil, Arlacel-60, polysorbate60, cetyl esters wax, palmityl alcohol, 2-Standamul G, phenylcarbinol and water.Pharmaceutical composition of the present invention also can be used for lower intestinal tract by rectal suppository preparation or with suitable enema forms.The present invention also comprises local through skin plaster.
Pharmaceutical composition of the present invention can nasal spray or the form administration of inhalation.These compositions are prepared by the technology of knowing according to field of pharmaceutical preparations, and can be prepared as salt brine solution with phenylcarbinol or other suitable preservatives, absorption enhancer, fluorocarbon and/or other solubility promoters as known in the art or the dispersion agent that strengthen bioavailability.
According to another aspect of the invention, provide a kind of generation according to the method for antibody of the present invention, described method comprises cultivates one or more according to the cell of separation of the present invention, thereby by antibody described in described cell expressing.Preferably, described antibody is by described emiocytosis.
According to another aspect of the invention, provide a kind of in mammalian subject or be derived from the method for the treatment of Tiroidina associated conditions in this experimenter's cell, described method comprises described experimenter or described cell is contacted with antibody of the present invention.
According to a further aspect in the invention, provide a kind of thyroid stimulating antibody that suppresses in the Tiroidina of mammalian subject to stimulate the method for TSHR, described method comprises described experimenter is contacted with antibody of the present invention.Preferably, stop the combination of thyroid stimulating antibody and TSHR.
According to a further aspect in the invention, provide a kind of thyroid stimulating antibody method that TSHR is combined outside Tiroidina that suppresses in mammalian subject, described method comprises described experimenter is contacted with antibody of the present invention.The outer TSHR of described Tiroidina can be present in after described experimenter's socket of the eye before tissue and/or shin bone in tissue.In the time being applied in the method, antibody of the present invention is preferably blocked the combination of TSHR autoantibody and the outer TSHR of Tiroidina.
According to a further aspect in the invention, a kind of method for the treatment of thyroid carcinoma in Tiroidina or that shift in experimenter or in the thyroid cell that is derived from experimenter is provided, described method comprises described cancer cells contacted with antibody of the present invention, and object is in described cell, to suppress composing type thyrotropin receptor activity.Preferably, stop or postpone the regrowth of thyroid carcinoma cell.
A kind of excessive method of Thyroid Activity that treatment is caused by composing type Tiroidina activity in experimenter or in the thyroid cell that is derived from experimenter is also provided, described method comprises described experimenter or thyroid cell contacted with antibody of the present invention, and object is that to suppress the Thyroid Activity that caused by composing type Tiroidina activity excessive.
The experimenter who treats in each method of the invention described above is preferably people.
According to a further aspect in the invention, provide the purposes of antibody according to the present invention in treatment Tiroidina associated conditions.Or, the purposes of antibody according to the present invention in the medicament for the preparation for the treatment of Tiroidina associated conditions is provided.
Also provide a kind of in medical therapy, use according to antibody of the present invention.Particularly, provide antibody according to the present invention to be used for the treatment of the purposes of Tiroidina associated conditions.Described Tiroidina associated conditions can be selected from Thyroid Activity excessively, hyperthyroidism, circumscribed myxedema, thyroid carcinoma and the thyroiditis of graves' ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotrophin induction.
According to a further aspect in the invention, a kind of method of the TSHR of sign antibody is provided, comprise the combination situation of determining TSHR antibody to be test and having the polypeptide of TSHR related amino acid sequence, wherein said method relates to a method steps, and this step comprises use antibody of the present invention.Preferably, described method comprises the effect of determining that antibody of the present invention is combined with this polypeptide to TSHR antibody.The polypeptide with the aminoacid sequence that TSHR is relevant preferably includes total length people TSHR.
According to a further aspect in the invention, provide a kind of for characterizing the method for TSH and associated molecule, comprise the combination situation of determining TSH or associated molecule to be test and having the polypeptide of TSHR related amino acid sequence, wherein said method relates to a step and comprises the method steps that uses antibody of the present invention.
Can be ELISA form for the method and the above-described methods involving that characterize TSHR antibody or TSH.
According to a further aspect in the invention, a kind of amino acid whose method of TSHR relating in the TSHR autoantibody process that is combined as antagonist of determining is provided, described method comprises: one article of polypeptide with the combinative TSHR related amino acid sequence of antibody of the present invention is provided, modifies at least one amino acid and determine the effect of such modification to described antibodies in the relevant aminoacid sequence of described TSHR.
Modify a method for antibody of the present invention, described method comprise modify at least one amino acid of described antibody and determine such modification for the effect of the combination of TSHR correlated series.Preferably, select the modified TSHR antibody of the avidity enhancing to TSHR.
According to a further aspect in the invention, provide a kind of qualification can suppress the method for the molecule that thyroid stimulating antibody is combined with TSHR, described method comprises provides at least one antibody according to the present invention as object of reference.Preferably, selection can stop the molecule to be test that thyroid stimulating antibody is combined with TSHR.
Also provide a kind of qualification can suppress the method for the molecule that Tiroidina blocking antibody is combined with TSHR, described method comprises provides at least one antibody according to the present invention as object of reference.Preferably, selection can stop the molecule that Tiroidina blocking antibody is combined with TSHR.
Brief description of the drawings
Now by reference to accompanying drawing 1-4, only antibody according to the present invention and method are described in the mode of illustration, wherein:
The 3 width series of drawing of Fig. 1 show from patient (n=40) and healthy tax blood person's (n=10) the serum pair of suffering from Graves disease
125i-5C9IgG,
125i-TSH or
125the comparison of the effect that I-M22 and the duct ligation applying through TSHR close.
A
125i-5C9IgG in conjunction with
125i-TSH combination
B
125i-5C9IgG in conjunction with
125i-M22IgG combination
C
125i-TSH in conjunction with
125i-M22IgG combination;
The sequence that Fig. 2 provides is the variable region sequences of 5C9 heavy chain (HC)
The oligonucleotide sequence of the 5C9HC that a shows with the form that does not annotate and annotate.In the form of annotation, be each complementary determining region (CDR) that frame shows for the sequence of PCR primer; And the constant region shown in black matrix.
B is derived from not annotate and the aminoacid sequence of the 5C9HC of the oligonucleotide sequence that annotation form shows;
The sequence that Fig. 3 provides is the variable region sequences of 5C9 light chain (LC):
The oligonucleotide sequence of 5C9LC that a shows not annotate and to annotate the form of (according to Fig. 2).
B is derived from not annotate and the aminoacid sequence of the 5C9LC of the oligonucleotide sequence that annotation form shows;
Has Fig. 4 shown consensus amino acid sequences (registration number P16473, the http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi of people TSHR? db=protein & val=62298994).
Embodiment
Method
Separation of lymphocytes and the cloning of human monoclonal TSHR autoantibody 5C9
Use is recorded in the method in WO2004/050708A2, separates routinely mono-clonal autoantibody 5C9.At first from collecting from suffering from hypothyroidism have patient's the blood sample of high-level TRAb and be separated to lymphocyte (obtaining the approval of local Ethics Committee) in postpartum.Described in WO2004/050708A2, with Epstein epstein-Barr virus (EBV) (European Collection of Cell Cultures-ECACC; Porton Down, SP40JG, UK) infect described lymphocyte, and be incubated on mouse macrophage feeder layer.Immortalization lymphocyte and mouse/people hybrid cell line K6H6/B5 (ECACC) fusion that will secretion TSHR autoantibody, by limited dilution cloning 4 times, to obtain single colony.By suppressing
125the TSH of I-mark and the combination of TSHR, detected the situation that exists of TSHR autoantibody (WO2004/050708A2) in the cell culture supernatant that is in the different cloning stages.The mono-clonal that produces TSHR autoantibody is expanded, and be used for carrying out autoantibody purifying from culture collection supernatant liquor.
The purifying of 5C9IgG goods and mark
Use a-protein affinity chromatography at MabSelect (GE Healthcare, UK) on, according to (Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premwardhana LD, Chirgadze DY, Nunez Miguel R, Blundell TL, Furmaniak J, Rees Smith B 2004 Characteristics of a human monoclonalautoantibody to the thyrotropin receptor:sequence structure andfunction.Thyroid 200414:560-570) record purifying 5C9IgG from culture supernatants, and by SDS-polyacrylamide gel electrophoresis (PAGE) assessment purity.
Use radial diffusion to measure (The Binding Site; Birmingham, B296AT, UK) determine the heavy chain isotype of 5C9, specificity mouse monoclonal antibody (the Sigma-Aldrich Company Ltd by Western blotting with anti-human κ chain and anti-human λ chain, Poole, UK) determine light chain isotype.
At room temperature, to be dissolved in 20mmol/L sodium acetate solution (pH 4.5) 10mg/mL 5C9IgG with according to manufacturer specification (Perbio Science UK Ltd, Cramlington, UK) the immobilized pepsin oscillation incubation prepared 4.5 hours.After this, remove immobilized pepsin by centrifugal (at room temperature, 1000 × g, 5 minutes), and supernatant liquor is used to 300mmol/L NaCl at 4 DEG C, 10mmol/LTris-HCl (pH 7.5) dialysed overnight.Use Sephacryl S-300High Resolution Matrix (GEHealthcare, Chalfont St Giles, UK), separate and comprise 5C9F (ab ')
2with mixture after the dialysis of a small amount of complete IgG.The 5C9F of purifying (ab ') by this way
2goods do not comprise complete IgG, and this analyzes to judge by SDS-PAGE and HPLC gel-filtration.
Moreover, at 37 DEG C, use the Cys of final concentration 100mmol/L to F (ab ')
2subdue 1 hour.At room temperature stop this reaction 30 minutes with the iodo-acetamide of final concentration 50mmol/L.As described above, use Sephacryl S-300 column purification F (ab ').The F of purifying (ab ') goods do not comprise F (ab ') by this way
2, this analyzes to judge by SDS-PAGE and HPLC gel-filtration.
In addition, with mercury papain (mercuripapain) (Sigma, UK) process 5C9IgG, dialysed into 50mmol/L NaCl with enzyme/protein ratio of 1: 100, in Tris-HCl (pH 9.0), and by anionresin Sepharose (purchased from the Q-Sepharose Fast flow of GE Healthcare) post, to separate complete IgG or Fc from Fab goods.By SDS-PAGE and gel-filtration (Sephacryl S-300 post; As above-mentioned) analysis show, in described Fab goods, complete IgG do not detected.
As Sanders J, Oda Y, Roberts S, Kiddie A, Richards T, Bolton J, McGrathV, Walters S, Jaskolski D, Furmaniak J, Rees Smith B 1999.The interactionof TSH receptor autoantibodies with
125described in I-labelled TSH receptor.Journalof Clinical Endocrinology and Metabolism.199984:3797-3802, with
125i mark 5C9IgG, or with vitamin H hydrazides (Perbio Science, Cramlington, UK) mark 5C9IgG.
Right
125i-TSH,
125i-M22 or
125the inhibition that I-5C9 is combined with TSHR
As the description in WO2004/050708A2, use the pipe applying through TSHR to carry out measuring in conjunction with suppressing.In this mensuration, at room temperature by 100 μ L test samples (Mab goods, patients serum or unlabelled TSH) and the initial damping fluid of 50 μ L (RSR Ltd) oscillation incubation 2 hours gently in the pipe applying through TSHR.After sucking-off, by described pipe washing, and add 100 μ L's
125the protein (5 × 10 of I-mark
4cpm) and at room temperature oscillation incubation 1 hour.Then, by described pipe sucking-off, washing and count in gamma counter.
Calculate according to the following formula the inhibition to the combination through labelled protein :-
Control material is that the health of mixing is contributed blood person serum, individual health tax blood person's serum or the other materials of indicating in each experimental result.
The Scatchard that 5C9IgG is combined with TSHR analyzes
At room temperature, 50 μ L be will be dissolved in and unlabelled 5C9IgG and 50 μ L in damping fluids (50mmol/L NaCl, 10mmol/L Tris (pH7.8) and 1%Triton X-100) measured
125the 5C9IgG of I-mark (be dissolved in and measure in damping fluid 30,000cpm) 2 hours (occurring under these conditions maximum combined) of oscillation incubation, sucking-off in the pipe applying through TSHR, measure damping fluid washed twice and count in gamma counter with 1mL.The concentration of IgG to combination and combination/concentration of free IgG maps (Scatchard G 1949 The attraction of proteins for small molecules andions.Annals of the New York Academy of Sciences 51:660-672), with the association constant of deriving.
The analysis of the stimulation that ring AMP is produced
Described in W02004/050708A2, test the ability that 5C9IgG and other goods produce at the Chinese hamster ovary with people TSHR transfection (CHO) cell moderate stimulation ring AMP.By about each cell expressing 5 × 10
4individual or about 5 × 10
5the Chinese hamster ovary celI of individual TSHR is with each hole 3 × 10
4individual cell is inoculated in 96-orifice plate, at (the Invitrogen Ltd of the DMEM without foetal calf serum, Paisley, UK) in, adapt to, then (100 μ L are diluted in ring AMP and measure damping fluid to add test sample (TSH, IgG or patients serum), without Hank ' the s buffer salt solution of NaCl, the 3-isobutyl-1-methylxanthine (pH 7.4) that comprises 1g/L glucose, 20mmol/L HEPES, 222mmol/L sucrose, 15g/L bovine serum albumin and 0.5mmol/L), and at 37 DEG C, hatch 1 hour.Removing after testing liquid, by lysis and by a kind of ring AMP concentration of measuring in described split product of following two methods: 1) use from GE Healthcare Chalfont St Giles, the Biotrak enzyme immunoassay system of UK; With 2) use from Assay Designs; Cambridge Bioscience, the Direct Cyclic AMP Correlate EIA test kit of UK.Result is expressed as the ring AMP of pmol/mL in product of cell lysis (200 μ L), or is expressed as fmol/ cell hole.
To the active measurement of antagonist (blocking-up)
Assess the ability that 5C9IgG and other goods suppress pig (p) TSH, natural human (h) TSH and recombinant chou people (rh) TSH, MAb M22 and the patients serum TRAb stimulating activity in the Chinese hamster ovary celI of expressing TSHR.The realization of this point is by the situation that existing and not having 5C9IgG (or other test products), the relatively stimulatory effect of TSH, M22 or TRAb.By this mensuration of carrying out mentioned above, except the 5C9 (or other test products) 50 μ L being diluted in ring AMP mensuration damping fluid is added into described cell hole, then add TSH, M22 or the patients serum's (suitably dilution in ring AMP measures damping fluid) of 50 μ L, and stimulate as described above mensuration to hatch and test.
Except 5C9, other MAb in this mensuration, are test and from patient's the serum with blocking-up type TRAb.
The analysis of variable region gene
Described in WO2004/050708A2, use from 1 × 10
7total RNA prepared by the hetero hybridoma cells of individual secretion 5C9IgG, to produce the mRNA for RT-PCR (reverse transcription PCR) reaction, has determined the variable region gene of 5C9 heavy chain and light chain.Use V-base (http://vbase.mrc-cpe.cam.ac.uk/) design specific IgG 1HC and κ LC justice and the antisense strand Oligonucleolide primers of Medical Research Council, and by Invitrogen (Paisley, PA49RF, UK) synthetic these primers.Carry out under the following conditions RT reaction: at 50 DEG C 15 minutes; Then carry out 40 PCR circulation, each circulation is: at 94 DEG C at 15 seconds, 50 DEG C at 30 seconds and 72 DEG C 30 seconds.By DNA product cloning to pUC18, and with Sanger-Coulson method (the Sanger F that checks order, Nicklen S, Coulson AR 1977 DNA sequencing with chain terminating inhibitors.Proceedings of the National Academy of Sciences of the USA 74:5463-5467).Use Ig blast (http://www.ncbi.nlm.nih.gov/igblast/) by V region sequence and existing people Ig Gene sequence comparison.
The analysis of the effect to the amino acid mutation in people TSHR sequence to 5C9 activity
Be described in patent application WO2006/016121A for the method for introducing TSHR sequence by specifically suddenling change.And, use Flp-In system that the TSHR construct transfection of sudden change is also described in WO2006/016121A to Chinese hamster ovary celI.
The Flp-In-CHO cell of expressing wild-type or mutation T SHR is inoculated in 96 orifice plates, and blocks as described above the ability of the stimulating activity of TSH, M22 or patients serum TRAb for testting 5C9 goods.
For the mensuration based on 5C9 of TSHR antibody, TSH and associated molecule
The 5C9IgG of 2.55mg/mL is dialysed to 100mmol/L sodium phosphate buffer (pH 8.5), and react with EZ-Link NHS-LC-Biotin (Perbio), the mol ratio of IgG and vitamin H is 1/10.At room temperature, test serum sample (75 μ L) vibration in the elisa plate hole (RSRLtd) applying through TSHR-(per minute vibration 500 times) is hatched 2 hours.Removing after described test sample washing, add biotin labeled 5C9IgG (2ng in 100 μ L), and at room temperature nonoscillatory continues to hatch 25 minutes.Hole is emptied, washed and add 100 μ L streptavidin-peroxidase (10ng in 100 μ L; RSR Ltd), and at room temperature nonoscillatory continues to hatch 20 minutes.Then,, by hole washing 3 times, add substrate tetramethyl benzidine (tetramethyl the benzidine) (TMB of peroxidase; 100 μ L; RSR Ltd), and under lucifuge and non-oscillating condition, at room temperature hatch 30 minutes.Then, add the 0.5mol/L H of 50 μ L
2sO
4to stop this reaction, use elisa plate readout instrument under 450nm, to read the absorbancy of each plate hole.Calculate according to the following formula the inhibition to the combination of 5C9IgG-vitamin H :-
Result
Separation and the cloning of 5C9 secretory cell system
By the lymphocyte obtaining from 20mL blood samples of patients (27 × 10
6) with EBV infect, and in 48 orifice plates with every hole 1 × 10
6individual cell spreads on mouse macrophage feeder layer.Infect latter the 13rd day at EBV, it is right in plate hole supernatant liquor, to monitor
125the inhibition of I-TSH combination.Cell from positive hole is expanded, merge with K6H6/B5 hybridoma cell line, and sprawl in 96 orifice plates.Having obtained stable a generation has
125the clone of the antibody of I-TSH binding inhibition activity, and carry out 4 times and clone again.From allos hybridoma culture supernatants, the monoclonal antibody that is named as 5C9 of purifying is the subclass IgG1 with κ light chain.
The TSHR of 5C9IgG is in conjunction with active and blocking-up activity
In table 1, shown, the 5C9IgG of different concns suppresses TSH, the M22 of mark of mark or the ability that the 5C9 self of mark is combined with TSHR.As shown in table 1, in the case of using as few as the 5C9IgG of 0.005 μ g/mL,
125there is 12% inhibition in the combination of I-TSH, and this inhibition increases in dose-dependent mode, suppresses promotion to 84% in the time of the 5C9 of 100 μ g/mL.This can with donor's serum IgG pair
125the inhibition of I-TSH combination is similar; In the time of 0.05mg/mL, suppress 13%, and increase to and suppress 94% in dose-dependent mode in the time of 1mg/mL.Under the situation of donor's blood plasma, when with 1: 160 healthy pooled serum of contributing blood person of dilution,
125i-TSH, in conjunction with having occurred 16% inhibition, suppresses as 95% when taking dilution in 1: 10.
5C9IgG is also right
125i-M22IgG be coated in TSHR on pipe be combined with effect (table 1).In the time of the 5C9IgG of 0.01 μ g/mL,
125having there is 9% inhibition in the combination of I-M22IgG, and causes that this inhibition increases in dose-dependent mode in the time increasing concentration, in the time of 100 μ g/mL, is up to 85%.Donor's serum IgG is effectively under 0.01mg/mL, causes 9% inhibition, and increase to 89% inhibition in dose-dependent mode in the time of 1mg/mL.When with dilution in 1: 320, it is right that donor's serum blood plasma demonstrates
12513% inhibition of I-M22IgG combination suppresses 91% when with dilution in 1: 10.
Unlabelled 5C9IgG can suppress in dose-dependent mode
125the combination (suppress 11% during at 0.005 μ g/mL, rise at most in the time of 100 μ g/mL and suppress 88%) (table 1) of I-5C9 and the pipe applying through TSHR.
125the combination of I-5C9 is also suppressed by donor's serum IgG (in the time of 0.05 μ g/mL, to suppress 15%, in the time of 1mg/mL, suppress 91%), and suppressed by the dilution of donor's blood plasma (suppress 10% when the extent of dilution of 1: 320, in the time of the extent of dilution of 1: 10 92%).
In table 2a-c, shown, 5C9IgG blocks the ability of the stimulation to ring AMP of TSH and M22-mediation in the Chinese hamster ovary celI of expressing TSHR.Pig TSH (3ng/mL) is the generation of stimulating ring AMP (19,020 ± 2154fmol/ cell hole consumingly; Mean value ± SD; N=3) (table 2a).In the case of there is the 5C9IgG of 0.1 μ g/mL, the stimulating activity of pig TSH is reduced to 11874 ± 4214fmol/ cell hole (mean value ± SD; N=3), and this retarding effect depends on 5C9 concentration, only produces the ring AMP (table 2a) of 2,208 ± 329fmol/ cell hole in the time there is the 5C9 of 1 μ g/mL.Lymphocyte donor's serum also stimulates and has potent answering the ring AMP of TSH mediation in CHO-TSHR cell.As show as shown in 2a, in the situation that there is the dilution donor's serum of 1:10 (the total serum IgG concentration under this extent of dilution is 1.43mg/mL), the inhibition that ring AMP is produced is reduced to about 6000fmol/ cell hole, and this is similar to the 19000fmol/ cell hole not existing under serum.This effect is corresponding to the effect of the purifying 5C9IgG of about 0.37 μ g/mL (utilize the extent of dilution curve calculation of the effect of different concns 5C9IgG shown in table 2a and obtain).This shows, the ability this respect producing at blocking-up TSH stimulating ring AMP, and specific activity donor's serum IgG of purifying 5C9IgG exceeds about 3900 times.
The fragment of 5C9, for example 5C9F (ab ')
2with 5C9Fab be also the inhibition that effective TSH stimulates.Particularly, 5C9IgG, 5C9F (ab ')
2tSH with 5C9Fab when the 100 μ g/mL stimulates that to suppress activity be identical (showing 2b).In the time of 10 μ g/mL, all three kinds of goods: 5C9IgG, 5C9F (ab ')
2with 5C9Fab be also the potent inhibitor of TSH stimulating activity, but 5C9IgG seems than 5C9F (ab ')
2or more effectively (table 2b) of 5C9Fab.
M22Fab (3ng/mL) is the potent stimulator (9 of a kind of AMP of ring, 432 ± 822fmol/ cell hole) (table 2c), and in the situation that there is 5C9, the stimulatory effect of M22Fab is suppressed in dose-dependent mode, the level of ring AMP is reduced to 1,298 ± 134fmol/ cell hole in the case of there is the 5C9IgG of 0.1 μ g/mL.When what M22 stimulated suppresses to come across the 5C9 of 100 μ g/mL completely (table 2c).
Scatchard analytical table is clear,
125the 5C9 of I-mark is with 4 × 10
10the association constant of L/mol is incorporated into TSHR.
Serum T RA b couple
125the inhibition that I-5C9IgG is combined with TSHR
In table 3 and Fig. 1, shown, serum T RAb suppresses
125i-5C9IgG and the ability of closing through the duct ligation of TSHR coating.Also for than show identical serum T RAb couple than object
125i-TSH in conjunction with and
125the effect of I-M22IgG combination.
125i-5C9IgG is not obviously suppressed (suppressing scope 3.4-18.9%) from 10 different healthy serum (N1-N10, table 3) of contributing blood person from the combination of TSHR.?
125i-TSH and
125during I-M22 suppresses to measure (table 3), right from 40 Robert Graves patients' that are TSHR autoantibodies serum (G1-G40, table 3)
125i-5C9 closes the inhibition of (suppressing scope 22.0-85.2%) with the duct ligation applying through TSHR, reached recently from the healthy larger degree (table 3) of serum of contributing blood person.Patients serum TRAb suppresses
125i-5C9IgG,
125i-TSH or
125the ability that I-M22IgG is combined with TSHR is suitable, Pearson correlation coefficient r=0.95 (
125i-5C9IgG with
125i-TSH; Fig. 1 a) and r=0.95 (
125i-5C9IgG with
125i-M22IgG; Fig. 1 b).Fig. 1 c shows identical serum pair
125i-TSH and
125the comparison (Pearson coefficient r=0.99) that I-M22 IgG suppresses.
These experiments show, the combination of 5C9IgG and TSHR can be suppressed effectively by serum T RAb, and serum T RAb is similar to their retarding effects to TSH or M22 combination to the retarding effect of 5C9IgG combination.
Table 4 shown the different dilution TSH of having block active patients serum TRAb (B1-B5) and there is the patients serum TRAb (S1, S2 and S4) of strong Tiroidina stimulating activity right
125the inhibition of I-5C9IgG combination.
125the combination of I-5C9IgG is by serum B1-B5, and serum S1, S2 and S4 suppress in dose-dependent mode.Same these blocking-up and stimulation serum also suppress in dose-dependent mode
125i-TSH and
125i-M22IgG combination, and under same serum dilution, all three kinds of inhibition percentage ratios through tagged ligand are that suitable (table 4a & is b).
These results show to have the combination that stimulating activity and the active TSHR autoantibody of blocking-up can suppress 5C9 and TSHR.
There is the mouse MAb couple of TSH binding inhibition activity
124the inhibition that I-5C9IgG is combined with TSHR
Test has
125the different mouse TSHR Mab of I-TSH binding inhibition activity suppress
125the ability that I-5C9 is combined with TSHR, and with right
125the effect of I-M22IgG combination compares (table 5).As shown in table 5, there is inhibition
125i-TSH combination and
125all Mab of the ability of I-M22IgG combination have also suppressed
125i-5C9 combination, but right in the situation that of some Mab
125i-5C9 combination and
125it is right that the retarding effect of I-M22 combination is weaker than
125the effect of I-TSH combination.
These experiments show, have a large amount of overlappingly between the binding site of 5C9 on TSHR and the binding site of mouse TSHR MAb on TSHR, and described mouse TSHR MAb has the ability that suppresses TSH combination.
The effect of the stimulation that 5C9IgG produces ring AMP patients serum in the Chinese hamster ovary celI of expressing TSHR
As shown in table 2,5C9IgG can block TSH or the stimulation of M22 to ring AMP level in the Chinese hamster ovary celI of expressing TSHR.In the experiment of different series, test the effect of the stimulating activity of 5C9IgG to patients serum TRAb, the results are shown in table 6a.Serum T 1-T9 and T11-T18 at CHO-TSHR cell moderate stimulation the generation of ring AMP, and with contrast hatching of MAb IgG (to the specific 2G4 of Anti-thyroid Peroxidase) their stimulating activity do not had to effect.But in the case of existing 5C9IgG (the 200 μ g/mL of 50 μ L), the stimulating activity of all test serum all significantly reduces (table 6a).
The dose response effect of 6 kinds of different serum (T1, T6, T3, T19, T20 and T21) is shown in table 6b-g.In these experiments, the 5C9IgG of concentration range 0.1 μ g/mL-100 μ g/mL has caused the dose-dependent reduction of serum stimulation activity, and in all test serum except serum T3, the effect of 5C9IgG is all suitable with the effect of 9D33, and the latter is the mouse monoclonal antibody (being recorded in WO2004/050708A2) with the active anti-TSHR of blocking-up.For the situation of T3 serum (table 6a & 6d), in the case of there is the 5C9IgG of 100 μ g/mL, observe approximately 50% the inhibition that ring AMP is produced, and the 9D33IgG of 100 μ g/mL causes almost suppressing completely.This shows, between the epi-position of identifying, may have some fine differences at 5C9 and 9D33.
The effect that 5C9IgG produces basis (not being excited) ring AMP in the Chinese hamster ovary celI of expressing TSHR
Except suppressing the stimulating activity of TSH and TSHR antibody, 5C9 has suppressed the generation of ring AMP in the situation that not there are not these thyroid stimulants.Particularly, table 6b has shown, the ring AMP of the 1207 ± 123fmol/ cell hole producing in the situation that existing 100 μ g/mL to contrast monoclonal IgG (2G4) is reduced to 301 ± 38fmol/ cell hole in the case of there is the 5C9IgG of 100 μ g/mL.The effect of 9D33IgG is less, in the situation of existence 100 μ g/mL 9D33IgG, produces 721 ± 183fmol/ cell hole.In the independent experiment that is shown in table 6d, 6e, 6f and 6g, obtain similar result.This shows, basis activity or the constitutive activity of 5C9IgG to TSHR has significant effect.
The effect of the stimulation in the Chinese hamster ovary celI of the TSHR that 5C9IgG comprises amino acid mutation in expression, ring AMP being produced
In Fig. 7, show, in CHO-TSHR cell, the effect of the ability of the ring AMP stimulating activity of the monamino acid sudden change of TSHR to 5C9 blocking-up pig TSH.Particularly, the effect of having studied the stimulation of 5C9 to ring AMP generation in such Chinese hamster ovary celI, the following residue of this expressing cho cell is mutated into TSHR:Lys 58, Ile 60, Arg 80, Tyr 82, Thr 104, Arg 109, Lys129, Phe 134, Asp 151, Lys 183, Gln 235, Arg 255, Trp 258 and the Ser 281 of L-Ala.In addition, the effect that charged sudden change is changed: Arg80Asp, Asp151Arg, Lys183Asp and Arg255Asp in the situation that of following TSHR residue, are studied, wherein, according to conventional representation, the amino-acid residue being replaced and the position in the polypeptide of original series thereof were indicated before replacing amino-acid residue.Research in the past shows, the charged sudden change of TSHR Asp160Lys changed and causes TSHR to lose the responsiveness to TSH, and to the response of M22 uninfluenced (patent application WO2006/016121A).Therefore, use the stimulator of M22 as ring AMP, in CHO-TSHR cell, studied TSHR Asp160Lys sudden change to the bioactive effect of 5C9 (table 7l).
In studied all TSHR sudden changes, only find that 3 sudden changes have impact on 5C9 as the ability of antagonist.Lys129 is mutated into Ala (table 7h) and causes 5C9IgG to completely lose the ability of the stimulation of blocking-up TSH to ring AMP generation.
And TSHR sudden change Lys183Ala has caused that 5C9IgG blocks active part and reduces; In the time testting with TSHR Lys183Ala sudden change, when 1 μ g/mL, observe 28% inhibition that TSH is stimulated, be different from 84% while using wild-type TSHR and suppress (table 7m).Even in the time of the 5C9IgG of 100 μ g/mL, in the experiment with TSHR Lys183Ala sudden change, only detect the part that TSH is stimulated and suppress (43%), and in the time of this concentration, in the experiment with wild-type receptor, observe the blocking-up completely (93%) (table 7m) to TSH stimulating activity.If the Lys183 of positive charge is mutated into the aspartic acid of negative charge, the effect (table 7m) of observing while so the bioactive effect of 5C9 being similar to use Lys183Ala sudden change.This has illustrated, Lys183 is important to the biological activity of 5C9.Under the situation of Asp151Ala, observe that 5C9IgG blocking-up is active has occurred slight reduction: 49% suppresses in the time of 1 μ g/mL, be different from 88% inhibition (table 7j) of wild-type.But in the case of there is the 5C9IgG of 100 μ g/mL, this activity is identical with wild-type TSHR.In the time that the Asp151 of negative charge is mutated into the arginine of positive charge, can not observe the active remarkable reduction of 5C9IgG blocking-up (table 7k).
TSHR sudden change can be compared to the effect of 9D33 activity with TSHR sudden change to the effect of 5C9 activity.Described in patent application W02006/016121A, TSHR sudden change Lys 58, Arg 80, Tyr 82, Arg 109, Lys 129 and Phe 134 all have effect to 9D33 activity.But, except Lys 129, in these sudden changes, do not have one also 5C9 activity to be had to effect.Moreover, described in patent application WO2006/016121A, affecting in the sudden change of M22 activity (Arg 80, Tyr 82, Glu 107, Arg 109, Lys 129, Phe 130, Lys 183, Tyr 185, Arg 255 and Trp 258), except Lys 129, do not have one to affect 5C9 activity.In addition, suddenly change active to 5C9 in M22 activity of Lys 183 has effect partly, but to 9D33 activity without effect.
These results show in following TSHR residue to exist different, and described residue is for important TSHR residue interacting with Tiroidina pungency people antibody M22, with mouse blocking antibody 9D33 and with people's blocking antibody 5C9.Therefore, 5C9 and other combinations with the TSHR antibody (as 9D33) of antagonistic activity may be a kind of special efficient manner, for suppressing the constitutive activity of stimulating activity, other stimulator and/or TSHR of patients serum TRAb.
The variable region sequences of 5C9
The sequential analysis of gene to coding 5C9 shows, HC V district gene is from VH3-53 family, and D gene is from D2-2 family, and J gene is from JH4 family.Under the situation of LC, V district gene from O12 family and J district gene from JK2 germline.The Nucleotide of HC and aminoacid sequence are shown in Fig. 2 a and 2b, and the Nucleotide of LC and aminoacid sequence are shown in Fig. 3 a and 3b.
Compared with germline sequence, in HC gene order, there is somatic mutation; Specifically 1 silent mutation in 1 silent mutation and 1 Substitution and FWR4 in 2 Substitutions, CDR3 in 1 silent mutation in FWR1, CDR2.But HC V region sequence is characterised in that two Insert Fragments; Long 6 base pairs between V gene and D gene, another is long 15 base pairs between D gene and J gene.Therefore, (Fig. 2 b) for long 5 amino acid of HC CDR1, long 16 amino acid of CDR2 and long 18 amino acid of CDR3.
In LC sequence, have: in 1 silent mutation in FWR1, CDR1 in 1 Substitution, CDR3 1 Substitution and between V gene and J gene 6 Insert Fragments that base pair is long.LC CDR1 by 11 amino acid form, CDR2 is made up of 7 amino acid and CDR3 is made up of 10 amino acid that (Fig. 3 b).
For detection of the mensuration based on 5C9 of TSH or TRAb
In table 8, shown the ELISA example for detection of TSHR autoantibody, this ELISA is the combination with the plate hole applying through TSHR based on 5C9IgG-vitamin H.In this mensuration, all also to 5C9IgG-vitamin H, combination is positive all samples being positive for the inhibition of TSH-vitamin H combination.Moreover unit/L value of absorbance signal, inhibition percentage ratio and derivation in TSH-determination of biotin and 5C9-determination of biotin is suitable (table 8).
5C9IgG is expressing in the Chinese hamster ovary celI of TSHR Mouse thyroid pungency monoclonal antibody (mouse TSHR MAb; The effect of stimulating activity TSMAb)
As shown in table 9,5C9IgG can block the stimulation that all 5 kinds of test TSMAb (1,2,4,5 and 7) produce ring AMP.For example, TSMAb1 (table 9) stimulating ring AMP level to 18.94 ± 7.4pmol/mL, and there is the ring AMP that only produces 1.24 ± 0.07pmol/mL in the 5C9IgG situation of 100 μ g/mL.This can with in the case of there is the ring AMP level that contrasts 16.5 ± 1.1pmol/mL MAb 2G4 of 100 μ g/mL compare (table 9).
In table 9 and the ring AMP level of below showing to show in 10-15 represent with pmol/mL, the ring AMP level of each cell hole is: pmol/mL ÷ 5 (representing the sample from each mensuration hole of 200 μ L).
The effect of 5C9IgG stimulating activity to natural human TSH and recombinant human TSH in the Chinese hamster ovary celI of expressing TSHR
In table 2 and table 10, show the ability that 5C9IgG blocking-up pig TSH stimulates ring AMP.In addition, 5C9IgG has shown that inhibition natural human TSH is (from National Institute for BiologicalStandards and Control, South Mimms, the NIBSC of Potters Bar EN6 3QG UK is with reference to goods 81/565) and both abilities (table 10) to ring AMP stimulation of restructuring TSH (NIBSC is with reference to goods 94/674).Particularly, the stimulation that the restructuring of 100ng/mL or natural human TSH produce ring AMP needs the 5C9IgG of 0.1-1.0 μ g/mL, to obtain the inhibition completely that ring AMP is produced.In the time gathering for separating of the blood of 5C9, the level of recycle system TSH in donor's serum is 160mU/L (approximately 32ng/mL), the result of gained (table 2a and table 10) shows, exists the 5C9IgG of 32-320ng/mL and will block the recycle system TSH of this level completely in serum.It is right to use
125the inhibition that I-M22 is combined with TSHR, assess the level of TSHR autoantibody in described donor's serum (as Nakatake N, Sanders J, Richards T, Burne P, Barrett C, Dal PraC, Presotto F, Betterle C, Furmaniak J, described in Rees Smith B 2006 Estimationof serum TSH receptor autoantibody concentration and affinity.Thyroid 16:1077-1084), for 1700ng/mL (120ng/mg), than stimulate required 5C9 concentration to exceed several times for the Tiroidina of blocking TSH.
5C9IgG has the effect to basis (not being excited) ring AMP activity in the Chinese hamster ovary celI of TSSHR of activation sudden change S281I, I568T and A623I in expression
In the time not there is not thyroid stimulant (being TSH or TSHR antibody), 5C9IgG has the generation that can reduce ring AMP in the Chinese hamster ovary celI that activates the TSHR suddenling change in expression.As show as shown in 11a, in the situation that not there is not 5C9, the concentration that discharge ring AMP has in the Chinese hamster ovary celI of TSHR of activation sudden change S281I in expression is 9.90 ± 1.51pmol/mL, and it is reduced to 4.17 ± 0.60pmol/mL in the case of there is the 5C9IgG of 0.01 μ g/mL, and be reduced to 3.44 ± 0.63pmol/mL in the case of there is the 5C9IgG of 1 μ g/mL.Barrier mouse TSHR MAb 9D33 and contrast MAb 2G4 almost do not have effect (table 11a).
Use TSHR activation sudden change I568T to obtain similar result (table 11b), this result has shown the discharge ring AMP concentration of 21.39 ± 5.31pmol/mL.In the time adding the 5C9IgG of 1 μ g/mL, it is reduced to 5.29 ± 0.75pmol/mL, is different from respectively to 20.52 ± 0.95pmol/mL and 21.65 ± 1.99pmol/mL under the situation of adding 2G4IgG and 5C9IgG.Under the situation of the third TSHR activation mutation research (being A623I) that is 36.89pmol/mL in discharge ring AMP concentration, ring AMP level is reduced to 16.43 ± 1.27pmol/mL by the 5C9IgG that adds 1 μ g/mL, and this is different from the contrast IgG 2G4 (28.96 ± 2.29pmol/mL) of 1 μ g/ml or the 9D33IgG (40.09 ± 7.73pmol/mL) of 1 μ g/mL (table 11c) without effect substantially.
These results show, different from mouse barrier MAb 9D33, the 5C9 couple of ring AMP relevant to TSHR activation sudden change produces has significant effect, even if in the time of the different piece of described sudden change at TSHR (A623I in encircling in the I568T in the S281I in extracellular domain, the second extracellular loop of membrane spaning domain and the 3rd cell of membrane spaning domain).
Comparison to following effect in the Chinese hamster ovary celI of expressing wild-type TSHR: the mixture of 5C9, mouse TSHR barrier monoclonal antibody 9D33 and these two kinds of antibody is for the effect of the stimulation that ring AMP is produced being mediated by TSH
As shown in experiment neutralization table 12 above, be low to moderate the people TSHR barrier MAb 5C9 of concentration of 1 μ g/mL and mouse TSHR barrier MAb 9D33 and have the ability of the TSHR ring AMP stimulating activity of blocking-up TSH in CHO-TSHR cell.As shown in the experiment 1-5 of table 12,9D33IgG and 5C9IgG can be added for the effect of the stimulation to ring AMP being mediated by TSH.As the TSH of two different concns (3ng/mL and 0.3ng/mL) experiment 1-3 and the experiment 4 and 5 of table 12 (respectively) when stimulating, there is same additive effect.
Comparison to following effect in the Chinese hamster ovary celI of expressing wild-type TSHR: the mixture of 5C9, mouse TSHR barrier monoclonal antibody 9D33 and these two kinds of antibody is for the effect of the stimulation that ring AMP is produced being mediated by M22
As indicated earlier, the stimulation to ring AMP that 5C9 and 9D33 can also suppress to be mediated by M22Fab in CHO-TSHR cell.9D33IgG and 5C9IgG are (table 13, the experiment 1-4) that can be added for the effect of the stimulation to ring AMP being mediated by M22.When the M22Fab of two different concns (3ng/mL and 0.3ng/mL) when stimulating (respectively the experiment 1 of table 13 and 2 and experiment 3 and 4), there is same additive effect.
5C9IgG and 9D33IgG are similar (tables 12 and 13) for the additive effect of the stimulation that ring AMP is produced by TSH and M22 mediation.
All express in the Chinese hamster ovary celI of a large amount of wild-type TSHR the effect of 5C9 to basis (not being excited) ring AMP activity at each cell
Each cell all expresses about 5 × 10
5standard C HO clone (each cell expressing about 5 × 10 that in the Chinese hamster ovary celI system of individual acceptor and experiment above, (for example showing 9-13) used
4individual TSHR) compare, show basis (not being excited) the ring AMP of higher level, be respectively 47.1 ± 11.7pmol/mL and about 1.0pmol/mL.The clone that uses each cell all to express a large amount of acceptors has been assessed 5C9IgG and the effect of 9D33IgG to wild-type TSHR basis activity.Cause the 0-5.3% of discharge ring AMP activity to suppress (table 14 with the hatching of negative control antibody of 9D33IgG and anti-GAD (5B3); Experiment 1), show that this barrier mouse MAb 9D33 or contrast MAb produce and there is no effect discharge ring AMP in the Chinese hamster ovary celI of expressing wild-type TSHR.But, under the situation of 5C9IgG, observe the obvious inhibition (table 14 to discharge ring AMP activity; Experiment 2), 0.1 μ g/mL and 10 μ g/mL cause respectively 45.7% and 74.6% inhibition.In addition, all express in the Chinese hamster ovary celI of a large amount of wild-type TSHR at each cell, 5C9Fab and 5C9F (ab ') are also effective inhibitor (table 14 tests 3) of discharge ring AMP activity.For example, the 5C9Fab of 1 μ g/mL and 100 μ g/mL has shown respectively 39% and 61% the inhibition that discharge ring AMP is produced, and is all different from 48% the inhibition (table 14 tests 3) of the 5C9F (ab ') of 100 μ g/mL.
There is the active patients serum TSHR autoantibody of antagonist (i.e. blocking-up) and there is the effect to basis (not being excited) ring AMP activity in the Chinese hamster ovary celI of TSHR of activation sudden change I568T in expression
In the case of existing the ring AMP of 20.5 ± 8.7pmol/mL to measure damping fluid; the discharge ring AMP of TSHR I568T cell produces and substantially can not be subject to adding the impact of contributing blood person's normal pooled serum (NPS) or the health of 3 kinds of Different Individual tax blood person's serum (N1-N3) from health, and described serum is to test with 1/10 and 1/50 extent of dilution.Compared with producing with the discharge ring AMP in the situation that existing ring AMP to measure damping fluid, the discharge ring AMP in the situation that there is NPS and N1-N3 serum produces the inhibition (table 15) that has shown 0-14%.But, there is the next inhibition (table 15) of observing the 23-89% that discharge ring AMP is produced of situation of 4 kinds of different serum (B2-B5) of high-level blocking-up type TRAb in existence.In the situation that there is 5C9IgG (1 μ g/mL), observe 83% the inhibition to TSHR I568T discharge ring AMP activity.In table 15, also shown, 2 kinds of barrier serum (B3 and B4) have in expression the dose response effect in the Chinese hamster ovary celI of TSHR of I 568T sudden change, discharge ring AMP being produced.
These results show, 5C9 has the TSHR blocking-up living features of patient's barrier TSHR autoantibody, the inhibition to discharge ring AMP generation in relevant TSHR activation mutant I 568T particularly.
The patients serum TSHR autoantibody with antagonistic activity has the effect to basis (not being excited) ring AMP activity in the Chinese hamster ovary celI of TSHR of activation sudden change S281I in expression
In the situation that existing ring AMP to measure damping fluid, the discharge ring AMP of TSHR S281I cell is produced as 11.2 ± 2.0pmol/mL, and there is no effect (table 16) with the hatching of health tax blood person serum (1/10 or 1/50 dilution) of health tax blood person's pooled serum or individuality.On the contrary, the 4 kinds of different serum (B2-B5) that there is high level blocking-up type TRAb in existence, observe the inhibition (table 16) of the 31-56% that discharge ring AMP is produced.Using in the experiment of TSHR S281I, the 5C9IgG of 1 μ g/mL has caused 71% the inhibition to discharge ring AMP activity.
The patients serum TSHR autoantibody with antagonistic activity has the effect to basis (not being excited) ring AMP activity in the Chinese hamster ovary celI of TSHR of activation sudden change A623I in expression
In the situation that existing ring AMP to measure damping fluid, the discharge ring AMP of TSHR A623I cell is produced as 43.5 ± 11.2pmol/mL, and is not substantially subject to contribute with health the impact (table 17) of hatching of the serum of blood person's pooled serum or individuality.In these experiments, from 4 kinds of different serum (B2-B5) with high-level blocking-up type TRAb hatch caused to ring AMP-inhibition (table 17) of 1%-56%.This can compare with 49% the inhibition being caused by the 5C9IgG of 1 μ g/mL in identical experiment.
All express 5 × 10 at each cell
5in the Chinese hamster ovary celI of individual wild-type TSHR, the effect of the patients serum TSHR autoantibody with antagonistic activity to basis (not being excited) ring AMP activity
In this serial experiment, the discharge ring AMP all expressing in the Chinese hamster ovary celI of higher number wild-type TSHR at each cell is produced as 28.1 ± 0.7pmol/mL.When described cell and 1/10 dilution healthy when contributing blood person's pooled serum or individual serum (N1-N3) and hatching, the scope of discharge ring AMP level is the 99%-146% of the ring AMP level in the situation that existing ring AMP to measure damping fluid, and this scope is 93%-137% in the time of 1/50 extent of dilution.In 4 kinds of test serum with blocking-up type TSHR autoantibody, a kind of serum (B2) produces without effect (table 18) discharge ring AMP.Under the situation of two kinds of serum (B3 and B5), the level of ring AMP is higher than the level (table 18) of observing in the situation that existing ring AMP to measure damping fluid.Well-grounded, serum B3 and B5 comprise a kind of mixture that has stimulating activity and block active TSHR autoantibody.On the contrary, with 1/10 and 1/50 dilution serum B4, discharge ring AMP is produced and has an obvious retarding effect, be respectively 31% and 61% (table 18) of discharge ring AMP level compared with level in the situation that there is ring AMP mensuration damping fluid.With exist ring AMP to measure compared with level damping fluid in the situation that, this can compare with 33% level in the case of the 5C9IgG that has 1 μ g/mL (table 18).
Total 5C9IgG is with wild-type TSHR or the effect discharge ring AMP is produced in having the Chinese hamster ovary celI of TSHR transfection of activation sudden change, with use from the patient to blocking-up type TSHR autoantibodies serum time the effect observed similar.But individual patients serum's effect is different (tables 19) under the situation of difference sudden change.Under the situation of wild-type TSHR, some serum have shown stimulatory effect, and described stimulatory effect is presumably by the existence of TSHR pungency antibody and blocking antibody and causes (table 19).
The effect of the stimulation in the Chinese hamster ovary celI of the TSHR that 5C9 comprises amino acid mutation in expression, ring AMP being produced
Have in the Chinese hamster ovary celI of TSHR of amino acid mutation in expression, the effect of the stimulation that 5C9 is produced ring AMP extends to the sudden change comprising with down to L-Ala: Asp43, Glu61, His105, Glu107, Phe130, Glu178, Tyr185, Asp203, Tyr206, Lys209, Asp232, Lys250, Glu251, Thr257, Arg274 and Asp276 (table 20a-p is also summarized in table 21).
Amino acid Asp43, Glu61, His105, Glu107, Tyr185, Asp232 and the Thr275 of TSHR are mutated into L-Ala, and the ability that 5C9IgG is suppressed to TSH stimulating ring AMP generation does not have effect.By TSHR Phe130, Glu178, Asp203, Tyr206, Lys250, Glu251 and Asp276 are mutated into L-Ala, reduce 5C9 and suppressed the ability that TSH stimulating ring AMP produces.Under the situation of Lys209Ala and these two sudden changes of Asp274Ala, 5C9IgG suppresses the ability rising that TSH stimulating ring AMP produces.
(table 7,20 and 21) in a word, with wild-type TSHR comparison, 10 TSHR residue Lys 129, Phe130, Asp151, Glu178, Lys183, Asp203, Tyr206, Lys250, Glu251 and Asp276 have reduced the ability of the ring AMP stimulation of 5C9 inhibition TSH.The Lys129 of TSHR and Asp203 sudden change have shown maximum effect and have caused the inhibition completely to 5C9 activity.
In the Chinese hamster ovary celI of expressing wild-type TSHR and in the Chinese hamster ovary celI of TSHR Asp203Ala of expressing sudden change, compare the effect of barrier serum B2-B5 for the stimulation to ring AMP generation being mediated by TSH
The blocking effect of the 5C9 of 1 μ g/mL to wild-type TSHR (92% that inhibition that the ring AMP of TSH induction is stimulated) is reduced to 4% (table 22) under the situation of TSHR Asp203Ala sudden change.
Barrier serum B4 activity is not subject to the impact of TSHR Asp203Ala sudden change, and compared with wild-type TSHR, under the situation of TSHR Asp203Ala, observes the inhibition percentage ratio that the ring AMP of TSH induction is stimulated slightly low while using barrier serum B2 and B3.
Under the situation of a kind of serum B5, observe the inhibition percentage ratio that the ring AMP of TSH induction is stimulated and occurred remarkable reduction; Different in the TSHR of wild-type and sudden change, suppress respectively 69% and 30%.
TSHR Asp203Ala sudden change is greater than the effect to barrier serum active to the effect of 5C9 activity, but the blocking-up activity of 3/4 test serum is affected to different degree.This may show, for block the binding site of TSHR autoantibody and 5C9 have overlapping, but there are some differences from the actual TSHR amino acid of different serum contacts.
Sum up and conclusion
Above-described experiment provides such evidence, for example, can block the stimulating activity of different thyroid stimulants according to antibody of the present invention (5C9), described thyroid stimulant comprises people and mouse TSHR pungency antibody, natural humans and animals TSH and recombinant human TSH.Moreover, such evidence is provided, the i.e. antibody of two kinds of different blocking-up types---people MAb 5C9 and mouse MAb 9D33, in the time being test separately, in the Chinese hamster ovary celI of expressing TSHR, there is the ability of blocking-up by the stimulation to ring AMP of TSH or M22 mediation, and in the time being blended in one, demonstrate the be added blocking effect to TSH or M22 stimulation.
New effect for example, according to antibody of the present invention (5C9) to TSHR basis (not being excited) ring AMP activity.By use have the TSHR transfection CHO cell of higher baseline ring AMP level more experimental study these effects, confirmed for example, according to antibody of the present invention (5C9) blocking effect to discharge ring AMP activity.Moreover, showing, some serum that contain the TSHR autoantibody with blocking-up (antagonist) activity have the ability of blocking-up discharge ring AMP activity in the cell of these TSHR transfections.These experiments also provide such evidence, for example, according to antibody of the present invention (5C9 and serum TSH R blocking antibody) the discharge ring AMP activity of suddenling change relevant to activation TSHR are had to blocking effect.
These results highlight, 5C9 is a kind of people Mab of the feature that shows the TSHR autoantibody of blocking type, it is the representative of the patients serum TSHR autoantibody relevant to autoimmune thyroid disease.
Described experiment also makes to identify some such TSHR amino acid, and these TSHR amino acid are important for the blocking-up activity according to antibody of the present invention.
Generally speaking, these results show, for example, compare from the TSHR blocking antibody occurring in the different serum from trouble patients with autoimmune thyroid disease according to antibody of the present invention (5C9), demonstrate similar TSHR in conjunction with the active and similar biological effect to TSHR function.Therefore, owing to thering is feature and the biological activity of serum barrier TSHR autoantibody, according to antibody of the present invention (for example 5C9) can be in various clinical illness for by TSHR inactivation.These illnesss comprise the TSHR activation that the TSHR activation of TSH mediation, TSHR activation by the mediation of Tiroidina pungency TSHR autoantibody, basis (be not excited, composing type) TSHR is active and suddenly change relevant to reactivity TSHR.Therefore, for example, can be applicable to treat and control activating relevant illness to TSHR mentioned above according to antibody of the present invention (5C9); For example Graves disease, graves' ophthalmopathy, the hyperthyroidism being caused by TSHR activation sudden change, hyperthyroidism, thyroid carcinoma and the metastasis of thyroid carcinoma being caused by TSH horizontal abnormality (pathological or pharmacological).
Table 1
5C9IgG, contribute lymphocyte person IgG, contribute lymphocyte person blood plasma for
125i-TSH,
125i-M22IgG or
125i-5C9IgG and the inhibition of closing through the duct ligation of TSHR coating
| Sample | 125I-TSH (suppressing %) | 125I-M22IgG (suppressing %) | 125I-5C9 (suppressing %) |
| Donor's blood plasma (being diluted in HBD mixture) | |||
| 1/5 | 97 | 92 | 93 |
| 1/10 | 95 | 91 | 92 |
| 1/20 | 82 | 85 | 82 |
| 1/40 | 57 | 75 | 56 |
| 1/80 | 30 | 53 | 29 |
| 1/160 | 16 | 29 | 13 |
| 1/320 | 8 | 13 | 10 |
| Donor IgG (being diluted in 1mg/mL HBD mixing IgG) | |||
| 1mg/mL | 94 | 89 | 91 |
| 0.5mg/mL | 90 | 86 | 89 |
| 0.25mg/mL | 63 | 79 | 64 |
| 0.1mg/mL | 29 | 51 | 26 |
| 0.05mg/mL | 13 | 29 | 15 |
| 0.025mg/mL | 2 | 17 | 5 |
| 0.01mg/mL | 5 | 9 | 1 |
| 5C9IgG (being diluted in 100 μ g/mL2G4) | |||
| 100μg/mL | 84 | 85 | 88 |
| 50μg/mL | 76 | 81 | 80 |
| 25μg/mL | 67 | 69 | 71 |
| 10μg/mL | 54 | 60 | 59 |
| 5μg/mL | 47 | 53 | 54 |
| 2.5μg/mL | 42 | 43 | 49 |
| 1μg/mL | 37 | 31 | 42 |
| 0.5μg/mL | 33 | 32 | 41 |
| 0.1μg/mL | 29 | 22 | 34 |
| 0.05μg/mL | 25 | 19 | 30 |
| 0.01μg/mL | 13 | 9 | 15 |
| 0.005μg/mL | 12 | 5 | 11 |
| 0.001μg/mL | 3 | 2 | 3 |
The tracer of mark with the % of being on average combined of the pipe applying through TSHR is:
Using
125in the experiment of I-TSH 14%;
Using
125in the experiment of I-M22IgG 21%;
And using
125in the experiment of I-5C9 20%;
HBD mixture=health is contributed the mixture of blood person serum;
2G4=contrast IgG (the people MAb of antithyroid peroxidase).
Table 2a
The effect that the TSH that 5C9IgG or tax lymphocyte person serum IgG produce for ring AMP in the Chinese hamster ovary celI of expressing people TSHR stimulates
| Sample | IgG concentration | Ring AMP (fmol/ cell hole; Mean value ± SD, n=3) |
| Only pTSH (3ng/mL) | 19020±2154 | |
| Only 5C9IgG | 1μg/mL | 141±5 |
| 5C9IgG+pTSH(3ng/mL) | 1μg/mL | 2208±329 |
| 5C9IgG+pTSH(3ng/mL) | 0.5μg/mL | 4754±876 |
| 5C9IgG+pTSH(3ng/mL) | 0.1μg/mL | 11874±4214 |
| 5C9IgG+pTSH(3ng/mL) | 0.08μg/mL | 14525±3690 |
| 5C9IgG+pTSH(3ng/mL) | 0.06μg/mL | 13290 1 |
| 5C9IgG+pTSH(3ng/mL) | 0.04μg/mL | 13928±1572 |
| 5C9IgG+pTSH(3ng/mL) | 0.02μg/mL | 16432±9286 |
| 5C9IgG+pTSH(3ng/mL) | 0.01μg/mL | 18969±5308 |
| Only encircle AMP and measure damping fluid | 207±51 | |
| Donor's serum a1/10 | 1.43mg/mL b | 1102±46 |
| Donor's serum 1/10+pTSH (3ng/mL) | 1.43mg/mL | 5931±350 |
| Donor's serum 1/20+pTSH (3ng/mL) | 0.715mg/mL | 16886±728 |
| Donor's serum 1/30+pTSH (3ng/mL) | 0.477mg/mL | 16453±3455 |
| Donor's serum 1/40+pTSH (3ng/mL) | 0.358mg/mL | 17716±1753 |
| Donor's serum 1/50+pTSH (3ng/mL) | 0.286mg/mL | 17928±4772 |
| Donor's serum 1/100+pTSH (3ng/mL) | 0.143mg/mL | 18226±2268 |
1unitary determination
aby shown in by donor's serum be diluted in ring AMP measure damping fluid
bundiluted serum IgG concentration by Nephelometric Determination is 14.3mg/mL
5C9IgG and TSH are diluted in ring AMP and measure in damping fluid.
Table 2b
5C9IgG, F (ab ')
2inhibition with the ring AMP generation to TSH induction in the Chinese hamster ovary celI of expressing people TSHR of Fab fragment
Embodiment 1
| Sample | Ring AMP (fmol/ cell hole; Mean value ± SD, n=3) |
| Only encircle AMP and measure damping fluid | 586±148 |
| Only TSH 3ng/mL | 18557±363 |
| Only 5C9Fab 100 μ g/mL | 235±35 |
| 5C9Fab?100μg/mL+TSH | 938±93 |
| 5C9Fab?10μg/mL+TSH | 1283±239 |
| Only 5C9F (ab ') 2100μg/mL | 204±12 |
| 5C9F(ab′) 2100μg/mL+TSH | 877±195 |
| 5C9F(ab′) 210μg/mL+TSH | 916±188 |
| Only 5C9IgG 100 μ g/mL | 237±54 |
| 5C9IgG?100μg/mL+TSH | 754±177 |
| 5C9IgG?10μg/mL+TSH | 247±115 |
| 2G4IgG?100μg/mL+TSH | 6082 1 |
Embodiment 2
| Sample | Ring AMP (fmol/ cell hole; Mean value ± SD, n=3) |
| Only encircle AMP and measure damping fluid | 584±111 |
| Only TSH 3ng/mL | 19363±5198 |
| Only 2G4IgG 100 μ g/mL | 608±169 |
| 2G4IgG?100μg/mL+TSH | 18147±972 |
| 2G4IgG?10μg/mL+TSH | 18114±6544 |
| Only 5C9F (ab ') 2100μg/mL | 414±22 |
| 5C9F(ab′) 2100μg/mL+TSH | 1058±223 |
| 5C9F(ab′) 210μg/mL+TSH | 1333±443 |
| Only 5C9IgG 100 μ g/mL | 338±108 |
| 5C9IgG?100μg/mL+TSH | 1109±375 |
| 5C9IgG?10μg/mL+TSH | 723±71 |
| 5C9Fab?100μg/mLonly | 212±37 |
| 5C9Fab?100μg/mL+TSH | 867±127 |
| 5C9Fab?10μg/mL+TSH | 4131±776 |
1the mean value of double sample
2G4=contrast IgG (the people MAb of antithyroid peroxidase).
Antibody and TSH goods are diluted in ring AMP and measure in damping fluid.
Table 2c
5C9IgG and 9D33IgG in the Chinese hamster ovary celI of expressing people TSHR for the effect of stimulation ring AMP being produced by TSH or M22Fab
| Test sample | Ring AMP produces (fmol/ cell hole; Mean value ± SD n=3) |
| Only encircle AMP and measure damping fluid | 473±21 |
| Only pTSH 3ng/mL | 12,270±980 |
| Only 5C9IgG 100 μ g/mL | 426±27 |
| Only 5C9IgG 10 μ g/mL | 360±53 |
| Only 5C9IgG 1 μ g/mL | 376±18 |
| Only 5C9IgG 0.1 μ g/mL | 404±42 |
| Only 5C9IgG 0.01 μ g/mL | 578±65 |
| Only 5C9IgG 0.001 μ g/mL | 554±47 |
| 5C9IgG?100μg/mL+pTSH?3ng/mL | 1094±70 |
| 5C9IgG?10μg/mL+pTSH?3ng/mL | 1028±47 |
| 5C9IgG?1μg/mL+pTSH?3ng/mL | 1872±168 |
| 5C9IgG?0.1μg/mL+pTSH?3ng/mL | 3920±464 |
| 5C9IgG?0.01μg/mL+pTSH?3ng/mL | 15,050±386 |
| 5C9IgG?0.001μg/mL+pTSH?3ng/mL | 14,147±1,310 |
| Only 9D33IgG 100 μ g/mL | 626±127 |
| 9D33IgG?100μg/mL+pTSH?3ng/mL | 2,218±5 |
| Only M22Fab 3ng/mL | 9,432±822 |
| 5C9IgG?100μg/mL+M22Fab?3ng/mL | 354±56 |
| 5C9IgG?10μg/mL+M22Fab?3ng/mL | 638 1±190 |
| 5C9IgG?1μg/mL+M22Fab?3ng/mL | 956±169 |
| 5C9IgG?0.1μg/mL+M22Fab?3ng/mL | 1,298±134 |
| 5C9IgG?0.01μg/mL+M22Fab?3ng/mL | 9,978±919 |
| 5C9IgG?0.001μg/mL+M22Fab?3ng/mL | 11,614±393 |
| 9D33IgG?100μg/mL+M22Fab?3ng/mL | 1,048±10 |
1the mean value of double sample
9D33 is a kind of mouse antibodies that can block in the Chinese hamster ovary celI of expressing TSHR by the stimulation that ring AMP is produced of TSH and TRAb mediation.
Antibody and TSH goods are diluted in ring AMP and measure in damping fluid.
Table 3
125i-5C9IgG,
125i-TSH and
125i-M22IgG and combination and the inhibition of patient serum sample to it of pipe applying through TSHR
| Serum sample | 125I-5C9 is in conjunction with suppressing (%) | 125I-TSH is in conjunction with suppressing (%) | 125I-M22 is in conjunction with suppressing (%) |
| N1 | 15.7 | 0 | 9.3 |
| N2 | 3.3 | 1.2 | 7.2 |
| N3 | 15.3 | 1.9 | 0.7 |
| N4 | 4.0 | 4.2 | 0 |
| N5 | 18.9 | 6.3 | 11.4 |
| N6 | 5.9 | 2.9 | 1.4 |
| N7 | 17.1 | 7.2 | 9.4 |
| N8 | 5.5 | 0 | 0 |
| N9 | 11.2 | 4.2 | 2.9 |
| N10 | 10.5 | 6.5 | 5.8 |
| G1 | 79.6 | 83.6 | 80.5 |
| G2 | 75.8 | 77.5 | 73.5 |
| G3 | 82.2 | 77.5 | 78.1 |
| G4 | 77.7 | 74.9 | 74.8 |
| G5 | 77.0 | 73.6 | 71.5 |
| G6 | 64.7 | 71.6 | 69.1 |
| G7 | 75.6 | 74.3 | 66.0 |
| G8 | 73.7 | 74.7 | 77 |
| G9 | 76.1 | 78.5 | 79.2 |
| G10 | 75.9 | 75.8 | 69.9 |
| G11 | 81.6 | 82.5 | 79.7 |
| G12 | 71.6 | 76.6 | 73.9 |
| G13 | 72.3 | 71.1 | 70.2 |
| G14 | 81.9 | 85.8 | 80.9 |
| G15 | 84.9 | 85.3 | 84.4 |
| G16 | 80.5 | 84.9 | 81.7 |
| G17 | 85.0 | 86.9 | 85.3 |
| G18 | 84.9 | 85 | 84.2 |
| G19 | 85.1 | 87.3 | 85.4 |
| G20 | 84.4 | 89.3 | 87.7 |
| G21 | 77.6 | 84.9 | 77.0 |
| G22 | 67.1 | 59.7 | 61.5 |
| G23 | 57.5 | 62.2 | 59.5 |
| G24 | 65.7 | 67.1 | 64.4 |
| G25 | 59.3 | 56.3 | 62.3 |
| G26 | 38.4 | 67.7 | 69.4 |
| G27 | 22.0 | 59.1 | 58.9 |
| G28 | 68.3 | 69.7 | 72.8 |
| G29 | 40.9 | 54.2 | 50.4 |
| G30 | 71.2 | 69.1 | 72 |
| G31 | 62.2 | 59.2 | 62.0 |
| G32 | 46.0 | 40.6 | 49.2 |
| G33 | 44.0 | 25.9 | 37.2 |
| G34 | 52.0 | 48 | 55.0 |
| G35 | 60.1 | 54.4 | 60.7 |
| G36 | 29.3 | 31.4 | 43.2 |
| G37 | 49.0 | 44.1 | 45.5 |
| G38 | 40.1 | 26.7 | 29.8 |
| G39 | 66.4 | 54.7 | 58.9 |
| G40 | 48.8 | 48.5 | 48.5 |
N1-10=contributes blood person's serum from health
G1-G40=has the patient's of Graves disease history serum by oneself
Result is the mean value of very consistent twice mensuration
There is the combination in the situation of testting serum in A=wherein; There is the combination in healthy situation of contributing blood person's serum mixture (HBD mixture) in B=.
125i-5C9 in the situation that there is HBD mixture, show 20% combination,
125i-TSH in the situation that there is HBD mixture, show 12% combination and
125i-M22 shows 17% combination in the situation that there is HBD mixture.
Table 4a
Relatively have blocking-up or the patients serum of stimulating activity for
125i-5C9IgG,
125i-TSH and
125the inhibition of combination of I-M22IgG and the pipe applying through TSHR
| Working sample | 125I-5C9 is in conjunction with suppressing (%) | 125I-TSH is in conjunction with suppressing (%) | 125I-M22 is in conjunction with suppressing (%) |
| Measure verification thing | |||
| 40U/L | 87 | 90 | 84.4 |
| 8U/L | 62 | 67 | 63.0 |
| 2U/L | 15 | 27 | 25.6 |
| 1U/L | 4.3 | 15 | 14.8 |
| Positive control serum | 34 | 35 | 38.7 |
| Barrier serum | |||
| B1 | |||
| 1/5 | 93 | 95 | 91.7 |
| 1/10 | 92 | 94 | 89.2 |
| 1/20 | 91 | 91 | 85.9 |
| 1/40 | 85 | 80 | 78.8 |
| 1/80 | 67 | 55 | 67.4 |
| 1/160 | 33 | 33 | 45.9 |
| 1/320 | 20 | 17 | 26.4 |
| B2 | |||
| 1/5 | 92 | 91 | 86.6 |
| 1/10 | 85 | 85 | 79.5 |
| 1/20 | NT | 73 | 66.2 |
| 1/40 | 68 | 51 | 50.0 |
| 1/80 | 42 | 34 | 29.7 |
| 1/160 | 19 | 22 | 20.3 |
| 1/320 | NT | 13 | 7.8 |
| B3 | |||
| 1/5 | 89 | 93 | 85.5 |
| 1/10 | 82 | 84 | 76.3 |
| 1/20 | 62 | 64 | 61.7 |
| 1/40 | 37 | 44 | 42.5 |
| 1/80 | 14 | 26 | 26.2 |
| 1/160 | NT | 12 | 16.6 |
| 1/320 | NT | 7 | 8.9 |
| B4 | |||
| 1/5 | 93 | 94 | 90.3 |
| 1/10 | 93 | 95 | 88.5 |
| 1/20 | 92 | 93 | 85.1 |
| 1/40 | 89 | 89 | 81.1 |
| 1/80 | 78 | 76 | 72.1 |
| 1/160 | 56 | 54 | 56.9 |
| 1/320 | 34 | 37 | 39.9 |
| B5 | |||
| 1/5 | 94 | 93 | 90.3 |
| 1/10 | 91 | 92 | 87.0 |
| 1/20 | 87 | 87 | 82.2 |
| 1/40 | 74 | 72 | 71.7 |
| 1/80 | 56 | 47 | 54.6 |
| 1/160 | 31 | 29 | 36.4 |
| 1/320 | 19 | 17 | 21.4 |
| Pungency serum | |||
| S1 | |||
| 1/5 | 89 | 91 | 84.9 |
| 1/10 | 80 | 84 | 75.4 |
| 1/20 | 63 | 67 | 62.9 |
| 1/40 | 42 | 50 | 46.2 |
| 1/80 | 26 | 34 | 31.9 |
| 1/160 | 9 | 21 | 16.3 |
| 1/320 | 16 | 4 | 10.6 |
Table 4b
| Test sample | 125I-5C9 is in conjunction with suppressing (%) | 125I-TSH is in conjunction with suppressing (%) | 125I-M22 is in conjunction with suppressing (%) |
| Measure verification thing | |||
| 40U/L | 87.2 | 90.4 | 82.2 |
| 8U/L | 62.0 | 67.2 | 63.3 |
| 2U/L | 21.3 | 22.0 | 27.9 |
| 1U/L | 15.0 | 13.0 | 21.8 |
| Positive control serum | 34.2 | 31.4 | 38.6 |
| HBD mixture | 6.2 | -0.6 | 12.5 |
| S2 | |||
| Sterling | 91.8 | 95.1 | 88.6 |
| 1/5 | 76.7 | 84.5 | 72.7 |
| 1/10 | 62.3 | 71.4 | 66.4 |
| 1/20 | 48.2 | 57.0 | 51.9 |
| 1/40 | 31.8 | 39.3 | 41.4 |
| 1/80 | 21.3 | 19.4 | 32.9 |
| 1/160 | 16.8 | 9.7 | 25.1 |
| S4 |
| Sterling | 91.0 | 92.9 | 86.2 |
| 1/5 | 71.0 | 72.4 | 68.7 |
| 1/10 | 55.5 | 55.6 | 57.4 |
| 1/20 | 39.9 | 34.0 | 46.3 |
| 1/40 | 27.3 | 16.4 | 35.4 |
| 1/80 | 17.0 | 8.6 | 25.4 |
| 1/160 | 16.1 | 2.1 | 19.5 |
B1-B5 is for containing the high-caliber patients serum with the active TRAb of antagonist (blocking-up).
B3 is from the 5C9 serum of contributing lymphocyte person
S1, S2 and S4 are for containing the high-caliber patients serum with the active TRAb of agonist (stimulation).
Measuring verification thing 40U/L, 8U/L, 2U/L and 1U/L is that M22IgG contributes the dilution in blood person's serum mixture (HBD mixture) in health, and the activity representing with the NIBSC 90/672 of U/L is to assess with the inhibition of the combination of the pipe applying through TSHR by the TSH to through mark.
Wherein A=test sample; B=HBD mixture
NT=not test (N.T.)
1/5,1/10 etc. represents the dilution factor of test serum in HBD mixture, sterling=undiluted serum
In the situation that there is HBD mixture, about warp of 20%, 17% and 12%
125m22IgG, the 5C9IgG of I-mark and TSH are incorporated into respectively the pipe applying through TSHR.
Table 5
The mouse MAb of the anti-TSHR of different concns is for warp
125the TSH of I-mark,
125i-M22IgG and
125the inhibition of combination of I-5C9IgG and the pipe applying through TSHR
Antibody dilution is contributed in blood person's serum mixture (HBD mixture) in health.
There is the combination % in the situation of testting sample in A=wherein; There is the combination % in the situation of HBD mixture in B=.
1there is the mouse TSHR MAb of Tiroidina stimulating activity
2the mouse TSHR MAb (in table 2) of the stimulation that ring AMP is produced by TSH and TRAb mediation capable of blocking
3the people MAb (negative control) of antithyroid peroxidase MAb
4there is TSH and block active mouse TSHR MAb (can identify the epi-position being formed by TSHR amino acid 381-385)
5there is TSH and block active mouse TSHR MAb (can identify the epi-position being formed by TSHR amino acid 36-42)
6there is TSH and block active mouse TSHR MAb (can identify the epi-position being formed by TSHR amino acid 246-260)
3mouse Tg MAb (negative control)
In the situation that there is HBD mixture, about warp of 13%, 24% and 15%
1255C9IgG, the M22IgG of I-mark and TSH are incorporated into respectively the pipe applying through TSHR.
Table 6a
The effect of the stimulation that 5C9IgG produces ring AMP for TRAb in by patients serum in the Chinese hamster ovary celI of expressing people TSHR
Experiment 1a
| Test sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 378±21 |
| Only HBD mixture | 352±38 |
| HBD mixture+2G4IgG | 316±54 |
| HBD mixture+5C9IgG | 136±46 |
| Only T1 | 13734±580 |
| T1+2G4IgG | 10928±740 |
| T1+5C9IgG | 142±4 |
| Only T2 | 1716±185 |
| T2+2G4IgG | 1362±190 |
| T2+5C9IgG | 146±4 |
| Only T3 | 11722±1280 |
| T3+2G4IgG | 11948±3200 |
| T3+5C9IgG | 5660±790 |
| Only T4 | 6388±820 |
| T4+2G4IgG | 6022±710 |
| T4+5C9IgG | 188±65 |
| Only T5 | 3084±990 |
| T5+2G4IgG | 2152±240 |
| T5+5C9IgG | 152±15 |
| Only T6 | 14802±1475 |
| T6+2G4IgG | 10878±675 |
| T6+5C9IgG | 232±25 |
Experiment 1b
| Test sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 434±52 |
| Only HBD mixture | 518±216 |
| HBD mixture+2G4IgG | 378±34 |
| HBD mixture+5C9IgG | 178±47 |
| Only T7 | 7388±1250 |
| T7+2G4IgG | 5696±715 |
| T7+5C9IgG | ud |
| Only T8 | 1736 1 |
| T8+2G4IgG | 1392 1 |
| T8+5C9IgG | ud |
| Only T9 | 5052 1 |
| T9+2G4IgG | 5000 1 |
| T9+5C9IgG | ud |
Experiment 1c
| Sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 366±316 |
| Only HBD mixture | 646±62 |
| HBD mixture+2G4IgG | 496±42 |
| HBD mixture+5C9IgG | 294±92 |
| Only T13 | 4030±1146 |
| T13+2G4IgG | 3330±63 |
| T13+5C9IgG | 540±36 |
| Only T14 | 5490±197 |
| T14+2G4IgG | 4470±1867 |
| T14+5C9IgG | 510±146 |
| Only T15 | 2130±387 |
| T15+2G4IgG | 2380±320 |
| T15+5C9IgG | ud |
| Only T16 | 4990±155 |
| T16+2G4IgG | 5270±941 |
| T16+5C9IgG | ud |
| Only T17 | 4410±470 |
| T17+2G4IgG | 4460±288 |
| T17+5C9IgG | ud |
| Only T18 | 910±126 |
| T18+2G4IgG | 830±21 |
| T18+5C9IgG | ud |
Experiment 1d
| Sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 487±75 |
| Only HBD mixture | 285±71 |
| HBD mixture+2G4IgG | 311±68 |
| HBD mixture+5C9IgG | 108±33 |
| Only T11 | 4052±233 |
| T11+2G4IgG | 4659±1260 |
| T11+5C9IgG | 154±33 |
| Only T12 | 4058±721 |
| T12+2G4IgG | 5556±593 |
| T12+5C9IgG | 145±24 |
Ud=does not detect
1=twice mensuration
HBD mixture is the healthy mixture of contributing blood person serum; Used in these experiments is the dilution with 1: 10 in ring AMP measures damping fluid.
T1-T9 and T11-T18 are the serum producing at the Chinese hamster ovary celI moderate stimulation ring AMP that expresses TSHR.T1-T9 and T11-T18 were diluted in ring AMP mensuration damping fluid and test with 1: 10.
2G4 is a kind of human monoclonal antibodies (negative control) of antithyroid peroxidase.
2G4IgG and 5C9IgG test under 100 μ g/mL.
Table 6b
The dose response effect of the stimulation that 5C9IgG and 9D33IgG produce ring AMP for TRAb in by patients serum in the Chinese hamster ovary celI of expressing people TSHR
Experiment 2
| Test sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 707±147 |
| Only HBD mixture | 503±80 |
| Only T1 | 20336±1539 |
| Only 100 μ g/mL2G4IgG (negative control IgG) | 1207±123 |
| T1+100μg/mL?2G4IgG | 22078±2546 |
| T1+10μg/mL?2G4IgG | 18868±1806 |
| T1+1μg/mL?2G4IgG | 19025±1450 |
| T1+0.1μg/mL?2G4IgG | 16659±1031 |
| T1+0.01μg/mL?2G4IgG | 20876±1887 |
| T1+0.001μg/mL?2G4IgG | 18134±2126 |
| Only 100 μ g/mL9D33IgG | 721±183 |
| T1+100μg/mL?9D33IgG | 1061±104 |
| T1+10μg/mL?9D33IgG | 1464±191 |
| T1+1μg/mL?9D33IgG | 4990±1670 |
| T1+0.1μg/mL?9D33IgG | 17867±2220 |
| T1+0.01μg/mL?9D33IgG | 19943±1834 |
| T1+0.001μg/mL?9D33IgG | 21648±502 |
| Only 100 μ g/mL 5C9IgG | 301±38 |
| T1+100μg/mL?5C9IgG | 724±28 |
| T1+10μg/mL?5C9IgG | 1119±348 |
| T1+1μg/mL?5C9IgG | 2428±594 |
| T1+0.1μg/mL?5C9IgG | 16152±3577 |
| T1+0.01μg/mL?5C9IgG | 20314±279 |
| T1+0.001μg/mL?5C9IgG | 16868±912 |
T1 is the patient serum sample producing at the Chinese hamster ovary celI moderate stimulation ring AMP that expresses TSHR; Be diluted in ring AMP mensuration damping fluid and test with 1: 10.
2G4 is a kind of human monoclonal antibodies (negative control) of antithyroid peroxidase.
9D33 is the mouse monoclonal antibody (in table 2) of the TSHR of the disconnected ring AMP generation being stimulated by TSH and TRAb of a kind of resistance
Table 6c
5C9IgG in the Chinese hamster ovary celI of expressing people TSHR for the dose response effect of stimulation ring AMP being produced by patients serum TRAb
Experiment 3
| Test sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 757±138 |
| Only HBD mixture | 512±76 |
| Only T6 | 14216±3985 |
| Only 100 μ g/mL9D33IgG | 729±31 |
| T6+100μg/mL?9D33IgG | 1052±702 |
| T6+10μg/mL?9D33IgG | 2256±1088 |
| T6+1μg/mL?9D33IgG | 5447±313 |
| T6+0.1μg/mL?9D33IgG | 8700±665 |
| T6+0.01μg/mL?9D33IgG | 10290±495 |
| T6+0.001μg/mL?9D33IgG | 10296 1 |
| Only 100 μ g/mL 5C9IgG | 360±38 |
| T6+100μg/mL?5C9IgG | 295±30 |
| T6+10μg/mL?5C9IgG | 1027±368 |
| T6+1μg/mL?5C9IgG | 2368±528 |
| T6+0.1μg/mL?5C9IgG | 9533±1679 |
| T6+0.01μg/mL?5C9IgG | 13883±1718 |
| T6+0.001μg/mL?5C9IgG | 11843±1241 |
1unitary determination
In the illustrative footnote of Table 6a and 6b.
Table 6d
The dose response effect of the stimulation that 5C9IgG produces ring AMP for TRAb in by patients serum in the Chinese hamster ovary celI of expressing people TSHR
| Sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 622±79 |
| Only HBD mixture | 479±53 |
| Only T3 | 14023±2487 |
| Only 100 μ g/mL 2G4IgG | 745±136 |
| T3+100μg/mL?2G4IgG | 12086±2613 |
| T3+10μg/mL?2G4IgG | 12862±250 |
| T3+1μg/mL?2G4IgG | 12931±891 |
| T3+0.1μg/mL?2G4IgG | 13853±1589 |
| T3+0.01μg/mL?2G4IgG | 11939±131 |
| T3+0.001μg/mL?2G4IgG | 13650±1679 |
| Only 100 μ g/mL 9D33IgG | 616±111 |
| T3+100μg/mL?9D33IgG | 1597±323 |
| T3+10μg/mL?9D33IgG | 4262±367 |
| T3+1μg/mL?9D33IgG | 7385±554 |
| T3+0.1μg/mL?9D33IgG | 11960±1390 |
| T3+0.01μg/mL?9D33IgG | 12178±1676 |
| T3+0.001μg/mL?9D33IgG | 12159±2970 |
| Only 100 μ g/mL 5C9IgG | 212±40 |
| T3+100μg/mL?5C9IgG | 6136±558 |
| T3+10μg/mL?5C9IgG | 7806±793 |
| T3+1μg/mL?5C9IgG | 8075±610 |
| T3+0.1μg/mL?5C9IgG | 10414±1094 |
| T3+0.01μg/mL?5C9IgG | 13743±1687 |
| T3+0.001μg/mL?5C9IgG | 11641±2168 |
Referring to the illustrative footnote of table 6a and 6b.
Table 6e
5C9IgG in the Chinese hamster ovary celI of expressing people TSHR for the dose response effect of stimulation ring AMP being produced by TRAb patients serum
| Sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 616±161 |
| Only HBD mixture | 312±56 |
| Only T19 | 6014±280 |
| Only 100 μ g/mL 2G4IgG | 1058±75 |
| T19+100μg/mL?2G4IgG | 7142±215 |
| T19+10μg/mL?2G4IgG | 6182±46 |
| T19+1μg/mL?2G4IgG | 7280±1052 |
| T19+0.1μg/mL?2G4IgG | 7275±145 |
| T19+0.01μg/mL?2G4IgG | 6820±729 |
| T19+0.001μg/mL?2G4IgG | 7620±870 |
| Only 100 μ g/mL 9D33IgG | 592±168 |
| T19+100μg/mL?9D33IgG | 550±65 |
| T19+10μg/mL?9D33IgG | 448±76 |
| T19+1μg/mL?9D33IgG | 404±36 |
| T19+0.1μg/mL?9D33IgG | 2394 1 |
| T19+0.01μg/mL?9D33IgG | 5765 1 |
| T19+0.001μg/mL?9D33IgG | 7088±668 |
| Only 100 μ g/mL 5C9IgG | 186 2 |
| T19+100μg/mL?5C9IgG | 220±90 |
| T19+10μg/mL?5C9IgG | 275±150 |
| T19+1μg/mL?5C9IgG | 187±34 |
| T19+0.1μg/mL?5C9IgG | 375±129 |
| T19+0.01μg/mL?5C9IgG | 5747±411 |
| T19+0.001μg/mL?5C9IgG | 6467 1 |
1the mean value of double sample
2unitary determination
Referring to the illustrative footnote of table 6a and 6b.
Table 6f
The dose response effect of the stimulation that 5C9IgG produces ring AMP for TRAb in by patients serum in the Chinese hamster ovary celI of expressing people TSHR
| Sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 255±40 |
| Only HBD mixture | 200±82 |
| Only T21 | 4764±732 |
| Only 100 μ g/mL 2G4IgG | 835±94 |
| T20+100μg/mL?2G4IgG | 6684±931 |
| T20+10μg/mL?2G4IgG | 4571±776 |
| T20+1μg/mL?2G4IgG | 5744±727 |
| T20+0.1μg/mL?2G4IgG | 4323±849 |
| T20+0.01μg/mL?2G4IgG | 6396±1314 |
| T20+0.001μg/mL?2G4IgG | 6789±893 |
| Only 100 μ g/mL 9D33IgG | 382±142 |
| T20+100μg/mL?9D33IgG | 287±164 |
| T20+10μg/mL?9D33IgG | 204±49 |
| T20+1μg/mL?9D33IgG | 980 1 |
| T20+0.1μg/mL?9D33IgG | 5362±574 |
| T20+0.01μg/mL?9D33IgG | 5389±1139 |
| T20+0.001μg/mL?9D33IgG | 7514±785 |
| Only 100 μ g/mL 5C9IgG | 224±109 |
| T20+100μg/mL?5C9IgG | NT |
| T20+10μg/mL?5C9IgG | NT |
| T20+1μg/mL?5C9IgG | 181 1 |
| T20+0.1μg/mL?5C9IgG | 2184±1078 |
| T20+0.01μg/mL?5C9IgG | 6486±436 |
| T20+0.001μg/mL?5C9IgG | 4856 1 |
1the mean value of double sample
NT=not test (N.T.)
Referring to the illustrative footnote of table 6a and 6b.
Table 6g
The dose response effect of the stimulation that 5C9IgG produces ring AMP for TRAb in by patients serum in the Chinese hamster ovary celI of expressing people TSHR
| Sample | Ring AMP (fmol/ cell hole mean value ± SD; N=3) |
| Only encircle AMP and measure damping fluid | 466±65 |
| Only HBD mixture | 390±118 |
| Only T21 | 9781±1672 |
| Only 100 μ g/mL 2G4IgG | 999±55 |
| T21+100μg/mL?2G4IgG | 10848±373 |
| T21+10μg/mL?2G4IgG | 10355±469 |
| T21+1μg/mL?2G4IgG | 10831±140 |
| T21+0.1μg/mL?2G4IgG | 12215±793 |
| T21+0.01μg/mL?2G4IgG | 13014±855 |
| T21+0.001μg/mL?2G4IgG | 10500±162 |
| Only 100 μ g/mL 9D33IgG | 534±89 |
| T21+100μg/mL?9D33IgG | 442±32 |
| T21+10μg/mL?9D33IgG | 605±254 |
| T21+1μg/mL?9D33IgG | 1383±66 |
| T21+0.1μg/mL?9D33IgG | 8719±389 |
| T21+0.01μg/mL?9D33IgG | 10772±799 |
| T21+0.001μg/mL?9D33IgG | 10229±714 |
| Only 100 μ g/mL 5C9IgG | 253±25 |
| T21+100μg/mL?5C9IgG | 210±60 |
| T21+10μg/mL?5C9IgG | 303±107 |
| T21+1μg/mL?5C9IgG | 418±65 |
| T21+0.1μg/mL?5C9IgG | 7483±415 |
| T21+0.01μg/mL?5C9IgG | 10441±122 |
| T21+0.001μg/mL?5C9IgG | 11281±911 |
Referring to the illustrative footnote of table 6a and 6b.
Table 7a
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Lys58 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
1the mean value of double sample
PTSH concentration=3ng/mL
All dilutions are all measured in damping fluid in ring AMP
2G4 is a kind of human monoclonal antibodies (negative control of 5C9) of antithyroid peroxidase.
Table 7b
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Ile60 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7c
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Arg80 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7d
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Arg80 and be mutated into aspartic acid, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7e
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Tyr82 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7f
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Thr104 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7g
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Arg109 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7h
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Lys129 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7i
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Phe134 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7j
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Asp151 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7k
In the Chinese hamster ovary celI of expressing wild-type TSHR and Asp151 and be mutated into arginic TSHR, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7l
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Asp160 and be mutated into Methionin, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
M22 concentration=3mg/mL
Referring to other illustrative footnotes of table 7a.
Table 7m
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Lys183 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7n
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Lys183 and be mutated into aspartic acid, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7o
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Gln235 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7p
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Arg255 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7q
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Arg255 and be mutated into aspartic acid, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7r
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Trp258 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 7s
In the Chinese hamster ovary celI of TSHR of expressing wild-type TSHR and Ser281 and be mutated into L-Ala, the ring AMP of TSH induction produces.The effect of different dilution 5C9IgG.
Referring to the illustrative footnote of table 7a.
Table 8
The serum T RAb measurement result that relatively uses the ELISA of the inhibition based on to the combination of TSH-vitamin H or the combination of 5C9IgG-vitamin H to obtain
Measuring verification thing 40U/L, 8U/L, 2U/L and 1U/L is that M22IgG contributes the dilution in blood person's serum mixture (HBD mixture) in health, and the activity representing with the NIBSC 90/672 of U/L is to assess with the inhibition of the combination of the pipe applying through TSHR by the TSH to through mark.Measuring negative control is HBD mixture.
Table 9
The effect that 5C9IgG stimulates the TSHR of Thyreoidine pungency mouse MAb (mTSMAb)
| Only damping fluid | 3.39±1.35 | 1.56±1.84 | 4.32±0.95 |
abeing diluted in ring AMP measures in damping fluid
b2G4 is a kind of human monoclonal antibodies of antithyroid peroxidase of contrast.
Table 10
The effect that 5C9IgG stimulates for the TSHR of different TSH goods
Experiment 1
| Be diluted in ring AMP and measure the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD in the Chinese hamster ovary celI of expression wild-type TSHR; N=3) |
| Only damping fluid | 0.37±0.15 |
| Only natural people TSH 100ng/mL | 36.14±1.83 |
| Natural people TSH 100ng/mL and 0.001 μ g/mL 5C9IgG. | 36.0±6.1 |
| Natural people TSH 100ng/mL and 0.01 μ g/mL 5C9IgG | 18.0±5.4 |
| Natural people TSH 100ng/mL and 0.1 μ g/mL 5C9IgG | 0.20±0.06 |
| Natural people TSH 100ng/mL and 1.0 μ g/mL 5C9IgG | 0.14±0.02 |
| Natural people TSH 100ng/mL and 10 μ g/mL 5C9IgG | 0.11±0.04 |
| Natural people TSH 100ng/mL and | 0.16±0.03 |
| 100μg/mL?5C9IgG | |
| Only recombinant chou people TSH 100ng/mL | 12.73±4.0 |
| Recombinant chou people TSH 100ng/mL and 0.001 μ g/mL 5C9IgG. | 13.63±1.7 |
| Recombinant chou people TSH 100ng/mL and 0.01 μ g/mL 5C9IgG | 8.13±2.13 |
| Recombinant chou people TSH 100ng/mL and 0.1 μ g/mL 5C9IgG | 0.12±0.06 |
| Recombinant chou people TSH 100ng/mL and 1.0 μ g/mL 5C9IgG | 0.10±0.03 |
| Recombinant chou people TSH 100ng/mL and 10 μ g/mL 5C9IgG | 0.12±0.05 |
| Recombinant chou people TSH 100ng/mL and 100 μ g/mL 5C9IgG | 0.19±0.08 |
Experiment 2
| Be diluted in ring AMP and measure the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD in the Chinese hamster ovary celI of expression wild-type TSHR; N=3) |
| Only damping fluid | 0.18±0.08 |
| Only natural people TSH 100ng/mL | 40.8±6.92 |
| Natural people TSH 100ng/mL and 0.001 μ g/mL 5C9IgG. | 37.6±6.35 |
| Natural people TSH 100ng/mL and 0.01 μ g/mL 5C9IgG | 34.4±2.28 |
| Natural people TSH 100ng/mL and 0.1 μ g/mL 5C9IgG | 15.8±1.39 |
| Natural people TSH 100ng/mL and 1.0 μ g/mL 5C9IgG | 0.13±0.03 |
| Natural people TSH 100ng/mL and 10 μ g/mL 5C9IgG | 0.18±0.05 |
| Natural people TSH 100ng/mL and 100 μ g/mL 5C9IgG | 0.21±0.10 |
| Only recombinant chou people TSH 100ng/mL | 16.72±2.90 |
| Recombinant chou people TSH 100ng/mL and 0.001 μ g/mL 5C9IgG. | 20.0±1.73 |
| Recombinant chou people TSH 100ng/mL and 0.01 μ g/mL 5C9IgG | 20.6±1.57 |
| Recombinant chou people TSH 100ng/mL and 0.1 μ g/mL 5C9IgG | 7.39±1.74 |
| Recombinant chou people TSH 100ng/mL and 1.0 μ g/mL 5C9IgG | 0.07±0.01 |
| Recombinant chou people TSH 100ng/mL and 10 μ g/mL 5C9IgG | 0.07±0.01 |
| Recombinant chou people TSH 100ng/mL and 100 μ g/mL 5C9IgG | 0.11±0.03 |
| Only natural pig TSH 0.3ng/mL | 40.2±4.0 |
| Natural pig TSH 0.3ng/mL and 0.001 μ g/mL 5C9IgG. | 32.7±4.0 |
| Natural pig TSH 0.3ng/mL and 0.01 μ g/mL 5C9IgG | 28.2±2.04 |
| Natural pig TSH 0.3ng/mL and 0.1 μ g/mL 5C9IgG | 18.0±1.36 |
| Natural pig TSH 0.3ng/mL and 1.0 μ g/mL 5C9IgG | 2.14±0.85 |
| Natural pig TSH 0.3ng/mL and 10 μ g/mL 5C9IgG | 0.20±0.14 |
| Natural pig TSH 0.3ng/mL and | 0.18±0.13 |
| 100μg/mL?5C9IgG |
Experiment 3
| Be diluted in ring AMP and measure the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD in the Chinese hamster ovary celI of expression wild-type TSHR; N=3) |
| Only damping fluid | 2.0±0.8 |
| Only natural people TSH 100ng/mL | 31.1±2.55 |
| Natural people TSH 100ng/mL and 0.001 μ g/mL 5C9IgG. | 36.0±3.27 |
| Natural people TSH 100ng/mL and 0.01 μ g/mL 5C9IgG | 28.4±3.55 |
| Natural people TSH 100ng/mL and 0.1 μ g/mL 5C9IgG | 7.22±3.20 |
| Natural people TSH 100ng/mL and 1.0 μ g/mL 5C9IgG | 0.67±0.46 |
| Natural people TSH 100ng/mL and 10 μ g/mL 5C9IgG | 0.8±0.45 |
| Natural people TSH 100ng/mL and 100 μ g/mL 5C9IgG | 1.35±1.0 |
| Only recombinant chou people TSH 100ng/mL | 26.4±3.53 |
| Recombinant chou people TSH 100ng/mL and 0.001 μ g/mL 5C9IgG. | 25.6±2.45 |
| Recombinant chou people TSH 100ng/mL and 0.01 μ g/mL 5C9IgG | 22.9±7.4 |
| Recombinant chou people TSH 100ng/mL and 0.1 μ g/mL 5C9IgG | 1.18±0.49 |
| Recombinant chou people TSH 100ng/mL and 1.0 μ g/mL 5C9IgG | 0.81±0.05 |
| Recombinant chou people TSH 100ng/mL and 10 μ g/mL 5C9IgG | 0.74±0.42 |
| Recombinant chou people TSH 100ng/mL and 100 μ g/mL 5C9IgG | 0.88±0.55 |
| Only natural pig TSH 0.3ng/mL | 35.7±4.82 |
| Natural pig TSH 0.3ng/L and 0.001 μ g/mL 5C9IgG. | 38.2±2.54 |
| Natural pig TSH 0.3ng/mL and 0.01 μ g/mL 5C9IgG | 26.6±3.22 |
| Natural pig TSH 0.3ng/mL and | 10.1±1.79 |
| 0.1μg/mL?5C9IgG | |
| Natural pig TSH 0.3ng/mL and 1.0 μ g/mL 5C9IgG | 2.28±0.71 |
| Natural pig TSH 0.3ng/mL and 10 μ g/mL 5C9IgG | 0.68±0.21 |
| Natural pig TSH 0.3ng/mL and 100 μ g/mL 5C9IgG | 1.03±0.63 |
Table 11a
5C9IgG and 9D33IgG are to having the effect of constitutive activity of TSHR of activation sudden change S281I
| Be diluted in ring AMP and measure the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD in the Chinese hamster ovary celI of expression TSHR S281I; N=3) |
| Only damping fluid | 9.90±1.51 |
| 0.001μg/mL?2G4IgG a | 6.83±0.37 |
| 0.01μg/mL?2G4IgG | 7.74±0.78 |
| 0.1μg/mL?2G4IgG | 8.58±1.12 |
| 1μg/mL?2G4IgG | 8.37±1.10 |
| 0.001μg/mL?5C9IgG | 4.31±0.16 |
| 0.01μg/mL?5C9IgG | 4.17±0.60 |
| 0.1μg/mL?5C9IgG | 3.20±0.63 |
| 1μg/mL?5C9IgG | 3.44±0.63 |
| 0.001μg/mL?9D33IgG | 5.97±0.94 |
| 0.01μg/mL?9D33IgG | 9.27±1.4 |
| 0.1μg/mL?9D33IgG | 8.13±0.72 |
| 1μg/mL?9D33IgG | 7.33±1.17 |
a2G4 is a kind of human monoclonal antibodies of antithyroid peroxidase of contrast.
Table 11b
5C9IgG and 9D33IgG are to having the effect of constitutive activity of TSHR of activation sudden change I568T
| Be diluted in ring AMP and measure the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD in the Chinese hamster ovary celI of expression TSHR I586T; N=3) |
| Only damping fluid | 21.39±5.31 |
| 0.001μg/mL?2G4IgG | 19.13±2.77 |
| 0.01μg/mL?2G4IgG | 16.67±1.87 |
| 0.1μg/mL?2G4IgG | 19.92±0.91 |
| 1μg/mL?2G4IgG | 20.52±0.95 |
| 0.001μg/mL?5C9IgG | 18.81±1.39 |
| 0.01μg/mL?5C9IgG | 9.24±0.83 |
| 0.1μg/mL?5C9IgG | 6.02±1.93 |
| 1μg/mL?5C9IgG | 5.29±0.75 |
| 0.001μg/mL?9D33IgG | 16.58±0.00 |
| 0.01μg/mL?RSR-B2IgG | 17.03±2.36 |
| 0.1μg/mL?RSR-B2IgG | 19.96±1.66 |
| 1μg/mL?RSR-B2IgG | 21.65±1.99 |
Table 11c
5C9IgG and 9D33IgG are to having the effect of constitutive activity of TSHR of activation sudden change A623I
| Be diluted in ring AMP and measure the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD in the Chinese hamster ovary celI of expression TSHR A621I; N=3) |
| Only damping fluid | 36.89 a |
| 0.001μg/mL?2G4IgG | 28.46±2.31 |
| 0.01μg/mL?2G4IgG | 33.44±1.12 |
| 0.1μg/mL?2G4IgG | 30.40±7.93 |
| 1μg/mL?2G4IgG | 28.96±2.29 |
| 0.001μg/mL?5C9IgG | 26.52±1.33 |
| 0.01μg/mL?5C9IgG | 27.03±2.13 |
| 0.1μg/mL?5C9IgG | 19.79±0.48 |
| 1μg/mL?5C9IgG | 16.43±1.27 |
| 0.001μg/mL?9D33IgG | 29.55±3.15 |
| 0.01μg/mL?9D33IgG | 27.64±3.49 |
| 0.1μg/mL?9D33IgG | 31.78±9.18 |
| 1μg/mL?9D33IgG | 40.09±7.73 |
atwice mensuration
Table 12
5C9 adds the effect that 9D33 stimulates for the pTSH of blocking-up ring AMP generation in the Chinese hamster ovary celI of expressing wild-type TSHR
Experiment 1
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 1.9±1.0 |
| TSH?3ng/mL | 48.5±14 |
| 10μg/mL?9D33 | 2.9±1.0 |
| 10μgmL?5C9 | 0.45±0.31 |
| 10μg/mL9D33+10μg/mL?5C9 | 1.12±0.4 |
| 10μg/mL?9D33+TSH | 23.8±8.7 |
| 10μg/mL?5C9+TSH | 3.7±2.7 |
| 10μg/mL?9D33+10μg/mL?5C9+TSH | 4.9(n＝2) |
| 1μg/mL?9D33+TSH | 28.7±1.8 |
| 1μg/mL?5C9+TSH | 13.9±3.1 |
| 1μg/mL?9D33+1μg/mL?5C9+TSH | 12.6±2.5 |
Experiment 2
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 0.72±0.63 |
| TSH?3ng/mL | 34.3±3.1 |
| 10μg/mL?9D33 | 1.6±1.3 |
| 10μg/mL?5C9 | 0.4±0.4 |
| 10μg/mL?9D33+10μg/mL?5C9 | 0.03(n＝1) |
| 10μg/mL?9D33+TSH | 9.5±1.7 |
| 10μg/mL?5C9+TSH | 3.1±1.0 |
| 10μg/mL?9D33+10μg/mL?5C9+TSH | 3.3±2.0 |
| 1μg/mL?9D33+TSH | 19.0±3.2 |
| 1μg/mL?5C9+TSH | 6.1±0.6 |
| 1μg/mL?9D33+1μg/mL?5C9+TSH | 5.9±0.6 |
Experiment 3
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 0.99±0.03 |
| TSH?3ng/mL | 51.3±5.0 |
| 100μg/mL?9D33 | 1.14±0.13 |
| 100μg/mL?5C9 | 0.50±0.04 |
| 100μg/mL?9D33+100μg/mL?5C9 | 0.66±0.9 |
| 100μg/mL?9D33+TSH | 10.53±0.84 |
| 100μg/mL?5C9+TSH | 1.4±0.18 |
| 100μg/mL?9D33+100μg/mL?5C9+TSH | 1.3±0.36 |
| 10μg/mL?9D33+TSH | 13.5±2.9 |
| 10μg/mL?5C9+TSH | 1.9±1.1 |
| 10μg/mL?9D33+10μg/mL?5C9+TSH | 1.2±0.1 |
| 1μg/mL?9D33+TSH | 27.8±2.2 |
| 1μg/mL?5C9+TSH | 5.4±1.8 |
| 1μg/mL?9D33+1μg/mL?5C9+TSH | 5.2±0.3 |
| 0.1μg/mL?9D33+TSH | 35.1±2.3 |
| 0.1μg/mL?5C9+TSH | 18.7±3.4 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+TSH | 14.4±1.0 |
| 0.01μg/mL?9D33+TSH | 47.1±1.9 |
| 0.01μg/mL?5C9+TSH | 33.9±7.8 |
| 0.01μg/mL?9D33+0.01μg/mL?5C9+TSH | 27.8±1.3 |
Experiment 4
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 1.4±0.5 |
| TSH?0.3ng/mL | 46.6±12.9 |
| 100μg/mL?9D33 | 0.99±0.62 |
| 100μg/mL?5C9 | 0.15±0.12 |
| 100μg/mL?9D33+100μg/mL?5C9 | 0.41±0.40 |
| 100μg/mL?9D33+TSH | 3.53±1.1 |
| 100μg/mL?5C9+TSH | 0.29±0.15 |
| 100μg/mL?9D33+100μg/mL?5C9+TSH | 0.63±0.38 |
| 10μg/mL?9D33+TSH | 4.23±0.81 |
| 10μg/mL?5C9+TSH | 0.23±0.08 |
| 10μg/mL?9D33+10μg/mL?5C9+TSH | 0.52±0.15 |
| 1μg/mL?9D33+TSH | 9.01±0.67 |
| 1μg/mL?5C9+TSH | 1.65±0.47 |
| 1μg/mL?9D33+1μg/mL?5C9+TSH | 1.21±0.67 |
| 0.1μg/mL9D33+TSH | 20.2±2.2 |
| 0.1μg/mL?5C9+TSH | 6.2±1.8 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+TSH | 7.6±1.3 |
Experiment 5
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 1.83±0.64 |
| TSH?0.3ng/mL | 17.26±1.5 |
| 10μg/mL?9D33 | 2.07±0.52 |
| 10μg/mL?5C9 | 0.38±0.2 |
| 10μg/mL?9D33+10μg/mL?5C9 | 0.96±0.14 |
| 10μg/mL9D33+TSH | 3.57±0.58 |
| 10μg/mL?5C9+TSH | 1.17±0.00 |
| 10μg/mL?9D33+10μg/mL?5C9+TSH | 1.58±0.20 |
| 5μg/mL?9D33+TSH | 3.71±0.10 |
| 5μg/mL?5C9+TSH | 1.21±0.36 |
| 1μg/mL?9D33+TSH | 6.38±1.35 |
| 1μg/mL?5C9+TSH | 2.57±0.65 |
| 1μg/mL?9D33+1μg/mL?5C9+TSH | 1.46±0.59 |
| 0.1μg/mL?9D33+TSH | 14.67±4.69 |
| 0.1μg/mL?5C9+TSH | 12.43±1.59 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+TSH | 9.99±3.78 |
Table 13
5C9 adds the effect of the stimulation that discharge ring AMP is produced that 9D33 mediates by M22Fab for blocking-up in the Chinese hamster ovary celI of expressing wild-type TSHR
Experiment 1
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 1.45±0.46 |
| M223ng/mL | 49.1±9.8 |
| 100μg/mL?9D33 | 1.51±0.22 |
| 100μg/mL?5C9 | 0.35±0.30 |
| 100μg/mL?9D33+100μg/mL?5C9 | 1.57±0.13 |
| 100μg/mL?9D33+M22 | 2.13±0.74 |
| 100μg/mL?5C9+M22 | 0.35±0.08 |
| 100μg/mL?9D33+100μg/mL?5C9+ M22 | 0.70±0.40 |
| 10μg/mL?9D33+M22 | 2.5±0.8 |
| 10μg/mL?5C9+M22 | 0.36±0.25 |
| 10μg/mL?9D33+10μg/mL?5C9+M22 | 0.52±0.12 |
| 1μg/mL?9D33+M22 | 5.25±0.55 |
| 1μg/mL?5C9+M22 | 0.93±0.07 |
| 1μg/mL?9D33+1μg/mL?5C9+M22 | 0.69±0.13 |
| 0.1μg/mL?9D33+M22 | 27.6±2.5 |
| 0.1μg/mL?5C9+M22 | 6.0±2.6 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+ M22 | 13.7±7.5 |
| 0.01μg/mL?9D33+M22 | 47.5±4.5 |
| 0.01μg/mL?5C9+M22 | 48.1±5.4 |
| 0.01μg/mL?9D33+0.01μg/mL?5C9+ M22 | 47.5±4.5 |
Experiment 2
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 1.69±0.47 |
| M223ng/mL | 68.3±6.3 |
| 100μg/L9D33 | 1.76±0.43 |
| 100μg/mL?5C9 | 0.69±0.18 |
| 100μg/mL?9D33+100μg/mL?5C9 | 1.16±0.35 |
| 100μg/mL?9D33+M22 | 1.42±1.20 |
| 100μg/mL?5C9+M22 | Can not survey |
| 100μg/mL?9D33+100μg/mL?5C9+ M22 | 0.14(n＝2) |
| 10μg/mL?9D33+M22 | 0.67(n＝2) |
| 10μg/mL?5C9+M22 | 0.14(n＝1) |
| 10μg/mL?9D33+10μg/mL?5C9+M22 | 2.41(n＝1) |
| 1μg/mL?9D33+M22 | 6.03±1.15 |
| 1μg/mL?5C9+M22 | 2.54(n＝1) |
| 1μg/mL?9D33+1μg/mL?5C9+M22 | 1.51(n＝1) |
| 0.1μg/mL?9D33+M22 | 38.7±8.1 |
| 0.1μg/mL?5C9+M22 | 4.17±1.7 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+ M22 | 5.17±2.85 |
| 0.01μg/mL?9D33+M22 | 60.2±8.0 |
| 0.01μg/mL?5C9+M22 | 57.3±13.7 |
| 0.01μg/mL?9D33+0.01μg/mL?5C9+ M22 | 40.4±5.3 |
| 0.001μg/mL?9D33+M22 | 79.1±26.3 |
| 0.001μg/mL?5C9+M22 | 63.3±19.0 |
| 0.001μg/mL?9D33+0.001μg/mL?5C9+ M22 | 40.2±8.7 |
Experiment 3
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 0.86±0.12 |
| M220.3ng/mL | 21.8±3.23 |
| 100μg/mL?9D33 | 0.88±0.30 |
| 100μg/mL?5C9 | 0.58±0.28 |
| 100μg/mL?9D33+100μg/mL?5C9 | 0.81±0.36 |
| 100μg/mL?9D33+M22 | 1.03±0.32 |
| 100μg/mL?5C9+M22 | 0.05(n＝2) |
| 100μg/mL?9D33+100μg/mL?5C9+ M22 | 0.06(n＝2) |
| 10μg/mL?9D33+M22 | 0.97±0.48 |
| 10μg/mL?5C9+M22 | 0.20(n＝2) |
| 10μg/mL?9D33+10μg/mL?5C9+M22 | 0.14±0.08 |
| 1μg/mL?9D33+M22 | 0.83±0.21 |
| 1μg/mL?5C9+M22 | 0.02(n＝2) |
| 1μg/mL?9D33+1μg/mL?5C9+M22 | 0.13±0.13 |
| 0.1μg/mL?9D33+M22 | 5.38±1.71 |
| 0.1μg/mL?5C9+M22 | 1.43±1.09 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+ M22 | 2.39±1.0 |
| 0.01μg/mL?9D33+M22 | 15.2±1.42 |
| 0.01μg/mL?5C9+M22 | 13.1±1.34 |
| 0.01μg/mL?9D33+0.01μg/mL?5C9+ M22 | 12.7±3.4 |
| 0.001μg/mL?9D33+M22 | 12.8±1.60 |
| 0.001μg/mL?5C9+M22 | 13.3±0.89 |
| 0.001μg/mL?9D33+0.001μg/mL?5C9+ M22 | 15.8±2.15 |
Experiment 4
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration (pmol/mL mean value ± SD; N=3) |
| Only damping fluid | 1.29±0.68 |
| M220.3ng/mL | 31.1±9.4 |
| 100μg/mL?9D33 | 2.38±1.11 |
| 100μg/mL?5C9 | 0.12±0.09 |
| 100μg/mL?9D33+100μg/mL?5C9 | 0.81±0.15 |
| 100μg/mL?9D33+M22 | 2.00±0.87 |
| 100μg/mL?5C9+M22 | 0.22±0.11 |
| 100μg/mL?9D33+100μg/mL?5C9+ M22 | 0.40±0.18 |
| 10μg/mL?9D33+M22 | 1.25±0.09 |
| 10μg/mL?5C9+M22 | 0.40±0.17 |
| 10μg/mL?9D33+10μg/mL?5C9+M22 | 0.45±0.31 |
| 1μg/mL?9D33+M22 | 2.66±0.64 |
| 1μg/mL?5C9+M22 | 0.21±0.18 |
| 1μg/mL?9D33+1μg/mL?5C9+M22 | 0.14±0.07 |
| 0.1μg/mL?9D33+M22 | 27.6±2.5 |
| 0.1μg/mL?5C9+M22 | 6.0±2.6 |
| 0.1μg/mL?9D33+0.1μg/mL?5C9+M22 | 7.2±3.9 |
| 0.01μg/mL?9D33+M22 | 38.9±6.4 |
| 0.01μg/mL?5C9+M22 | 31.5±4.0 |
| 0.01μg/mL?9D33+0.01μg/mL?5C9+ M22 | 20.6±5.3 |
| 0.001μg/mL?9D33+M22 | 43.2±7.9 |
| 0.001μg/mL?5C9+M22 | 33.1±4.0 |
| 0.001μg/mL?9D33+0.001μg/mL?5C9+ M22 | 25.3±6.0 |
Table 14
5C9 and 9D33IgG are expressing Chinese hamster ovary celI (each cell about 5 × 10 of wild-type TSHR
5individual acceptor) in to discharge ring AMP produce effect
Experiment 1
| Test sample | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 47.1±11.7 | 0 |
| 3ng/mL?TSH | 152.0±28.2 | -ve |
| 5B3 aIgG?100μg/mL | 44.5±5.2 | 5.3 |
| 5B3 aIgG?10μg/mL | 45.4±6.3 | 3.4 |
| 5B3 aIgG?1μg/mL | 52.0±12.1 | -ve |
| 9D33IgG?100μg/mL | 74.2±7.2 | -ve |
| 9D33IgG?10μg/mL | 56.5±4.0 | -ve |
| 9D33IgG?1μg/mL | 65.8±9.8 | -ve |
| 9D33IgG?0.1μg/mL | 61.7±13.3 | -ve |
| 9D33IgG?0.01μg/mL | 52.0±5.1 | -ve |
| 9D33IgG?0.001μg/mL | 61.6±3.8 | -ve |
In the contrast Chinese hamster ovary celI of not expressing TSHR, in the situation that existing ring AMP to measure damping fluid, discharge ring AMP generation is 1.02 ± 0.06pmol/mL (mean value ± SD; , and in the case of the TSH that has 3ng/mL, be 0.74 ± 0.32pmol/mL (mean value ± SD n=3); N=3).
-ve=feminine gender, does not suppress to encircle AMP and produces.
a5B3 is the human monoclonal antibodies (negative control of 5C9) of a kind of antiglutamic acid decarboxylase (GAD).
Experiment 2
| Test sample | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 58.0±15.2 | 0 |
| 3ng/mLTSH | 156.5±22.2 | -ve |
| 5B3 aIgG?100μg/mL | 52.7±7.8 | 9.1 |
| 5B3 aIgG?10μg/mL | 43.7±10.4 | 24.7 |
| 5B3 aIgG?1μg/mL | 55.9±12.2 | 3.7 |
| 5C9IgG?100μg/mL | 26.2±2.7 | 54.9 |
| 5C9IgG?10μg/mL | 14.7±1.7 | 74.6 |
| 5C9IgG?1μg/mL | 16.8±4.2 | 71.0 |
| 5C9IgG?0.1μg/mL | 31.5±8.8 | 45.7 |
| 5C9IgG?0.01μg/mL | 36.4±4.5 | 37.2 |
| 5C9IgG?0.001μg/mL | 51.7±9.8 | 10.8 |
In the contrast Chinese hamster ovary celI of not expressing TSHR, in the situation that existing ring AMP to measure damping fluid, discharge ring AMP generation is 0.03pmol/mL (n=2), and in the case of the TSH that has 3ng/mL, is 0.40 ± 0.09pmol/mL (mean value ± SD; N=3).
-ve=feminine gender, does not suppress to encircle AMP and produces.
a5B3 is the human monoclonal antibodies (negative control of 5C9) of a kind of antiglutamic acid decarboxylase (GAD).
Experiment 3
The effect that 5C9Fab and F (ab ') produce discharge ring AMP in the Chinese hamster ovary celI of expressing wild-type TSHR
| Ring AMP measures the test sample in damping fluid | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 59.4±8.6 | 0 |
| 9D33IgG?100μg/mL | 67.8±1.0 | -ve |
| 9D33IgG?10μg/mL | 79.4±9.8 | -ve |
| 9D33IgG?1μg/mL | 71.5±8.8 | -ve |
| 9D33IgG?0.1μg/mL | 75.6±8.9 | -ve |
| 9D33IgG?0.01μg/mL | 60.5±7.5 | -ve |
| 9D33IgG?0.001μg/mL | 52.3±6.3 | 12 |
| 5C9IgG?100μg/mL | 26.2±1.8 | 56 |
| 5C9IgG?10μg/mL | 24.5±5.8 | 59 |
| 5C9IgG?1μg/mL | 22.9±2.1 | 61 |
| 5C9IgG?0.1μg/mL | 59.1±2.6 | 1 |
| 5C9IgG?0.01μg/mL | 64.3±8.4 | -ve |
| 5C9IgG?0.001μg/mL | 67.3±9.8 | -ve |
| 5C9Fab?100μg/mL | 23.3±2.3 | 61 |
| 5C9Fab?10μg/mL | 32.1±4.8 | 46 |
| 5C9Fab?1μg/mL | 36.4±1.5 | 39 |
| 5C9Fab?0.1μg/mL | 52.8±1.9 | 11 |
| 5C9Fab?0.01μg/mL | 61.1±2.4 | -ve |
| 5C9Fab?0.001μg/mL | 62.5±7.3 | -ve |
| 5C9F(ab′)100μg/mL | 30.9±2.6 | 48 |
| 5C9F(ab′)10μg/mL | 36.5±3.4 | 39 |
| 5C9F(ab′)1μg/mL | 45.9±4.5 | 23 |
| 5C9F(ab′)0.1μg/mL | 48.2±3.1 | 19 |
| 5C9F(ab′)0.01μg/mL | 62.3±5.7 | -ve |
| 5C9F(ab′)0.001μg/mL | 57.9±9.1 | 3 |
In the contrast Chinese hamster ovary celI of not expressing TSHR, in the situation that existing ring AMP to measure damping fluid, discharge ring AMP generation is 0.03pmol/mL (n=2), and in the case of the TSH that has 3ng/mL, is 0.40 ± 0.09pmol/mL (mean value ± SD; N=3).
-ve=feminine gender, does not suppress to encircle AMP and produces.
F (ab ') is by reduction F (ab ')
2be prepared; Refer to text.
Table 15
There is patients serum TSHR autoantibody (B2-B5) effect to discharge ring AMP level in the Chinese hamster ovary celI of expressing the active sudden change of TSHR I568T of antagonistic activity
Experiment 1
| Test sample and serum dilution | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 20.5±8.7 | 0 |
| HBD mixture/10 | 19.5±3.4 | 5 |
| HBD mixture/50 | 25.7±2.8 | -ve |
| N1/10 | 20.7±5.5 | -ve |
| N1/50 | 18.5±1.5 | 10 |
| N2/10 | 23.3±1.7 | -ve |
| N2/50 | 17.6±1.8 | 14 |
| N3/10 | 20.3±2.4 | 1 |
| N3/50 | 23.6±5.9 | -ve |
| B2/10 | 5.3±1.3 | 74 |
| B2/50 | 9.6±2.8 | 53 |
| B3/10 | 8.3±3.1 | 60 |
| B3/50 | 10.5±2.5 | 49 |
| B4/10 | 2.2±0.5 | 89 |
| B4/50 | 3.0±0.3 | 86 |
| B5/10 | 15.9±3.3 | 23 |
| B5/50 | 14.5±1.3 | 29 |
Experiment 2
| Test sample and serum dilution | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 19.7±3.7 | 0 |
| HBD mixture/10 | 28.0±1.6 | -ve |
| HBD mixture/50 | 18.1±3.9 | 8 |
| HBD mixture/100 | 18.0±1.6 | 9 |
| HBD mixture/500 | 17.8±1.3 | 10 |
| HBD mixture/1000 | 20.7±2.9 | -ve |
| HBD mixture/5000 | 15.6±2.1 | 20 |
| B3/10 | 14.7±2.2 | 25 |
| B3/50 | 13.7±1.3 | 31 |
| B3/100 | 12.6±0.6 | 36 |
| B3/500 | 19.0±0.8 | 4 |
| B3/1000 | 18.4±4.6 | 7 |
| B3/5000 | 17.6±0.9 | 11 |
| B4/10 | 4.0±0.5 | 80 |
| B4/50 | 3.6±0.8 | 81 |
| B4/100 | 3.8±1.1 | 81 |
| B4/500 | 7.2±2.6 | 64 |
| B4/1000 | 12.0±0.6 | 39 |
| B4/5000 | 17.7±2.7 | 10 |
-ve=feminine gender, does not suppress to encircle AMP and produces.
HBD mixture=health is contributed the mixture of blood person serum
The health of N1-N3=individuality is contributed blood person serum
All serum is all diluted in ring AMP and measures in damping fluid.
Table 16
There is patients serum TSHR autoantibody (B2-B5) effect to discharge ring AMP level in the Chinese hamster ovary celI of expressing the active sudden change of TSHR S281I of antagonistic activity
| Test sample and serum dilution | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP output |
| Only encircle AMP and measure damping fluid | 11.2±2.0 | 0 |
| HBD/10 | 12.1±0.6 | -8 |
| HBD/50 | 10.0±2.0 | 11 |
| N1/10 | 8.0±1.6 | 28 |
| N1/50 | 10.8±3.3 | 4 |
| N2/10 | 8.8±1.4 | 21 |
| N2/50 | 8.8±2.3 | 22 |
| N3/10 | 10.0±0.8 | 17 |
| N3/50 | 9.3±1.7 | 17 |
| B2/10 | 7.7±039 | 31 |
| B2/50 | 5.7±1.3 | 49 |
| B3/10 | 5.4±0.5 | 52 |
| B3/50 | 6.6±1.1 | 41 |
| B4/10 | 5.4±1.1 | 52 |
| B4/50 | 4.9±0.7 | 56 |
| B5/10 | 9.1±2.5 | 18 |
| B5/50 | 7.6±0.8 | 32 |
Referring to the illustrative footnote of table 15.
In the experiment with TSHR S281I, the 5C9IgG of 1 μ g/mL causes 71% inhibition to discharge ring AMP activity.
Table 17
There is patients serum TSHR autoantibody (B2-B5) effect to discharge ring AMP level in the Chinese hamster ovary celI of expressing the active sudden change of TSHR A623I of antagonistic activity
| Test sample and serum dilution | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The inhibition % of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 43.5±11.2 | 0 |
| HBD mixture/10 | 34.7±4.5 | 20 |
| HBD mixture/50 | 49.9±5.7 | -15 |
| N1/10 | 32.1±2.5 | 26 |
| N1/50 | 43.9±12.0 | -1 |
| N2/10 | 51.1±8.4 | -17 |
| N2/50 | 32.6±2.1 | 26 |
| N3/10 | 47.2±7.1 | -10 |
| N3/50 | 57.3±16.5 | -32 |
| B2/10 | 28.8±1.1 | 34 |
| B2/50 | 43.9±2.7 | -1 |
| B3/10 | 33.5±3.5 | 23 |
| B3/50 | 44.2±12.7 | -1 |
| B4/10 | 27.2±6.6 | 37 |
| B4/50 | 23.9±1.0 | 45 |
| B5/10 | 19.2±6.3 | 56 |
| B5/50 | 40.6±10.9 | 7 |
Referring to the illustrative footnote of table 15.
In the experiment with TSHR A623I, the 5C9IgG of 1 μ g/mL causes 49% inhibition to discharge ring AMP activity.
Table 18
There is the patients serum TSHR autoantibody (B2-B5) of antagonistic activity at Chinese hamster ovary celI (each cell about 5 × 10 of expressing wild-type TSHR
5individual acceptor) in effect to discharge ring AMP level
| Test sample and serum dilution | Ring AMP concentration pmol/mL (mean value ± SD; N=3) | The variation (%) of discharge ring AMP generation |
| Only encircle AMP and measure damping fluid | 28.1±0.7 | 100 |
| HBD/10 | 37.5±6.9 | 133 |
| HBD/50 | 37.2±2.4 | 132 |
| N1/10 | 27.7±5.7 | 99 |
| N1/50 | 26.0±4.8 | 93 |
| N2/10 | 41.0±2.7 | 146 |
| N2/50 | 27.0±1.2 | 96 |
| N3/10 | 34.3±2.7 | 122 |
| N3/50 | 38.5±7.8 | 137 |
| B2/10 | 39.7±1.7 | 141 |
| B2/50 | 41.4±3.8 | 147 |
| B3/10 | 74.0±11.2 | 263 |
| B3/50 | 46.5±8.7 | 165 |
| B4/10 | 8.7±0.3 | 31 |
| B4/50 | 17.2±1.9 | 61 |
| B5/10 | 54.2±6.0 | 193 |
| B5/50 | 48.0±10.5 | 171 |
In the case of existing the 5C9IgG of 1 μ g/mL, discharge ring AMP level is reduced to 33% of level in the situation that existing ring AMP to measure damping fluid.
Table 20a
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Asp43 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
1the mean value of twice mensuration
2unitary determination
PTSH concentration=3ng/mL
All dilutions are all to measure in damping fluid at ring AMP
a5B3 is the human monoclonal antibodies (negative control of 5C9) of a kind of antiglutamic acid decarboxylase (GAD).
Table 20b
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Glu61 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20c
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and His105 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20d
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Glu107 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Experiment 1
Referring to the illustrative footnote of table 20a.
Experiment 2
Referring to the illustrative footnote of table 20a.
Table 20e
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Phe130 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20f
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Glu178 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20g
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Tyr185 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
UD=is lower than measuring the limit value detecting.
Referring to the illustrative footnote of table 20a.
Table 20h
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Asp203 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20i
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Tyr206 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20j
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Lys209 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20k
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Asp232 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20l
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Lys250 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20m
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Glu251 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20n
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Thr257 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20o
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Arg274 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Referring to the illustrative footnote of table 20a.
Table 20p
The effect that 5C9IgG produces at the ring AMP that expresses wild-type TSHR and Asp276 and pTSH is stimulated in being mutated into the Chinese hamster ovary celI of TSHR of L-Ala
Experiment 1
Referring to the illustrative footnote of table 20a.
Experiment 2
Referring to the illustrative footnote of table 20a.
Table 21
TSHR sudden change (with respect to wild-type) is the summary to the effect aspect following in the Chinese hamster ovary celI of TSHR transfection: the ability of the stimulation that 5C9IgG and 9D33IgG blocking-up TSH produce ring AMP
| TSHR sudden change | The stimulation (with respect to wild-type) that pTSH produces ring AMP | The blocking-up (with respect to wild-type) of the stimulation that 5C9IgG produces ring AMP for TSH | The blocking-up (with respect to wild-type) of the stimulation that 9D33IgG produces ring AMP for TSH |
| Wild-type | +++++ | +++++ | +++++ |
| Asp?43Ala | +++ | +++++ | +++++ |
| Lys?58Ala | +++++ | +++++ | 0 |
| Ile?60Ala | +++++ | +++++ | +++++ |
| Glu?61Ala | ++++ | +++++ | +++++ |
| Arg?80Ala | +++++ | +++++ | 0 |
| Tyr?82Ala | +++++ | +++++ | 0 |
| Thr?104Ala | +++++ | +++++ | NT |
| His?105Ala | +++++ | +++++ | NT |
| Glu?107Ala | +++ | +++++ | +++++ |
| Arg?109Ala | +++++ | +++++ | 0 |
| Lys?129Ala | +++++ | 0 | 0 |
| Phe?130Ala | +++++ | +++ | +++++ |
| Phe?134Ala | +++++ | +++++ | ++ |
| Asp?151Ala | +++++ | ++++ | NT |
| Glu?178Ala | ++++ | +++ | ++++ |
| Lys?183Ala | +++++ | + | +++++ |
| Tyr?185Ala | ++++ | +++++ | +++++ |
| Asp?203Ala | ++++ | 0 | +++++ |
| Tyr?206Ala | ++++ | +++ | +++++ |
| Lys?209Ala | ++++ | +++++++ | +++++ |
| Asp?232Ala | +++ | +++++ | +++++ |
| Gln?235Ala | +++++ | +++++ | +++++ |
| Lys?250Ala | +++++ | ++++ | ++++ |
| Glu?251Ala | +++++ | +++ | +++++ |
| Arg?255Ala | +++++ | +++++ | +++++ |
| Thr?257Ala | +++++ | +++++ | +++++ |
| Trp?258Ala | +++++ | +++++ | +++++ |
| Arg?274Ala | +++++ | +++++++ | +++++++ |
| Asp?276Ala | +++++ | ++++ | +++++ |
| Ser?281Ala | ++++ | +++++ | ++++ |
| Arg?80Asp | ++++ | +++++ | 0 |
| Asp?151Arg | +++++ | +++++ | NT |
| Lys?183Asp | +++ | + | +++++ |
| Arg?255Asp | +++++ | +++++ | +++++++ |
| Asp?160Lys | 0 | +++++ a | NT |
PTSH concentration=the 3ng/mL using.
The relative effect of TSHR sudden change is expressed as and uses the viewed percentage ratio of wild-type, as follows :-+++ ++=100% wild-type activity; The wild-type activity of the < 100-80% of +++ +=; +++ the wild-type activity of=< 80-60%; ++ the wild-type activity of=< 60-40%; The wild-type activity of +=< 40-20%; The wild-type activity of 0=< 20%, and with respect to the rising activity of wild-type: > 100%=+++++++.NT=not test (N.T.).
adue to TSH nonreply, use M22 to test the stimulation (referring to text) to ring AMP in this experiment.
Table 22
The TSHR Asp203Ala effect to following aspect of suddenling change: the ability with the stimulation that patients serum TSHR autoantibody (B2-B5) blocking-up of antagonistic activity produces ring AMP by TSH
| test sample and serum dilution a | wild-type TSHR ring AMP concentration fmol/ cell hole.Mean value ± SD; N=3 | the inhibition % of the ring AMP level that wild-type TSHR stimulates TSH b | tSHR Asp203Ala ring AMP concentration fmol/ cell hole.Mean value ± SD; N=3 | the inhibition % of the ring AMP level that TSHR Asp203Ala stimulates TSH b |
| ring AMP measures damping fluid | 242 ± 130 | 292 ± 89 | ||
| tSH c | 9357 ± 1155 | 7591 ± 832 | ||
| tSH c+ 1 μ g/mL 5C9 | 1110 ± 811 | 92 | 9400 1 | 4 |
| hBD mixture/10 | 183 ± 67 | 496 ± 76 | ||
| tSH c+ HBD pool/10 | 13303 ± 1819 | 0 | 9756 1 | 0 |
| b2/10 | 110 ± 36 | 270 ± 72 | ||
| tSH c+ B2/10 | 454 ± 381 | 97 | 1963 ± 357 | 80 |
| b3/10 | 329 1 | 582 ± 74 | ||
| tSH c+ B3/10 | 3407 ± 1341 | 74 | 4027 ± 278 | 59 |
| b4/10 | 59 ± 22 | 161 ± 36 | ||
| tSH c+ B4/10 | 278 ± 73 | 98 | 150 ± 41 | 98 |
| b5/10 | 647 ± 170 | 1064 ± 228 | ||
| tSH c+ B5/10 | 4173 ± 515 | 69 | 6871 ± 618 | 30 |
abeing diluted in ring AMP measures in damping fluid.
bthe inhibition % that the ring AMP of TSH induction is stimulated
1the mean value of double sample
cpTSH uses with the final concentration of 3ng/mL.
HBD mixture=health is contributed the mixture of blood person serum.
Sequence table
<110>RSR company limited
<120> is as the human monoclonal antibodies of the thyrotropin receptor which act of antagonist
<130>P108503PCT
<150>GB?0702990.3
<151>2007-02-15
<150>GB?0714036.1
<151>2007-07-18
<160>11
<170>PatentIn?version?3.3
<210>1
<211>5
<212>PRT
<213> people
<400>1
Ser?Asn?Tyr?Met?Ser
1 5
<210>2
<211>16
<212>PRT
<213> people
<400>2
Val?Thr?Tyr?Ser?Gly?Gly?Ser?Thr?Ser?Tyr?Ala?Asp?Ser?Val?Lys?Gly
1 5 10 15
<210>3
<211>18
<212>PRT
<213> people
<400>3
Gly?Gly?Arg?Tyr?Cys?Ser?Ser?Ile?Ser?Cys?Tyr?Ala?Arg?Ser?Gly?Cys
1 5 10 15
Asp?Tyr
<210>4
<211>11
<212>PRT
<213> people
<400>4
Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Tyr?Leu?Asn
1 5 10
<210>5
<211>7
<212>PRT
<213> people
<400>5
Ala?Ala?Ser?Ser?Leu?Gln?Ser
1 5
<210>6
<211>10
<212>PRT
<213> people
<400>6
Gln?Gln?Ser?Tyr?Ser?Ser?Pro?Ser?Thr?Thr
1 5 10
<210>7
<211>699
<212>DNA
<213> people
<400>7
gaagtgcagc?tggtggagtc?tggaggaggc?ctgatccagc?ctggggggtc?cctgagactc 60
tcctgtgcag?cctctgggtt?caccgtcagt?agcaactaca?tgagctgggt?ccgccaggct 120
ccagggaagg?ggctggagtg?ggtctcagtt?acttatagcg?gtggtagcac?atcctacgca 180
gactccgtga?agggccgatt?caccatctcc?agagacaatt?ccaagaacac?gctgtatctt 240
caaatgaaca?gcctgagagc?cgaggacacg?gccgtgtatt?actgtgcgag?aggggggcga 300
tattgtagta?gtataagctg?ctacgcgagg?agcgggtgtg?actactgggg?ccagggaacc 360
ctggtcaccg?tctcctcagc?ctccaccaag?ggcccatcgg?tcttccccct?ggcaccctcc 420
tccaagagca?cctctggggg?cacagcggcc?ctgggctgcc?tggtcaagga?ctacttcccc 480
gaaccggtga?cggtgtcgtg?gaactcaggc?gccctgacca?gcggcgtgca?caccttcccg 540
gctgtcctac?agtcctcagg?actctactcc?ctcagcagcg?tggtgaccgt?gccctccagc 600
agcttgggca?cccagaccta?catctgcaac?gtgaatcaca?agcccagcaa?caccaaggtg 660
gacaagagag?ttgagcccaa?atcttgtgac?aaaactagt 699
<210>8
<211>617
<212>DNA
<213> people
<400>8
gccatccaga?tgacccagtc?tccttcctcc?ctgtctgcat?ctgtaggaga?cagagtcacc 60
atcacttgcc?gggcaagtca?gagcattagc?aactatttaa?attggtatca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
aggttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?caacttacta?ctgtcaacag?agttacagtt?ccccctccac?cacttttggc 300
caggggacca?agctggagat?caaacgaact?gtggctgcac?catctgtctt?catcttcccg 360
ccatctgatg?agcagttgaa?atctggaact?gcctctgttg?tgtgcctgct?gaataacttc 420
tatcccagag?aggccaaagt?acagtggaag?gtggataacg?ccctccaatc?gggtaactcc 480
caggagagtg?tcacagagca?ggacagcaag?gacagcacct?acagcctcag?cagcaccctg 540
acgctgagca?aagcagacta?cgagaaacac?aaagtctacg?cctgcgaagt?cacccatcag 600
ggcctgagct?cgcccgt 617
<210>9
<211>233
<212>PRT
<213> people
<400>9
Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Ile?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Val?Ser?Ser?Asn
20 25 30
Tyr?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Val?Thr?Tyr?Ser?Gly?Gly?Ser?Thr?Ser?Tyr?Ala?Asp?Ser?Val?Lys
50 55 60
Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr?Leu
65 70 75 80
Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Arg?Gly?Gly?Arg?Tyr?Cys?Ser?Ser?Ile?Ser?Cys?Tyr?Ala?Arg?Ser?Gly
100 105 110
Cys?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser
115 120 125
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro
145 150 155 160
Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val
165 170 175
His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser
180 185 190
Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile
195 200 205
Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Arg?Val
210 215 220
Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?Ser
225 230
<210>10
<211>205
<212>PRT
<213> people
<400>10
Ala?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr?Ser?Ser?Pro?Ser
85 90 95
Thr?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala
100 105 110
Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser
115 120 125
Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu
130 135 140
Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser
145 150 155 160
Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu
165 170 175
Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val
180 185 190
Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro
195 200 205
<210>11
<211>764
<212>PRT
<213> people
<400>11
Met?Arg?Pro?Ala?Asp?Leu?Leu?Gln?Leu?Val?Leu?Leu?Leu?Asp?Leu?Pro
1 5 10 15
Arg?Asp?Leu?Gly?Gly?Met?Gly?Cys?Ser?Ser?Pro?Pro?Cys?Glu?Cys?His
20 25 30
Gln?Glu?Glu?Asp?Phe?Arg?Val?Thr?Cys?Lys?Asp?Ile?Gln?Arg?Ile?Pro
35 40 45
Ser?Leu?Pro?Pro?Ser?Thr?Gln?Thr?Leu?Lys?Leu?Ile?Glu?Thr?His?Leu
50 55 60
Arg?Thr?Ile?Pro?Ser?His?Ala?Phe?Ser?Asn?Leu?Pro?Asn?Ile?Ser?Arg
65 70 75 80
Ile?Tyr?Val?Ser?Ile?Asp?Val?Thr?Leu?Gln?Gln?Leu?Glu?Ser?His?Ser
85 90 95
Phe?Tyr?Asn?Leu?Ser?Lys?Val?Thr?His?Ile?Glu?Ile?Arg?Asn?Thr?Arg
100 105 110
Asn?Leu?Thr?Tyr?Ile?Asp?Pro?Asp?Ala?Leu?Lys?Glu?Leu?Pro?Leu?Leu
115 120 125
Lys?Phe?Leu?Gly?Ile?Phe?Asn?Thr?Gly?Leu?Lys?Met?Phe?Pro?Asp?Leu
130 135 140
Thr?Lys?Val?Tyr?Ser?Thr?Asp?Ile?Phe?Phe?Ile?Leu?Glu?Ile?Thr?Asp
145 150 155 160
Asn?Pro?Tyr?Met?Thr?Ser?Ile?Pro?Val?Asn?Ala?Phe?Gln?Gly?Leu?Cys
165 170 175
Asn?Glu?Thr?Leu?Thr?Leu?Lys?Leu?Tyr?Asn?Asn?Gly?Phe?Thr?Ser?Val
180 185 190
Gln?Gly?Tyr?Ala?Phe?Asn?Gly?Thr?Lys?Leu?Asp?Ala?Val?Tyr?Leu?Asn
195 200 205
Lys?Asn?Lys?Tyr?Leu?Thr?Val?Ile?Asp?Lys?Asp?Ala?Phe?Gly?Gly?Val
210 215 220
Tyr?Ser?Gly?Pro?Ser?Leu?Leu?Asp?Val?Ser?Gln?Thr?Ser?Val?Thr?Ala
225 230 235 240
Leu?Pro?Ser?Lys?Gly?Leu?Glu?His?Leu?Lys?Glu?Leu?Ile?Ala?Arg?Asn
245 250 255
Thr?Trp?Thr?Leu?Lys?Lys?Leu?Pro?Leu?Ser?Leu?Ser?Phe?Leu?His?Leu
260 265 270
Thr?Arg?Ala?Asp?Leu?Ser?Tyr?Pro?Ser?His?Cys?Cys?Ala?Phe?Lys?Asn
275 280 285
Gln?Lys?Lys?Ile?Arg?Gly?Ile?Leu?Glu?Ser?Leu?Met?Cys?Asn?Glu?Ser
290 295 300
Ser?Met?Gln?Ser?Leu?Arg?Gln?Arg?Lys?Ser?Val?Asn?Ala?Leu?Asn?Ser
305 310 315 320
Pro?Leu?His?Gln?Glu?Tyr?Glu?Glu?Asn?Leu?Gly?Asp?Ser?Ile?Val?Gly
325 330 335
Tyr?Lys?Glu?Lys?Ser?Lys?Phe?Gln?Asp?Thr?His?Asn?Asn?Ala?His?Tyr
340 345 350
Tyr?Val?Phe?Phe?Glu?Glu?Gln?Glu?Asp?Glu?Ile?Ile?Gly?Phe?Gly?Gln
355 360 365
Glu?Leu?Lys?Asn?Pro?Gln?Glu?Glu?Thr?Leu?Gln?Ala?Phe?Asp?Ser?His
370 375 380
Tyr?Asp?Tyr?Thr?Ile?Cys?Gly?Asp?Ser?Glu?Asp?Met?Val?Cys?Thr?Pro
3853 90 395 400
Lys?Ser?Asp?Glu?Phe?Asn?Pro?Cys?Glu?Asp?Ile?Met?Gly?Tyr?Lys?Phe
405 410 415
Leu?Arg?Ile?Val?Val?Trp?Phe?Val?Ser?Leu?Leu?Ala?Leu?Leu?Gly?Asn
420 425 430
Val?Phe?Val?Leu?Leu?Ile?Leu?Leu?Thr?Ser?His?Tyr?Lys?Leu?Asn?Val
435 440 445
Pro?Arg?Phe?Leu?Met?Cys?Asn?Leu?Ala?Phe?Ala?Asp?Phe?Cys?Met?Gly
450 455 460
Met?Tyr?Leu?Leu?Leu?Ile?Ala?Ser?Val?Asp?Leu?Tyr?Thr?His?Ser?Glu
465 470 475 480
Tyr?Tyr?Asn?His?Ala?Ile?Asp?Trp?Gln?Thr?Gly?Pro?Gly?Cys?Asn?Thr
485 490 495
Ala?Gly?Phe?Phe?Thr?Val?Phe?Ala?Ser?Glu?Leu?Ser?Val?Tyr?Thr?Leu
500 505 510
Thr?Val?Ile?Thr?Leu?Glu?Arg?Trp?Tyr?Ala?Ile?Thr?Phe?Ala?Met?Arg
515 520 525
Leu?Asp?Arg?Lys?Ile?Arg?Leu?Arg?His?Ala?Cys?Ala?Ile?Met?Val?Gly
530 535 540
Gly?Trp?Val?Cys?Cys?Phe?Leu?Leu?Ala?Leu?Leu?Pro?Leu?Val?Gly?Ile
545 550 555 560
Ser?Ser?Tyr?Ala?Lys?Val?Ser?Ile?Cys?Leu?Pro?Met?Asp?Thr?Glu?Thr
565 570 575
Pro?Leu?Ala?Leu?Ala?Tyr?Ile?Val?Phe?Val?Leu?Thr?Leu?Asn?Ile?Val
580 585 590
Ala?Phe?Val?Ile?Val?Cys?Cys?Cys?Tyr?Val?Lys?Ile?Tyr?Ile?Thr?Val
595 600 605
Arg?Asn?Pro?Gln?Tyr?Asn?Pro?Gly?Asp?Lys?Asp?Thr?Lys?Ile?Ala?Lys
610 615 620
Arg?Met?Ala?Val?Leu?Ile?Phe?Thr?Asp?Phe?Ile?Cys?Met?Ala?Pro?Ile
625 630 635 640
Ser?Phe?Tyr?Ala?Leu?Ser?Ala?Ile?Leu?Asn?Lys?Pro?Leu?Ile?Thr?Val
645 650 655
Ser?Asn?Ser?Lys?Ile?Leu?Leu?Val?Leu?Phe?Tyr?Pro?Leu?Asn?Ser?Cys
660 665 670
Ala?Asn?Pro?Phe?Leu?Tyr?Ala?Ile?Phe?Thr?Lys?Ala?Phe?Gln?Arg?Asp
675 680 685
Val?Phe?Ile?Leu?Leu?Ser?Lys?Phe?Gly?Ile?Cys?Lys?Arg?Gln?Ala?Gln
690 695 700
Ala?Tyr?Arg?Gly?Gln?Arg?Val?Pro?Pro?Lys?Asn?Ser?Thr?Asp?Ile?Gln
705 710 715 720
Val?Gln?Lys?Val?Thr?His?Asp?Met?Arg?Gln?Gly?Leu?His?Asn?Met?Glu
725 730 735
Asp?Val?Tyr?Glu?Leu?Ile?Glu?Asn?Ser?His?Leu?Thr?Pro?Lys?Lys?Gln
740 745 750
Gly?Gln?Ile?Ser?Glu?Glu?Tyr?Met?Gln?Thr?Val?Leu
755 760
Claims (34)
1. human monoclonal or the recombinant antibodies of the thyrotropin receptor which act of separation (TSHR), described antibody is the antagonist of a kind of thyrotropic hormone (TSH), has 10
9the binding affinity to people's total length TSHR of L/mol, wherein said antibody comprises V
hdistrict, described V
hdistrict comprises following complementary determining region (CDR):
a)SNYMS(CDR1)；
B) VTYSGGSTSYADSVKG (CDR2); With
c)GGRYCSSISCYARSGCDY(CDR3)，
And V
ldistrict, described V
ldistrict comprises following CDR:
a)RASQSISNYLN(CDR1)；
B) AASSLQS (CDR2); With
c)QQSYSSPSTT(CDR3)。
2. according to the antibody of claim 1, described antibody comprises nucleotide sequence coded aminoacid sequence that SEQ ID No.9 represents or SEQ ID No.7, and SEQ ID No.10 nucleotide sequence coded aminoacid sequence that represent or SEQ ID No.8.
3. according to the antibody of claim 1, described antibody has 10
10the binding affinity to people's total length TSHR of L/mol.
4. separation is anti-according to an antibody of claim 1,2 or 3 TSHR, and described antibody can suppress the constitutive activity of TSHR.
5. according to claim 1,2,3 or 4 antibody, described antibody is a kind of antagonist of thyroid stimulating antibody.
6. according to claim 1,2,3 or 4 antibody, described antibody has the TSH antagonist properties of patients serum TSHR autoantibody, and described patients serum TSHR autoantibody is TSH antagonist.
7. according to the antibody of claim 5, described antibody is the antagonist of a kind of TSH, and is a kind of antagonist of thyroid stimulating antibody.
8. according to the antibody of claim 6, described antibody is the antagonist of a kind of TSH, and is a kind of antagonist of thyroid stimulating antibody.
9. according to the antibody of claim 1-4,7 and 8 any one, described antibody has the antagonist feature of patients serum TSHR autoantibody, and described patients serum TSHR autoantibody is the antagonist of thyroid stimulating antibody.
10. according to the antibody of claim 1-4,7 and 8 any one, described antibody is a kind of TSH, hMAb TSHR1(M22) or TSHR is had the antibody of stimulating activity or has the active antibody of blocking-up and the inhibitor of TSHR or its a part of combination.
11. according to the antibody of claim 10, and wherein said TSHR part comprises the leucic structural domain of being rich in of TSHR (LRD) or its sizable part.
12. 1 kinds of Nucleotide, comprise
A kind of coding is according to the nucleotide sequence of the antibody of claim 1-11 any one.
13. 1 kinds comprise according to the carrier of the Nucleotide of claim 12.
14. one kind comprises according to the cell of the separation of the antibody of claim 1-11 any one.
15. 1 kinds of compositions, described composition comprises the TSHR autoantibody of determining concentration, and comprises a kind of according to the antibody of claim 1-11 any one.
16. 1 kinds of compositions, described composition comprises the TSHR autoantibody with TSH antagonistic activity of determining concentration, comprises a kind of according to the antibody of claim 1-11 any one.
17. 1 kinds of compositions, described composition comprises determines the TSHR autoantibody of concentration, comprise a kind of according to the antibody of claim 1-11 any one, the antagonist that described TSHR autoantibody is thyroid stimulating antibody.
18. 1 kinds of compositions, described composition comprises determines the TSHR autoantibody with TSH antagonistic activity of concentration, comprise a kind of according to the antibody of claim 1-11 any one, the antagonist that described TSHR autoantibody is thyroid stimulating antibody.
19. a pharmaceutical composition, comprises according to antibody and pharmaceutically acceptable carrier of claim 1-11 any one.
20. comprise the purposes in the medicine for the preparation for the treatment of Tiroidina associated conditions according to the pharmaceutical composition of the antibody of claim 1-11 any one and pharmaceutically acceptable carrier, and wherein said Tiroidina associated conditions is selected from: Thyroid Activity excessively, hyperthyroidism, circumscribed myxedema, thyroid carcinoma and the thyroiditis of graves' ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotrophin induction.
21. according to the pharmaceutical composition of claim 19 or 20, and described composition comprises the thyrotropin receptor antagonist that one or more are other.
22. 1 kinds of generations are according to the method for the antibody of claim 1-11 any one, and described method comprises that cultivation is a kind of according to the cell of claim 14, thereby described antibody is by described cell expressing.
23. according to the method for claim 22, and wherein said antibody is by described emiocytosis.
24. according to the antibody of claim 1-11 any one the purposes in the medicine for the preparation of a kind of Tiroidina associated conditions for the treatment of, described Tiroidina associated conditions is selected from: Thyroid Activity excessively, hyperthyroidism, circumscribed myxedema, thyroid carcinoma and the thyroiditis of graves' ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotrophin induction.
Determine the method for the combination situation of a kind of TSHR antibody to be test and a kind of polypeptide with TSHR-related amino acid sequence for 25. 1 kinds, wherein said method relates to a step and uses according to the method steps of the antibody of claim 1-11 any one.
26. according to the method for claim 25, and described method comprises determines according to the antibody of claim 1-11 any one the effect for the combination of TSHR antibody and described polypeptide.
27. according to the method for claim 26, the people TSHR that wherein said TSHR-related polypeptide comprises total length.
Determine the method for the combination situation of TSH to be test or an associated molecule and a kind of polypeptide with TSHR-related amino acid sequence for 28. 1 kinds, wherein said method relates to a step and uses according to the method steps of the antibody of claim 1-11 any one.
29. according to the method for claim 28, and wherein said method is ELISA.
30. determine the amino acid whose method of TSHR that participates in the TSHR autoantibody that is combined as antagonist for one kind, described method comprises provides one to have according to the polypeptide of relevant the first aminoacid sequence of the combinative TSHR-of antibody of claim 1-11 any one, in the relevant aminoacid sequence of described TSHR-, modify at least one amino acid, and determines the effect of such change to described antibodies.
31. 1 kinds of changes are a kind of according to the method for the antibody of claim 1-11 any one, and described method comprises at least one amino acid of modifying described antibody, and determine that such modification is to being incorporated into the effect of a TSHR-correlated series.
32. 1 kinds of qualifications can suppress the method for the molecule that thyroid stimulating antibody is combined with TSHR, described method comprise provide at least one according to the antibody of claim 1-11 any one as object of reference.
33. 1 kinds of qualifications can suppress the method for the molecule that Tiroidina blocking antibody is combined with TSHR, and described method comprises provides at least one according to the antibody of claim 1-11 any one, as object of reference.
34. according to the method for claim 33, wherein selects the molecule that stops Tiroidina blocking antibody to be combined with TSHR.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US90133207P | 2007-02-15 | 2007-02-15 | |
| US60/901,332 | 2007-02-15 | ||
| GB0702990.3 | 2007-02-15 | ||
| GB0702990A GB0702990D0 (en) | 2007-02-15 | 2007-02-15 | Products |
| GB0714036.1 | 2007-07-18 | ||
| GB0714036A GB0714036D0 (en) | 2007-07-18 | 2007-07-18 | Products |
| PCT/GB2008/000518 WO2008099185A1 (en) | 2007-02-15 | 2008-02-14 | Human monoclonal antibodies to the thyrotropin receptor which act as antagonists |
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| Publication Number | Publication Date |
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| CN101657468A CN101657468A (en) | 2010-02-24 |
| CN101657468B true CN101657468B (en) | 2014-09-03 |
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| CN200880011577.5A Active CN101657468B (en) | 2007-02-15 | 2008-02-14 | Human monoclonal antibodies to the thyrotropin receptor which act as antagonists |
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| JP (1) | JP5345556B2 (en) |
| CN (1) | CN101657468B (en) |
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| JP5997446B2 (en) * | 2011-02-03 | 2016-09-28 | アークレイ株式会社 | Liquid reagent for immobilizing thyroid hormone and use thereof |
| JP7025041B2 (en) * | 2019-12-26 | 2022-02-24 | ヤマサ醤油株式会社 | Mutant-introduced thyrotropin receptor |
| CN112269026A (en) * | 2020-09-04 | 2021-01-26 | 首都医科大学附属北京朝阳医院 | Method suitable for accurately preparing TSH powdery international standard substance in concentration |
| CN119731208A (en) * | 2022-08-31 | 2025-03-28 | 长春金赛药业有限责任公司 | Antibodies that bind thyroid stimulating hormone receptor and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB9909661D0 (en) * | 1998-06-06 | 1999-06-23 | Rsr Ltd | Assays for TSH receptor autoantibodies |
| DE19907094C1 (en) * | 1999-02-19 | 2000-04-13 | Brahms Diagnostica Gmbh | Use of antibodies capable of blocking human thyroid-stimulating hormone receptor to treat hyperthyroidism and endocrine orbitopathy |
| US7084257B2 (en) * | 2001-10-05 | 2006-08-01 | Amgen Inc. | Fully human antibody Fab fragments with human interferon-gamma neutralizing activity |
| CN1717418B (en) * | 2002-11-29 | 2011-03-30 | Rsr有限公司 | Antibody for the thyrotropin receptor and uses thereof |
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- 2008-02-14 CN CN200880011577.5A patent/CN101657468B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| KOHN L D.ET AL.characterization of monoclonal thyrotropin binding-inhibition autoantibodies from a hashimoto"s patient whose chindren ha inteauterine and neonatal throid disease..《THE JOURNAL OF CLINICAL EMDOCRINOLOGY AND METABOLISM》.1997,第82卷(第2期),3998-4009. * |
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| Publication number | Publication date |
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| JP5345556B2 (en) | 2013-11-20 |
| JP2010521139A (en) | 2010-06-24 |
| CN101657468A (en) | 2010-02-24 |
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