CN101506659B - Anti-drug antibody assay - Google Patents
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- CN101506659B CN101506659B CN200780030744.6A CN200780030744A CN101506659B CN 101506659 B CN101506659 B CN 101506659B CN 200780030744 A CN200780030744 A CN 200780030744A CN 101506659 B CN101506659 B CN 101506659B
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Abstract
本发明提供了与食蟹猴IgG特异性结合的抗体，特征在于其不与人IgG结合，以及用双抗原桥联免疫测定法对猴种样品中药物抗体(DA)和针对所述药物抗体的抗体(抗药物抗体，ADA)的免疫复合物(DA/ADA复合物)进行免疫测定的方法。
The present invention provides an antibody that specifically binds to cynomolgus monkey IgG, which is characterized in that it does not bind to human IgG, and uses a double-antigen bridging immunoassay method to detect drug antibody (DA) in a monkey sample and the antibody against the drug. A method for immunoassay of an immune complex (DA/ADA complex) of an antibody (anti-drug antibody, ADA).
Description
Invention field
The present invention comprises the method for measuring anti-drug antibodies and is used for the kit of such determination method.
Background of invention
Use the standard solid-phase immunoassay of monoclonal antibody relate to be adsorbed on antibody (capture antibody) on the solid phase, antigen and to another epi-position of antigen, puted together between the antibody (tracer antibody) of enzyme or detectable label and formed compound.Form sandwich body thus: solid phase-capture antibody-antigen-tracer antibody.In sandwich body, the enzymatic activity that antibody is puted together (or amount of detectable label) is proportional with the antigen concentration of hatching in the medium.A kind of sandwich assay is two antigen bridging immunoassays (double antigen bridging immunoassay), wherein catches with tracer antibody to combine with the different epi-positions of antigen.Hoesel, people such as W. have reported the two antigen bridging determination methods of anti-EPO in J.Immunol.Methods 294 (2004) 101-110, used with amino and with the potpourri of the immobilization rhEPO of glycosyl coupling.Immunoassay such as two antigen bridging ELISA be the research patient for the immunogenic response of antibody drug in the common determination method type of application of institute.Mire-Sluis, people such as A.R. in J.Immunol.Methods 289 (2004) 1-16, have summed up design and have optimized the suggestion of detection to the immunoassay of host's antibody of biological technology products.According to people such as Mire-Sluis, well-known anti-drug antibody assay form demonstrates many shortcomings.Anti-drug antibody assay has mentioned in WO for example 2005/045058 and WO 90/006515.The anti-idiotype determination method has mentioned in for example US 5,219,730, WO 87/002778, EP 0139389 and EP 0170302.Wadhwa, people such as M. have reported the method for the unnecessary antibody that detection, measurement and sign are induced by the therapeutic biological products in J.Immunol.Methods 278 (2003) 1-17.The immunoassay of the antibody that is directed against drug antibody in the sample being carried out immunoassays with two antigen bridging immunoassays has been described among the PCT/EP2007/001935.The immunoassay of measuring the people's antibody in the monkey has been described among the WO 2006/066912.The mensuration system that (for example US 2003/0068664) can the detection of active therapeutic antibodies in this area is known equally.Such system needs antigen to combine with solid phase, and the antigen that therapeutic antibodies combines therewith combines, and detects the therapeutic antibodies that combines with solid phase through antigen.
The invention summary
The invention provides the antibody that combines, do not combine with machin (Cynomolgus) IgG specificity with human IgG.Preferred said antibody is monoclonal antibody.
In preferred embodiments, antibody according to the present invention is produced with clone 3.25.12 (DSMACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSM ACC2802) or 7.72.32 (DSM ACC2803).
The invention provides and use sandwich assay, (anti-drug antibodies, immune complex ADA) carries out the method for immunoassays to promptly two antigen bridging immunoassays to the sample Chinese traditional medicine antibody (DA) of monkey kind with to the antibody of said drug antibody.
Said immune complex further is abbreviated as the DA/ADA compound.
The invention provides the method for DA/ADA compound in the immunoassays sample; Its use comprises sandwich type or two antigen bridging immunoassay of capture antibody and tracer antibody; Be characterised in that one of said antibody; Being tracer antibody or capture antibody, is the antibody that combines with machin IgG specificity, and another antibody is the antibody that combines with the human immunoglobulin(HIg) specificity.
In a preferred embodiment of the invention, capture antibody is the anti-people Ig antibody that combines with the human immunoglobulin(HIg) specificity, and tracer antibody is the anti-machin IgG antibody that combines with machin IgG specificity.In a preferred embodiment of the invention, capture antibody is the anti-machin IgG antibody that combines with machin IgG specificity, and tracer antibody is the anti-people Ig antibody that combines with people Ig specificity.
Preferred anti-people Ig antibody combines with the human IgG specificity.Preferred anti-machin IgG antibody and/or anti-people Ig antibody are monoclonal antibody.Preferred said specificity combines the antibody of human immunoglobulin(HIg) not combine with machin IgG.The antibody of preferred said combination human immunoglobulin(HIg) is produced by clone DSMACC2708.The antibody of preferred said combination machin IgG does not combine with human IgG.The antibody of preferred said combination machin IgG is produced by clone 3.25.12 (DSM ACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSM ACC2802) or 7.72.32 (DSM ACC2803).
In said mensuration process, between anti-machin IgG antibody, DA/ADA compound and anti-people Ig antibody, form compound, the amount of the compound of formation is associated with the concentration of DA/ADA compound, DA and/or ADA.
According to the present invention, can carry out direct sample analysis for the detection of the DA/ADA compound that forms.In such determination method, have only and contain drug antibody in the sample and anti-drug antibodies just can be seen positive signal.
According to the present invention, alternatively, sample analysis (can) with the drug antibody preincubate of scheduled volume after carry out.In such determination method, if contain anti-drug antibodies in the sample, just can see positive signal, exist/there is not drug antibody in the sample and rely on.
Preferred capture antibody is puted together through passive absorption and solid phase, therefore puts together in two different antibody sites and solid phase at least.Butler for example, J.E., Solid Phases in Immunoassay, in: Immunoassay, Diamandis, E.P. and Christopoulos, T.K. (volume) academic press, Santiago (1996) are described passive absorption in the 205-225 page or leaf to some extent.
In a preferred embodiment of the invention, capture antibody combines immobilization through specificity.Such combination to (first component/second component) does; For example streptavidin or avidin/biotin, antibody/antigen (are seen for example Hermanson; G.T. wait the people; BioconjugateTechniques, academic press, 1996), agglutinin/polysaccharide, steroids/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/ albumin A and/or G etc.Preferred capture antibody and biotin-conjugated are fixed through immobilization avidin or streptavidin.
Preferred antibody is puted together puting together of gametophyte with it be to realize through chemical bond, combines via N end and/or epsilon-amino (lysine), the epsilon-amino of different lysines, carboxyl, sulfydryl, hydroxyl and/or the phenol functional group of antibody amino acid backbone and/or the sugar alcohol base of antibody sugar structure.
In a preferred embodiment of the invention, tracer antibody and detectable mark are puted together, and preferably combine puting together through specificity.Such combination to (first component/second component) does, for example streptavidin or avidin/biotin, antibody/antigen, agglutinin/polysaccharide, steroids/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/ albumin A and/or G etc.Preferred tracer antibody is puted together through foxalin with to the antibody and the detectable mark of foxalin.Alternatively, tracer antibody and electrochemiluminescence mark are puted together, like the ruthenium bipyridyl complexes.
Provide in other embodiment of the present invention with the antibody (anti-drug antibodies, method ADA) that are directed against drug antibody in sandwich type or the two antigen bridging immunoassay immunoassays monkey kind sample.
The invention provides the method for ADA in the immunoassays sample; Use comprises sandwich type or two antigen bridging immunoassay of capture antibody and tracer antibody; Be characterised in that one of said antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, another antibody is drug antibody.
In the preferred embodiment of immunoassays ADA, capture antibody is a drug antibody, and tracer antibody is the anti-machin IgG antibody that combines, do not combine with human IgG with machin IgG specificity.In other preferred embodiment of immunoassays ADA, capture antibody is the anti-machin IgG antibody that combines, do not combine with human IgG with machin IgG specificity, and tracer antibody is a drug antibody.In said mensuration process, between drug antibody, ADA and anti-machin IgG antibody, form compound, the amount of the said compound of formation is associated with the concentration of ADA.In the preferred embodiment of immunoassays ADA, said anti-machin IgG antibody is monoclonal antibody (anti-machin mAb).
Another embodiment of the present invention is hybridoma cell line 3.25.12 (DSM ACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSMACC2802), 7.72.32 (DSM ACC2803).
Another aspect of the present invention is the antibody compositions that is used for according to the method for the invention, comprises the potpourri of the antibody of being produced by clone DSM ACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSM ACC 2802 and/or clone DSM ACC2803.
Detailed Description Of The Invention
The invention provides the antibody that combines, do not combine with machin IgG specificity with human IgG.
According to the present invention, term " drug antibody " expression can be used the antibody with the treatment disease to individuality.In a kind of determination method of carrying out according to the present invention, drug antibody and capture antibody, or drug antibody and tracer antibody comprise " identical " antibody molecule respectively, for example use identical expression vector recombinant production, and comprise identical amino acid sequence.Drug antibody (therapeutic monoclonal antibodies) is widely used in treating multiple disease such as tumor disease (for example blood and solid malignant comprise NHL, breast cancer and colorectal cancer), immunity disease, nervous centralis disease, vascular diseases or infectious disease.For example at Levene, people such as A.P., Journal of the Royal Society ofMedicine 98 (2005) 145-152 describe such antibody to some extent.Such antibody is the antibody that for example is directed against CD20, CD22, HLA-DR, CD33, CD52, EGFR, G250, GD3, HER2, PSMA, CD56, VEGF, VEGF2, CEA, Levis Y antigen, IL-6 acceptor or IGF-1 acceptor.Therapeutic antibodies is at Groner, people such as B., Curr.Mol.Meth.4 (2004) 539-547; Harris, M. also describes among Lancet Oncol. (2004) 292-302 to some extent.
The instance of therapeutic/drug antibody (preferred monoclonal antibody) is the antibody (mAb IL-6R) to the IL-6 acceptor.Such antibody is at for example Mihara, people such as M., Clin.Immunol.98 (2001) 319-326; Nishimoto, people such as N., Blood 106 (2005) 2627-2632; Clinical trial NCT00046774; Describe to some extent among the WO 2004/096274.
The instance of therapeutic/drug antibody (preferred monoclonal) is the antibody (mAbIGF-1R) to the IGF-1 acceptor.Such antibody is described among the WO 2005/005635 at for example WO 2004/087756 to some extent.
" anti-drug antibodies " is antibody, and it can be to any zone of drug antibody, the for example variable domains of drug antibody, constant domain or sugared structure.Such anti-drug antibodies possibly the immunogenic response as the patient (see Pan, people such as Y., FASEB be (1995) 43-49 J.9) occur in the Antybody therapy process.Most " anti-drug antibodies " combines with one or more complementary determining regions of drug antibody.Affinity between the antigen of anti-drug antibodies and its drug antibody is usually less than the affinity of drug antibody and its target antigen.
" anti-machin IgG antibody " is the antibody that combines with machin IgG (machin immunoglobulin G) specificity, is preferably monoclonal antibody (being monoclonal anti machin antibody, mAb < CynoIgG >).The dissociation constant that such antibody combines with machin IgG (=KDiss.) be at least 10
-9Mol/L, more preferably KDiss. is few 10
-10Mol/L.Simultaneously, through 10
-8Mol/L or poorer KDiss. for example 10
-5Mol/L guarantees the character that it does not combine with human IgG.Equally preferably, with the antibody that machin IgG combines, do not combine with human IgG, the difference to KDiss. between the reactivity of machin IgG and human IgG is at least 100 times respectively.
Preferred anti-machin IgG antibody also combines with marmoset IgG, rhesus macaque IgG and baboon IgG, dissociation constant (=KDiss.) be at least 10
-8Mol/l is preferably 10
-9Mol/L, more preferably KDiss. is at least 10
-10Mol/L.
In one embodiment, be monoclonal antibody according to antibody of the present invention.The antibody population by single kind cell or its filial generation production represented in the term " monoclonal " that uses in this application, and combine with the single antigenic determinant of its target antigen.The term that uses in this application " does not combine with human IgG " or it is equal to the grammer term and representes the antibody that do not combine with the human IgG specificity, and promptly its KDiss. is 10
-7Mol/L or poorer, for example 10
-5Mol/L.This does not comprise the polyclonal antibody crowd that machin is such, and it intersects absorption to remove the machin IgG that combines with human IgG with human immunoglobulin(HIg).Because the binding equilibrium that forms in this process, the absorption that intersects does not provide the polyclonal antibody that does not combine with human IgG crowd, let alone monoclonal antibody.Because many antibody that combine with human IgG do not intersect absorption under this balance, and stay in the solution, so stay in the antibody preparation that obtains.Therefore, immunoglobulin (Ig) can not got rid of the combination of any anti-human IgG fully, and still show the human IgG combination in case intersects absorption with human IgG.
Others of the present invention are clone DSM ACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSM ACC 2802, clone DSM ACC2803, and the monoclonal antibody of being produced by clone DSM ACC2799 or clone DSM ACC2800 or clone DSMACC2801 or clone DSM ACC 2802 or clone DSM ACC2803.Another aspect of the invention is antibody compositions, comprise the potpourri of the antibody of producing by clone DSMACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSMACC 2802 and/or clone DSM ACC2803.The present invention also comprises composition, and it comprises the antibody by clone DSM ACC2799 or clone DSM ACC2800 or clone DSM ACC2801 or clone DSM ACC 2802 or clone DSMACC2803 production.
Use is according to antibody of the present invention, can minimize or even gets rid of the change of background of individual human serum.
According to the present invention, term " monkey " refers to machin, marmoset, rhesus macaque and baboon." monkey " preferably represented machin, rhesus macaque and baboon.
Preferred anti-people Ig antibody (Ig representes immunoglobulin (Ig)) is such antibody, and it combines with epitope specificity on not being present in the machin immunoglobulin (Ig), in WO 2006/066912, this is described to some extent.This epi-position is characterised in that itself and MAB < H-Fc γ pan>M-R10Z8E9 also claims MAB < h-Fc gamma>M-R10Z8E9, or is called for short MAB M-R10Z8E9 and combines.In according to the preferred embodiments of the invention, anti-people Ig antibody is further characterized in that the epi-position of its combination is identical with MAB M-R10Z8E9.MAB M-R10Z8E9 is by such clone production, and this cell lies in and was preserved in German microbial preservation center (DSMZ) on Dec 22nd, 2004, and preserving number is DSM ACC2708.Preferred anti-people Ig antibody comprises variable heavy chain and the light chain domain of MAB M-R10Z8E9.More preferably anti-people Ig antibody comprises the CDR district and the inhuman framework of MAB M-R10Z8E9 variable heavy chain and light chain domain.Preferred anti-people Ig antibody is monoclonal antibody (anti-people Ig mAb).
Preferably use the combination character, particularly KDiss. of
instrument assessment antibody.In the method, through the variation evaluation combination character of surface plasma body resonant vibration (SPR).Can be easily with the antibodies of being studied on solid phase (being called chip), and assessment monoclonal antibody, polyclonal antibody or even comprise the combination of the chip that the serum of IgG encapsulates therewith.
According to the present invention, the solid support that is used for immunoassay has extensive description (seeing for example Butler, J.E., Methods 22 (2000) 4-23) in state of the art.
Hage for example, D.S. has described different immunoassays ratio juris in Anal.Chem.71 (1999) 294R-304R.Lu, people such as B. have reported that in Analyst.121 (1996) 29R-32R the orientation of antibody fixes, to be used for immunoassay.Wilchek for example, M. and Bayer, E.A., Methods Enzymol.184 (1990) 467-469 have reported the immunoassay of avidin-biotin mediation.
The term that uses among the present invention " two antigen bridging immunoassay " expression sandwich type immunoassay, wherein antigen combines with two kinds of different antibodies, its separately with antigen in the different epi-positions combinations of non-overlapping or interference.In this determination method, form the sandwich body that comprises capture antibody, antigen and tracer antibody, two kinds of antibody bridgings that antigen will combine with it thus.
As protein, monoclonal antibody comprises many reactive side chains and binding partners coupling with its constant domain, and binding partners is like surface, protein, polymkeric substance (for example PEG), cellulose or polystyrene, enzyme or combine right member.The chemical reaction group of antibody is for for example, amino (epsilon-amino of lysine, alpha-amido), mercapto (cystine, halfcystine, methionine), carboxylic acid group's (aspartic acid, glutamic acid) and sugar alcohol base.Aslam for example, M. and Dent, A., Bioconjuation, wheat Courlene publishing company (MacMillan Ref.Ltd.) (1999), the 50-100 page or leaf is described such method to some extent.
Term " sample " comprises the material from any amount of monkey.Such material includes but not limited to from the whole blood of such individuality, serum or blood plasma, and these are the most widely used sample sources in the routine before clinical.
Term " solid phase " refers to comprise non-fluid substance: particle (comprising particulate and pearl), its by as process such as materials such as polymkeric substance, metal (paramagnetic, ferromagnetic particle), glass and potteries; Gelatinous mass such as silica gel, aluminium oxide and polymer gel; Kapillary, it can be processed by polymkeric substance, metal, glass and/or pottery; Zeolite and other porous mass; Electrode; Microwell plate; Solid detector bar (strip); Cuvette, pipe or other spectrometer sampling receptacle.The difference on the inert solid surface that possibly touch in solid phase element in the determination method and the determination method is that " solid phase " element surface comprises at least one expection and the interactional part of capture antibody.Solid phase can be a retaining element, like pipe, bar, cuvette or microwell plate, maybe can be unfixed element, like pearl and particulate.Particulate can also be used for even phase determination method form as solid phase.Can use the multiple particulate that allows the non-covalent or covalent attachment of protein and other material.Such particle comprises: polymer beads such as polystyrene and polymethylmethacrylate; Gold grain such as gold nano grain and aurosol; With ceramic particle such as silica gel, glass and metal oxide particle.See for example Martin, people such as C.R., Analytical Chemistry-News&Features70 (1998) 322A-327A, it incorporates this paper by reference into.
The instance of detectable mark is chromogen (fluorescence or luminophore and dyestuff), enzyme, NMR-reactive group or metallic particles, haptens, for example foxalin.Detectable mark can also be the photoactivated cross-linking group, for example azido or aziridinyl (azirine).Metallo-chelate that can the electricity consumption chemiluminescence detection also is preferred signal emission group, preferred especially ruthenium chelate, for example ruthenium (dipyridine)
3 2+Chelate.The ruthenium labelling groups that is fit to has for example been described among EP 0580979, WO 90/05301, WO 90/11511 and the WO92/14138.
Immunoassay is known by the technician.Summed up the method for implementing such determination method in the relevant textbook, and practical use and operation.The instance of relevant textbook has Tijssen, P., Preparation of enzyme-antibody or other enzyme-macromoleculeconjugates; In: Practice and theory of enzyme immunoassay; Burdon, R.H. and v.Knippenberg, P.H. (volume); Like to think only your company (Elsevier), Amsterdam (1990) 221-278 page or leaf; Colowick, S.P. and Caplan, N.O. (volume), " Methodsin Enzymology ", the academic press, in relate to the multireel of immunologic detection method, particularly roll up 70,73,74,84,92 and 121.
Can be produced by hybridoma cell line 3.25.12 (DSM ACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSMACC2802), 7.72.32 (DSM ACC2803) according to antibody of the present invention, these hybridoma cell lines itself also are one side of the present invention.According to other antibody of the present invention, the antibody that promptly combines, do not combine with machin IgG specificity with human IgG, can use-case such as embodiment 3 in the method for general introduction obtain.
Alternatively, for example can use such method, wherein the epi-position of two kinds of antibody combining with same target antigen with the competition experiments system measurement earlier of the first step is overlapping.For this purpose, for example by enzyme immunoassay, the antibody studied of test combines the degree of immobilization target antigen with the known antibodies competition, for example according to the competition combination degree of the antibody of clone production of the present invention.For this purpose, with suitably the known antibodies and the excessive antibody of studying of immobilized target antigen and mark pattern are hatched.Under the antibody existence of studying through being determined at and non-existent situation, the amount of the antibodies of mark pattern can be assessed the degree that the antibody of being studied can be replaced the combination of known antibodies.If replace more than 20%, preferably more than 30% in same concentration; The antibody of perhaps being studied is in higher concentration, and preferred 10
3-10
5When doubly being in excess in known antibodies, if displacement is then overlapping for epi-position takes place preferably more than 80% more than 70%, two kinds of antibody all combine with the identical or lap of same epi-position.In second step of said method, use the antibody of identifying like this.Measure combining of the antibody identified in the first step and human IgG this moment.Can carry out such mensuration with ELISA for example, immunoassay or with surface plasma body resonant vibration.Do not combine with human IgG if measure it, then defining such antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity.Alternatively, can be according to antibody of the present invention through measuring it respectively for the KDiss. of machin IgG and human IgG and contrast these numerical value and obtain identifying.
The present invention has reported the method for immunity of measuring the compound of drug antibody and anti-drug antibodies, and said compound is expressed as the DA/ADA compound hereinafter.In more detail; The present invention comprises with the sandwich type immunoassay to monkey kind sample Chinese traditional medicine antibody (DA) with to the antibody (anti-drug antibodies of said drug antibody; ADA) method that immune complex (DA/ADA compound) carries out immunoassays; This method comprises capture antibody and tracer antibody; One of wherein said antibody is the antibody that combines with machin IgG specificity, preferably do not combine with human IgG, and another said antibody is the antibody that combines with the human IgG specificity, preferably do not combine with machin IgG.In one embodiment, capture antibody is the antibody that combines with the human IgG specificity, do not combine with machin IgG, and tracer antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity.In different embodiments, capture antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and tracer antibody is the antibody that combines with the human IgG specificity, do not combine with machin IgG.In another embodiment of the present invention, antibody that combines with machin IgG specificity and/or the antibody that combines with the human IgG specificity are monoclonal antibody.
In the preferred embodiment of this method, the drug antibody preincubate of sample and scheduled volume.This makes no matter whether to have drug antibody in the sample, all can detect anti-drug antibodies, is DA/ADA compound (seeing for example Fig. 5 and 6) because this method detects.
According to the height homogeneity between people or humanization drug antibody and the machin IgG, the major antigen determinant of drug antibody is its complementary determining region.Anti-drug antibodies is preferably discerned these antigenic determinants.So, thereby most anti-drug antibodies is discerned the complementary determining region of relative medicine antibody, also combination with it.The formation of anti-drug antibodies-drug antibody compound shielding epitope, thus in the antagonism sandwich assay to the mensuration of said compound.
For example as described in WO 2006/066912, anti-human IgG antibody is the antibody that combines with such epitope specificity, and said epi-position is not present on the immunoglobulin (Ig) that from machin, obtains.In preferred embodiments; Anti-human IgG antibody is further characterized in that; Its epi-position with the antibodies of being produced by clone DSM ACC 2708 is identical, just combines with such epi-position, and said epi-position is present in all subclass of G class human immunoglobulin(HIg); And except on the IgG that is present in chimpanzee, on the immunoglobulin (Ig) of most experiments animal, do not exist.In other preferred embodiment, anti-human IgG antibody is the antibody of being produced by clone DSM ACC2708.
Find amazingly, use to have prevented incomplete detection according to the method for the invention for anti-drug antibodies.For example; If capture molecules and anti-drug antibodies combine identical antigenic determinant, i.e. the CDR of drug antibody, then drug antibody and capture molecules combine can seal these epi-positions; Be about to them and shield, thereby stoped the combination of anti-drug antibodies and be detected with respect to anti-drug antibodies.This causes the incomplete detection to the anti-drug antibodies in the sample.Anti-drug antibodies has low binding affinity for drug antibody usually, so bound drug antibody needs the cooperation of two antigen binding domains of anti-drug antibodies.So, the low detection that can not combine also possibly cause resisting drug antibody with capture molecules.Therefore, the CDR through anti-drug antibodies catches and is not suitable for measuring.
Therefore; One side of the present invention is to monkey kind sample Chinese traditional medicine antibody (DA) be directed against the antibody (anti-drug antibodies of said drug antibody with the sandwich immunoassay method that comprises capture antibody and tracer antibody; ADA) method that immune complex (DA/ADA compound) carries out immunoassays; One of wherein said antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and another antibody is the antibody that combines with the human IgG specificity, do not combine with machin IgG.In the embodiment in this regard, the antibody that combines, do not combine with human IgG with machin IgG specificity is produced by clone DSM ACC2799 or DSM ACC2800 or DSM ACC2801 or DSM ACC2802 or DSM ACC2803.The antibody that in another embodiment in this regard, combine with the human IgG specificity, does not combine with machin IgG is produced by clone DSM ACC2708.
In an embodiment of this method, the amount of the compound of formation is associated with the concentration of DA/ADA compound, DA and/or ADA.
In another embodiment, capture antibody combines with anti-drug antibodies or DA/ADA compound, not with the CDR of anti-drug antibodies or combining with CDR next-door neighbour's framework region on the sequence or on the geometry.
Another aspect of the present invention is to being directed against the antibody (anti-drug antibodies of drug antibody in the monkey kind sample with the sandwich immunoassay method that comprises capture antibody and tracer antibody; ADA) carry out the method for immunoassays; Wherein capture antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and tracer antibody is a drug antibody.Also have is to being directed against the antibody (anti-drug antibodies of drug antibody in the monkey kind sample on the one hand with the sandwich immunoassay method that comprises capture antibody and tracer antibody; ADA) carry out the method for immunoassays; Wherein tracer antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and capture antibody is a drug antibody.
According to preferred hybridoma cell line 3.25.12 of the present invention, 3.29.15,4.38.30,7.57.41 and 7.72.32 according to Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure be preserved in German microbial preservation center (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, DSMZ):
| The clone | Preserving number | Preservation day |
| 3.25.12 | DSM?ACC2799 | 2006.08.24 |
| 3.29.15 | DSM?ACC2800 | 2006.08.24 |
| 4.38.30 | DSM?ACC2801 | 2006.08.24 |
| 7.57.41 | DSM?ACC2802 | 2006.08.24 |
| 7.72.32 | DSM?ACC2803 | 2006.08.24 |
| MAB?M-R10Z8E9 | DSM?ACC2708 | 22.12.2004 |
The antibody that can be got by said clone is embodiment of the present invention.
Embodiment and the accompanying drawing that hereinafter is provided is helping understanding of the present invention, and true scope of the present invention is illustrated by the claims of enclosing.Should understand under the situation that does not depart from spirit of the present invention and can make modification the method for listing.
Description of drawings
Fig. 1: the determination method that detects the DA/ADA compound---do not add DA.
Biotinylated anti-people Ig antibody is fixed on the microwell plate (SA-MTP) that streptavidin encapsulates.Drug antibody/anti-drug antibodies (DA/ADA) compound is immobilized anti-people Ig antibody (Bi; Biotinylated) institute catch.(Dig with the foxalin mark; The digoxigenin glycosidation) anti-machin IgG antibody and anti-foxalin antibody horseradish peroxidase (POD) conjugate (anti-DIG antibody of polyclone and POD put together, pAb < Dig>POD) detect the DA/ADA compound that combines.Use with the chemically conjugated human IgG of machin IgG as standard.
Fig. 2: the determination method that detects the DA/ADA compound---add DA.
Before sample analysis, use damping fluid dilution monkey serum sample, the admixture drug antibody based on PBS.After 15 minutes preincubates, with above-mentioned elisa assay sample (seeing the explanation of Fig. 1).
Fig. 3: with the determination method of anti-machin antibody test ADA.
The microwell plate (SA-MTP) that biotinylated drug antibody and streptavidin are encapsulated combines (Bi; Biotinylated).Anti-drug antibodies (ADA) combines with the immobilization drug antibody.(Dig with the foxalin mark; The digoxigenin glycosidation) anti-machin IgG antibody and anti-foxalin antibody horseradish peroxidase conjugate (pAb < Dig>POD) detect the ADA that combines.Use with the chemically conjugated anti-human IgG antibody of machin IgG as standard.
Fig. 4: the typical curve of DA/ADA compound determination method.
Damping fluid dilution and the chemically conjugated human IgG of machin IgG based on PBS with containing 1% (v/v) machin serum have provided its optical density (OD) under a plurality of concentration among the figure.
Fig. 5: in machin, carry out HuMab < IGF-1R>single dose of drug dynamics research (3mg/kg; Intravenous injection), detect DA/ADA compound in this study sample.
8 time points between 0h after the administration and 1176h are collected blood serum sample, with elisa assay (as shown in Figure 1), do not add drug antibody.The amount (the OD signal of 405nm) of DA/ADA compound was mapped with respect to the time after the administration.
Fig. 6: in machin, carry out HuMab < IGF-1R>single dose of drug dynamics research (3mg/kg; Intravenous injection), detect DA/ADA compound in this study sample.
8 time points between 0h after the administration and 1176h are collected blood serum sample, with elisa assay (as shown in Figure 2), add drug antibody.The amount (the OD signal of 405nm) of DA/ADA compound was mapped with respect to the time after the administration.
Fig. 7: the contrast that monoclonal anti machin IgG and polyclone machin IgG detect for machin IgG.
Damping fluid dilution and the chemically conjugated human IgG of machin IgG based on PBS with containing 1% (v/v) machin serum have provided its optical density (signal (median)) under a plurality of concentration among the figure.
Embodiment
Preparation machin IgG and machin Fc segment
A) preparation machin IgG
Machin serum precipitates with ammonium sulfate (ad 2.0M) through
380 degreasings.To be deposited in phosphate buffer and homogenize, with phosphate buffer, pH 7.0 dialysis.With the DEAE ion-exchange chromatography at pH 7.0 separating mixtures, through the IgG in the concentrated and purified effluent of gel filtration.
B) preparation machin Fc
In the presence of the 15mM halfcystine, at 37 ℃, pH 7.0 usefulness papains (the every mg IgG of 4mU papain) are with the IgG segmentization of purifying in a).After 80 minutes, potpourri and iodoacetamide (ad 30mM) are hatched at 25 ℃, subsequently to contain 30mM NaCl, the 10mM HEPES damping fluid dialysis of pH 7.5.With Q-agarose ion-exchange chromatography separating mixture.The Fab level is divided in effluent, reaches the salt gradient wash-out Fc level branch of 1M sodium chloride with concentration.At last, the Fc level is divided with the phosphate buffer dialysis, through the gel filtration purifying.
Produce monoclonal anti machin IgG antibody
A) immune mouse
With 100 μ g machin IgG (machin immunoglobulin G) or the machin Fc that mixes with CFA (Freund's complete adjuvant), the female Balb/c or the NMRI mouse in 8-12 age in week carried out initial immunity in the peritonaeum respectively.After 4,7 and 10 weeks, the machin IgG that every mouse mixes with IFA (incomplete Freund) with 100 μ g carries out immune step in three peritonaeums again.Merging first three day then, each is in PBS (PBS with 100 μ g; Add antihistamine and adrenaline) in machin IgG carry out the intravenous booster immunization.
B) fusion and clone
According to Galfr é, G. and Milstein, C., Methods Enzymol.73 (1981) 3-46 will be according to a) splenocyte and the myeloma cell of mice immunized are merged.With about 1 * 10
8Splenocyte and about 2 * 10
7Myeloma cell (P3x63-Ag8.653, ATCC CRL1580) mixes, centrifugal (10 minutes, 300 * g, 4 ℃).With the RPMI 1640 nutrient culture media washed cells of no FCS (hyclone) once, centrifugal once more with 400 * g in the sharp bottom tube of 50mL afterwards.After this, (polyglycol, molecular weight 4 000g/mol), mix through imbibition to add 1mL PEG.After 1 minute, dropwise add the RPMI 1640 that 5mL does not have FCS 37 ℃ of water-baths, mix suspension, fill it up with to 50mL with the RPMI 1640 that contains 10% (v/v) FCS, centrifugal then.The cell of deposition is resuspended with the RPMI1640 that contains 10%FCS; Containing growth factor interleukin 6 (IL-6; Hypoxanthine 100U/mL)-azaserine is selected bed board in the nutrient culture media (100mmol/L hypoxanthine, 1 μ g/mL azaserine is among the RPMI 1640 that contains 10%FCS).After about 10 days, measure the antibodies specific synthetic (referring to embodiment 3) of primary culture.With show combine with machin IgG and not with the primary culture of the normal IgG cross reaction of people with flow cytometer (FACSAria; BD Biological Science Co., Ltd (BD Biosciences)) carries out individuation through unicellular being deposited in the 96 porocyte culture plates, contain growth factor interleukin 6 (100U/mL) in the nutrient culture media.Produce clone's (table 1) of preservation according to this experimental program.Be used for clone of the present invention and be deposited in German microbial preservation center (DSMZ) (table 1).
Table 1:
Anti-machin mAb clone
| The clone | IgG class and subclass | Immunogene | Preserving number | Preservation day |
| 3.25.12 | ?IgG1，κ | IgG | DSM?ACC2799 | 2006.08.24 |
| 3.29.15 | ?IgG1，κ | IgG | DSM?ACC2800 | 2006.08.24 |
| 4.38.30 | ?IgG1，κ | IgG | DSM?ACC2801 | 2006.08.24 |
| 7.57.41 | ?IgG2a，κ | Fc | DSM?ACC2802 | 2006.08.24 |
| 7.72.32 | ?IgG1，κ | Fc | DSM?ACC2803 | 2006.08.24 |
C) produce immunoglobulin (Ig) by cell culture supernatant
The hybridoma cell line that produces is with 1.0 * 10
5To 2.2 * 10
5The initial cell density (living cells) of the every mL of individual cell (depending on individual cell lines) is at the RPMI that adds 10%FCS 1640 inoculation of mediums, with the time of stirring technique amplification 9 to 16 days (depending on individual cell lines).In the culture supernatant of results, the monoclonal anti bulk concentration reaches every mL 36 to 61 μ g.The purifying antibody that the culture supernatant obtains according to the standard protein chemical method for example according to Bruck, people such as C., Methods Enzymol.121 (1986) 587-596 carries out.
Embodiment 3
Detect the screening method of anti-machin IgG antibody
A) antibody that preferentially combines of preliminary screening and machin IgG
For measuring the specificity of antibody in the Hybridoma Cell Culture thing supernatant; Streptavidin (MicroCoat will recombinate; Bernried company; Batch MC 1098) MTP (microwell plate) that encapsulates in advance is respectively with being in the biotinylated machin IgG among the PBS that adds 1.0% (w/v) BSA II; 250ng/mL or biotinylated human IgG, 250ng/mL encapsulate (the every hole of 100 μ L, incubated at room 60 minutes; Follow vibration), use 0.9% (w/v) NaCl/0.05%
, 20 washings three times afterwards.Next step, every hole adds 100 μ L antibody-solutions to be measured (culture supernatant), and incubated at room 60 minutes is followed vibration.Process is with 0.9% (w/v) NaCl/0.05%
After three times the step of 20 washings, every hole adds the F (ab ') of the polyclone sheep anti mouse Fc gamma antibodies of 100 μ L horseradish peroxidase-labeled
2Segment, to detect the sample antibody that combines, incubated at room 60 minutes is followed vibration.Subsequently, wash by preceding text.At last; Every hole adds 100 μ L
(German Luo Shi diagnostic products company limited (Roche Diagnostics GmbH), catalog number (Cat.No.) 1684302).After the incubated at room 30 minutes, read in the plate device to measure 405 and the delustring (OD) located of 492nm [405/492] at commercial microwell plate ELISA.This screening is selected with machin IgG combine well and to human IgG and is shown not have/hang down the antibody of cross reactivity.The antibody of selecting is proceeded the mensuration of step b).
B) screening does not have the antibody of detectable cross reactivity to human IgG
In order identifying in the antibody of selecting a) from preliminary screening human IgG to be shown the antibody that does not have detectable cross reactivity, to carry out the determination method that hereinafter is described.Streptavidin (MicroCoat will recombinate; Bernried company; Batch MC 1098) MTP that encapsulates in advance is with being in the biotinylated machin IgG among the PBS (PBS) that contains 1.0%BSA II; 250ng/mL encapsulates (the every hole of 100 μ L; Incubated at room 60 minutes is followed vibration), use 0.9% (w/v) NaCl/0.05%
, 20 washings three times subsequently.Next step adds every hole 100 μ L antibody-solutions (culture supernatant) to be measured and 50 μ L PBS (contrast signal) respectively, or 50 μ L human IgG solution (80mg/mL; Final concentration in the determination method: 27mg/mL; Measured signal), incubated at room 60 minutes is followed vibration.Process is with 0.9% (w/v) NaCl/0.05%
After three times the step of 20 washings, every hole adds the F (ab ') of the polyclone sheep anti mouse Fc gamma antibodies of 100 μ L horseradish peroxidase-labeled
2Segment, to detect the sample antibody that combines, incubated at room 60 minutes is followed vibration.Subsequently, wash by preceding text.At last; Every hole adds 100 μ L
(German Luo Shi diagnostic products company limited, catalog number (Cat.No.) 1684302).After the incubated at room 30 minutes, the delustring of reading in the plate device to measure [405/492] nm place at commercial microwell plate ELISA.Selecting measured signal to show does not have the antibody of significant difference to make further purposes with relevant contrast signal.Quantizing angle, this equals cross reactivity with human IgG and is estimated as＜and 0.001%.
(the definition of " not having significant difference ": the 90-110% of measured signal=contrast signal.)
Embodiment 4
The conjugate of preparation machin IgG (Cyno-IgG) and human IgG (H-IgG)
A) preparation Cyno-IgG-SATP
The machin IgG that from machin serum, passes through ion-exchange chromatography and gel filtration purifying is with the 30mM kaliumphosphate buffer, and pH 7.1 dialyses, and protein solution to the protein concentration that adjustment obtains is about 10mg/ml.N-succinimide-3-acetyl thio propionic ester (SATP) is dissolved in DMSO, with 1: 5 (IgG: in mol ratio adding antibody-solutions SATP).Potpourri is at 25 ℃, and pH 7.1 was hatched 60 minutes.Through adding L-lysine to final concentration is that 10mM stops reaction, and pH is adjusted into pH 6.1, and to contain 200mM NaCl and 1mM EDTA, the 10mM kaliumphosphate buffer of pH 6.1 is dialysed, and removes excessive SATP.
B) preparation H-IgG-MH
The human IgG that from human serum, passes through the ion-exchange chromatography purifying is with the 30mM kaliumphosphate buffer, and pH 7.1 dialyses, and protein solution to the protein concentration that adjustment obtains is about 20mg/ml.(Maleimidohexanoyl-N-hydroxysuccinimide ester MHS) was dissolved in DMSO, with 1: 6 (IgG: in mol ratio adding antibody-solutions MHS) with maleimide hexanoyl-N-hydroxy-succinamide ester.Potpourri is at 25 ℃, and pH 7.1 was hatched 60 minutes.Through adding L-lysine to final concentration is that 10mM stops reaction, and pH is adjusted into pH 6.1, and to contain 200mMNaCl and 1mM EDTA, the 10mM kaliumphosphate buffer of pH 6.1 is dialysed, and removes excessive MHS.
C) put together Cyno-IgG-SATP and H-IgG-MH
Add 2.5% (v/v) 1M hydroxylamine solution, pH 7.5, hatched 60 minutes at 25 ℃, and be Cyno-IgG-SH with the Cyno-IgG-SATP deacetylation.The antibody of deacetylation mixes (mol ratio of Cyno-IgG-SH: H-IgG-MH=1: 1) to total IgG with H-IgG-MH final concentration is about 7mg/ml.PH is adjusted into 7.1,25 ℃ of mixtures incubated.Put together process with analytical solvent resistant column (for example TSK 3000) analysis.Generally speaking, after 40 minutes, be that 2mM stops to put together process through adding halfcystine to final concentration.After hatching 30 minutes, adding N-methyl maleimide (NMM) to final concentration is 5mM, and pH is adjusted into 7.5.25 ℃ hatch 60 minutes after, separate conjugate with propylene sephadex S-300 through gel filtration chromatography, remove unconjugated antibody.
The monoclonal anti machin Fc antibody of preparation digoxigenin glycosidation
A) preparation monoclonal anti machin Fc antibody
The fermentation supernatant of monoclonal anti machin Fc antibody is concentrated about 10 times, move to and contain 20mMTRIS, 1M ammonium sulfate, the damping fluid of pH 9.0, last kind to albumin A-agarose column.To use the 0.2M sodium citrate, the eluent of pH 3.0 wash-outs is with phosphate buffer, and pH 7.5 dialyses.Separate ox IgG pollutant (from the FCS in the fermentation liquor) through carrying out immunoadsorption with immobilized antibody to ox IgG.
B) the digoxigenin glycosidation of monoclonal anti machin Fc antibody
The monoclonal anti machin Fc antibody-solutions that will be in the phosphate buffer is adjusted into pH 8.1, and concentration is about 2mg/ml.Foxalin-3-O-methyl carbonyl-EACA-N-hydroxy-succinamide ester is dissolved in DMSO, adds in the antibody-solutions with 1: 5 mol ratio.Stop reaction through adding L-lysine after 60 minutes, to contain 150mM NaCl, excessive labelled reagent is removed in the 50mM kaliumphosphate buffer dialysis of pH 7.5.
Embodiment 6
Combination/specificity through
system evaluation antibody
All measurements are all carried out with
2000 instruments that use the CM5 chip.Realize antibody encapsulating through the standard amine coupling to chip.Except as otherwise noted, all incubation step are all carried out in HBS damping fluid (pH 7.4 for HEPES, NaCl) in 25 ℃.Different monoclonal anti machin IgG antibody, MAB M-R10Z8E9 and the anti-people Fc of polyclone gamma antibodies (Dianova company, Germany) with saturating capacity are separately fixed at through the amine coupling on the different slots (channel) of same CM5 chip.All animal blood serums all are diluted to final concentration in containing the HBS damping fluid of 1mg/ml Sensor Chip CM 5 be 1%.Serum through injecting 1 to 100 dilution and hatching analyze to combine in 60 seconds.Dissociate through measuring in 180 seconds with HBS damping fluid washing chip surface.With the BIAevaluation software of
, with 1: 1 blue Mil's model of fit (Langmuir fitting model) computational solution from constant value (=KDiss.).For all animal blood serums, this calculating all is the hypothesis of 15mg/ml based on the IgG level.Selection begins to inject the amount (RU of table 2) that the signal value of test antibody after 80 seconds is used for contrasting the IgG of combination.
Table 2:
Animal blood serum and monoclonal anti machin IgG antibody and the sero-fast binding signal of the anti-people Fc of polyclone γ [RU]
Table 2 show monoclonal anti machin IgG antibody not with the human serum cross reaction.Only detected combining of its IgG that comprises with machin, rhesus macaque and baboon serum.Different with monoclonal anti machin IgG antibody, the high response of the serum of the anti-people Fc of polyclone antibody demonstration and people, dog and all test monkey kinds.
Embodiment 7
Measure the DA/ADA compound---do not add DA
In the first step, biotinylated MAB M-R10Z8E9 is bonded in the hole of the microwell plate (SA-MTP) that streptavidin encapsulates.Remove excessive not binding antibody through washing.Afterwards, in the hole, hatch monkey serum sample and reference standard (the machin IgG in 1% machin serum is chemically conjugated for human IgG and admixture).After the unconjugated material of flush away, in conjunction with the DA/ADA compound with the anti-machin antibody test of digoxigenin glycosidation, then hatch with the anti-foxalin antibody of horseradish peroxidase-labeled.The chromogenic reaction of antibody-enzyme conjugate catalysis
substrate.With ELISA read the plate device at wavelength 405nm place (with reference to wavelength: 490nm ([405/490] nm)) measuring-signal.Light absorption value with duplicate each blood serum sample of mensuration.Fig. 1 shows the scheme of this test macro of illustration.
Embodiment 8
Measure the DA/ADA compound---add DA
Before the sample analysis, the monkey serum sample is diluted the admixture drug antibody in the damping fluid based on PBS.After 15 minutes preincubates, through above-mentioned elisa assay sample.Fig. 2 shows the scheme of this test macro of illustration.
Embodiment 9
Detect the DA/ADA compound in the machin pharmacokinetic study sample
With (WO 2005/005635 to IGF-1R; 3mg/kg; People's antibody iv) carries out machin blood serum sample single dose of drug dynamics research (PK=pharmacokinetics); Analyze with above-mentioned ELISA: i) detect the DA/ADA compound; Do not add drug antibody (Fig. 5) and ii) detect the DA/ADA compound, add drug antibody (Fig. 6).8 time points between 0h after the administration and 1176h are collected blood serum sample and analysis.The amount (the OD signal at 405nm place) of DA/ADA compound was mapped with respect to the time after the administration.As shown in Figure 5, if form the AD/ADA compound in the body, and without external adding drug antibody, then in the blood serum sample only between 336h and 672h (peak shape) detect positive signal.Do not exist under the situation of anti-drug antibodies, do not have immune complex to form, detect less than positive signal (time point before the 336h).672h or afterwards after the administration owing to do not have medicine in the sample, can not detect compound.When in blood serum sample, adding drug antibody, all can detect/can detect the DA/ADA compound of interior formation of body and external formation as the preincubate step.As shown in Figure 6, positive signal only depends on the generation of ADA.Two width of cloth figure (Fig. 5 and 6) are all relevant well with medicine-time curve.
Carry out the determination method that ADA detects with anti-machin antibody
In the first step, biotinylated drug antibody (to people's antibody of IGF-1R) is attached in the hole of the microwell plate (SA-MTP) that streptavidin encapsulates.Excessive unconjugated antibody is removed in washing.Afterwards, hatch monkey serum sample (dilution is 20 times in based on the damping fluid of PBS) and reference standard.After the unconjugated material of flush away,, then use the anti-foxalin antibody incubation of horseradish peroxidase-labeled with the anti-drug antibodies (ADA) that the anti-machin antibody test of digoxigenin glycosidation combines.The chromogenic reaction of antibody-enzyme conjugate catalysis
substrate.Read the plate device with ELISA and measure the signal at wavelength 405nm place (with reference to wavelength: 490nm).Light absorption value with triplicate each blood serum sample of mensuration.Fig. 3 shows the scheme of this test macro of illustration.
Embodiment 11
Preparation is to the polyclonal antibody of machin IgG
A) purifying polyclonal antibody from rabbit anteserum
According to standard method, rabbit is carried out immunity with machin Fc.For from five original serum, remove fat composition wherein with Aerosil (1.5% (w/v)) degreasing, with ammonium sulfate (1.7M) deposition immunoglobulin (Ig) with the rabbit of machin Fc immunity.Through acid treatment (30min.; PH 5.5) and to add 50mM NaCl; After the 15mM kaliumphosphate buffer dialysis of pH 7.0, in pH 7.0 separating mixtures, the IgG level branch in the effluent (the anti-machin IgG of=rabbit polyclonal antibody) is concentrated into about 25mg/ml with the DEAE ion-exchange chromatography.
B) the anti-machin IgG of the multi-clone rabbit antibody (pAb < Cyno-IgG >) of preparation affinity purification, it does not have cross reactivity to human IgG
The concentrated IgG level branch of step a) moved to add 150mM NaCl, the 50mM kaliumphosphate buffer system (PBS) of pH 7.5.With prior art machin IgG and NHS agarose are puted together, prepare immunosorbent, be filled in the post, with the 50mM kaliumphosphate buffer balance of adding 150mM NaCl pH 7.5 with immobilization machin IgG.
In appearance to post on the 10mg IgG/ml immunosorbent with the PBS balance.Wash post with PBS, the 0.5M NaCl and the 30mM NaCl that add 0.05% (w/v)
20 successively.IgG with 3mMHCl and 1M propionic acid wash-out combine with the affinity substrate specificity dialyses with PBS.
For removing the antibody that human IgG is had cross reactivity, to the affinity column with immobilization human IgG, this affinity column gets for prior art non-specific human IgG and NHS agarose being puted together to prepare with appearance on the antibody of affinity purification.Use the PBS balance columns.Appearance is to post on will about 6mg IgG/ml immunosorbent.Specific polyclonal IgG level is divided in effluent.After using the 0.5M NaCl, 30mM NaCl, 1M propionic acid and the PBS that add 0.05% (w/v)
20 to make column regeneration; Repeat immunoadsorption twice, to remove the antibody that human IgG is had cross reactivity fully for the IgG of non-specific binding.
The anti-machin IgG of the purifying polyclone antibody that human IgG is not had a cross reactivity that obtains is concentrated into about 4mg/ml, is stored in-80 ℃.
Embodiment 12
Antibodies/specificity with
anti-machin Fc of system evaluation multi-clone rabbit antibody (pAb < Cyno-Fc >)
Whole measurements is all carried out with
2000 instruments that use the CM5 chip.Realize antibody encapsulating through the standard amine coupling to this chip.Except as otherwise noted, all hatching all carried out at 25 ℃ in HBS damping fluid (pH 7.4 for HEPES, NaCl).The anti-machin IgG of the polyclone of saturating capacity antibody is fixed on the CM5 chip through the amine coupling.All animal blood serums all are diluted to final concentration in containing the HBS damping fluid of 1mg/ml Sensor Chip CM 5 be 1%.Serum through injecting 1 to 100 dilution and hatching analyze to combine in 60 seconds.Dissociate through measuring in 180 seconds with HBS damping fluid washing chip surface.With the BIAevaluation software of
, with 1: 1 blue Mil's model of fit computational solution from constant value (=KDiss.).For all animal blood serums, this calculating all is the hypothesis of 15mg/ml based on the IgG level.Selection begins to inject the amount (RU of table 3) that the signal value of test antibody after 80 seconds is used for contrasting the IgG of combination
Table 3:
The binding signal [RU] of the anti-machin IgG of animal blood serum and polyclone antibody
Table 3 show the anti-machin IgG of polyclone antibody not with the human serum cross reaction.Only detected combining of its monkey IgG that comprises with marmoset, machin, rhesus macaque and baboon serum.Different with monoclonal anti machin IgG antibody, the anti-machin IgG of polyclone antibody shows the reactivity with the NMRI mice serum.And the anti-machin IgG of polyclone antibody do not combine with human IgG, and and monkey IgG and mouse IgG between reactive difference be at least 100 times.
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| HRP20151023T1 (en) * | 2010-04-29 | 2015-11-06 | Theradiag Sa | METHODS FOR DETECTION OF ANTIBODIES ON MEDICINES |
| EP2823310B1 (en) * | 2012-03-08 | 2018-06-20 | F.Hoffmann-La Roche Ag | Multiplexed chromatography-immunoassay method for the characterization of circulating immune complexes |
| RU2015140955A (en) * | 2013-03-20 | 2017-04-21 | Ф. Хоффманн-Ля Рош Аг | SPECIFIC DETERMINATION OF RAT ANTIBODIES IN SERUM OF MICE |
| SG11201908154RA (en) * | 2017-03-31 | 2019-10-30 | Ablynx Nv | Improved immunogenicity assays |
| EP3665203B1 (en) * | 2017-08-08 | 2022-06-29 | F. Hoffmann-La Roche AG | Method for determining anti-drug antibodies in a minipig sample |
| CN109358192B (en) * | 2018-02-08 | 2020-05-12 | 中国科学院上海药物研究所 | Device and method for removing free drugs in anti-drug antibody detection sample, preparation method and application of device |
| CN114902052B (en) * | 2020-01-02 | 2025-07-18 | 豪夫迈·罗氏有限公司 | ADA response specificity assay |
| CN114044828A (en) * | 2021-11-03 | 2022-02-15 | 湖州中科湖兴生物科技有限公司 | Mouse anti-monkey IgG species specific antibody, application thereof and universal detection method of anti-drug antibody based on mouse anti-monkey IgG species specific antibody |
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| WO2006066912A2 (en) * | 2004-12-23 | 2006-06-29 | F. Hoffmann-La Roche Ag | Detection of a therapeutic antibody in an experimental animal |
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