CN1924579A - Cross-linked composite used as standard diagnosing reagent replacing positive serum and method for use as standard reagent - Google Patents

Cross-linked composite used as standard diagnosing reagent replacing positive serum and method for use as standard reagent Download PDF

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CN1924579A
CN1924579A CNA2005100274765A CN200510027476A CN1924579A CN 1924579 A CN1924579 A CN 1924579A CN A2005100274765 A CNA2005100274765 A CN A2005100274765A CN 200510027476 A CN200510027476 A CN 200510027476A CN 1924579 A CN1924579 A CN 1924579A
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antigen
antibody
human immunoglobulin
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杨子义
倪健
罗鹏
魏国兰
陈闻卓
司徒维娜
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SHANGHAI FUCHUN ZHONGNAN BIOTECHNOLOGY CO Ltd
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Priority to CNA2005100274765A priority Critical patent/CN1924579A/en
Priority to PCT/CN2006/001199 priority patent/WO2007003090A1/en
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    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

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Abstract

This invention relates to one positive serum as diagnose agent standard compound, which is processed through single clone antigen technique or antigen immune animal and is the crossed compound of antigen and human immune globulin. This invention also discloses one use method of the compound, which comprises the following steps: a, using antigen immune globulin to get the specific antigen; b, crossing the specific antigen and human immune globulin by the crossing agent; c, diluting the said cross antigen gradient to draw standard curve; d, using the said antigen as standard product for quantitative diagnose test method.

Description

A kind of cross-linked composite of used as standard diagnosing reagent replacing positive serum and as the using method of standard items
Technical field
The invention belongs to biomedical sector, relevant for a kind of new compound that can be used as standard diagnosing reagent and as the using method of standard diagnosing reagent, especially relevant for a kind of antigen-specific antibodies and the cross-linked composite of human immunoglobulin(HIg) and that can used as standard diagnosing reagent replacing positive serum as the using method of standard diagnosing reagent.
Background technology
Antibody is in the immune response to antigenic stimulus, class glycoprotein by the bone-marrow-derived lymphocyte generation, molecule basic structure comprises that two identical heavy chains (H chain) and two identical light chains (L chain) form, between light, the heavy chain and connected by disulfide bond between two heavy chains.Difference according to CH, antibody falls into 5 types, be IgG, IgM, IgA, IgE and IgD, the heavy chain molecule of IgG is the γ chain, the heavy chain of IgM, IgA, IgE and IgD is respectively μ, α, ε and δ chain, polypeptide heavy chain difference makes the specific period of each antibody-like in dissimilar immune responses and immune response process bring into play different effects.Although 5 kinds of different heavy chains are arranged, light chain has only two kinds, promptly κ and λ chain (E. breathes out the Lip river, D. Lay grace. " antibody technique experiment guide " .).
Nearly 220 amino acid residues of light chain are divided into two districts, and 110 amino acid residues are approximately contained in every district, are heterogeneous variable regions (V district) near N-terminal district, and near c-terminus is constant region (C district).Nearly 440 amino acid residues of IgG heavy chain comprise a variable region and 3 constant regions, and each zone is made up of 110 amino acid residues, and inhomogeneous heavy chain has the constant region of varying number, as IgM 1 variable region and 4 constant regions are arranged.
Article one, the variable region of a heavy chain and a light chain is in conjunction with forming an antigen-binding site.The inhomogeneity of variable region can be used for starting effective immune response, and forms huge antibody type.But the inconsistency of sequence and nonrandom all variable regions that occur in, be distributed in the hypervariable region that some are made up of 5-10 amino acid residue but concentrate, heavy chain and light chain respectively have 3 hypervariable regions, the main contact residues of antibody and antigen combination has been formed in these hypervariable regions, and is positioned on the short amino acid residue ring with AI.Because the hypervariable region is and the real position that combines of antigen, thus be called again complementary determining region (complementarydetermining regions, CDRs).Antigen binding site and specificity are improved in the variable region, the function of constant region decision antibody, constant region at all antibody all provides a series of binding site, the performance critical function, as functional areas with the combination of Fc recipient cell are provided, promoted macrophage and the granulocytic cytotoxicity of engulfing and having promoted lymphocyte and the mediation of NK cell generation antibody dependent cellular.The important area of conjugated complement chain reaction also is provided, has promoted the dissolving of exotic invasive antigen.
The characteristic of the high degree of specificity combination between antigen and the antibody makes them become desirable reagent for clinical diagnosis.According to statistics, in the existing clinical diagnosis, 50% test item is realized by antibody or detection of antigens.Immunoassay provides the experimental technique of the quick and responsive detection infectious disease original, physical signs, allergy, autoimmune disease, cancer, drug metabolism etc.Manual and automated immunochemistry analysis uses specific antibodies to detect specific antigen or related antigen usually.A variety of immune analysis methods have been arranged now, but mainly can be divided into two big classes: competitive and noncompetitive analysis.Antibody or antigen pass through covalently or non-covalently crosslinked on solid phase carrier, for example, and porous or pore-free material, latex particle, magnetic material, microcarrier, beaded glass, film, micropore and plastic tube etc.Characteristic decision solid phase material and antigen/antibody labeling method according to analytical approach.As, in some immunoassays, need not mark, as when detecting the antigen on observable erythrocyte surface, can judge by agglutinating reaction.In addition, antigen-antibody reaction also can generate observable variation.As a rule, the antigen or the antibody that all comprise a kind of signaling molecule or " label " mark in the immune detection.Sort signal molecule or " label " can detect self, or by reacting and produce detectable signal with other a kind of/several other compounds.Signaling molecule comprise dyestuff, isotope (as 125I, 131I, 32P, 3H, 35S, 14C), fluorescent material, chemiluminescent material, particulate (visible or fluorescence), nucleic acid, compound or catalyzer such as enzyme etc. (as alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta galactosidase etc.).When using enzyme labeling, also need chemistry, fluorescence or visible light to generate substrate and produce detectable signal.Time-resolved fluoroimmunoassay, internal reflection fluorescence method, nucleic acid amplification (PCR) and Raman spectrum analysis method also have application.
Immunoassay has been used for the detection of body fluid such as blood plasma, serum, celiolymph, saliva, tear, nose liquid or tissue and cellular water solution extract.Two kinds of forms commonly used are used for the detection of human body specific antibodies: (1) is antigen coated, and human body fluid comprises specific antibody, can react with antigen, and then, the antigen of antibodies bag quilt is with anti-people's antibody (antiantibody claims that again two is anti-) detection of mark; (2) antibody sandwich, human body fluid comprises specific antigen, can react with coated antibody, then, with second antibody test at the different epi-positions of same antigen of mark; In these two kinds of methods, antibody can be that how anti-monoclonal antibody also can be.
Antibody is the immunoglobulin (Ig) that body produces foreign molecules, microorganism or other factor immune responses, under certain conditions, immune system gets muddled, also can produce immune response at autoantigen, produce autoantibody, cause the autologous tissue and the organ of body the pathologic change to occur and corresponding clinical manifestation occurs, this class disease is referred to as autoimmune disease.Autoimmune disease at Europe and the incidence of disease of North America up to 5%, total number of patients is about 13,000,000 in the U.S., autoimmune disease is the third-largest class disease of cancer and angiocardiopathy of continuing, wherein the incidence of disease of rheumatoid arthritis is 1% (Advances inthe Laboratory Diagnosis of Autoimmune Diseases, Business Briefing Global Health Care2002, Issue 3.), the diagnosis of these diseases is except different separately clinical symptoms, and the detection of antibodies among the patients serum is the critical index of diagnosis.
Because of the titre of many antibody and generation, development and the prognosis of disease have very big relevance, (Liver Cytosol Antigen type 1 Autoantibodies, LC-1) titre is obviously relevant with disease activity degree, serum transaminase and gamma globulin level as anti-liver cytoplasm I type antigen-antibody.Anti--LC1 that height is tired only detects (Lenzi M in chronic hepatitis that high mobility is arranged or cirrhosis, Mantotti P, Muratori L, Cataleta M, et al.Liver cytosolic 1antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C Virusinfection.Gut, 1995,36:749-754.).Anti--LC1 does not have prediction significance to the curative effect of corticosteroid and imuran, and LC-1 is reflection disease process and result of treatment one a sero-immunity index preferably.Therefore to the qualitative of autoantibody and further quantitatively most important.The detection of antagonist, except clearly existing, also tackle its class and subclass and identify, in general, the IgM class mainly is common in acute infectious diseases, the IgG4 subclass is mainly seen in the organ specificity autoimmunity disease, and IgG1, IgG3 is mainly seen in the non-specific autoimmunity disease of organ (Deng Anmei, Zhong Renqian, but Kong Xiantao. the relation of autoimmunity patient's extracting antinuclear antibodies spectrum and humoral immunity. Chinese journal of medical examination, 1999,22:299-300.).
The detection kit generally all comprises standard items or feminine gender and positive reference substance, and quantitatively the standard items of autoantibody diagnostic kit often come from the serial dilution product of antibody titer high positive serum, or directly use the patients serum of different titers.But make standard items with the patients serum a lot of shortcomings are arranged: 1) be difficult to obtain to have high titre, the patients serum of high specific; 2) antibody titer between the individuality and the poor specificity opposite sex are bigger, are difficult to standardization; 3) because how anti-serum be, have different antibody type, compatibility and specificity, limited its application; 4) cost an arm and a leg,, may not guarantee the output of standard items owing to the restriction of resource.Therefore, the method for Development and Production standard items, significant in order to the level of detection specificity antibody.
Human mouse chimeric antibody can be used for the standard items of human serum antibody test, be used for preparation standard curve (United States Patent (USP): 6,015,662), but this method has significant limitation: 1) technology is very complicated, comprise animal immune, Fusion of Cells, the screening of monoclonal antibody, antibody cloning, the structure of people mouse mosaic gene engineered antibody and expression etc., the time is long, the expense height; 2) each antibody that makes up includes only a kind of constant region of antibody, and detecting other antibody types need rebuild.
Cross-linking antibody has very big advantage at user's mask of standard items, inhuman non-IgM and the crosslinked (United States Patent (USP): 5 of people's IgM, 478,753), as diagnosis IgM serum reagent box standard items, the inhuman non-IgM of cross-linking antibody is to detecting antigen-specific, and people's IgM can close with mark two resistive connections, these kits have well solved the difficulty of communicable disease in acute infection Phase I gM positive serum source, make the large-scale production of standard items become possibility.Other crosslinked also comprise heavy chain of antibody and light chain by disulfide bond and IgG heavy chain and light chain or Fc crosslinked (United States Patent (USP): 5,523,210), the bivalent antibody of structure can be used for chemical analyses such as enzyme; Two antibody to different antigen-specifics are crosslinked by crosslinking chemical, and (United States Patent (USP): 4,433,059), the allos cross-linking antibody can be used for the aggegation analysis.
Summary of the invention
One object of the present invention is to provide a kind of antigen-specific antibodies of used as standard diagnosing reagent replacing positive serum and the cross-linked composite of human immunoglobulin(HIg), and it can solve a certain antigentic specificity in conjunction with reaching and the reaction of (anti-people two resists) of anti-human immunoglobulin(HIg) antibody.
Another object of the present invention is to provide a kind of cross-linked composite, the antibody technique that wherein prepares specific antibody can be a monoclonal antibody technique (hybridoma technology, comprising mouse, rat and rabbit hybridoma etc.) technology is the antigen-specific antibodies of main preparation, the antiserum that also can use antigen-immunized animal (as rabbit, sheep, horse, ox etc.) to obtain, the antigentic specificity polyclonal antibody for preparing through the antigentic specificity affinity chromatography again.
Another object of the present invention is to provide a kind of cross-linked composite, when wherein antibody complex is used for the level of the specific antibody that human body produces at a certain antigen, can be used as that standard items substitute the serum standard panel that derives from human body or with reference to product.
Another object of the present invention is to provide a kind of cross-linked composite, utilize the method for its human body antigen-specific antibodies level can be used for medical diagnosis on disease, disorder in screening, as infectious diseases (anti-hepatitis B antibody, anti-c-hepatitis antibody, anti-AIDS antibody etc.), autoimmune disease (autoantibodies such as rheumatoid factor, antinuclear antibodies), anaphylactia (serum IgE level etc.) can also be used for blood group detection, tissue matching and health examination etc.
A further object of the present invention provides a kind of antigen-specific antibodies and the human immunoglobulin(HIg) cross-linked composite method as standard items, use the standard items of this method preparation, antibody specificity and affinity homogeneous, the preparation method is simple, it is unrestricted to originate, can large-scale production.
In order to reach above purpose, the present invention has disclosed a kind of compound that can used as standard diagnosing reagent replacing positive serum, it is characterized in that this compound is the cross-linked composite of antigen-specific antibodies and human immunoglobulin(HIg), antigen-specific antibodies is the Antiserum Preparation that obtains by monoclonal antibody technique or by antigen-immunized animal.
In order to reach above purpose, the present invention has also disclosed a kind of method, and this method comprises the steps: at least
A. use antigen-immunized animal, the specific antibody of acquisition;
B. with crosslinking chemical that described specific antibody and described human immunoglobulin(HIg) is crosslinked;
C. with described antibody gradient dilution after crosslinked, drawing standard curve; And,
D. described described antibody after crosslinked is applied in the quantitative Diagnosis detection method as standard items.
Description of drawings
Fig. 1 is the typical curve of preferred embodiment of the present invention.And,
Fig. 2 is the statistics figure of preferred embodiment of the present invention.
Embodiment
The present invention includes a kind of method, this method comprises the steps: at least
(a). use antigen-immunized animal, the specific antibody of acquisition;
(b). with crosslinking chemical that described specific antibody and described human immunoglobulin(HIg) is crosslinked;
(c). with described antibody gradient dilution after crosslinked, drawing standard curve; And,
(d). described described antibody after crosslinked is applied in the quantitative Diagnosis detection method as standard items.
A kind of preferred embodiment of the present invention is to make to be used for anti-cyclic citrullinated peptide (CCP) antibody class rheumathritis diagnosis kits.This kit is the enzyme-linked immunologic detecting kit that is used for the anti-CCP IgG of quantitative measurement patients serum type antibody.Anti-CCP IgG type detection of antibodies can the auxiliary diagnosis rheumatoid arthritis.Bag is by CCP antigen on 96 hole reaction plates, serum to be checked after the dilution and reference substance positive serum are added in the reaction plate hole, if there is the antibody of anti-CCP antigenic component in the tested serum, behind incubation, then the specific antibody in the serum combines with solid phase CCP antigen, forms the solid phase antigen antibody complex.The unconjugated antibody component of flush away adds the anti-human IgG antibody of enzyme labeling (anti-people two anti-) incubation, the solid phase antigen antibody complex again with the anti-IgG antibodies of enzyme labeling.The unconjugated enzyme labelled antibody composition of flush away adds the substrate of enzyme again.Substrate is become coloured product by enzymatic, can detect the numerical value of the color reaction that reads sample and reference substance positive serum by microplate reader.Positive product control serum with different titers, make a microplate reader and detect the typical curve that is mutually related between color reaction numerical value and the anti-CCP antibody titer of reference substance positive serum, detect the position of numerical value on typical curve according to serum microplate reader to be checked, just can judge corresponding anti-CCP antibody horizontal in the serum to be checked.
As the standard items positive serum of drawing standard curve, be the composition of kit most critical in this diagnostic kit.The standard items positive serum is taken from the patient that anti-CCP antibody horizontal increases at present, has a lot of problems like this.The one, positive serum is to take from existing diagnostic kit to detect the patient that anti-CCP antibody horizontal increases, and existing diagnostic kit positive serum is taken from the patient who meets the rheumatoid disease diagnostic criteria, increases and meet the anti-CCP antibody of may not all having of rheumatoid disease diagnostic criteria.The 2nd, the anti-CCP antibody horizontal of patient's positive serum can change with change of illness state, and therefore " standard items " are also nonstandard.The 3rd, each patients serum originates limited, possible each lot number serum source all inequality, variant between standard items are criticized, therefore to same patient's same a serum, have different results with the diagnostic kit detection of different batches.The cross-linking agent replacing positive serum that utilizes anti-CCP monoclonal antibody and human IgG then can not produce aforesaid shortcoming as the standard items of kit.
In step (a), CCP and bovine serum albumin(BSA) (BSA) cross-linking method is breathed out the Lip river with reference to E., and D. Lay grace is shown " antibody technique experiment guide ".Select Balb/c mouse in age in 6-8 week for use, get the CCP of 1-100 μ g after crosslinked and inject with the fully emulsified pneumoretroperitoneum of the complete freund adjuvant of equivalent.Add the fully emulsified pneumoretroperitoneum injection of the incomplete freund adjuvant of equivalent, 3-5 time altogether every 2 weeks with same doses of antigen later on.Merge preceding 3 days abdominal cavities or intravenous injection and do not have adjuvant antigen 50-100 μ g booster immunization once.
Get and finish mice immunized, pluck eyeball and get blood, separation of serum is standby.Draw neck to put to death mouse, be soaked in 75% the alcohol 3-5 minute.Spleen is taken out in sterile working, the preparation splenocyte suspension.Get the P3-653 cell that is in exponential phase and mouse boosting cell in 1: 5-1: 10 ratio is mixed, and adds 20ml RPMI-1640 liquid, 1000r/min, and 5 minutes are centrifugal, abandon supernatant.Beat the centrifuge tube bottom gently, sedimentation cell is disperseed, centrifuge tube is put in 37 ℃ of water-baths, the 50%PEG that gets 37 ℃ of pre-temperature of 1ml slowly splashes in the centrifuge tube, adds in 1 minute.37 ℃ left standstill 2 minutes, and Dropwise 5 0mLRPMI-1640 liquid stops the PEG effect in 5 minutes.800r/ minute, 8 minutes centrifugal, abandons supernatant.Sedimentation cell is suspended in gently in the HAT nutrient culture media of required volume, is inoculated in 96 well culture plates.Culture plate is put into 37 ℃ of 5%CO 2Cultivate in the incubator.After 7-10 days, change the HT nutrient culture media.Change behind the liquid and to draw culture supernatant in three days, be ELISA and detect.Choose positive porocyte, with limiting dilution assay cell is spread monoclonal, promptly do the cell counting, the concentration that makes cell is 50/ml, in 96 orifice plates inoculation, three rows, does doubling dilution with nutrient solution, is paved with 1 96 orifice plate.Cultivate after 7-10 days, choose single clone and do the ELISA detection, choose positive porocyte, clone again.Generally need clone 3-5 time repeatedly, through 100% positive hole can be with the cell enlarged culture, and is frozen.
The hybridoma that screening is finished is made suspension cultured with serum free medium, collect culture supernatant in order to being further purified.
Do affinity chromatography, antibody purification with ProteinG or ProteinA Sepharose.With reference to (E. breathes out the Lip river, D. Lay grace. and " antibody technique experiment guide ").Carry out antibody screening at last.
Step of the present invention (b) is the crosslinked of mouse-anti CCP monoclonal antibody and human immunoglobulin(HIg) (IgG).Taking by weighing human IgG 25mg is dissolved among the pH 7.2 0.1mol/L PBS that 0.5ml contains 1.25% glutaraldehyde, in 2-8 ℃ of standing over night.Reacted solution is fully dialysis in pH 7.2 0.1mol/L PBS, removes unnecessary glutaraldehyde, adds pH 7.2 0.1mol/L PBS to 1.5ml.Place in the 25ml small beaker, slowly stir.Mouse-anti CCP antibody 12.5mg is diluted to 0.5ml with pH 7.2 0.1mol/L PBS, dropwise joins in the small beaker under stirring.Add in 1mol/L PH 9.6 carbonate buffer solutions, continue to stir 3-4 hour.The lysine solution that adds 0.25mL 0.2mol/L at last, room temperature were put 2 hours.Reacted solution, is collected first and is gone out the peak with PH7.2 0.1mol/L PBS wash-out through the SephacrylS-300HR chromatographic column, and this solution is the cross-linking agent of mouse-anti CCP antibody and human immunoglobulin(HIg) (IgG) just.
With containing 1.59g/L Na 2CO 3, 2.93g/L NaHCO 3Bag to be cushioned liquid (CBS solution) be 5 μ g/ml with polyclonal anti-human IgG dilution.To dilute good solution then and add in each hole of ELISA Plate every hole 100 μ l; 2-8 ℃ is spent the night, and takes out ELISA Plate afterwards, gets rid of coating buffer, washes plate three times, pats dry, and adds and contains 1% bovine serum albumin(BSA), 8.7g/L NaCl, 1.15g/L Na 2HPO 412H 2O, 0.2g/L NaH 2PO 42H 2O, the confining liquid of PH 7.4, every hole 200 μ l; Place 37 ℃ following 2 hours, take out ELISA Plate afterwards, discard confining liquid, wash plate three times, pat dry.CCP monoclonal antibody after crosslinked is carried out the two-fold dilution, and dilution ratio can also be 1: 100,1: 200, and until 1: 1000.Monoclonal antibody after the dilution is added to bag by in each hole in the plate, every hole 100 μ l, the dilutability of each the crosslinked monoclonal antibody hole of writing in reply, room temperature (18-25 ℃) reaction 30 minutes.After reaction finished, bag was washed 3 times with washing lotion by plate, pats dry.The goat anti-human igg's working fluid 100 μ l that add the HRP mark in each reacting hole, room temperature (18-25 ℃) reaction 30 minutes.After reaction finished, bag was washed 5 times with washing lotion by plate, pats dry.Every hole adds colour developing liquid A and (contains citric acid 35.8g/L, Na 2HPO 412H 2O 9.34g/L, 30% hydrogen peroxide, 660 μ l) and each 50 μ l of colour developing liquid B (containing 3,3,5,5 ,-tetramethyl biphenyl ammonia 0.2g/L, dimethyl sulfoxide (DMSO) 5ml/L, 6N HCl 1ml/L), mixing, room temperature lucifuge reaction 30 minutes.Every hole, reaction back adds stop buffer (2N H 2SO 4) 50 μ l, microplate reader OD 450Reading.
Because of the world of nonreactive CCP antibody with reference to Quality Control, press the OD of antibody 450The relative unit of self-defined its antibody of value, OD 450During=2.0-2.2, its corresponding dilutability is 1: 400, and the setting unit is 400RU.Other dilutabilitys by that analogy, as 1: 800 be 200RU, be 100RU etc. at 1: 1600.
Step of the present invention (c) is the different dilutability drawing standard curves with the CCP monoclonal antibody after crosslinked.
Being cushioned liquid with bag is 5 μ g/ml with the CCP dilution.Then CCP solution is added in each hole of ELISA Plate every hole 100 μ 1; Place 2-8 ℃ following 17 hours, take out ELISA Plate afterwards, get rid of coating buffer, wash plate three times, pat dry, add confining liquid, every hole 200 μ l; Place 37 ℃ following 2 hours, take out ELISA Plate afterwards, discard confining liquid, wash plate three times, pat dry; The ELISA Plate lath is placed on carries out in the vacuum drying chamber that vacuum is drained and in vacuum tank, preserved 1 hour; The aluminium foil bag of at last ELIAS strip being packed into, vacuum sealer seals, and labels and adds a cover lot number.
CCP monoclonal antibody after crosslinked is carried out the two-fold dilution, and initial concentration unit is 400RU, totally 9 dilutabilitys.Dilution is by 1% bovine serum albumin(BSA), 8.7g/L NaCl, 1.15g/L Na 2HPO 412H 2O, 0.2g/L NaH 2PO 42H 2O, Tween-20 0.5ml, PH 7.4 forms.
Control serum is the anti-CCP antibody positive serum that comprehensive research and development and clinical effectiveness are determined, weak positive serum (near the serum the critical value) and negative serum.Monoclonal antibody after the dilution and control serum (1: 100 dilution) are added to the CCP bag by in each hole in the plate, every hole 100 μ l, the dilutability of each crosslinked monoclonal antibody and the control serum hole of writing in reply, room temperature (18-25 ℃) reaction 30 minutes.After reaction finished, bag was washed 3 times with washing lotion by plate, pats dry.The goat anti-human igg's working fluid 100 μ l that add the HRP mark in each reacting hole, room temperature (18-25 ℃) reaction 30 minutes.After reaction finished, bag was washed 5 times with washing lotion by plate, pats dry.Every hole adds colour developing liquid A and each 50 μ l of colour developing liquid B, mixing, room temperature (18-25 ℃) lucifuge reaction 30 minutes.Every hole, reaction back adds stop buffer 50 μ l, microplate reader OD 450Reading.
Calculate the OD of the different dilutability replicate determinations of the CCP monoclonal antibody after crosslinked 450Mean value is with each dilution OD 450Value is ordinate, and each self-corresponding concentration is horizontal ordinate, draws out typical curve.
The typical curve of this preferred embodiment as shown in Figure 1.From the whole trend of curve, linearity range is 0-100RU, and more than the 100RU, curve has trended towards saturated.In linearity range, get 5 points (100RU, 50RU, 25RU, 12.5RU, 0), with this typical curve as kit.Calculate the OD of three parts of serum sample replicate determinations 450Mean value is read corresponding unit then on typical curve.The unit of positive serum correspondence is that the unit of 65RU, weak positive serum correspondence is 18RU, and the unit of negative serum correspondence is 2RU.Find out from data, three reference substance serum drop in the range of linearity entirely, especially weak positive serum (critical value) drops on the middle part of line segment, illustrates that this line segment can cover the sensing range of whole yin and yang attribute, further specifies the quantitative curve that this typical curve can be used as this kit.The critical value that can make kit from the unit of weak positive serum correspondence is 18RU, and is promptly positive more than or equal to 18RU, negative less than 18RU.
Step of the present invention (d) is that typical curve is applied in the anti-CCP antibody assay kit.Kit 200 portions of normal human serums of test and 200 parts of RA patients serums make above typical curve simultaneously at 5, and operating process is the same.Calculate the OD of sample replicate determination 450Mean value is read corresponding unit then on typical curve.With 18RU is critical value, and statistics as shown in Figure 2.
In this statistics, the true positives sample refers to that 200 parts of rheumatoid arthritis patients serum samples are positive samples with the test of this kit, and the false negative sample refers to that 200 parts of rheumatoid arthritis patients serum samples are negative samples with the test of this kit.The true negative sample refers to that 200 parts of normal human serum samples are negative samples with this product test, and the false positive sample refers to that 200 parts of normal human serum samples are positive samples with this product test.

Claims (17)

  1. One kind can used as standard diagnosing reagent replacing positive serum compound, it is characterized in that:
    Described compound is the cross-linked composite of antigen-specific antibodies and human immunoglobulin(HIg).
  2. 2. compound according to claim 1 is characterized in that antigen-specific antibodies prepares by monoclonal antibody technique.
  3. 3. compound according to claim 1 is characterized in that, antigen-specific antibodies is the Antiserum Preparation that obtains by antigen-immunized animal.
  4. 4. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is human immunoglobulin(HIg) IgG.
  5. 5. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is human immunoglobulin(HIg) IgM.
  6. 6. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is human immunoglobulin(HIg) IgE.
  7. 7. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is human immunoglobulin(HIg) IgA.
  8. 8. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is the total length of human immunoglobulin(HIg).
  9. 9. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is the constant region of human immunoglobulin(HIg).
  10. 10. compound according to claim 1 is characterized in that, described human immunoglobulin(HIg) is the Fc fragment of human immunoglobulin(HIg).
  11. 11. the using method of a compound as claimed in claim 1 is characterized in that this method may further comprise the steps:
    A. use antigen-immunized animal, the specific antibody of acquisition;
    B. with crosslinking chemical that described specific antibody and described human immunoglobulin(HIg) is crosslinked;
    C. with described antibody gradient dilution after crosslinked, drawing standard curve; And,
    D. described described antibody after crosslinked is applied in the quantitative Diagnosis detection method as standard items.
  12. 12. method according to claim 11 is characterized in that, described detection method is a kit.
  13. 13. method according to claim 11 is characterized in that, described detection method is a detectable.
  14. 14. method according to claim 11 is characterized in that, described detection method is an instrument detecting.
  15. 15. method according to claim 11 is characterized in that, the described antigen-specific antibodies of step a is a monoclonal antibody.
  16. 16. method according to claim 11 is characterized in that, the described antigen-specific antibodies of step a is a polyclonal antibody.
  17. 17. method according to claim 11 is characterized in that, the described crosslinking chemical of step b is glutaraldehyde and sodium periodate.
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