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WO1999001477A1 - Methode de diagnostic du lupus erythemateux systemique - Google Patents

Methode de diagnostic du lupus erythemateux systemique Download PDF

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Publication number
WO1999001477A1
WO1999001477A1 PCT/US1998/012684 US9812684W WO9901477A1 WO 1999001477 A1 WO1999001477 A1 WO 1999001477A1 US 9812684 W US9812684 W US 9812684W WO 9901477 A1 WO9901477 A1 WO 9901477A1
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WO
WIPO (PCT)
Prior art keywords
sample
antibody
serum
reagent
bound
Prior art date
Application number
PCT/US1998/012684
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English (en)
Inventor
Marica M. Bieber
Neelima M. Bhat
Nelson H. Teng
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Palingen, Inc.
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Publication date
Application filed by Palingen, Inc. filed Critical Palingen, Inc.
Priority to AU79770/98A priority Critical patent/AU7977098A/en
Publication of WO1999001477A1 publication Critical patent/WO1999001477A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]

Definitions

  • the invention pertains to methods of diagnosing and monitoring systemic lupus erythematosus. More particularly, the invention involves an improved immuno-diagnostic assay method for diagnosing and for determining of the stage of severity of the disease.
  • SLE Systemic lupus erythematosus
  • SLE is a chronic, inflammatory disease of unknown cause which may affect the skin, joints, kidneys, nervous system, serous membranes and other organs.
  • the classic clinical course of the disease is characterized by periods of remissions and relapses.
  • Therapeutic agents including steroids, immunosuppresants, and cytotoxins, such as prednisone, azathioprine, methotrexate and cyclophosphamide, as well as therapeutic treatments such as radiation, are used to treat SLE.
  • steroids such as prednisone, azathioprine, methotrexate and cyclophosphamide
  • therapeutic treatments such as radiation
  • the dosages administered require careful control.
  • a test which can be used to diagnose the disease and to monitor the severity of SLE so that the dosages of such therapeutic agents can be adjusted, discontinued or resumed.
  • serological markers are in clinical use for systemic lupus erythematosus (SLE), including diagnostic tests and markers for disease activity (Ward, Arthritis Rheum. 38:1555 (1995)).
  • the former include the anti-nuclear antibody titer which has high sensitivity but poor specificity (Slater, et al., Arch. Intern. Med. 156:1421 (1996)); and the anti- double stranded DNA antibody test (anti-DNA), which has high specificity but low sensitivity for SLE (Pisetsky, Rheum. Dis. Clin. NA 18:437 (1992)).
  • markers of disease activity include the erythrocyte sedimentation rate and complement levels C3 and C4.
  • VH4-34 encoded antibodies VH4-34 Abs
  • mAb monoclonal antibody
  • 9G4 rat monoclonal antibody
  • mAb monoclonal antibody
  • serum Ig serum Ig
  • Various auto-antibodies using the VH4-34 gene have been described, including the anti-l/i cold agglutinins in autoimmune hemolytic anemia (Pascual, et al., J. Immunol. 146:4385 (1991); Pascual et al., Arthr. Rheum.
  • VH4- 34 Abs are known to be elevated in mononucleosis (Roelcke, Transfusion Med. Rev. 2:140 (1989); Chapman, et al., J. Immunol.
  • One object of the present invention is to provide a test for the diagnosis of SLE which has high accuracy, sensitivity, and specificity.
  • a further object is to provide an improved diagnostic test for SLE which includes determination of the serum level of VH4-34 Ab.
  • Yet another object is to provide an immunoassay which is simple to use, inexpensive, convenient, and suitable for use in a medical office or clinic.
  • the invention provides a method for diagnosing systemic lupus erythematosus (SLE) from a sample of serum from a patient including the steps of (a) combining the sample with a VH4-34 binding anti-idiotypic antibody, e.g.
  • 9G4 monoclonal antibody wherein the sample is prepared by diluting serum with aqueous buffer at a volume ratio of sample to buffer of up to 1 :1000; (b) determining the proportion of VH4-34 binding anti-idiotypic antibody which has bound to VH4-34 antibody in the sample; and (c) comparing the result of step (b) to a standard (e.g., results from control group serum samples), to determine if said proportion is sufficient to positively diagnose SLE in the patient.
  • the volume ratio of sample to buffer is up to 1 :100.
  • the sample can be adjusted by dilution with aqueous buffer to yield a total IgG level within a selected range, preferably within the range for normal serum.
  • the method can include the step of determining the sample has been determined to be substantially free from rheumatoid factor antibody, in order to reduce false positive results from patients having rheumatoid arthritis.
  • step (a) the sample is subjected to an ELISA immunoassay using a labeled reagent and a reagent bound to an insoluble phase material, wherein the labeled reagent is enzyme- labeled VH4-34 binding anti-idiotypic antibody, the reagent bound to the insoluble phase material is VH4-34 antibody, and said step (b) includes determining the enzyme-labeled VH4-34 binding anti-idiotypic antibody which is bound to the insoluble phase material.
  • step (a) the sample is subjected to an ELISA immunoassay using a labeled reagent and a reagent bound to an insoluble phase material, wherein the labeled reagent is enzyme-labeled VH4-34 antibody, the reagent bound to the insoluble phase material is VH4-34 binding anti-idiotypic antibody, and said step (b) includes determining the enzyme-labeled VH4-34 antibody bound to the insoluble phase material.
  • step (a) the sample is subjected to an ELISA immunoassay using a first reagent, and a second reagent bound to an insoluble phase material, wherein the first reagent is VH4-34 binding anti-idiotypic antibody, the reagent bound to the insoluble phase material is VH4-34 antibody, and said step (b) includes determining the VH4-34 binding anti-idiotypic antibody which is bound to the insoluble phase material by contacting the insoluble phase material with a labeled antibody which binds with the VH4-34 binding anti-idiotypic antibody.
  • the instant invention discloses a method for diagnosing SLE from a sample of serum from a patient, with reduced false positive cross-reactions from rheumatoid arthritis, including the steps of (a) combining the sample with a binding fragment, e.g.
  • (Fab') 2 , Fab', Fab or Fv of 9G4 monoclonal antibody wherein the sample is prepared by diluting serum with aqueous buffer at a volume ratio of sample to buffer of up to 1 :1000; (b) determining the proportion of the binding fragment of 9G4 monoclonal antibody which has bound to VH4-34 antibody in the sample; (c) comparing the result of step (b) to a standard to determine if said proportion is sufficient to positively diagnose SLE in the patient.
  • the volume ratio of sample to dilution buffer is up to 1 :100.
  • the sample is adjusted by dilution with aqueous buffer to yield a total IgG level within a selected range, preferably within the range for normal serum.
  • the invention is a method for determining the severity stage of SLE from a sample of serum from a patient comprising the steps of (a) combining the sample with a VH4-34 binding anti-idiotypic antibody, wherein the sample is prepared by diluting serum with aqueous buffer at a volume ratio of sample to buffer of up to 1 :1000; (b) determining the VH4-34 binding anti-idiotypic antibody which has bound to VH4-34 antibody in the sample; (c) comparing the result of step (a) to a severity stage standard to determine the severity stage of the SLE in the patient.
  • the severity stage standard can be the Visual Analog Scale for Severity or the Lupus Severity Disease Index, for example.
  • the invention is a composition consisting essentially of a binding fragment of 9G4 monoclonal antibody.
  • the binding fragment of 9G4 monoclonal antibody can be labeled, e.g. enzyme-labeled.
  • the present invention provides a diagnostic assay set of inter-calibrated reagents comprising a binding fragment of 9G4 monoclonal antibody and VH4-34 reagent antibody.
  • the set includes labeled, e.g. enzyme-labeled, binding fragment of 9G4 monoclonal antibody.
  • the set includes VH4-34 antibody bound to an insoluble phase material.
  • FIG. 1 VH4-34 Ab levels (as % inhibition of 9G4 binding ) in patients with SLE, healthy controls, and clinical (inpatient and outpatient) controls.
  • FIG. 2. VH4-34 Ab levels (as % inhibition of 9G4 binding) in patients with SLE, healthy controls, arthritis controls, and other clinical controls (outpatients only).
  • FIG. 3. Correlation between VH4-34 Ab level and lupus severity by Visual Analog Scale (VAS-S).
  • FIG. 4. Correlation between VH4-34 Ab level and lupus severity by Lupus Severity of Disease Index (LSDI).
  • FIG. 5. Correlation between VAS-S and LSDI.
  • FIG. 6 Percentage of patients with severe SLE by VH4-34 Ab binding fertile.
  • the present invention provides improved methods for diagnosing and monitoring systemic lupus erythematosus (SLE) by detecting VH4-34 Ab.
  • the present invention discloses immunoassys for VH4-34 Ab using improved methods of sample preparation and improved immnoassay reagents which reduce artifacts such as false positive results.
  • the normal immune system has the ability to generate millions of antibodies with different antigen binding abilities. The diversity is brought about by the complexities of constructing immunoglobulin molecules. These molecules consist of paired polypeptide chains (heavy and light) each containing a constant and a variable region.
  • variable regions of the heavy and light chains are specified by immunoglobulin V genes.
  • the heavy chain variable region is derived from three gene segments known as VH, D and JH. In humans there are about 100 different VH segments over 20 D segments and six JH segments.
  • the light chain genes have only two segments, the VL and JL segments.
  • Antibody diversity is the result of random combinations of VH/D/JH segments with VL/JL components superimposed on which are several mechanisms including junctional diversity and somatic mutation.
  • VH4-34 Ab is a product of the VH4-34 gene which is one of the 53 identified functional VH germline genes (Cook, et al., Nat. Genet. 7:162
  • VH4-34 gene is present in all haplotypes and no sequence variation has been reported in germline DNA isolated from unrelated individuals (van der Maarel, et al., J. Immunol. 150:2858 (1993)).
  • Rat 9G4 mAb has been shown to recognize VH4-34 Ab (Stevenson, et al. Blood 68:430 (1986)).
  • the VH4-34 idiotope identified by mAb 9G4 is conformation restricted and dependent on a unique sequence near amino acids #23-25 in the framework 1 region of the variable heavy chain.
  • the VH4- 34 gene has low incidence of mutation, allowing the reliable detection by anti- idiotypic mAb 9G4 of VH4-34 Abs by standard immunoassay methods.
  • Sensitivity is the probability of a test having a positive result in a patient with a particular disease.
  • a diagnostic test with perfect sensitivity will detect all of the patients that have the disease.
  • the sensitivity value is derived from results of the tests done on subjects with known disease, and hence is completely dependent on the population of those with the disease.
  • Specificity is the probability of finding a negative test in a patient who does not have that particular disease. A diagnostic test with perfect specificity will be negative in all persons without the disease. The value for specificity is derived from subjects free of the disease, and is solely dependent on characteristics of the disease-free population.
  • test sample or “body fluid sample” typically refer to a naturally occurring or artificially formed liquid test medium suspected of containing the analyte of interest.
  • the test sample is generally a biological fluid or a dilution thereof.
  • Biological fluids from which an analyte can be determined include aqueous fluids such as serum, plasma, lymph fluid, synovial fluid, follicular fluid, seminal fluid, milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears, perspiration, mucous, tissue culture medium, tissue extracts, and cellular extracts.
  • aqueous fluids such as serum, plasma, lymph fluid, synovial fluid, follicular fluid, seminal fluid, milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears, perspiration, mucous, tissue culture medium, tissue extracts, and cellular extracts.
  • indicator reagent refers to an assay reagent comprising a detectable label directly or indirectly attached to a specific binding member which is capable of directly or indirectly binding to the analyte to indicate the presence, absence or amount of the analyte.
  • a variety of different indicator reagents can be formed by varying either the label or the specific binding member. In general, the indicator reagent is detected after it has formed a complex with either the analyte or a complementary specific binding member, but the unbound indicator reagent can also be detected.
  • specific binding member refers to a member of a specific binding pair, i.e., two different molecules wherein one of the molecules through chemical or physical means specifically binds to the second molecule.
  • other specific binding pairs include, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme
  • specific binding pairs can include members that are analogs of the original specific binding member, for example an analyte-analog. If the specific binding member is an immunoreactant it can be, for example, an antibody, antigen, hapten, or complex thereof.
  • an antibody can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a mixture(s) or fragment(s) thereof, as well as a mixture of an antibody and other specific binding members.
  • the details of the preparation of such antibodies and their suitability for use as specific binding members are well-known to those skilled- in-the-art.
  • VH4-34 binding anti-idiotypic antibody is meant to encompass intact molecules, as distinguished from “binding fragments” thereof, such as, for example, (Fab') 2 , Fab', Fab and Fv. These binding fragments lack the Fc fragment of an intact antibody molecule.
  • binding fragments e.g. from the 9G4 monoclonal antibody (mAb)
  • mAb monoclonal antibody
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce (Fab') 2 fragments) or by reducing the disulfide bridges.
  • label refers to any substance which is attached to a specific binding member and which is capable of producing a signal that is detectable by visual or instrumental means.
  • suitable labels for use in the present invention can include chromogen; catalysts; fluorescent compounds; chemiluminescent compounds; radioactive labels; direct visual labels including colloidal metallic and non-metallic particles, dye particles, enzymes or substrates, or organic polymer latex particles; liposomes or other vesicles containing signal producing substances; and the like.
  • the label can be a fluorescent compound where no enzymatic manipulation of the label is required to produce a detectable signal.
  • Fluorescent molecules such as fluorescein, phycobiliprotein, rhodamine and their derivatives and analogs are suitable for use as labels in this reaction.
  • An especially preferred class of labels includes the visually detectable, colored particles which enable a direct colored readout of the presence or concentration, of the analyte in the sample without the need for using additional signal producing reagents.
  • Materials for use as such particles include colloidal metals, such as gold, and dye particles as disclosed in U.S. Pat. Nos. 4,313,734 and 4,373,932.
  • the preparation and use of non-metallic colloids, such as colloidal selenium particles and organic polymer latex particles, are also suitable for use.
  • signal producing component refers to any substance capable of reacting with another assay reagent or the analyte to produce a reaction product or signal that indicates the presence of the analyte and that is detectable by visual or instrumental means.
  • Signal production system refers to the group of assay reagents that are needed to produce the desired reaction product or signal.
  • one or more signal producing components can be used to react with a label and generate the detectable signal, i.e., when the label is an enzyme, amplification of the detectable signal is obtained by reacting the enzyme with one or more substrates or additional enzymes to produce a detectable reaction product.
  • capture binding member refers to a specific binding member which can directly or indirectly bind the analyte or indicator reagent and which is bound or is capable of being bound to an insoluble phase material, or is capable of being precipitated, such that the capture binding member can be separated from the test sample and other assay reagents by any means.
  • capture reagent refers to a capture binding member which is directly or indirectly attached to an insoluble phase material to enable the separation of the capture binding member, and analyte or indicator reagent that is bound thereto, from unbound analyte and assay reagents.
  • the attachment of the capture binding member to the insoluble phase material is substantially irreversible and can include covalent mechanisms.
  • the capture reagent of the present invention is not limited to a capture binding member bound to an insoluble phase material. In an agglutination assay, the capture binding member of the capture reagent can be bound to a soluble carrier material such as bovine serum albumin.
  • the term "insoluble phase material” refers to any suitable chromatographic, bibulous, porous or capillary material or other conventional insoluble material, well-known to those skilled-in-the-art, used to immobilize specific binding members.
  • the insoluble phase material can include a fiberglass, cellulose or nylon pad for use in a flow- through assay device having one or more layers containing one or more of the assay reagents; a dipstick for a dip and read assay; a test strip for chromatographic (e.g., paper or glass fiber) or thin layer chromatographic (e.g., nitrocellulose) techniques in which one or all of the reagents are contained in separate zones of a single strip of insoluble phase material; or an absorbent material well-known to those skilled-in-the-art.
  • chromatographic e.g., paper or glass fiber
  • thin layer chromatographic e.g., nitrocellulose
  • the insoluble phase material can also include, without limitation, polyacrylamide beads, polystyrene beads or tubes, magnetic beads, a microtitre plate or a glass or plastic test tube.
  • Natural, synthetic or naturally occurring materials that are synthetically modified can be used as an insoluble phase material including polysaccharides, e.g., cellulose materials such as paper and cellulose derivatives such as diazobenzyloxymethylcellulose, nitrocellulose, 2- aminophenylthioethercellulose, and cellulose acetate; silica; silicon particles; inorganic materials such as deactivated alumina, or other inorganic finely divided material uniformly dispersed in a porous polymer matrix, with polymers such as vinyl chloride, vinyl chloride polymer with propylene, and vinyl chloride polymer with vinyl acetate; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran, and gelatin; polymeric films such as polyacrylates;
  • the insoluble phase material should have reasonable strength or strength can be provided by means of a support, and it should not interfere with the production of a detectable signal.
  • the specific binding member of the capture reagent can be affixed to particles, e.g., microparticles. These microparticles can serve as the insoluble phase material and be retained in a column, suspended in a mixture of soluble reagents and test sample, or retained and immobilized by another insoluble phase base material.
  • retained and immobilized is meant that the microparticles, associated with the insoluble phase base material, are not capable of substantial movement to positions elsewhere within that material.
  • microparticles can be selected by one skilled-in-the-art from any suitable type of particulate material including those composed of polystyrene, polymethylacrylate, polypropylene, polytetrafluoroethylene, polyacrylonitrile, polycarbonate or similar materials.
  • the size of the microparticles is not critical, although it is preferred that the average diameter be smaller than the average pore size of the insoluble phase base material if such is used.
  • ancillary specific binding member refers to a specific binding member used in addition to the capture binding member and the indicator reagent which becomes a part of the detectable binding complex. One or more ancillary specific binding members can be used in an assay.
  • an ancillary specific binding member can be used in an assay where the indicator reagent is capable of binding the ancillary specific binding member which is in turn capable of binding the analyte.
  • the selection of any given label, binding member, ancillary binding member or insoluble phase material is generally not critical to the present invention. The materials are chosen to optimize the results provided by the chosen assay configuration.
  • VH4-34 Ab can be readily detectable in serum samples by means of binding assays which are generally categorized into one of two major classes, homogeneous and heterogeneous assays. These assays may be further divided into sandwich and competitive assay formats, and variations thereof.
  • the capture reagent typically involves a specific binding member which has been bound to an insoluble phase material.
  • the specific binding member can be an immobilized antibody which will bind to an analyte in the test sample.
  • the capture reagent is contacted to a test sample, suspected of containing the analyte, and to an indicator reagent comprising a second specific binding member that has been labeled, for example, a labeled anti-analyte antibody.
  • the reagents can be mixed simultaneously or added sequentially, either singly or in combination.
  • a binding reaction results in the formation of a capture reagent/analyte/indicator reagent complex immobilized upon the insoluble phase material.
  • the assay can also comprise the step of separating the resultant complex from the excess reagents and test sample.
  • the complex retained on the insoluble phase material is detected by examining the insoluble phase material for the indicator reagent. If analyte is present in the sample, then label will be present on the insoluble phase material. The amount of label on the insoluble phase material is a function of the amount of analyte in the sample.
  • the assays of the present invention can be carried out using any of the sandwich assay formats, including the forward, reverse and simultaneous techniques. Typically, a forward assay involves the contact of the test sample to the capture reagent, followed by a certain incubation period, which is in turn followed by the addition of the indicator reagent.
  • a reverse assay involves the addition of the indicator reagent to the test sample followed by the addition of the capture reagent after a certain incubation period.
  • a simultaneous assay involves a single incubation step as the capture reagent and indicator reagent are both contacted to the test sample at the same time.
  • the present invention can be used in an indirect sandwich assay with the formation of a complex of capture reagent/analyte/analyte-specific binding member/indicator reagent.
  • the additional analyte-specific binding member is the ancillary specific binding member.
  • the methods of the present invention can also be carried out using competitive assay formats.
  • the capture reagent again typically involves a specific binding member which has been affixed to an insoluble phase material and which is contacted with both test sample and an indicator reagent.
  • the indicator reagent can be formed from an analyte or analyte-analog which has been conjugated with a label.
  • a binding reaction occurs and results in the formation of complexes of (1) immobilized capture reagent/analyte complex and (2) immobilized capture reagent/indicator reagent complex.
  • the amount of label on the insoluble phase material is inversely related to the amount of analyte in the sample.
  • One embodiment of the present invention is an isotype specific sandwich enzyme-linked immunosorbent assay (ELISA) for VH4-34 Ab in sera.
  • the capture reagent in this embodiment is monoclonal VH4-34 IgM bound to an insoluble phase material.
  • the serum is diluted in aqueous buffer.
  • the sample is mixed with a VH4-34 Ab binding anti-idiotypic antibody, e.g. mAb 9G4, under conditions allowing an immunocomplex to form between the analyte and the anti-idiotypic antibody.
  • mAb 9G4 acts as an ancillary specific binding member in this example.
  • the test sample in the present invention typically refers to a serum sample, but the detection of analyte need not be limited thereto. A variety of other biological fluids can be tested for the analyte. Because the assays of the present invention are very sensitive, only small amounts of test sample are required. For example, the assay can be performed with 0.0005 mL of serum. However, it is usually impractical to work with such small volumes, necessitating a dilution. In general, any dilution of the test sample will give a less accurate result because of errors introduced while measuring and transferring liquid samples. In one aspect, the present invention provides an assay having improved accuracy in the quantification of serum VH4-34 Ab by the use of low dilution of sample prior to analysis.
  • the dilution is in an aqueous buffer at volume ratio of sample to buffer of up to 1 :1000. In a more preferred embodiment, the dilution is at a volume ratio of sample to buffer of up to 1 :100.
  • An example of a suitable dilution buffer is Hanks' balanced salt solution (HBSS) containing 0.1% non-fat dry milk.
  • HBSS Hanks' balanced salt solution
  • the diluted sample is contacted with a capture binding member consisting of bound VH4-34 Ab, e.g. monoclonal VH4-34 IgM, under conditions that allow any unbound anti-idiotypic antibody to form a complex with the bound VH4-34 Ab.
  • unbound material is separated, e.g. by washing, from the insoluble phase material, and the amount of anti-idiotypic antibody bound to the insoluble phase material is determined by a signal production system.
  • a signal producing component such as an enzyme- labeled antibody which recognizes the anti-idiotypic antibody, is allowed to contact the insoluble phase material, and the enzyme is exposed to a substrate.
  • the anti-idiotypic antibody is rat mAb 9G4 IgG
  • the signal producing component can be labeled anti-rat IgG, e.g., goat anti-rat IgG.
  • VH4-34 Ab the level of VH4-34 Ab in the serum sample, the less anti-idiotypic antibody will be available to bind to the insoluble phase material.
  • the percent inhibition in binding of anti-idiotypic antibody to the insoluble phase material is proportional to the VH4-34 Ab present in the test sample.
  • the test sample is also assayed in a separate immunoassay, e.g. ELISA, for binding to rat IgG.
  • a separate immunoassay e.g. ELISA
  • rheumatoid factor antibody if present, can inhibit the binding of anti-rat IgG to the anti- idiotypic antibody, and give rise to a false positive result.
  • a fragment of the mAb 9G4, such as (Fab') 2 is utilized in place of mAb 9G4.
  • the fragment lacks the binding site for rheumatoid factor antibody and the use of the fragment thus avoids this source of interference.
  • Another embodiment of the present invention is a competitive immunoassay utilizing an indicator reagent and a capture reagent.
  • the capture reagent is a VH4-34 binding anti-idiotypic antibody, such as 9G4 mAb or fragments thereof
  • the indicator reagent is a labeled reagent such as enzyme-labeled VH4-34 IgM.
  • the diluted serum sample is contacted with the capture reagent in the presence of labeled VH4-34 IgM for a time sufficient to allow an immunocomplex to form between the bound reagent and either unlabeled VH4-34 Ab present in the sample or labeled VH4-34 IgM.
  • non-bound materials are removed, and the amount of labeled VH4-34 Ab bound to the insoluble phase material is determined by adding signal producing component, e.g., enzyme substrate.
  • the serum level of VH4-34 Ab is inversely proportional to the amount of bound label.
  • an insoluble phase sandwich assay can be used to detect the presence or amount of VH4-34 Ab in a test sample.
  • the bound capture reagent includes a VH4-34 binding anti- idiotypic antibody, such as 9G4 mAb, or individual fragments thereof.
  • the test sample is incubated with the capture reagent for a period of time and under conditions sufficient for the formation of specific complexes between VH4-34 Abs in the sample and the anti-idiotypic antibody.
  • the insoluble phase material can then be washed with a buffer solution to remove unbound test sample.
  • the buffer solution can be any buffer conventionally known and used by those skilled-in-the-art.
  • the resultant complexes are then incubated with a signal producing component for a period of time and under conditions sufficient for the formation of a ternary complex.
  • the signal producing component can comprise a labeled reagent second antibody which recognizes VH4-34 Ab, such as enzyme labeled anti-human IgG.
  • the unreacted labeled reagent is then removed by again washing the insoluble phase material with a buffer solution.
  • the quantity of labeled reagent bound to the insoluble phase material is then measured by a technique compatible with the label component of the labeled reagent. If quantitated, the amount of labeled reagent bound to the insoluble phase material is proportional to the quantity of test sample VH4-34 Ab bound to the insoluble phase material. It should be noted that the specific binding member of the capture reagent and indicator reagent in an assay will usually be different.
  • the capture reagent can be a VH4-34 binding anti-idiotypic antibody or antibody fragment, etc., used to immobilize the VH4-34 Ab upon the insoluble phase material
  • the indicator reagent can be any labeled binding member which will also bind to serum VH4-34 Ab, but at a different binding site.
  • Such indicator reagents include, but are not limited to, anti-human IgG, IgM, IgA etc.
  • the homogeneous assay configurations do not require the separation of the test solution and the labeled reagent prior to the detection of the labeled reagent or binding complexes.
  • This broad classification includes many formats such as agglutination and precipitation assays as well as others known to those skilled-in-the-art for the detection of analytes.
  • a homogeneous assay is fluorescence excitation transfer immunoassay as described by Rubenstein in Immunology for the 80s pp. 127-132, University Park Press (1981).
  • the method is based on the increased efficiency of energy transfer from a f luorescer to a quencher when the two are brought into close proximity by virtue of an antigen-antibody reaction.
  • the quencher, rhodamine can accept energy from the excited fluorescer, fluorescein, with about 50% efficiency at a distance of 5 nm.
  • the binding of fluorescein-labeled reagent VH4-34 Ab to rhodamine-labeied 9G4 anti-idiotypic antibody would result in significant quenching of fluorescence.
  • Addition of serum containing the VH4-34 Ab analyte would result in displacement of labeled anti-idiotypic antibody from labeled VH4-34 Ab which when free exhibits fluorescence emission. Therefore increased analyte in a sample results in increased fluorescence signal.
  • the reagent VH4-34 Ab and the anti-idiotypic antibody can be labeled with N-hydroxysuccinimide ester derivatives of the fluorescer and quencher, respectively.
  • immunoassays include precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, protein A immunoassays, and immunoelectrophoresis assays.
  • VH4-34 Ab assays By observing the results of VH4-34 Ab assays, an accurate diagnosis of SLE can be made, and a determination of the severity of the disease is made possible.
  • the results of the assays used to detect VH4-34 Abs are interpreted as described below.
  • serum samples are obtained from SLE patients.
  • immunoassays were designed and performed wherein VH4-34 Ab was detected in serum from patients who had been previously diagnosed as having SLE based upon alternative diagnostic methods. The level of the antibody was well above those levels found in healthy individuals. Using this methodology, serum from normal healthy controls gave inhibition values of 6.5% + 6.7% (mean + standard deviation). Values greater than the normal mean + 3 standard deviations, i.e., greater than 26.6%, were considered significantly elevated.
  • the present invention provides for adjusting the serum samples to a concentration of IgG within a selected range prior to analysis for VH4-34 Ab.
  • the serum samples are adjusted to a concentration of IgG within the range for normal serum prior to analysis. Adjustment for total IgG provides a more reliable interpretation of the level of VH4-34 Ab.
  • Total serum IgG levels can be elevated in patients with SLE due to polyclonal activation of B cells or lowered due to nephrotic syndrome. It is preferable to determine whether the actual level of VH4-34 Ab is selectively altered among the IgGs present in the serum sample and that apparent changes in VH4-34 Ab are not merely a result of a change in total IgG. While in the Examples presented hereinbelow, samples were adjusted to a concentration of IgG within the range for normal serum, another range can be selected, as long as all samples are diluted to be within the same range of IgG.
  • An advantage of adjusting the serum for total IgG is an increase in sensitivity of elevated VH4-34 for a diagnosis SLE.
  • Sensitivity is the percent of SLE patients from the total SLE patient population tested who test positive. In the Examples presented hereinbelow, the sensitivity was 55% when unadjusted and 59% when adjusted for total IgG. The percent specificity is 100 minus the percent of controls testing positive out of the total number of controls tested, i.e. 100 minus percent false positives. In the Examples presented hereinbelow, the specificity of elevated VH4-34 Ab for a diagnosis of SLE is 95%. The specificity was 94% when the samples were adjusted for total IgG.
  • the total serum concentration of IgG is determined by conventional immunoassay, and each serum sample is adjusted by dilution with aqueous buffer to yield a total IgG level within a selected range, most preferably within the range for normal serum, of IgG prior to immunoassay for VH4-34 Ab.
  • Diagnostic sets comprising one or more components for carrying out the assays of the present invention are also within the scope of the invention.
  • a set can comprise 9G4 mAb or fragment thereof.
  • the set can contain labeled 9G4 mAb (or fragment thereof), e.g. enzyme-labeled 9G4 (Fab') 2 .
  • the set can contain a combination of components such as 9G4 mAb or fragment thereof and reagent VH4-34 Ab, e.g., IgM.
  • the reagent VH4-34 Ab can be bound to an insoluble phase material as capture reagent.
  • a set can contain 9G4 mAb, reagent VH4-34 Ab bound to an insoluble phase material, and a labeled specific binding member which recognizes 9G4 mAb, such as an anti-rat IgG.
  • a set can comprise a 9G4 mAb (or fragment thereof) and reagent VH4-34 Ab.
  • the 9G4 mAb (or fragment thereof) can be a capture reagent pre-absorbed to an insoluble phase material.
  • the 9G4 mAb (or fragment thereof) can be conjugated to a carrier protein which is pre-absorbed to an insoluble phase material.
  • the reagent VH4-34 Ab can be labeled or the set can contain unlabeled reagent VH4-34 Ab and a labeled indicator reagent which recognizes human VH4-34 Ab, e.g. anti- human IgG or a combination of isotype specific antibodies.
  • the set preferably contains the other necessary washing reagents well-known in the art.
  • the set contains the signal production system including chromogenic substrate as well as a reagent for stopping the enzymatic reaction when color development has occurred.
  • the substrate included in the set is one appropriate for the enzyme label. These are well-known in the art, and some are exemplified hereinbelow.
  • the set also comprises a VH4-34 Ab standard; e.g., an amount of purified VH4-34 IgM corresponding to a normal amount of VH4-34 Ab in a standard sample.
  • Other components such as stabilizers and preservative agents can also be present in the set and/or in the reagents.
  • a set of the invention comprises in one or more containers: 1) An insoluble phase material, such as a microtiter plate coated with 9G4 anti-idiotpyic mAb (or portion of the molecule) or coated with a carrier protein to which is conjugated 9G4 anti-idiotpyic mAb (or portion of the molecule); 2) a detectably labeled VH4-34 Ab, e.g., VH4-34 IgM, (or portion of the molecule); 3) a standard sample of VH4-34 Ab (or portion of the molecule) recognized by 9G4 mAb.
  • the set optionally includes an appropriate supply of a suitable indicator reagent, buffers and washing solutions.
  • the components of the diagnostic assay sets are inter-calibrated, i.e., the relative proportions of components have been pre-tested for operability and compatibility.
  • the sets are convenient for use without undue optimization and testing.
  • Components can be in separate containers, or combined, and include reagents which would react with the labels on the antibodies to emit a signal.
  • the antibodies are preferably in solution, such as a buffer, which has no adverse effect on immunoassay.
  • the reagents are lyophilized for reconstitution prior to use.
  • the above sets may additionally have containers calibrators and/or controls or standards.
  • the preferred calibrator and standard contains a known amount of VH4-34 IgM.
  • the present invention is based on the surprising discovery of a correlation between VH4-34 Ab serum level and the severity stage of the disease.
  • the invention provides a means for correlating the level of VH4-34 Ab with severity stage of systemic lupus erthematosus.
  • the analyte level can provide a progress parameter for monitoring the disease and allow prediction of severity
  • Severity stage standards are instruments used for clinical assessment of severity as exemplified by the Lupus Severity of Disease Index (LSDI) as described by Katz et al. in Lupus 2:119-123 (1993) and the Visual Analog Scale for Severity (VAS-S).
  • LSDI Lupus Severity of Disease Index
  • VAS-S Visual Analog Scale for Severity
  • LSDI measures disease severity in the entire history of the patient's disease using the criteria shown in TABLE I.
  • VAS-S is a visual analog scale used by the physician to indicate the severity stage.
  • the physician is instructed to indicate by marking on a linear scale of 0 (not severe at all) to 10 cm (most severe have ever seen) the maximum severity of the patient's lupus at any time during the course of the disease. This is a subjective rating based on the physician's global experience with lupus patients.
  • An aspect of the present invention is a method for determining the severity stage of SLE from a sample of serum from a patient comprising determining the level of VH4-34 Ab in the serum using the improved methods as described herein and comparing the result to at least one of the severity stage standards to determine the severity stage of SLE in the patient.
  • the plate was blocked with 4% milk/2% bovine serum albumin for 1 h at room temperature, then washed with 0.1% Tween 20 in phosphate buffered saline. Serum was diluted in Hanks' balanced salt solution with 0. 1 % non fat dry milk. In some experiments, the serum was adjusted to the normal serum IgG concentration in the same medium, using the value for total IgG as
  • VH4-34 Ab levels were determined and expressed as percent inhibition of optical density.
  • VH4-34 IgM mAb was serially diluted in normal sera to determine total inhibition of 9G4 binding to the ELISA plate. Each serum sample was assayed at least twice and the inhibition value rounded to the nearest multiple of 5.
  • 9G4 mAb was purified from concentrated culture supernatant using immobilized Protein G (Pierce, Rockford, IL) according to the manufacture's instructions.
  • (Fab') 2 fragments were prepared from the purified mAb using immobilized papain (Pierce) according to manufacture's instructions.
  • 9G4 mAb IgG and (Fab') 2 were labeled using N-hydroxy succinimide biotin- amidocaproate (B2643, Sigma).
  • the exemplary assays of the present invention typically involve the addition and incubation of several different reagents.
  • a variety of different buffer and washing solutions can be used to stabilize the reagents and to remove excess reagents or test sample from the reaction.
  • modifications can be made in the buffer and washing solutions, as well as in the reaction times.
  • EXAMPLE 2 Serum levels of VH4-34 antibodies in patients with systemic lupus erythematosus and in controls
  • serum were obtained from 34 healthy individuals (28 female), 28 patients with other autoimmune diseases (22 female; 19 patients had rheumatoid arthritis; the other diagnoses were: psoriatic arthritis (2 patients), Wegener's granulomatosis, Sjogren's syndrome, Crohn's disease, dermatomyositis, Hashimoto's thyroiditis, polymyalgia rheumatica and primary antiphospholipid antibody syndrome)
  • 112 sera were obtained from clinic outpatients (through the gynecology clinic at Stanford Medical Center, the Immunology Clinic at Stanford, or from the Stanford clinical laboratory; 77 were female); and 170 sera were obtained from inpatients.
  • FIG. 1 shows VH4-34 Ab levels (as % inhibition of 9G4 binding) in patients with SLE, healthy controls, and clinical (inpatient and outpatient) controls.
  • the VH4-34 Ab levels were not adjusted for total serum immunoglobulin. Filled circles represent patients with mononucleosis or HIV infection. Only 18 of 310 non-SLE patient sera had elevated levels of VH4-34 Ab, and none of 34 healthy individuals, whereas 52 of 95 patients with SLE had elevated levels. These results are summarized in Table II. The diagnoses of the 18 non-SLE patients with elevated VH4-34 Abs are given in Table III. Four patients had mononucleosis or HIV infection, conditions known to be associated with elevated VH4-34 Ab levels.
  • Controls in this study were chosen from 3 different sources: healthy individuals, serum samples from the clinics and the clinical laboratory at Stanford Medical Center, and patients with other autoimmune syndromes. While the controls were not specifically matched, the age and gender distribution was similar to that seen in the patients with SLE, and age and gender did not appear independently to affect VH4-34 Ab levels (not shown). Importantly, the control sera, particularly from the inpatients, were likely to reflect a very wide range of pathologies, including infectious and neoplastic, besides a (probably small) number of healthy individuals.
  • VH4-34 antibodies adjusted for total serum immunoglobulin, in patients with SLE, and controls Because total serum IgG levels can be elevated in patients with SLE due to polyclonal activation of B cells, or significantly lowered due to the presence of nephrotic syndrome, determinations of VH4-34 Ab levels were adjusted with respect to total IgG levels for all SLE patients and all outpatient.
  • the amount of total IgG in the serum samples was determined by isotype-specific sandwich ELISA, using affinity-isolated Fab 2 anti-human IgG antibodies bound to the microtiter plate, HRPO labeled anti-human IgG as second antibodies, and a human IgG standard (CalTag, South San Francisco, CA).
  • ABTS Sigma
  • Multiple determinations of IgG level in 34 normal sera gave serum IgG values of 10.5 +_1.5 mg/mL in this ELISA.
  • As an example of the adjustment procedure for total IgG if a sample dilution of 1 :100 is used, then
  • a range for normal serum for IgG is about 105 + 15 ⁇ g/mL, and the serum
  • sample is diluted to this range of total IgG prior to assay.
  • FIG. 2 shows VH4-34 Ab levels (as % inhibition of 9G4 binding) in patients with SLE, healthy controls, arthritis controls, and other clinical controls (outpatients only).
  • the VH4-34 Ab levels were adjusted for total serum IgG. Filled circles represent patients with mononucleosis or HIV infection.
  • Severe lupus defined by physician's VAS-S >50, was seen in 37 patients. Twenty-one of 30 (70%) of patients in the highest fertile of VH4-34 Ab binding, as opposed to 4 of 30 (13%) patients in the lowest fertile of VH4- 34 Ab binding, had severe SLE (FIG. 6). Thus the relative risk for having severe SLE for a patient with VH4-34 Ab in the highest fertile was 5.25 compared to the lowest tertile.

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Abstract

L'invention concerne des nouvelles méthodes de dosage immunologique et des ensembles de diagnostic pour le diagnostic du lupus érythémateux systémique et pour la détermination du degré de gravité de la maladie. On combine un échantillon d'essai, comme par exemple du sérum dilué, selon un rapport volume entre l'échantillon et une solution tampon pouvant atteindre 1:1 000, avec un anticorps anti-idiotypique fixant VH4-34, par exemple un anticorps monoclonal 9G4 (mAb), et la proportion d'anticorps anti-idiotypique fixant VH4-34, qui se lie à l'anticorps de VH4-34 dans l'échantillon, est déterminée et est comparée à une référence. L'invention porte également sur: un dosage immunologique, comme ELISA, au moyen de VH4-34 réactif immobilisé et de 9G4 mAb non lié; un dosage immunologique comparatif à l'aide de 9G4 mAb et VH4-34 Ab réactif marqué; et un dosage immunologique en sandwich au moyen de 9G4 mAb immobilisé. Des échantillons de sérum peuvent être ajustés à l'aide d'une solution tampon pour la production d'un taux d'IgG total dans une plage sélectionnée, de préférence dans la plage du sérum normal, avant l'analyse. Ledit procédé peut consister à vérifier que l'échantillon est sensiblement exempt de l'anticorps du facteur rhumatoïde. L'interférence dans le dosage par anticorps du facteur rhumatoïde peut être réduite au moyen d'un fragment de fixation de 9G4 mAb, comme (Fab')2.
PCT/US1998/012684 1997-06-17 1998-06-17 Methode de diagnostic du lupus erythemateux systemique WO1999001477A1 (fr)

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WO2001027621A3 (fr) * 1999-10-07 2001-08-30 Pharmacia Corp DOSAGE IMMUNOENZYMATIQUE D'INHIBITION (iELISA) PERMETTANT DE DETECTER ET QUANTIFIER LES ANTICORPS PRESENTS DANS DES PRELEVEMENTS BIOLOGIQUES
WO2007035857A3 (fr) * 2005-09-19 2007-06-21 Palingen Inc Traitement d'affections des lymphocytes b avec des agents se liant a des anticorps antigerminaux
CN1328583C (zh) * 2004-06-30 2007-07-25 中国人民解放军军事医学科学院野战输血研究所 血清或血浆样品稀释液及其应用
US20100227415A1 (en) * 2007-07-24 2010-09-09 Iss Immune System Stimulation Ab Method and means for prediction of systemic lupus erythematosus susceptibility

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MILLER J. J., ET AL.: "VH4-34 (VH4.21) GENE EXPRESSION IN THE CHRONIC ARTHRITIDES OF CHILDHOOD: STUDIES OF ASSOCIATIONS WITH ANTI-LIPID A ANTIBODIES, HLA ANTIGENS, AND CLINICAL FEATURES.", JOURNAL OF RHEUMATOLOGY, JOURNAL OF RHEUMATOLOGY PUBLISHING COMPANY., CA, vol. 23., no. 12., 1 January 1996 (1996-01-01), CA, pages 2132 - 2139., XP002914737, ISSN: 0315-162X *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027621A3 (fr) * 1999-10-07 2001-08-30 Pharmacia Corp DOSAGE IMMUNOENZYMATIQUE D'INHIBITION (iELISA) PERMETTANT DE DETECTER ET QUANTIFIER LES ANTICORPS PRESENTS DANS DES PRELEVEMENTS BIOLOGIQUES
CN1328583C (zh) * 2004-06-30 2007-07-25 中国人民解放军军事医学科学院野战输血研究所 血清或血浆样品稀释液及其应用
WO2007035857A3 (fr) * 2005-09-19 2007-06-21 Palingen Inc Traitement d'affections des lymphocytes b avec des agents se liant a des anticorps antigerminaux
US20100227415A1 (en) * 2007-07-24 2010-09-09 Iss Immune System Stimulation Ab Method and means for prediction of systemic lupus erythematosus susceptibility

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