CN101726588B - Composition for externally detecting rheumatoid arthritis antibody and application thereof - Google Patents

Abstract

The invention discloses an antigenic composition which comprises a polypeptide I and a polypeptide II, wherein the molar ratio of the polypeptide I to the sum of the polypeptide I and the polypeptide II is 0-1. An annular polypeptide is generated in a mode of forming a disulfide bond from two nonadjacent cysteine side chain hydrosulphonyls on a sequence. The polypeptide not only can be combined with a rheumatoid arthritis autoimmune antibody, but also can simultaneously have high affinity for HLA-DR. Through experimental verification, the specificity of the polypeptide combined with the rheumatoid arthritis autoimmune antibody is larger than 95 percent, and the detection sensitivity for the rheumatoid arthritis autoimmune antibody is larger than 75 percent. Compared with like products sold on the market, the sensitivity of the composition is markedly improved, thereby being more beneficial to externally detecting the rheumatoid arthritis autoimmune antibody.

Description

The composition of externally detecting rheumatoid arthritis antibody and application thereof

Technical field

The present invention relates to detect the antigen of autoimmune antibody, relate in particular to the antigen that detects rheumatoid arthritis autoimmunity antibody, relate to more specifically a kind of antigen composition that detects rheumatoid arthritis autoimmunity antibody.

Background technology

Autoimmune disease mainly contains systemic loupus erythematosus, rheumatoid arthritis, Sjogren syndrome, dermatomyositis, polymyositis, system (multiple) property sclerosis, chorionitis etc. clinically, these diseases once were named as " connective tissue disease (CTD) ", all they were classified as rheumatic disease with domestic abroad afterwards.

Rheumatoid arthritis (rheumatoid arthritis, RA) be a kind of more common systemic autoimmune disease take chronic polyarthritis disease as main manifestations, how repeatedly its course of disease is long,, and be irreversible to tissue damage, cause very large misery to the patient; This sick incidence of disease worldwide is 0.5-1%, and the incidence of disease of China is about 0.36%.Patient's age of onset is many, and the women was more than the male sex at 20～50 years old, and men and women's ratio is 1: 3.Epidemiology statistics has recently increases trend year by year, its outstanding early clinical manifestation is symmetry redness and swelling of joints heat pain, the little joint of common four limbs, near-end arthroncus between finger, painful swelling of joints and the movable difficulties such as the palm refers to, wrist, elbow, ankle, the daystart anchylosis alleviates in the afternoon gradually, and symptom has 20% patient subcutaneous nodule can occur approximately outside the joint; The permanent symptom in late period that does not heal then has in various degree joint deformity and tetanic, and function of joint forfeiture etc. can damage internal organs, many histoorgans, cause the bone ischemic necrosis, and are large to human consumption, and disability rate is high.

Because the rheumatoid arthritis cause of disease is still not fully aware of so far, and early clinical manifestation is not true to type yet.At home and abroad in the clinical practice, the diagnostic tool commonly used of this disease is (the American College of Rheumatology of U.S. rheumatism association, ACR) rheumatoid disease criteria for classification (the Arthritis Rheum1988 that formulated in 1987,31,315-24), this standard more depends on the clinical disease that rheumatoid arthritis shows.Early stage in this disease, these clinical indices are normally not easy-operating.Therefore, generally believe that at present this standard can not better be adapted to early stage Diagnosis of Rheumatoid Arthritis (Arthritis Rheum2001,44,2485-91; Neth J Med 2002,60,383-8).And the method for the treatment rheumatoid arthritis that uses at present mainly is to adopt the anti-inflammatory method, though this method can so that the degree of arthroncus (swelling) and erosion (erosive) is slowed down, but can't make disease obtain curing.The common viewpoint of current people is, this disease adopt in early days the several different methods treatment can obtain best result for the treatment of (Autoimmunity Reviews2006,6,37-41).Thus, serology certification mark with high selectivity (Specificity) and high sensitivity (Sensitivity) is particularly necessary (the ClinApplied Immunol Rev2004 that just seems of the early diagnosis before the bone joint is damaged for rheumatoid arthritis, 4,239-62).

Although research finds that some autoimmune antibodies interrelate with the rheumatoid arthritis disease, as: anti-calpastatin antibody (Proc Natl Acad Sci USA1995,92,7267-71), anti-neutrophil leucocyte cytoplasmic antibody (anti-neutrophil cytoplasmic antibodies, ANCA) (Int ArchAllergy Immunol1996; 109:201-6), nuclear antigen antibody (anti-nuclear antigens, ANA) (Scand J Rheumatol1986,15,185-92), anti-II Collagen Type VI antibody (anti-collagentype II) (J Immunol Methods2001,247,191-203) with anti-glucose 6 phosphoric acid isomerases (anti-glucose-6-phosphate isomerase, anti-GPI) (Nat Immunol2001,2,746-53) etc. also can both be in suffering from the patient of other autoimmune disease, in healthy individual, detect even (Clin Applied Immunol Rev2004,4,239-62).Although some antibody wherein can be applied to the identification of rheumatic disease, and screen these antibody and also help disease surveillance and prediction, but because it lacks selectivity to rheumatoid arthritis, the overwhelming majority in these antibody seldom is applied to rheumatoid arthritis early diagnosis (Clinica Chimica Acta2004,350,17-34).

Ideal mark (Marker) for the rheumatoid arthritis early diagnosis should satisfy 4 standards at least: (1) high sensitivity, and the patient that can detect reaches high number percent; (2) high selectivity, the as much as possible generation of limit erroneous positive findings; (3) can be applicable to early diagnosis; (4) some patient can be predicted and aggressivity disease (erosive disease) (AutoimmunityReviews2006,6,37-41 can be developed into; MEDICINE2006,34,441-4).

Rheumatoid factor (rheumatoid factor, RF) antibody is that the serology that is applied in the ACR standard detects, and is acknowledged as the direct antibody of Fc domain on the IgG molecule.But its selectivity for rheumatoid arthritis is also more general, is 60%, and the range of sensitivity can reach 60-80%.RF can also be in other suffers from the patient of autoimmune disease, suffer among the patient of infectious diseases and in 3-5% healthy population (wherein 10-30% the elderly) detect (Ann Rheum Dis2003,62,261-3).

Anti-BiP (p68), anti-Sa and antioxidant cyclic guanidine propylhomoserin protein antibodies (APF, AKA, anti-filaggrin, anti-CCP) then have better selectivity to rheumatoid arthritis.64% Patients With Rheumatoid Arthritis can produce direct antibody for BiP, report that in addition it has the selectivity of height to this disease, there is not at present Data support BiP having effect aspect the prediction rheumatoid arthritis, and the BiP antibody of these reports then needs products for further to confirm (Clinica ChimicaActa2004 by clinical research independently, 350,17-34).Other has research to disclose Sa antigen can be up to 92-99% for the selectivity of rheumatoid arthritis, but its sensitivity then seems generally, only is 30-40% (J Rheumatol1994,21,1027-33; J Rheumatol1999; 26:7-13).

Antiperinuclear factor (antiperinuclear factor, APF) is disclosed in 1964 first.These autoimmune antibodies have selectivity to rheumatoid arthritis after deliberation, and can indirectly record by the immunofluorescence technique take human cheek mucosa's cell as the antigen substrate (Ann Rheum Dis1964; 23,302-5).

The researcher had described a kind of rheumatoid arthritis selectivity keratoprotein antibody in 1979, it can directly suppress cuticula epithelial cell keratinization, and be anti-keratoprotein antibody (anti-keratin andtibody with the title of this antibody-like is tentative, AKA) (Young BJ et al BMJ1979,2,97-9).Studies confirm that afterwards, although the discovery of AKA and APF is mutually independently, both directly corresponding antigen be consistent (Sebbag M et al J.Clin.Invest1995,95,2672-9).Many researchs show that further AKA and APF have many common characteristics.Filaggrin (filament aggregating protein) is a kind of mutually crosslinked thread body of keratin, effect is to form very firm cytoskeletal structure, it also is the public antigen (ClinApplied Immunol Rev2004,4,239-62) of AKA and APF.

Although AKA and APF have selectivity for rheumatoid arthritis, its defective is also very outstanding.The detection sensitivity of these two kinds of antibody depends critically upon the purification process of filaggrin.In the practice, owing to be difficult to separate and obtain pure and guanidine radicals content has repeatable antigen, it is not easy to add its detection means, and fluorescence immunoassay need to expend larger workload, thereby so that be difficult to form standardization between the laboratory, cause AKA and APF not to become the main stream approach (ClinApplied Immunol Rev2004,4,239-62) that rheumatoid arthritis detects.

Be the knowledge of AKA and APF antibody target spot based on filaggrin and profilaggrin, people have synthesized the polypeptide that contains citrulline to detect the activity of AKA and APF antibody in the rheumatoid arthritis serum.Because citrulline is not primary amino acid and can't adding in protein translation, so usual way is to take off imines enzyme (peptidylargnine deiminine, PAD, EC3.5.3.15) catalysis arginine residues deaminizating by peptide arginine to obtain.According to these facts, the arginine side chain that some are derived from the filaggrin sequence is modified, and the polypeptide that especially contains citrulline is synthesized out, and is used for the vitro detection (US6,858,438) of rheumatoid arthritis antibody.Using Enzyme-linked Immunosorbent Assay (Enzyme-Linked immunosorbent Assay, ELISA) in the method test experience, this method is synthetic contains citrulline rectilinearity polypeptide can detect anti-citrulline peptide antibody from rheumatoid arthritis patients serum, in the situation that keeps high selectivity (greater than 90%), greatly improve the sensitivity (reaching 63%) that detects.Experiment is proof also, and the polypeptide that only contains citrulline could be realized this reaction, if the citrulline in the polypeptide is replaced then fully not reaction generation with other amino acid.These results show, citrulline partly be determine can by the antigenic determining factor of AKA and APF antibody recognition (J Clin Invest1998,101,273-81).

Other researcher finds to contain the rectilinearity polypeptide of citrulline by the mode energy analogy β-corner structure of disulfide bond (S-S key) formation loop configuration, thereby imitate β-corner structure of initial antigenic determinant, and can increase affinity (the FASEB J1995 of polypeptide-antibody, 9,37-42; Mol Immunol1985,22,1255-64).Thus, a kind of cyclic peptide, i.e. first generation cyclic citrullinated peptide (the first generation cyclic citrullinated peptide, CCP1) (the Schellekens GA et al Arthritis Rheum2000 that manually is synthesized, 43,155-63).The researcher replaces with halfcystine with two serines in the citrulline peptide that is comprised of 19 amino acid residues and forms the disulfide bond that has analog structure with β-corner, is manually synthesized CCP, and the testing result of CCP1 and straight line peptide is contrasted.The result shows that employing CCP1 is that the polypeptide of antigen uses the ELISA method to detect RA patient's anti-CCP antibody, and sensitivity is that antigen is significantly improved with rectilinearity citrulline peptide, is respectively 68% and 49%, and the two selectivity is similar.

In containing the reaction pattern of different citrulline polypeptides, the rheumatoid arthritis serum detected representation goes out huge changeability.Outside the pale of civilization by citrulline except the filaggrin arginine residues in the serum/plasma of rheumatoid arthritis patients, citrulline also appears in Sa antigen, collagen (I and II type), histone, myelin basic protein, fibronectin etc., and produces anti-CCP antibody.Further investigation shows, is not that all arginine deaminizatings are converted into citrulline in the autoantigen of citrulline; The citrulline of citrulline also not exclusively participates in forming epitope, namely produces anti-CCP antibody.To sum up, generation and the citrulline of anti-citrulline antibody are closely related, and the generation of the flanking sequence of citrulline antagonism citrulline antibody plays an important role, this fact is hinting that all skirt amino acids of citrulline residue have vital role for epitope (antigen episode), and anti-citrulline albumen (as: AKA and APF antibody) activity is polyclone reaction (J Clin Invest1998,101,273-81).Based on the attribute of filaggrin, it is considered to simulate the natural stock (ClinApplied Immunol Rev2004,4,239-62) of citrulline epitope.

Second generation CCP (CCP2) detection method results from 2002, obtain the second generation CCP irrelevant with filaggrin by peptide library and the rheumatoid arthritis serum screening that will contain citrulline, it has epi-position (Report on the5th Dresden symposium onautoantibodies.Lengerich, the Germany:Pabst Science Publishers that more is conducive to antibody test; 2000.p.140-5).CCP1 and CCP2 are shown that CCP2 has not only kept higher selectivity (96%) after same group of patient's test, and its sensitivity for analysis also increase significantly (Ann.Rheum.Dis.2005,64,1510-2).Many researchers studies have shown that recent five years the anti-sensitivity that contains the test of citrulline polypeptide has higher changeability, and scope is at 40%-94% (Clin.ExpRheumatol.2005,23 (Supp139), 569-76; Ann.Rheum.Dis.2006,65,845-51).CCP2 is applied in the rheumatoid arthritis autoimmunity antibody vitro detection and has illustrated that the screening at the detectable antigens in this field does not need only to rely on filaggrin, and also the explanation associative list potential energy different from filaggrin more is conducive to vitro detection.

People (the Clin Chem2007 such as Bizzaro N, 53,1527-33) and people (the ClinicaChimica Acta2007 such as Lutteri L, 386,76-81) compare for the second generation of using on the present market and third generation CCP kit respectively, found that, in three kinds of detection methods of her Nova diagnosis (Inova Diagnostics) company exploitation, its CCP3 kit with use CCP2 only to compare as the method for antigen slightly to be improved, sensitivity separately is respectively 67% and 64%.On the contrary, the CCP3.0 that is used for anti-IgG compares rear discovery with the detection method that can detect IgG and can detect again the CCP3.1 of IgA, and both do not have a bit difference unexpectedly.As seen, the method that detects simultaneously IgG and IgA antibody does not resemble has remarkable improvement looking.Their conclusion is because the diagnostic kit sensitivity of test may be relevant with position and the quantity of guanidinated arginine residues, selectivity then may be subject to the impact of protein or assorted peptide sequence, therefore, for this two, the kind of antigen seems even more important.Both consider, and experimental data shows that the preparation of antigen is the most important variable factor that determines the assay method quality.

The people such as Lutteri L (Clinica Chimica Acta2007,386, another value of anti-CCP has also been found in research 76-81), they point out that IgM-RF is the sensitiveest mark, can find in 77.9% rheumatoid arthritis patients.Should be noted that the RF in the ACR standard, its Diagnosis of Rheumatoid Arthritis medium sensitivity can not directly be compared with those other methods that are not included in the diagnostic criteria.IgM-RF is considered to slightly be weak aspect the disease selectivity.Use IgM-RF to detect rheumatoid arthritis patients, wherein have 13-19% people negative for RF, by comparison, all positive when these patients use anti-CCP method to detect.

The inherent cause of rheumatoid arthritis can affect appearance and the generation of citrulline albumen, also can affect the antibody product (Clinica Chimica Acta2004,350,17-34) for these modified proteins.A series of correlative studys show that rheumatoid arthritis and some HLA-DR allele have close ties, especially HLA-DRB1 ^*0401 and HLA-DRB1 ^*0404 (Cell1996,85,307-10).Among the crowd of these different regions, these allele are also slightly different.DRB1 ^*0401 mainly appears among the crowd of Northern Europe and north America region, evidence suggests that " especially big joint " that this gene and rheumatoid arthritis cause characterizes (extraarticular manifestations) and be related, at DRB1 ^*0401/DRB1 ^*Particularly outstanding in the 0404 heterozygous genes type.DRB1 ^*0403, DRB1 ^*0406 and DRB1 ^*0407 and DRB1 ^*0401, ^*0404 or ^*The development that can increase the rheumatoid arthritis state of an illness after 0101 assortment of genes may.DRB1 ^*0404 and DRB1 ^*As long as 0408 is present among the white race.DRB1 ^*0405 seldom in the crowd of Northern Europe, but in the Asia with go back among the crowd of Mediterranean country extremely general.DRB1 ^*0101 mainly is present among Northern Europe and the North America crowd.Then be DRB1 among the American Indian group ^*1402 Hes ^*1406 (Hum Immuno2000,61,1254-1261).Although these genes relevant with rheumatoid arthritis are had nothing in common with each other in the crowd of each department, but research finds to have shared epi-position (sharedepitope in the third high degree variable region (HV3) of DRB1, SE), all be QKRAA, QRRAA or RRRAA (Clinica Chimica Acta2004 at 70-74 amino acids residues, 350,17-34).

There are the genotypic rheumatoid arthritis patients of several shared epi-positions and the patient's comparative studies with anti-CCP2 antibody to show a group, the generation of a kind of shared epi-position allele and anti-CCP2 antibody has close inner link (Arthritis Rheum2000,50,2113-21; Arthritis ResTher2004,6, R303-8).Verified, this association is because the MHC molecule is shared 70-74 amino acids residue (Q/R, K/R of epi-position β chain, R, A, A) formation the 4th grappling bag (P4), itself and electric neutrality or negative charge amino acid have high affinity (J Immuno2003,171,538-541).When positively charged arginine after converting electroneutral citrulline under the effect of PAD enzyme, the posttranslational modification protein/polypeptide affinity that obtains is far longer than the affinity of arginine and P4, and can activate CD4 ⁺The T cell, these " the special T cells of citrulline " can help generation (J Immuno2003,171,538-541 of anti-citrulline albumen (polypeptide) antibody; Arthritis Rheum1987,30,1205-13; Rheum Dis Clin North Am1992,18,741-59; ArthritisRheum2005,52,1063-68).These results show that the DBR1 allele with shared epi-position can activate the rheumatoid arthritis patients autoimmune response.

The rheumatoid arthritis detection method is used single polypeptide antigen usually at present.Although annular or the linear pattern polypeptide of that these derive from filaggrin or artificial design all show higher selectivity (〉 90% rheumatoid arthritis detects), but because there is numerous HLA-DR allele in rheumatoid arthritis patients, consequent antibody is difference to some extent correspondingly also, and this is so that detection sensitivity then can not get obvious raising (＜70%) all the time.

Summary of the invention

One object of the present invention is to provide a kind of antigen composition that detects rheumatoid arthritis autoimmunity antibody.

Another object of the present invention is to provide a kind of rheumatoid arthritis autoimmunity antibody to detect reagent composition.

Another purpose of the present invention is to provide a kind of external detection method of rheumatoid arthritis autoimmunity antibody.

A further object of the present invention is to provide a kind of ELISA external detection method of rheumatoid arthritis autoimmunity antibody.

Antigen composition of the present invention (polypeptide) includes following amino acid sequence:

His—Gln—Cys—Xaa1—Xaa2—Phe—Xaa3—Xaa4—Xaa5—Xaa6—Xaa7—Xaa8—Xaa9—Xaa10—Xaa11—Cys—Gly，

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Arg; Xaa6 is Gly or His; Xaa7 is Arg; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg, wherein has at least an arginine to be modified.

Say that further described polypeptide specifically includes following amino acid sequence:

His—Gln—Cys—Xaa1—Xaa2—Phe—Xaa3—Xaa4—Xaa5—Xaa6—Xaa7—Xaa8—Xaa9—Xaa10—Xaa11—Cys—Gly，

Wherein, Xaal is His or A1a; Xaa2 is G1n or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is the Arg of modification or unmodified; Xaa6 is Gly or His; The Arg of Xaa7 for modifying; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg.

Say that further described polypeptide comprises following concrete amino acid sequence:

His—Gln～Cys—Xaa1—Xaa2—Phe—Xaa3—Xaa4—Xaa5—Xaa6—Xaa7—Xaa8—Xaa9—Xaa10—Xaa11—Cys—Gly，

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is the Arg of modification or unmodified; Xaa6 is Gly or His; The Arg of Xaa7 for modifying; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg.

The arginine of modification of the present invention is the Arg that side chain is modified, and has the described structure of general formula (I).

Formula I

Wherein G1 is O, NH or CH ₂G2 is NH ₂, CH ₃, NHCH ₃Or N (CH ₃) ₂G3 is O, NH, NHCH ₃, NHCH ₃N is 2,3 or 4;

Wherein working as G1 is NH, and G2 is NH ₂The time, G3 is not NH.

Say that further the arginine of described modification is citrulline (Cit), namely working as G1 is NH, and G2 is NH ₂, G3 is O, and n is 3.

Specifically, to comprise concrete amino acid sequence be SEO ID No1-261 to described polypeptide.

Polypeptide of the present invention can combine with autoimmune antibody, forms antigen one antibody complex (Complex).

Advance one by one and say that described polypeptide can have high-affinity with HLA-DR molecule to the step, combining with HLA-DR forms HLA-DR one polypeptide complex.

Say that further described polypeptide is by forming disulfide bond between non-conterminous two Cys in the sequence.Formed disulfide bond can make this polypeptide have ring texture and can simulate β-corner structure, has high-affinity so that this polypeptide not only has with HLA-DR molecule, combines with HLA-DR to form HLA-DR-polypeptide complex; Can also with the efficient combination of anti-CCP antibody, form antigen-antibody complex.

Say that further the amino acid sequence that comprises of described polypeptide is His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Xaa-Xaa5-Xaa-Xaa6-Xaa7-Xaa8-Xaa9-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Gly or His; Xaa6 is Ser or Arg; Xaa7 is Arg or Leu; Xaa8 is Ala or Ile; Xaa9 is Ala or Arg, and Xaa is citrulline; Described polypeptide forms disulfide bond between the 3rd Cys and sixteen bit Cys.

Specifically, the amino acid sequence that comprises of described polypeptide is His-Gln-Cys-His-Gln-Phe-Arg-Phe-Xaa-Gly-Xaa-Ser-Arg-Ala-Ala-Cys-Gly, and wherein Xaa is citrulline; Form disulfide bond between the 3rd Cys of described polypeptide and the sixteen bit Cys.

Specifically, described polypeptide (SEQ.ID No2-118) forms disulfide bond between the 3rd Cys and sixteen bit Cys.

Say that further described polypeptide comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Xaa5-Xaa-Xaa6-Xaa7-Xaa8-Xaa9-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Glu or Arg; Xaa3 is Arg or Glu; Xaa4 is Phe or Met; Xaa5 is Gly or His; Xaa6 is Ser or Arg; Xaa7 is Arg or Leu; Xaa8 is Ala or Ile; Xaa9 is Ala or Arg, and Xaa is citrulline; Described polypeptide forms disulfide bond between the 3rd Cys and sixteen bit Cys.。

Specifically, the amino acid sequence that comprises of described polypeptide is His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Arg-His-Xaa-Arg-Leu-Ile-Arg-Cys-Gly, and wherein Xaa is citrulline; Described polypeptide forms disulfide bond between the 3rd Cys and sixteen bit Cys.

Specifically, described polypeptide (SEQ.ID No120-261) forms disulfide bond between the 3rd Cys and sixteen bit Cys.

Polypeptide of the present invention also is included in the product that carries out gained behind chemical modification or the bio-modification on its amino acid sequence skeleton.Described modification comprises Pegylation (PEGylation) (Adv.Drug deliv.Rev.28,275-299; Adv.Drug deliv.Rev.54,453-609; Adv.Drug deliv.Rev.60,1-88), acyl group (Acylation) (J.Pharma.Sci.86,768-773; J.Pharma.Sci.86,1365-1368), glycosylation (Glycosylation) (J.Pharma.Sci.87,326-332; Adv.Drug deliv.Rev.6,103-131; Adv.Drug deliv.Rev.13,251-267) and the organic molecule modification etc.

The molecular weight that described organic molecule refers to have C, H, O or C, H, O, N, several elements such as P, S form is less than the organic molecule of 2000Da.Refer to particularly each amino acid, all kinds of monose or polysaccharide, each biostearin (Vitamin), biotin and molecular weight at the polymkeric substance of 20-2000Da, as: polyglycol, polypeptide, oligosaccharide and polysaccharide (glucosan, starch, chitin etc.) and nucleosides etc.

Antigen composition of the present invention comprises polypeptide I and polypeptide II, and the mol ratio ratio of polypeptide I and polypeptide I and polypeptide II sum is 0-1;

Described polypeptide I comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; The Arg of Xaa5 for modifying; Xaa6 is Gly or His; Xaa7 is Arg; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg, wherein has at least two arginine to be modified;

Described polypeptide II comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Gly or His; Xaa6 is Arg; Xaa7 is Ser or Arg; Xaa8 is Arg or Leu; Xaa9 is Ala or Ile; Xaa10 is Ala or Arg, wherein has at least an arginine to be modified.

Say that further the mol ratio ratio of described polypeptide I and polypeptide I and polypeptide II sum is 0.3-0.7; Concrete, described mol ratio ratio is 0.5.

Described polypeptide I comprises following amino acid sequence: His-Gln-Cys-HisGln-Phe-Arg-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; The Arg of Xaa5 for modifying; Xaa6 is Gly or His; The Arg of Xaa7 for modifying; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg;

Described polypeptide II comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Gly or His; The Arg of Xaa6 for modifying; Xaa7 is Ser or Arg; Xaa8 is Arg or Leu; Xaa9 is Ala or Ile; Xaa10 is Ala or Arg.

Specifically, described polypeptide I comprises following amino acid sequence:

His—Gln—Cys—His—Gln—Phe—Arg—Phe—Xaa1—Gly—Xaa2—Ser—Arg—Ala—Ala—Cys—Gly，

Wherein, the Arg of Xaa1 for modifying; The Arg of Xaa2 for modifying;

Described polypeptide II comprises following amino acid sequence: His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Arg-His-Xaa1-Arg-Leu-Ile-Arg-Cys-Gly,

Wherein, the Arg of Xaa1 for modifying;

Specifically, to comprise concrete amino acid sequence be SEQ ID No1-118 to described polypeptide I; It is SEQ ID No119-261 that described polypeptide II comprises concrete amino acid sequence.

Say that further described polypeptide I and polypeptide II can combine with autoimmune antibody, form antigen-antibody complex.Described polypeptide can have high-affinity with HLA-DR molecule, combines with HLA-DR to form HLA-DR-polypeptide I or II compound.

Say that further described antigen composition is by forming disulfide bond between non-conterminous two Cys in the sequence.Formed disulfide bond can make this polypeptide have ring texture and can simulate β-corner structure, has high-affinity so that this polypeptide not only has with HLA-DR molecule, combines with HLA-DR to form HLA-DR-polypeptide I or II compound; Can also with the efficient combination of anti-CCP antibody, form antigen-antibody complex.

Say that further described polypeptide I or II are by forming disulfide bond between two non-conterminous Cys in the sequence; Concrete, described polypeptide I or II are by forming disulfide bond between the 3rd Cys in the sequence and the sixteen bit Cys.

Say that further described antigen composition (SEQ.ID No1-261) forms disulfide bond between the 3rd Cys and sixteen bit Cys.

Polypeptide of the present invention is for directly synthetic or first by chemosynthesis or gene engineering method by chemical method, be about to that separation and purification will obtain from expression vector polypeptide again the step of process modification arginine side chain obtain.

Say that further described polypeptide is directly synthetic for passing through chemical method, or for after synthesizing by chemical method first, obtained by PAD enzyme modification arginine side chain.

Antigen composition of the present invention can combine with rheumatoid arthritis autoimmunity antibody.

Say that further the selectivity that described antigen composition combines with rheumatoid arthritis autoimmunity antibody is specially greater than 95% greater than 90%.

Say that further described antigen composition, is specially greater than 75% greater than 70% the sensitivity of rheumatoid arthritis autoimmunity antibody.

Antigen composition of the present invention has high-affinity to HLA-DR.

The method that described polypeptide I or II combine with biotin labeling can be by first or gene engineering expression and purifying synthetic by solid phase, and the gained polypeptide is connected also purifying (be 200310108264.0 Chinese invention patents disclosed technical schemes such as application number) again with biotin.

The method that described polypeptide I or II and biotin labeling combine can also be connected first the mode (Pept.Res.1989,2,189-194) of repurity with biotin in the solid phase building-up process of polypeptide.Concrete, described biotin labeled polypeptide I or the II of being combined with, biotin combines with the N end of polypeptide I or II on it.

Antigen composition of the present invention can be applied to the vitro detection of rheumatoid arthritis disease autoimmune antibody.

The external detection method of rheumatoid arthritis disease autoimmune antibody of the present invention is with test serum (serums) and includes above-mentioned polypeptide (being antigen) reagent mix, detects the formed compound of described polypeptide and Serum Antibody.Testing result is positive and shows, has rheumatoid arthritis disease autoimmune antibody in this serum.

Specifically, described external detection method is that ELISA detects.

Immune antiboidy vitro detection reagent composition of the present invention, comprise biotin labeled antigen composition, hydrophobicity equipment, Avidin or Streptavidin, the anti-human IgG antibody of enzyme labeling and chromogenic substrate, described antigen composition is specially polypeptide I and II, and wherein the mol ratio ratio of polypeptide I and polypeptide I and polypeptide II sum is 0-1.

Say that further the mol ratio ratio of described polypeptide I and polypeptide I and polypeptide II sum is 0.3-0.7; Concrete, described mol ratio ratio is 0.5.

Described polypeptide I comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; The Arg of Xaa5 for modifying; Xaa6 is Gly or His; Xaa7 is Arg; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg, wherein has at least two arginine to be modified;

Described polypeptide II comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Gly or His; Xaa6 is Arg; Xaa7 is Ser or Arg; Xaa8 is Arg or Leu; Xaa9 is Ala or Ile; Xaa10 is Ala or Arg, wherein has at least an arginine to be modified.

Say that further described polypeptide I comprises following amino acid sequence: His-Gln-Cys-His-Gln-Phe-Arg-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; The Arg of Xaa5 for modifying; Xaa6 is Gly or His; The Arg of Xaa7 for modifying; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg;

Described polypeptide II comprises following amino acid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Gly,

Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Gly or His; The Arg of Xaa6 for modifying; Xaa7 is Ser or Arg; Xaa8 is Arg or Leu; Xaa9 is Ala or Ile; Xaa10 is Ala or Arg.

Say that further described polypeptide I comprises following amino acid sequence: His-Gln-Cys-His-Gln-Phe-Arg-Phe-Xaa1-Gly-Xaa2-Ser-Arg-Ala-Ala-Cys-Gly,

Wherein, the Arg of Xaa1 for modifying; The Arg of Xaa2 for modifying;

Described polypeptide II comprises following amino acid sequence: His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Arg-His-Xaa1-Arg-Leu-Ile-Arg-Cys-Gly,

Wherein, the Arg of Xaa1 for modifying;

Specifically, to comprise concrete amino acid sequence be SEQ ID No1-118 to described polypeptide I; It is SEQ ID No119-261 that described polypeptide II comprises concrete amino acid sequence.

Say that further described hydrophobicity equipment is selected from plastics or glass.

Plastics should be understood to all or part organic and inorganic elements chemical combination forms by carbon and oxygen, hydrogen, nitrogen and other, become solid in the final stage of making, some stage is that liquid (must want and can flow in some stage becoming before the final products by plastic material in the mill.), thereby can heat or plus-pressure, or the mode of the two and usefulness, make it form various shapes, any in this huge and protean material same clan, such as resin, thermoset resin, cellulose derivative etc., in the molecular structure of a long-chain, there are numerous repetition atom or molecule.Plastics comprise artificial synthetic or nature organic material.

Glass is appreciated that frangible amorphous material, these materials can be transparent also can be translucent, usually merged by molten silicon and silicon-carbon hydrochlorate and form.Glass can also think that a class does not have crystallization process, but is solidified and next material by molten state, substantially by Na ₂O, CaO and 6SiO ₂Chemical oxide forms, and has optical properties and various mechanical attributes.

Specifically, described hydrophobicity equipment is made by polystyrene, tygon, Polyvinylchloride, polycarbonate or glass etc., has the ELISA Plate of vesicular structure.

Say that further described Avidin (Avidin) is that a kind of relative molecular mass is 68,000Da, pI is a kind of alkaline glycoprotein of 10.5, claims again avidin, and content is abundanter in egg, generally extracts from albumin.Described Streptavidin (Streptavidin) is a kind of protein that extracts from streptomycete (Streptomyces avidinii) culture, relative molecular mass 60, and 000Da does not have sugar chain.Its characteristic is the same with Avidin, also has 4 biotin binding sites, has high-affinity with biotin.The described polypeptide that is combined with biotin, biotin labeled polypeptide I or II can with Avidin or the non-covalent combination of Streptavidin.

Specifically, the anti-human IgG antibody of described enzyme labeling uses horseradish peroxidase (horseradish peroxidase, HRP) mark, and described chromogenic substrate is tetramethyl biphenyl ammonia (tetramethyl benzidine, TMB).

The application of immune antiboidy vitro detection reagent composition of the present invention in rheumatoid arthritis ELISA vitro detection.

Say that further the concrete experimental procedure of described ELISA detection method can be referring to US6, the appropriate section that detects about ELISA in 858,438.

Its ultimate principle is: polypeptide (antigen) is coated on the ELISA Plate surface, serum/plasma to be checked after the dilution and tester are added in the reaction plate hole, if there is anti-CCP antibody in the tested serum/plasma, behind incubation, then the CCP antigen polypeptide of specific antibody in reaction plate hole is combined in the serum/plasma, form solid phase antigen-antibody complex, other composition in the unconjugated serum/plasma of flush away, the anti-human IgG antibody that adds enzyme labeling, incubation, the anti-CCP antibody of solid phase CCP antigen (polypeptide) combination is combined with the anti-human IgG antibody of enzyme labeling again, the unconjugated enzymic-labelled antibody composition of flush away, add zymolyte, and be catalyzed into and be coloured product, add at last the stop buffer cessation reaction.According to the tester in the kit, can resist CCP antibody and carry out quantitatively or qualitative determination.

The preparation method of detection reagent composition of the present invention comprises following concrete steps successively:

1, configuration coating buffer;

2, coating buffer is added hydrophobicity equipment surface;

3, under 0-40 ℃ of conditions, preserve more than 6 hours

4, washing hydrophobicity equipment surface is more than 2 times;

5, add the hydrophobicity equipment surface of solution after washing of containing through biotin labeled antigen composition;

6, preserve more than 1 hour under 10-30 ℃ of conditions;

7, washing hydrophobicity equipment surface is more than 2 times.

Say that further described coating buffer pH is 7.0-8.0 damping fluid, wherein be dissolved with the Avidin that concentration is 0.1-10 μ g/mL, can select 0.5,1,2 or 5 μ g/mL.Described damping fluid is specifically as follows phosphate buffer (Phosphate Buffer, PB), phosphate buffered saline(PBS) (Phosphate Buffer Saline, PBS), citrate buffer solution, carbonate buffer solution, Tris-HCl, TRIS buffer (TBS) and barbitol buffer solution.Described Avidin is specially Streptavidin.

Specifically, described pH of buffer selects 7.4, and described damping fluid is selected phosphate buffered saline(PBS).

Say that further described hydrophobicity equipment is container or the straight plate with certain volume, can be ELISA Plate, is specially 96 or 48 hole ELISA Plate.

Say that further described 0-40 ℃ of temperature ranges are selected 0-10 ℃.

Say that further described 0-10 ℃ of temperature ranges are selected 2-8 ℃, are specially 4 ℃.

Say that further described incubation time more than 6 hours can be selected to be specially 12-18 hours more than 12 hours.

Say that further described cleansing solution pH is 7.4 damping fluid.Described damping fluid is specifically as follows phosphate buffer, phosphate buffered saline(PBS), citrate buffer solution, carbonate buffer solution, Tris-HCl, TRIS buffer (TBS) and barbitol buffer solution.Select particularly phosphate buffered saline(PBS).

Say that further described antigen composition is selected from protein, polypeptide, nucleic acid or organic molecule.Be selected from particularly polypeptide I and polypeptide II, the mol ratio ratio of polypeptide I and polypeptide I and polypeptide II sum is 0-1.

Described protein and nucleic acid also are included in the product that carries out gained behind chemical modification or the bio-modification on its amino acid or the nucleotide sequences skeleton.Described modification comprises Pegylation (PEGylation) (Adv.Drug deliv.Rev.28,275-299; Adv.Drug deliv.Rev.54,453-609; Adv.Drug deliv.Rev.60,1-88), acyl group (Acylation) (J.Pharma.Sci.86,768-773; J.Pharma.Sci.86,1365-1368), glycosylation (Glycosylation) (J.Pharma.Sci.87,326-332; Adv.Drug deliv.Rev.6,103-131; Adv.Drug deliv.Rev.13,251-267) and the organic molecule modification etc.

Say that further described 10-30 ℃ of temperature ranges are selected 15-25 ℃, preferred 20-25 ℃.

The preparation method of detection reagent composition of the present invention, the subsequent step that also comprises has: drying, vacuum preservation etc.

The application of the preparation method of detection reagent composition of the present invention in ELISA detects.

The application of the preparation method of detection reagent composition of the present invention in the immune antibody of rheumatoid arthritis vitro detection.

Peptide composition of the present invention comprises polypeptide I and polypeptide II, and the mol ratio ratio of polypeptide I and polypeptide I and polypeptide II sum is 0-1.By the mode of two non-conterminous cysteine side chain sulfydryl formation disulfide bond on the sequence, produce the polypeptide of ring-type.This class peptide composition can not only combine with rheumatoid arthritis autoimmunity antibody, can also have high-affinity to HLA-DR simultaneously.Through experimental verification, the selectivity that such polypeptide combines with rheumatoid arthritis autoimmunity antibody is greater than 95%, to the detection sensitivity of rheumatoid arthritis autoimmunity antibody greater than 75%.Compare with the like product of selling on the market, its sensitivity is significantly improved, and more is conducive to the vitro detection of rheumatoid arthritis autoimmunity antibody.

Description of drawings

The polypeptide 1 purity chromatogram that Fig. 1: RP-HPLC detects, transverse axis represents the time, unit: minute (min), the longitudinal axis represents voltage, unit: millivolt (mv);

The polypeptide 2 purity chromatograms that Fig. 2: RP-HPLC detects;

Polypeptide 1 mass spectrogram that Fig. 3: ESI detects, transverse axis represents that matter/nuclear is than (m/z);

Polypeptide 2 mass spectrograms that Fig. 4: ESI detects.

Biotin-polypeptide 1 purity chromatogram that Fig. 5: RP-HPLC detects;

Biotin-polypeptide 2 purity chromatograms that Fig. 6: RP-HPLC detects;

Biotin-polypeptide 1 mass spectrogram that Fig. 7: ESI detects;

Biotin-polypeptide 2 mass spectrograms that Fig. 8: ESI detects.

Embodiment

Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.The embodiment of the invention is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.

If the used reagent of the present invention does not clearly indicate, then all available from Sigma-aldrich (Sigma-Aldrich).

Synthesizing of embodiment 1 polypeptide

Polypeptide adopts the Fmoc chemical method, and is synthetic by solid phase synthesis technique.The concrete steps of the method are referring to Eur.J.Immunol.1994, and 24,3188-3193; J.Org.Chem.1972,37,3404-3409; Huang Weide, old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985.

The formation method of disulfide bond and step thereof can be referring to document: Huang Weide, and old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985, p85; Michael W.Pennington Peptide SynthesisProtocols (Methods in Molecular Biology), Humana Press, 1994, p91-169

By above-mentioned steps, the concrete sequence of synthetic polypeptide is:

Polypeptide 1:His-Glu-Cys-His-Glu-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Ala-Ala-Cys-Glu (SEQ ID No1), the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make polypeptide have ring texture, and can simulate β-corner structure.

Polypeptide 2:His-Glu-Cys-Ala-Arg-Phe-Glu-Met-Arg-His-Cit-Arg-Leu-Ile-Arg-Cys-Glu (SEQ ID No119), the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make polypeptide have ring texture, and can simulate β-corner structure.

The purifying of embodiment 2 polypeptide

With protecting group deprotection in dimethylformamide (DMF) solvent of protein or polypeptide end, neutralize first.With hydrogen fluoride polypeptide is downcut from synthetic resin again, the crude product that obtains is through reverse-phase chromatography C18 or C8 post (as: 5 μ m, 250 * 4.6mm), take mobile phase A (0.1% (v/v) trifluoroacetic acid acetonitrile solution) and Mobile phase B (0.1% (v/v) trifluoroacetic acid aqueous solution) as the gradient elution solvent.In 45 minutes, mobile phase A accounts for A, B two-phase cumulative volume 0% (v/v) changes to 100% (v/v) collection and obtains target polypeptides, remove organic solvent by desalination chromatographic column (GE Healthcare) or rotary evaporation, target polypeptides can be determined by the molecular weight that the ESI mass spectrum obtains by the mode that LC-MS is used in conjunction the polypeptide direct injected that maybe collection is obtained.(referring to: Chinese analytical chemistry 2002,30,1126-9)

Purity and the molecular weight detection of embodiment 3 polypeptide

The above-mentioned polypeptide that synthesizes 1 and polypeptide 2 are measured by reverse-phase chromatography (RP-HPLC) respectively, and its concrete grammar is:

4.6 * 250mm5 μ m C18 analytical column (Kromasil);

Mobile phase A is that trifluoroacetic acid (trifluoroacetic, TFA) adds in 100% acetonitrile (acetonitrile, ACN), so that TFA concentration is 0.1% (v/v);

Mobile phase B is that TFA adds in 100% water, so that TFA concentration is 0.1% (v/v);

Flow velocity is 1.0ml/min;

The detection wavelength is 220nm;

Gradient: the proportional balancing method that accounts for two cumulative volumes 15% of A, B (v/v) with mobile phase A, behind the sample introduction, adopt linear gradient elution (Gradient Elution), mobile phase A accounts for two cumulative volumes of A, B and changes to the ratio of 50% (v/v) from 15% (v/v) in 25 minutes, afterwards with 100% (v/v) mobile phase A balance 5 minutes.

The retention time of polypeptide 1 (Retention Time) is 12.5 (Fig. 1), and its purity is greater than 95%.

The retention time of polypeptide 2 (Retention Time) is 13.2 (Fig. 2), and its purity is greater than 95%.

Measure respectively the molecular weight (Fig. 4) of polypeptide 1 (Fig. 3) and polypeptide 2 by the ESI mass spectrum, its actual conditions is:

Mass spectrum (Probe) ESI

Mass spectrum voltage (Probe bias)+4.5kv

Sprayer flow velocity (Nebulizer Gas Flow) 1.5L/min

Detecting device (Detector) 1.5kv

Sample ionization gasifying device (CDL)-20.0v

Flow rate of mobile phase (T.Flow) 0.2ml/min

250 ℃ of CDL temperature (CDL Temp)

Buffer concentration (B.conc) 50%H ₂O, 50%ACN

200 ℃ of heat block temperature (Block Temp)

Polypeptide 1 molecular weight is 2017.26Da; Polypeptide 2 molecular weight are 2167.59Da.

The biotin labeling of embodiment 4 polypeptide

Biotin-polypeptide 1 (SEQ ID No1) is connected polypeptide 2 (SEQ ID No119) and is all adopted in the solid phase building-up process mode that is connected first repurity with biotin to carry out respectively with biotin, its concrete mode is referring to Deibel, M.R., Jr., Lob1, T.J.and Yem, A.W., A technique for rapidpurification of low yield products:Biotinylation of chemically synthesizedproteins on-resin.Pept.Res.1989,2,189-194.

When whether biotin behind the direct polycondensation, uses Kaiser test detection condensation reaction complete by condensation agent when polypeptide is synthetic.The detailed description of this reaction sees also Sigma-Aldrich60017 description of product (60017Data Sheet), the method can not only be applied to qualitative (Analytical Biochemistry1970,34,595), can also carry out quantitatively (Analytical Biochemistry1981,117,147) detect.

Embodiment 5 polypeptide separating and purifying after biotin is combined

Carry out biotin labeling at resin.At first, protein or polypeptide end be deprotection in dimethylformamide (DMF) solvent, neutralization; Then with N-hydroxy-succinamide biotin reaction (being biotinylation), reaction conditions be 45 ℃ 24 hours; To be marked with again the polypeptide cutting-out of biotin with hydrogen fluoride, the crude product that obtains is through the affinity column (GEHealthcare) of Avidin coupling agar, wash with phosphate buffer, remove unconjugated composition, use at last glycine solution (pH2.0) wash-out of 0.1M, obtain biotinylated protein or polypeptide.

Synthetic and the biotin labeling of embodiment 6 polypeptide (SEQ.ID No2-118, SEQ.ID No120-261)

Same method such as embodiment 1 can obtain annular polypeptide (SEQ.ID No2-118, SEQ.ID No120-261).

Same method such as embodiment 4 can obtain through biotin labeled annular polypeptide (SEQ.IDNo2-118, SEQ.ID No120-261).

The purity of embodiment 7 biotins-polypeptide and molecular weight detection

Above-mentioned biotin-the polypeptide that synthesizes 1 and biotin-polypeptide 2 are measured by reverse-phase chromatography (RP-HPLC) respectively, and its concrete grammar is:

4.6 * 250mm5 μ m C18 analytical column (Kromasil);

Mobile phase A is that trifluoroacetic acid (trifluoroacetic, TFA) adds in 100% acetonitrile (acetonitrile, ACN), so that TFA concentration is 0.1% (v/v);

Mobile phase B is that TFA adds in 100% water, so that TFA concentration is 0.1% (v/v);

Flow velocity is 1.0ml/min;

The detection wavelength is 220nm;

Gradient: the proportional balancing method that accounts for two cumulative volumes 15% of A, B (v/v) with mobile phase A, behind the sample introduction, adopt linear gradient elution (Gradient Elution), mobile phase A accounts for two cumulative volumes of A, B and changes to the ratio of 50% (v/v) from 15% (v/v) in 25 minutes, afterwards with 100% (v/v) mobile phase A balance 5 minutes.

The retention time of biotin-polypeptide 1 (Retention Time) is 8.4 (Fig. 1), and its purity is greater than 95%.

The retention time of biotin-polypeptide 2 (Retention Time) is 13.5 (Fig. 2), and its purity is greater than 95%.

Measure respectively the molecular weight of polypeptide 1 and polypeptide 2 by the ESI mass spectrum, its actual conditions is:

Mass spectrum (Probe) ESI

Mass spectrum voltage (Probe bias)+4.5kv

Sprayer flow velocity (Nebulizer Gas Flow) 1.5L/min

Detecting device (Detector) 1.5kv

Sample ionization gasifying device (CDL)-20.0v

Flow rate of mobile phase (T.Flow) 0.2ml/min

250 ℃ of CDL temperature (CDL Temp)

Buffer concentration (B.conc) 50%H ₂O, 50%ACN

200 ℃ of heat block temperature (Block Temp)

According to peak value and its with electric charge calculate, biotin-polypeptide 1 actual measurement molecular weight is that [M+3H+] 3+ of 748.80 peak values is calculated as 748.80 * 3-3=2243.40Da; The actual measurement molecular weight is that [M+4H+] 4+ of 599.20 peak values is calculated as 599.20 * 4-4=2392.80Da in biotin-polypeptide 2.

This numerical value is consistent with the theoretical molecular of biotin-polypeptide 1 (Mw=2243.56Da) and biotin-polypeptide 2 (Mw=2393.86Da).

Embodiment 8 detects the preparation of carrier

The preparation of concentrated cleaning solution

First pH7.4 phosphate buffer according to 10 times of concentration of following table preparation (be called for short 10 * PBS),

Add afterwards

-20 to make its final concentration be 0.5% (v/v), finally is mixed with to be added with

10 * PBST.

Sample diluting liquid is used for diluting sample to be checked, and its proportioning is as follows:

The preparation of coating buffer:

The Tween phosphate buffer of 1 times of concentration (1 * PBST, pH7.4,0.05% (v/v)

-20) be used for dilution and coated Avidin to the ELISA Plate surface; 1 * PBST (pH7.4,0.05% (v/v)

-20) be used for diluting biotin labeled hydrophilic molecule (protein, polypeptide or nucleic acid).

The concrete method for coating of hydrophilic molecule is as follows:

With phosphate buffer (1 * PBS, pH7.4) dilution Streptavidin (5 μ g/ml), then join in (140 μ l/ hole) in each hole of ELISA Plate, 4 ℃ spend the night (12-18 hours), take out ELISA Plate, remove coating buffer, with cleansing solution 1 * PBST (pH7.4,0.05% (v/v)

20) wash 2 times, then add 1 * PBST (pH7.4,0.05% (v/v) -20) dilute biotin labeled polypeptide, mixing on decolorization swinging table, incubated at room 1 hour discards unconjugated biotin labeled polypeptide, 350 μ l1 * PBST (pH7.4,0.05% (v/v)

-20) wash plate 2 times, drying.Preserve stand-by in the vacuum tank.During production, ELIAS strip is packed in the bag into vacuum seal.

The preparation of embodiment 9 testers

1., proofread and correct the preparation of product

This kit adopts the correction product of 6 different relative concentrations, and concentration unit is relative unit (RU/ml), and its establishing method is as follows:

OD ₄₅₀=1.9～2.0 o'clock, the setting concentration unit was 100RU/ml, as the upper limit;

OD ₄₅₀=0.9～1.0 o'clock, the setting concentration unit was 50RU/ml

OD ₄₅₀=0.4～0.5 o'clock, the setting concentration unit was 25RU/ml

OD ₄₅₀=0.2～0.3 o'clock, the setting concentration unit was 12RU/ml

OD ₄₅₀=0.1～0.15 o'clock, the setting concentration unit was 6RU/ml

OD ₄₅₀=0.05～0.08 o'clock, the setting concentration unit was 3RU/ml

OD ₄₅₀=0.02～0.04 o'clock, the setting concentration unit was 0RU/ml, as lower limit.

The drawing standard curve is set 6 measuring points, and (0,6,12,25,50,100RU/ml) are proofreaied and correct product 1～No. 6 in preparation respectively.

Proofread and correct the preparation of product No. 6: get anti-CCP antibody positive and OD ₄₅₀=1.9～2.0 RA patients serum/blood plasma 1ml is with the dilution of 100ml sample diluting liquid, fully mixing; Get 50ml wherein, in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-6 ", 2～8 ℃ of lower preservations.

Proofread and correct the preparation of product No. 5: get the 50ml of above-mentioned remainder, add sample diluting liquid 50ml, fully mixing is got 50ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-5 ", 2～8 ℃ of lower preservations.

Proofread and correct the preparation of product No. 4: get the 50ml of above-mentioned remainder, add sample diluting liquid 50ml, fully mixing is got 50ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-4 ", 2～8 ℃ of lower preservations.

Proofread and correct the preparation of product No. 3: get the 50ml of above-mentioned remainder, add sample diluting liquid 50ml, fully mixing is got 50ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-3 ", 2～8 ℃ of lower preservations.

Proofread and correct the preparation of product No. 2: get the 50ml of above-mentioned remainder, add sample diluting liquid 50ml, fully mixing is got 50ml wherein, and in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-2 ", 2～8 ℃ of lower preservations.

Proofread and correct the preparation of product No. 1: the sample thief dilution, in the packing standard vial, every bottle of 1ml is labeled as " proofreading and correct product-1 ", 2～8 ℃ of lower preservations.

2., the preparation of weak positive reference substance

Get anti-CCP antibody positive and OD ₄₅₀=0.1～0.15 RA patients serum/blood plasma 1ml, the centrifuging and taking supernatant, with 0.22 μ m biofilm filtration degerming, with the dilution of 100ml sample diluting liquid, abundant mixing, in the packing standard vial, every bottle of 1ml is labeled as " weak positive reference substance ", 2～8 ℃ of lower preservations.

3., the preparation of negative control product

Get many parts of normal human serums, equal proportion is mixed, and gets 1ml, the centrifuging and taking supernatant, and with 0.22 μ m biofilm filtration degerming, with the dilution of 100ml sample diluting liquid, abundant mixing, in the packing standard vial, every bottle of 1ml is labeled as " negative control product ", 2～8 ℃ of lower preservations.

Embodiment 10 2 anti-reagent configurations

1., the anti-human IgG antibody preparation of HRP mark:

This kit adopts rabbit anti-human igg's antibody of HRP mark, purchases (the article No.: BA1070) in Wuhan Boster Biological Technology Co., Ltd..Get 20 μ L storage liquid, add the 800ml antibody diluent and do the 1:40000 dilution, mixing, packing standard vial, every bottle of 15ml, 2～8 ℃ of lower preservations.Antibody diluent is available from Pierce company (article No.: 37552).

2., the anti-human IgG antibody substrate A preparation of HRP mark:

Take by weighing citric acid 17.9g and Na ₂HPO ₄12 ₂O4.67g is dissolved in the 400ml deionized water, then slowly adds 30% (w/w) H ₂O ₂330 μ L after stirring, add water to 500ml.Be distributed into the 10ml/ bottle, 2～8 ℃ of lower preservations.

3., the anti-human IgG antibody substrate B preparation of HRP mark:

Take by weighing TMB0.1g in the 100ml deionized water, add DMSO2.5ml, when stirring, slowly add 0.5ml6M HCl, until fully dissolving adds water to 500ml at last.Be distributed into the 10ml brown vial, 2～8 ℃ of lower preservations.

4., the preparation of the anti-human IgG antibody substrate reactions stop buffer of HRP mark:

Measure respectively 98%H ₂SO ₄Then 55ml and deionized water 445ml slowly join the concentrated sulphuric acid in the deionized water, and mixing is distributed into the 10ml/ bottle, 2～8 ℃ of lower preservations.

Embodiment 11 biotin labeling polypeptide are used for the vitro detection of rheumatoid arthritis antibody

Collection obtains 183 samples of Patients with Rheumatoid Arthritis, 104 samples of non-RA autoimmune disease patients serum, 181 samples of normal human serum.

Form disulfide bond between the 3rd Cys that uses that above-described embodiment synthesized and the sixteen bit Cys, and through biotin labeled annular polypeptide I (SEQ ID No1) and polypeptide II (SEQ ID No119).Be that 0.5 mixtures of polypeptides is coated in 96 hole ELISA Plate through embodiment 8 described methods with the mol ratio ratio of polypeptide I and polypeptide I and polypeptide II sum.Prepare two decorrelation reagent according to embodiment 9 modes, the result who measures is as follows:

Annotate: in the bracket numeral actual measurement sample number.

Sensitivity=true positives/(true positives+false negative)=144/183=78.7%

Selectivity=true negative/(true negative+false positive)=271/285=95.1%

Embodiment 12 immune antibody of rheumatoid arthritis vitro detection reagent composition standard practice instructions

(1), preliminary work

1., experiment will test with reagent and to take out from refrigerator in front 30 minutes, behind temperature and equilibrium at room temperature, use.Each reagent is abundant mixing before use.

2., preparation uses cleansing solution: according to the needed test sample book hole count of each test, get the concentrated cleaning solution (10 *) of respective amount in the kit, press the dilution proportion of 1:10 (such as 100ml → 900ml).

3., dilute serum/plasma to be checked: with serum/plasma sample diluting liquid to be checked, press the dilution proportion (i.e. 5 μ L → 500 μ L) of 1:101, abundant mixing, every part of sample must be done multiple hole and detect, to add negative control product and positive reference substance simultaneously, if quantitatively detect, must do simultaneously a series of correction product and detect.

(2), operation steps

1., will test required micropore lath and be placed on the ELISA Plate frame, the lath that needs should be put back in the pouch of drying agent immediately, and with its good seal, reduces and the contacting of water vapor as far as possible.

2., wash plate: every hole adds the application cleansing solution of 300 μ L, dries.So triplicate pats dry the lath back-off at last.

3., add sample: add respectively the serum/plasma to be checked after correction product, reference substance and the dilution in the hole, every hole 100 μ L, with shrouding paper lath is sealed, room temperature (20 ℃～25 ℃) is hatched one hour (beginning timing after adding last sample).

4., wash plate: incline to putting the solution of answering in the hole, every hole adds the application cleansing solution of 300 μ L, dries.So triplicate pats dry the lath back-off at last.

5., add ELIAS secondary antibody: every hole 100 μ L, with shrouding paper lath is sealed, room temperature (20 ℃～25 ℃) is hatched half an hour.

6., wash plate: incline to putting the solution of answering in the hole, every hole adds the application cleansing solution of 300 μ L, dries.So triplicate pats dry the lath back-off at last.

7., colour developing: every hole adds colour developing A, each 50 μ L of B liquid, and mixing was room temperature lucifuge reaction 30 minutes.

8., cessation reaction: every hole adds 50 μ L stop buffers, light shaking mixing, cessation reaction.

9., microplate reader is measured wavelength set at 450nm, each hole OD of mensuration in 30 minutes ₄₅₀Value.

10., the drawing standard curve, analysis result.

(3), the result calculates and judges

1., qualitative analysis

The result calculates: ratio=sample OD ₄₅₀/ weak positive control OD ₄₅₀

The result judges:

Ratio＜0.95 feminine gender

0.95＜Bi Zhi＜=1.0 critical values, suggestion redeterminates

Ratio〉1.0 positives

2., quantitative test

Drawing standard curve: with 6 OD that proofread and correct product ₄₅₀Value is ordinate, and each self-corresponding concentration is horizontal ordinate, draws out typical curve.

The result calculates: calculate the OD that sample is measured ₄₅₀Then mean value read respective concentration (RU/ml) at typical curve.

The result judges:

＞=25RU/ml is positive

＜25RU/ml is negative

Sequence table

＜110〉ShangHai RongSheng Biology Pharmacy Co., Ltd

＜120〉composition of externally detecting rheumatoid arthritis antibody and application thereof

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＜213〉artificial synthetic

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＜213〉artificial synthetic

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＜213〉artificial synthetic

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＜213〉artificial synthetic

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＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<212>PRT

＜213〉artificial synthetic

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<212>PRT

＜213〉artificial synthetic

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＜213〉artificial synthetic

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＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No14

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<212>PRT

＜223〉Xaa is citrulline

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＜213〉artificial synthetic

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<400>15

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<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>16

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<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>17

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<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>18

<210>SEQ?ID?No19

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>19

<210>SEQ?ID?No20

<211>17

<212>PRT

＜213〉artificial synthetic

<400>20

<210>SEQ?ID?No21

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>21

<210>SEQ?ID?No22

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No24

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>24

<210>SEQ?ID?No25

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<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>25

<210>SEQ?ID?No26

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>26

<210>SEQ?ID?No27

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>27

<210>SEQ?ID?No28

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>28

<210>SEQ?ID?No29

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>29

<210>SEQ?ID?No30

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>30

<210>SEQ?ID?No31

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>31

<210>SEQ?ID?No32

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>32

<210>SEQ?ID?No33

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>33

<210>SEQ?ID?No34

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>34

<210>SEQ?ID?No35

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>35

<210>SEQ?ID?No36

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>36

<210>SEQ?ID?No37

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>37

<210>SEQ?ID?No38

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>38

<210>SEQ?ID?No39

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>39

<210>SEQ?ID?No40

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>40

<210>SEQ?ID?No41

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>41

<210>SEQ?ID?No42

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>42

<210>SEQ?ID?No43

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>43

<210>SEQ?ID?No44

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>44

<210>SEQ?ID?No45

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No46

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No47

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No48

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No49

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No50

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No56

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>56

<210>SEQ?ID?No57

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>57

<210>SEQ?ID?No58

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>58

<210>SEQ?ID?No59

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>59

<210>SEQ?ID?No60

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>60

<210>SEQ?ID?No61

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>61

<210>SEQ?ID?No62

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>62

<210>SEQ?ID?No63

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>63

<210>SEQ?ID?No64

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>64

<210>SEQ?ID?No65

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>65

<210>SEQ?ID?No66

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

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<210>SEQ?ID?No69

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>69

<210>SEQ?ID?No70

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>70

<210>SEQ?ID?No71

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>71

<210>SEQ?ID?No72

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>72

<210>SEQ?ID?No73

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>73

<210>SEQ?ID?No74

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>74

<210>SEQ?ID?No75

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>75

<210>SEQ?ID?No76

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>76

<210>SEQ?ID?No77

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>77

<210>SEQ?ID?No78

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>78

<210>SEQ?ID?No79

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>79

<210>SEQ?ID?No80

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>80

<210>SEQ?ID?No81

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>81

<210>SEQ?ID?No82

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>82

<210>SEQ?ID?No83

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>83

<210>SEQ?ID?No84

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>84

<210>SEQ?ID?No85

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>85

<210>SEQ?ID?No86

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>86

<210>SEQ?ID?No87

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>87

<210>SEQ?ID?No88

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>88

<210>SEQ?ID?No89

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>89

<210>SEQ?ID?No90

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>90

<210>SEQ?ID?No91

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>91

<210>SEQ?ID?No92

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>92

<210>SEQ?ID?No93

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>93

<210>SEQ?ID?No94

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>94

<210>SEQ?ID?No95

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>95

<210>SEQ?ID?No96

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>96

<210>SEQ?ID?No97

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>97

<210>SEQ?ID?No98

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>98

<210>SEQ?ID?No99

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>99

<210>SEQ?ID?No100

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>100

<210>SEQ?ID?No101

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>101

<210>SEQ?ID?No102

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>102

<210>SEQ?ID?No103

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>103

<210>SEQ?ID?No104

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>1

<210>SEQ?ID?No105

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>105

<210>SEQ?ID?No106

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>106

<210>SEQ?ID?No107

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>107

<210>SEQ?ID?No108

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>108

<210>SEQ?ID?No109

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>109

<210>SEQ?ID?No110

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>110

<210>SEQ?ID?No111

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>111

<210>SEQ?ID?No112

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>112

<210>SEQ?ID?No113

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>113

<210>SEQ?ID?No114

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>114

<210>SEQ?ID?No115

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>115

<210>SEQ?ID?No116

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>116

<210>SEQ?ID?No117

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>117

<210>SEQ?ID?No118

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>118

<210>SEQ?ID?No119

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>119

<210>SEQ?ID?No120

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>120

<210>SEQ?ID?No121

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>121

<210>SEQ?ID?No122

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>122

<210>SEQ?ID?No123

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>35

<210>SEQ?ID?No124

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>36

<210>SEQ?ID?No125

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>125

<210>SEQ?ID?No126

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>126

<210>SEQ?ID?No127

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>127

<210>SEQ?ID?No128

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>128

<210>SEQ?ID?No129

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>129

<210>SEQ?ID?No130

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>131

<210>SEQ?ID?No132

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>132

<210>SEQ?ID?No133

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>133

<210>SEQ?ID?No134

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>134

<210>SEQ?ID?No135

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>135

<210>SEQ?ID?No136

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>136

<210>SEQ?ID?No137

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>137

<210>SEQ?ID?No138

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>138

<210>SEQ?ID?No139

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>139

<210>SEQ?ID?No140

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>140

<210>SEQ?ID?No141

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>141

<210>SEQ?ID?No142

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>142

<210>SEQ?ID?No143

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>143

<210>SEQ?ID?No144

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>144

<210>SEQ?ID?No145

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>145

<210>SEQ?ID?No146

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>146

<210>SEQ?ID?No147

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>147

<210>SEQ?ID?No148

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>148

<210>SEQ?ID?No149

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>149

<210>SEQ?ID?No150

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>150

<210>SEQ?ID?No151

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>151

<210>SEQ?ID?No152

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>152

<210>SEQ?ID?No153

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>153

<210>SEQ?ID?No154

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>154

<210>SEQ?ID?No155

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>155

<210>SEQ?ID?No156

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>156

<210>SEQ?ID?No157

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>157

<210>SEQ?ID?No158

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>158

<210>SEQ?ID?No159

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>159

<210>SEQ?ID?No160

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>160

<210>SEQ?ID?No161

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>161

<210>SEQ?ID?No162

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>162

<210>SEQ?ID?No163

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>163

<210>SEQ?ID?No164

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>164

<210>SEQ?ID?No165

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>165

<210>SEQ?ID?No166

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>166

<210>SEQ?ID?No167

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>167

<210>SEQ?ID?No168

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>168

<210>SEQ?ID?No169

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>169

<210>SEQ?ID?No170

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>170

<210>SEQ?ID?No171

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>171

<210>SEQ?ID?No172

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>172

<210>SEQ?ID?No173

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>173

<210>SEQ?ID?No174

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>174

<210>SEQ?ID?No175

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>175

<210>SEQ?ID?No176

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>176

<210>SEQ?ID?No177

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>177

<210>SEQ?ID?No178

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>178

<210>SEQ?ID?No179

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>179

<210>SEQ?ID?No180

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>180

<210>SEQ?ID?No181

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>181

<210>SEQ?ID?No182

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>182

<210>SEQ?ID?No183

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>183

<210>SEQ?ID?No184

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>184

<210>SEQ?ID?No185

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>185

<210>SEQ?ID?No186

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>186

<210>SEQ?ID?No187

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>187

<210>SEQ?ID?No188

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>188

<210>SEQ?ID?No189

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>189

<210>SEQ?ID?No190

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>190

<210>SEQ?ID?No191

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>191

<210>SEQ?ID?No192

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>192

<210>SEQ?ID?No193

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>193

<210>SEQ?ID?No194

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>1

<210>SEQ?ID?No195

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>195

<210>SEQ?ID?No196

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>196

<210>SEQ?ID?No197

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>197

<210>SEQ?ID?No198

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>198

<210>SEQ?ID?No199

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>199

<210>SEQ?ID?No200

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>200

<210>SEQ?ID?No201

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>201

<210>SEQ?ID?No202

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>202

<210>SEQ?ID?No203

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>203

<210>SEQ?ID?No204

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>204

<210>SEQ?ID?No205

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>205

<210>SEQ?ID?No206

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>206

<210>SEQ?ID?No207

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>207

<210>SEQ?ID?No208

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>208

<210>SEQ?ID?No209

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>209

<210>SEQ?ID?No210

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>210

<210>SEQ?ID?No211

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>211

<210>SEQ?ID?No212

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>212

<210>SEQ?ID?No213

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>213

<210>SEQ?ID?No214

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>214

<210>SEQ?ID?No215

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>215

<210>SEQ?ID?No216

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>216

<210>SEQ?ID?No217

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>217

<210>SEQ?ID?No218

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>218

<210>SEQ?ID?No219

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>219

<210>SEQ?ID?No220

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>220

<210>SEQ?ID?No221

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>221

<210>SEQ?ID?No222

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>222

<210>SEQ?ID?No223

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>223

<210>SEQ?ID?No224

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>224

<210>SEQ?ID?No225

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>225

<210>SEQ?ID?No226

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>226

<210>SEQ?ID?No227

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>227

<210>SEQ?ID?No228

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>228

<210>SEQ?ID?No229

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>229

<210>SEQ?ID?No230

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>230

<210>SEQ?ID?No231

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>231

<210>SEQ?ID?No232

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>232

<210>SEQ?ID?No233

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>233

<210>SEQ?ID?No234

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>234

<210>SEQ?ID?No235

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>235

<210>SEQ?ID?No236

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>236

<210>SEQ?ID?No237

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>237

<210>SEQ?ID?No238

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>238

<210>SEQ?ID?No239

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>239

<210>SEQ?ID?No240

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>240

<210>SEQ?ID?No241

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>241

<210>SEQ?ID?No242

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>242

<210>SEQ?ID?No243

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>243

<210>SEQ?ID?No244

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>244

<210>SEQ?ID?No245

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>245

<210>SEQ?ID?No246

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>246

<210>SEQ?ID?No247

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>247

<210>SEQ?ID?No248

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>248

<210>SEQ?ID?No249

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>249

<210>SEQ?ID?No250

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>250

<210>SEQ?ID?No251

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>251

<210>SEQ?ID?No252

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>252

<210>SEQ?ID?No253

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>253

<210>SEQ?ID?No254

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>47

<210>SEQ?ID?No255

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>255

<210>SEQ?ID?No256

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>256

<210>SEQ?ID?No257

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>257

<210>SEQ?ID?No258

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>258

<210>SEQ?ID?No259

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>259

<210>SEQ?ID?No260

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>260

<210>SEQ?ID?No261

<211>17

<212>PRT

＜213〉artificial synthetic

＜223〉Xaa is citrulline

<400>261

Claims (16)

1. an antigen composition comprises polypeptide I and polypeptide II, it is characterized in that the mol ratio ratio of described polypeptide I and polypeptide I and polypeptide II sum is 0-1;

The amino acid sequence of described polypeptide I is as follows: His-Gln-Cys-His-Gln-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Ala-Ala-Cys-Gly, and the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make circlewise structure of polypeptide;

The amino acid sequence of described polypeptide II is as follows: His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Arg-His-Cit-Arg-Leu-Ile-Arg-Cys-Gly, the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make circlewise structure of polypeptide.

2. antigen composition as claimed in claim 1 is characterized in that the mol ratio ratio of polypeptide I and polypeptide I and polypeptide II sum is 0.5.

3. antigen composition as claimed in claim 1 is characterized in that described polypeptide I or II can be combined with HLA-DR, forms HLA-DR-polypeptide I or II compound.

4. antigen composition as claimed in claim 1 is characterized in that described polypeptide I or II can be combined with immune antibody of rheumatoid arthritis, forms antigen-antibody complex.

5. an immune antiboidy vitro detection reagent composition that comprises the described antigen composition of claim 1 characterized by further comprising Avidin or Streptavidin, the anti-human IgG antibody of enzyme labeling and chromogenic substrate.

6. vitro detection reagent composition as claimed in claim 5 is characterized in that described anti-human IgG antibody is through horseradish peroxidase-labeled.

7. vitro detection reagent composition as claimed in claim 5 is characterized in that described chromogenic substrate is tetramethyl biphenyl ammonia.

8. such as claim 6 or 7 described vitro detection reagent compositions, it is characterized in that described polypeptide is combined with biotin.

9. vitro detection reagent composition as claimed in claim 8 is characterized in that described polypeptide N end is combined with biotin labeling.

10. vitro detection reagent composition as claimed in claim 5, it is characterized in that described Avidin or Streptavidin directly are incorporated into hydrophobicity equipment surface, described polypeptide I or II biotin and described Avidin or the Streptavidin by mark on it is non-covalent affine.

11. the preparation method of a vitro detection reagent composition is characterized in that by following concrete steps preparation:

A) configuration coating buffer;

B) coating buffer is added hydrophobicity equipment surface;

C) under 0-40 ℃ of condition, preserve more than 6 hours;

D) washing hydrophobicity equipment surface is more than 2 times;

E) add the hydrophobicity equipment surface of solution after washing of containing through biotin labeled antigen composition claimed in claim 1;

F) preserve more than 1 hour under 10-30 ℃ of condition;

G) washing hydrophobicity equipment surface is more than 2 times;

Described coating buffer pH is the damping fluid of 7.0-8.0, and wherein being dissolved with concentration is Avidin or the Streptavidin of 0.1-10 μ g/mL; Described hydrophobicity equipment is container or straight plate.

12. preparation method as claimed in claim 11 is characterized in that described coating buffer is phosphate buffer or the phosphate buffered saline(PBS) of pH7.4, described Streptavidin concentration is 2 μ g/mL.

13. preparation method as claimed in claim 11 is characterized in that described hydrophobicity equipment is ELISA Plate.

14. preparation method as claimed in claim 11 is characterized in that described hydrophobicity equipment made by polystyrene, tygon or Polyvinylchloride.

15. preparation method as claimed in claim 11 is characterized in that selecting 0-10 ℃ described 0-40 ℃ of temperature range.

16. preparation method as claimed in claim 11 is characterized in that selecting 20-25 ℃ described 10-30 ℃ of temperature range.

Images