WO2007003090A1

WO2007003090A1 - A composition instead of positive sera used as control in diagnostic agent and its application

Info

Publication number: WO2007003090A1
Application number: PCT/CN2006/001199
Authority: WO
Inventors: Ziyi Yang; Peng Luo; Guolan Wei; Wenzhuo Chen; Weina Situ; Jian Ni
Original assignee: Shanghai Fuchun Zhongnan Biotech Co. , Ltd.
Priority date: 2005-07-04
Filing date: 2006-06-02
Publication date: 2007-01-11
Also published as: CN1924579A

Abstract

A composition instead of positive sera used as control in diagnostic agent is a composition of cross-linked specific antibody to antigen and human immunoglobulin. The specific antibody to antigen is obtained from technology of monoclonal antibody or antiserum prepared by immunizing animal with antigens. The application of the composition comprises antibody preparing, cross-linking with immunoglobulin, standard curve plotting and applying the composition used as standard in quantitative measurement of diagnosis.

Description

Composite for replacing positive serum as a diagnostic reagent standard and use thereof

Technical field

The invention belongs to the field of biomedicine, relates to a novel compound which can be used as a standard for diagnostic reagents and a method for using the same as a diagnostic reagent standard, and particularly relates to a standard which can be used as a diagnostic reagent instead of positive serum. A cross-linked complex of an antigen-specific antibody and a human immunoglobulin and a method of using the same as a diagnostic reagent standard. Background technique

An antibody is a glycoprotein produced by B lymphocytes in an immune response to antigen stimulation. The basic structure of the molecule consists of two identical heavy chains (H chains) and two identical light chains (L chains). , between the light and heavy chains and between the two heavy chains are connected by disulfide bonds. According to the constant region of the heavy chain, the antibodies are classified into five classes, namely IgG, IgM, IgA, IgE and IgD. The heavy chain molecules of IgG are Y chains, and the heavy chains of IgM, IgA, IgE and IgD are μ, α, respectively. The ε and δ chains differ in the heavy chain of the polypeptide, allowing various antibodies to play different roles in different types of immune responses and at specific times during the immune response. Although there are five different heavy chains, there are only two light chains, the κ and λ chains (Ε. Harlow, D. Lane, “Guide to Experimental Techniques for Antibody Technology.”).

The light chain has approximately 220 amino acid residues and is divided into two regions, each region containing approximately 110 amino acid residues, the region near the amino terminus is a heterogeneous variable region (V region), and the region near the carboxy terminus is a constant region ( Area C). The IgG heavy chain has approximately 440 amino acid residues, including one variable region and three constant regions, each of which consists of 110 amino acid residues. Different types of heavy chains may have different numbers of constant regions, such as IgM. Variable zone and 4 constant zones.

The variable regions of one heavy chain and one light chain combine to form an antigen binding site. The heterogeneity of the variable regions can be used to initiate an effective immune response to form a large antibody species. However, the sequence inconsistency does not occur randomly in all variable regions, but is concentrated in some hypervariable regions consisting of 5-10 amino acid residues, and each of the heavy and light chains has three metamorphic regions. The region constitutes the major contact residue for binding of the antibody to the antigen and is located on the short amino acid residue loop that interacts with the antigen. Because Gao variable region is the antigen binding site of the real, also called complementarity determining regions (complementary determining regions, CDRs) ₀ variable region and improve the specificity of the antigen binding site, the antibody constant region determining function, all the antibodies The constant regions provide a range of binding sites that serve important functions, such as providing a functional region that binds to Fc receptor cells, promoting phagocytosis of macrophages and granulocytes and promoting antibody-dependent lymphocyte and NK cells. Cell-mediated cytotoxicity. It also provides an important region of the complement chain reaction that promotes the dissolution of foreign invading antigens. The highly specific knot characteristics between antigen and antibody make them ideal clinical diagnostic reagents. According to statistics, in the existing clinical diagnosis, 50% of the test items are realized by the detection of antibodies or antigens. Immunoassays provide rapid and sensitive assays for detecting infectious agents, physiological indicators, allergies, autoimmune diseases, cancer, drug metabolism, and the like. Manual and automated immunoassays typically use specific antibodies to detect specific antigens or related antigens. There are many methods of immunoassay available, but they can be divided into two main categories: competitive and non-competitive. The antibody or antigen is cross-linked to the solid support by covalent or non-covalent, for example, porous or non-porous materials, latex particles, magnetic materials, microcarriers, glass beads, membranes, confirmation Micropores and plastic tubes, etc. The solid phase material and the antigen/antibody labeling method are determined according to the characteristics of the analytical method. For example, in some immunoassays, no labeling is required, such as detection of an observable antigen on the surface of red blood cells, which can be judged by agglutination. In addition, antigen-antibody reactions can also produce observable changes. In most cases, a signal molecule or "marker" labeled antigen or antibody is included in the immunoassay. Such signal molecules or "markers" are detectable by themselves or by reacting with one or more other compounds to produce a detectable signal. Signal molecules include dyes, isotopes (such as 1251, 1311, 32P, 3H, 35S, 14C), fluorescent materials, chemiluminescent materials, microparticles (visible or fluorescent), nucleic acids, complexes or catalysts such as enzymes (such as alkaline phosphatase) , acid phosphatase, horseradish peroxidase, β-galactosidase, etc.). When an enzyme label is used, a chemical, fluorescent or visible light generating substrate is also required to produce a detectable signal. Time-resolved fluorescence analysis, internal reflection fluorescence, nucleic acid amplification (PCR), and Raman spectroscopy are also useful.

Immunoassays have been used for the detection of body fluids such as plasma, serum, cerebrospinal fluid, saliva, tears, nasal fluid or tissue and cell aqueous extracts. Two commonly used forms are used for the detection of specific antibodies in humans: (1) antigen coating, human body fluids contain specific antibodies, can react with antigens, and then antibodies bind to coated antigens, using labeled anti-human antibodies ( Anti-antibody, also known as secondary antibody) detection; (2) antibody coating, human body fluids include specific antigens, can react with coated antibodies, and then, with a labeled second antibody against different epitopes of the same antigen In both methods, the antibody may be a monoclonal antibody or a polyclonal antibody.

An antibody is an immunoglobulin produced by the body's immune response to foreign molecules, microorganisms or other factors. Under certain conditions, the immune system is disordered, and an immune response is generated against the autoantigen, producing autoantibodies, resulting in the body's own tissues and organs. Pathological changes occur and corresponding clinical manifestations appear. These diseases are collectively referred to as autoimmune diseases. The incidence of autoimmune diseases in Europe and North America is up to 5%, and the total number of patients in the United States is about 13 million. Autoimmune diseases are the third largest category of diseases such as cancer and cardiovascular diseases. The incidence of rheumatoid arthritis is 1% (Advances in the Laboratory Diagnosis of Autoimmune Diseases, Business Briefing Global Health Care 2002, Issue 3.). The diagnosis of these diseases is in addition to the different clinical symptoms, the antibodies in the patient's serum. The detection is a key indicator of diagnosis.

Because many antibody titers are highly correlated with the occurrence, development and prognosis of diseases, such as liver cell cytoplasmic antigen type 1 autoantibodies (LC-1) titer and disease activity, serum transaminase Significantly correlated with gamma globulin levels. High titer anti-LC1 is only detected in chronic hepatitis or cirrhosis with high activity (Lenzi M, Mantotti P, Muratori L, Cataleta M, et al. Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and Hepatitis C Virus infection. Gut, 1995, 36:749-754.). Anti-LCI has no predictive effect on the efficacy of corticosteroids and azathioprine. LC-1 is a good serum immunological indicator reflecting disease progression and therapeutic effects. Therefore, the qualitative and further quantification of autoantibodies is essential. For the detection of antibodies, in addition to the explicit presence, their classes and subclasses should be identified. In general, IgMs are mainly found in acute infectious diseases, and IgG4 subclasses are mainly found in organ-specific autoimmune diseases, while IgG1 and IgG3 Mainly found in organ non-specific autoimmune diseases (Deng Anmei, Zhong Renqian, Kong Xiantao. The relationship between nuclear antibody spectrum and humoral immunity can be extracted from patients with autoimmune diseases. Chinese Journal of Medical Laboratory Science, 1999, 22: 299-300.).

Clinical diagnostic kits generally include standard or negative and positive controls, and quantify the label of the autoantibody diagnostic kit. The quasi-products are often derived from serial dilutions of positive serum with higher antibody titers, or directly using patient sera at different titers. However, there are many disadvantages in using patient serum as a standard: 1) It is difficult to obtain patient serum with high titer and high specificity; 2) The antibody titer and specificity between individuals are large and difficult to standardize; 3) Because serum is polyclonal, it has different antibody types, affinities and specificities, which limits its application; 4) It is expensive, and due to resource constraints, the yield of standards may not be guaranteed. Therefore, it is important to develop methods for producing standards to detect the level of specific antibodies. Human and mouse chimeric antibodies can be used as standards for human serum antibody detection for the preparation of standard curves (US Patent No. 6,015,662), but this method has great limitations: 1) The process is very complex, including animal immunity, cell fusion Screening of monoclonal antibodies, cloning of antibody genes, construction and expression of chimeric genetically engineered antibodies in humans, etc., long time and high cost; 2) Each time the antibody is constructed, only one constant region of the antibody is included, and other antibody types are required for detection. rebuild.

Cross-linked antibodies have great advantages in the use of standards, non-human non-IgM and human IgM cross-linking (US Patent: 5,478,753), used as a diagnostic IgM serum kit standard, cross-linking antibody non-human non-human IgM is specific for detecting antigens, and human IgM can bind to labeled secondary antibodies. These kits are a good solution to the difficulty of IgM-positive serum sources in infectious diseases during acute infection, making large-scale production of standards possible. Other cross-linking also includes the cross-linking of a heavy chain and a light chain of an antibody through a disulfide bond and an IgG heavy chain and a light chain or Fc (U.S. Patent No. 5,523,210), and the constructed bivalent antibody can be used for chemical analysis such as enzyme; Antibodies specific for different antigens are cross-linked by a cross-linking agent (U.S. Patent No. 4,433,059), and heterologous cross-linked antibodies can be used for agglutination analysis. Summary of the invention

An object of the present invention is to provide a cross-linked complex of an antigen-specific antibody and a human immunoglobulin instead of a positive serum as a diagnostic reagent standard, which can solve specific antigen binding and anti-human immunity to an antigen. The response of a globulin antibody (anti-human secondary antibody).

Another object of the present invention is to provide a cross-linking complex, wherein the antibody technology for preparing a specific antibody can be a monoclonal antibody technology (hybridoma technology, including mouse, rat and rabbit hybridoma, etc.) The prepared antigen-specific antibody can also be used to immunize an anti-serum obtained by an animal (such as rabbit, sheep, horse, cow, etc.) with an antigen, and then an antigen-specific polyclonal antibody prepared by antigen characteristic affinity chromatography.

Another object of the present invention is to provide a cross-linking complex, wherein when the antibody complex is used for detecting the level of a specific antibody produced by a human body against an antigen, it can be used as a standard instead of a serum standard derived from a human body or Reference product.

Another object of the present invention is to provide a cross-linked complex, which can be used for disease diagnosis and disease screening, such as infectious diseases (anti-hepatitis B antibody, anti-HC antibody, anti-drug) by using the method for detecting human antigen-specific antibody levels. AIDS antibodies, etc., autoimmune diseases (autoantibodies such as rheumatoid factor and anti-nuclear antibodies), allergic diseases (serum IgE levels, etc.) can also be used for blood group testing, tissue matching, and health checkups.

A further object of the present invention is to provide a method for using an antigen-specific antibody and a human immunoglobulin cross-linking complex as a standard. The standard prepared by the method has uniform antibody specificity and affinity, and the preparation method is simple, and the source is simple. Unrestricted, it can be produced on a large scale.

In order to achieve the above object, the present invention discloses a complex which can be used as a diagnostic reagent standard instead of positive serum. The complex is characterized in that the complex is a cross-linked complex of an antigen-specific antibody and a human immunoglobulin, and the antigen-specific antibody is prepared by monoclonal antibody technology or an antiserum obtained by immunizing an animal with an antigen.

In order to achieve the above object, the present invention also discloses a method, the method comprising at least the following steps:

a. immunizing an animal with an antigen, obtaining a specific antibody;

b. crosslinking the specific antibody and the human immunoglobulin with a crosslinking agent;

c gradient dilution of the cross-linked antibody to prepare a standard curve;

d. The cross-linked antibody is used as a standard in a quantitative diagnostic test method. DRAWINGS

Figure 1 is a standard curve of a preferred embodiment of the present invention. as well as,

2 is a graph showing statistical results of a preferred embodiment of the present invention. detailed description

The invention includes a method comprising at least the following steps:

(a) . Immunization of animals with antigens, specific antibodies obtained;

(b) crosslinking the specific antibody and the human immunoglobulin with a crosslinking agent;

(c). The cross-linked antibody is diluted in a gradient to prepare a standard curve;

(d). The cross-linked antibody is used as a standard in a quantitative diagnostic test method.

The CCP antigen is coated on a 96-well reaction plate, and the diluted serum to be tested and the positive serum of the control substance are added to the well of the reaction plate. If the antibody of the anti-CCP antigen component is present in the serum to be tested, after incubation, the serum is in the serum. The specific antibody binds to the solid phase CCP antigen to form a solid phase antigen-antibody complex. The unbound antibody component is washed away, and an enzyme-labeled anti-human IgG antibody (anti-human secondary antibody) is added thereto, and the solid phase antigen-antibody complex is further bound to the enzyme-labeled anti-IgG antibody. The unbound enzyme-labeled antibody component is washed away, and the substrate of the enzyme is added. The substrate is catalyzed by the enzyme to become a colored product, and the value of the color reaction of the specimen and the positive serum of the control can be detected by a microplate reader. Using a different titer of positive control serum, a standard curve between the color reaction value and the positive serum anti-CCP antibody titer of the reference substance was determined by a microplate reader, and the value was determined on the standard curve according to the serum microplate reader to be tested. The position of the corresponding anti-CCP antibody in the serum to be tested can be determined.

The diagnostic kit is used as a standard positive serum for the standard curve and is the most critical component of the kit. Currently, standard positive serum is taken from patients with elevated levels of anti-CCP antibodies, which can cause problems. First, the positive serum is taken from patients who have increased the level of anti-CCP antibody by using the existing diagnostic kit. The positive serum of the existing diagnostic kit is taken from the patient who meets the diagnostic criteria for rheumatoid, and the anti-rheumatic diagnostic criteria may not be resistant. CCP antibodies increased. Second, the level of anti-CCP antibody in the patient's positive serum will change with the condition, so the "standard" is not standard. Third, the serum source of each patient is limited. The serum source of each batch may be different. There is a difference between the standard batches. Therefore, the same serum of the same patient may be detected by different batches of diagnostic kits. result. The use of a cross-linker of an anti-CCP monoclonal antibody with human IgG instead of a positive serum as a standard for the kit does not suffer from the disadvantages described above.

In step (a), the cross-linking method between CCP and bovine serum albumin (BSA) is described in E. Harlow, D. Lane. Physical Technology Experiment Guide. Balb/c mice of 6-8 weeks old were selected, and the CCP after cross-linking with l-100p _{g was} fully emulsified and injected intraperitoneally with the same amount of complete Freund's adjuvant. After the same dose, the antigen was added in equal amounts every 2 weeks. The Freund's adjuvant is fully emulsified and injected intraperitoneally, a total of 3-5 times. The booster immunization was performed once orally in the first 3 days of fusion without adjuvant antigen 50-100 g.

Take the immunized mice, take the eyeball to take blood, and separate the serum for use. The mice were sacrificed by necking and soaked in 75% alcohol for 3-5 minutes. The spleen was removed aseptically to prepare a spleen cell suspension. P3-653 cells in the logarithmic growth phase were mixed with mouse spleen cells in a ratio of 1:5-1:10, and 20 ml of RPM-1640 solution was added thereto, 1000 r/min, centrifuged for 5 minutes, and the supernatant was discarded. Gently tap the bottom of the centrifuge tube to disperse the precipitated cells. Place the centrifuge tube in a 37 ° C water bath and take 1 ml of 37 ° C pre-warmed 50% PEG slowly into the centrifuge tube and add in 1 minute. After standing at 37 ° C for 2 minutes, 50 mL of RPMI-1640 solution was added dropwise over 5 minutes to terminate the PEG action. Centrifuge at 800 r/min for 8 minutes and discard the supernatant. The precipitated cells were gently suspended in a desired volume of HAT medium and seeded in a 96-well culture plate. The plate was incubated in a 37 ° C 5% CO 2 ± incubator. After 7-10 days, change the HT medium. The culture supernatant was aspirated three days after the exchange, and an ELISA test was performed. Pick out the positive pore cells, and use the limiting dilution method to spread the cells in a single cell, that is, do the cell count, so that the concentration of the cells is 50/ml, inoculate three rows in the 96-well plate, and dilute with the culture solution. One 96-well plate. After 7-10 days of culture, a single clone was selected for ELISA, and the positive well cells were picked and cloned again. It is generally necessary to clone 3-5 times repeatedly, and the cells can be expanded and cultured by direct passage to 100% positive wells.

The screened hybridoma cells were cultured in suspension with serum-free medium, and the culture supernatant was collected for further purification. The antibody was purified by affinity chromatography using ProtdnG or ProteinA Sepharose. Reference (E. Harlow, D. Ryan. Antibody Technology Experiment Guide). Finally, antibody screening was performed.

Step (b) of the present invention is the crosslinking of a murine anti-CCP monoclonal antibody with human immunoglobulin (IgG). Human IgG 25 mg was weighed and dissolved in 0.5 mi of 1.25% glutaraldehyde in pH 7.2 0.lmoI/L PBS, and allowed to stand overnight at 2-8 Torr. The reacted solution was dialyzed extensively in H 7.2 O.lmol/L PBS to remove excess glutaraldehyde, and pH 7.2 O.lmol/L PBS was added to 1.5 ml. Place in a small 25ml beaker and stir slowly. 12.5 mg of the mouse anti-CCP antibody was diluted to 0.5 ml with pH 7.2 ΐ.ΐηιοΐ/L·PBS, and added dropwise to the small beaker with stirring. Add 1 mol/L PH 9.6 carbonate buffer and continue stirring for 3-4 hours. Finally, 0.25 mL of a 0.2 mol/L lysine solution was added and allowed to stand at room temperature for 2 hours. The reaction solution was subjected to Sephacryl S-300 HR column and eluted with PH7.2 0.1 mol/L PBS to collect the first peak. This solution was the interaction between the mouse anti-CCP antibody and human immunoglobulin (IgG). Linkage.

The polyclonal anti-human IgG was diluted to 5 g/ml with 1.59 g/L Na2CO3, 2.93 g/L NaHC03 coating buffer (CBS solution). Then add the diluted solution to each well of the microtiter plate, ΙΟΟ μ 每 per well; 2-8 Ό overnight, then remove the ELISA plate, remove the coating solution, wash the plate three times, pat dry, add 1% cattle Serum albumin, 8.7g/L NaCl, 1.15 g/ L Na2HP04* 12H20, 0.2 g/ L NaH2P04* 2H20, pH 7.4 blocking solution, 200 μl per well; placed at 37 ° C for 2 hours, then remove the enzyme label Plate, discard the blocking solution, wash the plate three times, pat dry. The cross-linked dilution of the cross-linked CCP monoclonal antibody may also be 1:100, 1:200, up to 1:1000. The diluted monoclonal antibody was added to each well of the coated plate, and each well was ΙΟΟ μ Ι, and the dilution of each conjugated monoclonal antibody was doubled, and the reaction was carried out at room temperature (18-25 ° C) for 30 minutes. After the reaction was completed, the coated plate was washed three times with a washing solution and patted dry. HRP-labeled goat anti-human IgG working solution was added to each well for 100 μl at room temperature (18-25 Torr) for 30 minutes. After the reaction was completed, the coated plate was washed 5 times with a washing solution and patted dry. Join in each hole Coloring solution A (containing 35.8g/L of citric acid, Na2HP04'12H209.34g/L, 30% hydrogen peroxide 660μΐ) and coloring liquid Β (containing 3,3,5,5, -tetramethyl Benzene 0.2g / L, dimethyl sulfoxide 5ml / L, 6 N HC1 lml / L) 50μ 1, mixed hook, room temperature in the dark for 30 minutes. After the reaction, a stop solution (2 Ν H2S04 ) 50 μ 1 was added to each well, and the OD450 reading of the microplate reader was performed. Because there is no international reference quality control for anti-CCP antibodies, the relative units of the antibodies are customized according to the OD450 value of the antibody. When OD450-2.0-2.2, the corresponding dilution is 1:400, and the setting unit is 400RU. Other dilutions are analogous, such as 1:800 for 200 RU, 1:1600 for 100 RU, and so on.

Step (c) of the present invention is to draw a standard curve with different dilutions of the cross-linked CCP mAb.

Dilute the CCP to 5 μg/ml with a coating buffer. Then, the CCP solution was added to each well of the microplate, 100 μl per well; and placed at 2-8 ° C for 17 hours, then the enzyme plate was taken out, the coating solution was removed, the plate was washed three times, patted dry, and added. Blocking solution, 200 μ 每 per well; placed at 37 ° C for 2 hours, then remove the enzyme plate, discard the blocking solution, wash the plate three times, pat dry; place the ELISA plate in a vacuum drying oven Vacuum dry and store in a vacuum box for 1 hour; finally, insert the enzyme label strip into the aluminum foil bag, seal it with a vacuum sealer, label and stamp the batch number.

The cross-linked CCP monoclonal antibody was double-diluted to a concentration of 400 RU for a total of 9 dilutions. The dilution consisted of 1% bovine serum albumin, 8.7 g/L NaCl, 1.15 g/L Na2HP04* 12H20, 0.2 g/L NaH2P04« 2H20, Tween-20 0.5 ml, pH 7.4. '

Control sera were anti-CCP antibody-positive sera, weak-positive sera (serum near the critical value) and negative sera as determined by comprehensive development and clinical outcomes. The diluted monoclonal antibody and control serum (1:100 release) were added to each well of the CCP coated plate, 100 μl per well, and the dilution of each conjugated monoclonal antibody and the control serum were duplicated at room temperature. (18-25 Ό) The reaction was carried out for 30 minutes. After the reaction was completed, the coated plate was washed 3 times with a washing solution and patted dry. HRP-labeled goat anti-human IgG working solution 100 μl was added to each well for 30 minutes at room temperature (18-25 ° C). After the reaction was completed, the coated plate was washed 5 times with a washing solution and patted dry. Add 50 μl of each of the coloring solution A and the color developing solution B to each well, and mix at room temperature (18-25 Ό) for 30 minutes in the dark. After the reaction, a stop solution of 50 μl and a OD450 reading of the microplate reader was added to each well.

The average OD450 of the different dilutions of the cross-linked CCP monoclonal antibody was calculated. The OD450 value of each dilution was plotted on the ordinate, and the corresponding concentration was plotted on the abscissa, and a standard curve was drawn.

The standard curve of the preferred embodiment is shown in FIG. From the overall trend of the curve, the linear segment is 0-100RU, above 100RU, and the curve has become saturated. Take 5 points (100RU, 50RU, 25RU, 12.5RU, 0) in the linear segment as a standard curve for the kit. Calculate the average OD450 of the three serum samples in parallel and read the corresponding units on the standard curve. The unit corresponding to the positive serum was 65 RU, the unit corresponding to the weak positive serum was 18 RU, and the unit corresponding to the negative serum was 2 RU. From the data, the serum of the three control products all fall within the linear range, especially the weak positive serum (threshold value) falls in the middle of the line segment, indicating that this line segment can cover the entire negative positive detection range, further explaining the standard curve. Can be used as a quantitative curve for this kit. The threshold value of the kit can be determined from the unit corresponding to the weak positive serum to be 18 RU, that is, greater than 18 RU is positive, and less than 18 RU is negative.

Step (d) of the present invention is to apply a standard curve to an anti-CCP antibody detection kit. The kit tested 200 normal human blood sputum and 200 RA patient sera, and made the above five standard curve, the operation process is the same as above. Calculate the average OD450 of the samples in parallel and read the corresponding units on the standard curve. With 18RU as the threshold, statistical results as shown in picture 2.

In this statistic, a true positive sample refers to a sample in which 200 serum samples of rheumatoid arthritis patients are tested positive by this kit, and a sample of false negative samples refers to a sample in which 200 serum samples of rheumatoid arthritis patients are negative by this kit. . The true negative sample refers to a sample in which 200 normal human serum samples are tested negative by this product, and the false positive samples refer to 200 normal human serum samples which are tested positive by this product. Industrial applicability

A preferred embodiment of the invention is a kit for the diagnosis of rheumatoid arthritis against an anti-cyclic citrullinated peptide (CCP) antibody. The kit is an enzyme-linked immunoassay kit for quantifying anti-CCP IgG type antibodies in patient serum. Detection of anti-CCP IgG-type antibodies can aid in the diagnosis of rheumatoid arthritis.

The present invention is described in the most common and preferred embodiments, and is not limited to the disclosed embodiments; instead, modifications and equivalent arrangements are intended to be included within the scope of the invention.

Claims

Rights request

A complex which can be used as a diagnostic reagent standard instead of a positive serum, characterized in that - the complex is a cross-linked complex of an antigen-specific antibody and a human immunoglobulin.

2. The complex according to claim 1, wherein the antigen-specific antibody is prepared by monoclonal antibody technology.

The complex according to claim 1, wherein the antigen-specific antibody is prepared by an antiserum obtained by immunizing an animal with an antigen.

The complex according to claim 1, wherein the human immunoglobulin is human immunoglobulin IgG.

The complex according to claim 1, wherein the human immunoglobulin is human immunoglobulin IgM.

The complex according to claim 1, wherein the human immunoglobulin is human immunoglobulin IgE.

The complex according to claim 1, wherein the human immunoglobulin is human immunoglobulin IgA.

8. The complex of claim 1 wherein said human immunoglobulin is the full length of a human immunoglobulin.

The complex according to claim 1, wherein the human immunoglobulin is a constant region of human immunoglobulin.

The complex according to claim 1, wherein the human immunoglobulin is an Fc fragment of human immunoglobulin.

11. A method of using a complex according to claim 1, characterized in that the method comprises the steps of: a. immunizing an animal with an antigen, obtaining a specific antibody;

c gradient dilution of the cross-linked antibody to prepare a standard curve;

d. The cross-linked antibody is used as a standard in a quantitative diagnostic test method.

12. The method according to claim 11, wherein the detecting method is a kit.

13. The method according to claim 11, wherein the detecting method is a detecting reagent.

14. The method according to claim 11, wherein the detecting method is an instrument detecting.

15. The method according to claim 11, wherein the antigen-specific antibody of step a is a monoclonal antibody.

16. The method according to claim 11, wherein the antigen-specific antibody of step a is a polyclonal antibody.

17. The method according to claim 11, wherein the crosslinking agent in step b is glutaraldehyde and sodium periodate.