CN102004154B - Composition for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay - Google Patents

Abstract

The invention relates to a composition for detecting immune antibodies of rheumatoid arthritis by a dot immunogold filtration assay, which comprises a cyclic citrullinated peptide (CCP) composition, a high molecular polymer, a microporous filtering film, a water absorption medium and a carrier medium, wherein the high molecular polymer is combined on the microporous filtering film in a covalent form or a non-covalent form; the CCP composition comprises CCP I and CCP II and is combined with the high molecular polymer in a covalent form or a non-covalent form; and the molar ratio of the CCP I to the CCP composition is 0-1. The composition not only can realize the purpose of quickly detecting the immune antibodies of rheumatoid arthritis in vitro but also is convenient for patients to carry out self-diagnosis, and provides a reference for knowing the progression of the disease in time.

Description

The composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay

Technical field

The present invention relates to a kind of composition of detection type rheumatic arthritis antibody, relate in particular to a kind of composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay, realize quick, accurately detection to immune antibody of rheumatoid arthritis.

Background technology

Autoimmune disease mainly contains systemic lupus erythematous, rheumatoid arthritis, sjogren syndrome, dermatomyositis, polymyositis, system (multiple) property sclerosis, scleroderma etc. clinically, these diseases once were named as " connective tissue disease (CTD) ", all they were classified as rheumatism 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 can cause and organize irreversible damage, and cause very large misery to the patient.This sick sickness rate is worldwide 0.5-1%, and the sickness rate 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.Nearest epidemiology statistics, rheumatoid arthritis has year by year the trend that increases, arthralgia and movable difficulty, the daystart ankylosises such as its outstanding early clinical manifestation is symmetry redness and swelling of joints heat pain, common four limbs little joints, refer to that a near-end arthroncus, the palm refer to, wrist, elbow and ankle, in the afternoon alleviate gradually, symptom approximately has 20% patient subcutaneous nodule can occur outside the joint; The permanent symptom in late period that does not heal has in various degree joint deformity and tetanic, and the phenomenon such as function of joint forfeiture, also can damage internal organs and many histoorgans, causes the bone ischemic necrosis, and large to human consumption, disability rate is high.

The cause of disease of rheumatoid arthritis is still not fully aware of so far, and early clinical manifestation is not true to type yet.At home and abroad in clinical practice, the diagnostic tool commonly used of this disease is (the American College of Rheumatology of U.S. rheumatism association, ACR) similar rheumatism criteria for classification (the Arthritis Rheum 1988 that formulated in 1987,31,315-24), this standard more depends on the clinical disease that rheumatoid arthritis shows.But 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 diagnosis (Arthritis Rheum 2001,44, the 2485-91 of early stage rheumatoid arthritis; Neth J Med 2002,60,383-8).And the method for the treatment rheumatoid arthritis that uses at present is mainly to adopt the anti-inflammatory method, though this method can make the degree of arthroncus (swelling) and erosion (erosive) be slowed down, disease is cured.The common viewpoint of current people thinks, this disease adopt in early days the several different methods treatment can obtain best result for the treatment of (Autoimmunity Reviews 2006,6,37-41).Thus, serology certification mark with high specificity (Specificity) and highly sensitive (Sensitivity) is particularly necessary (the Clin Applied Immunol Rev 2004 that just seems of the early diagnosis before osteoarthrosis 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 USA 1995, 92, 7267-71), anti-neutrophil leucocyte cytoplasmic antibody (anti-neutrophil cytoplasmic antibodies, ANCA) (IntArch Allergy Immunol 1996, 109, 201-6), nuclear antigen antibody (anti-nuclearantigens, ANA) (Scand J Rheumatol 1986, 15, 185-92), anti-II Collagen Type VI antibody (anti-collagen type II) (J Immunol Methods 2001, 247, 191-203) with anti-glucose 6 phosphoric acid isomerases (anti-glucose-6-phosphate isomerase, anti-GPI) (Nat Immunol 2001, 2, 746-53) etc. also can be in suffering from the patient of other autoimmune disease, detect even (Clin Applied Immunol Rev 2004 in healthy individuals, 4, 239-62).Although some antibody wherein can be applied to the identification of rheumatism, and screen these antibody and also help disease surveillance and prediction, but because it lacks specificity to rheumatoid arthritis, the overwhelming majority in these antibody seldom is applied to rheumatoid arthritis early diagnosis (Clinica Chimica Acta 2004,350,17-34).

Ideal mark (Marker) for the rheumatoid arthritis early diagnosis should satisfy 4 standards at least: (1) highly sensitive, and the patient that can detect reaches high per-cent; (2) high specificity, the generation of limit erroneous positive findings as much as possible; (3) can be applicable to early diagnosis; (4) can predict some patient can develop into aggressiveness disease (erosive disease) (AutoimmunityReviews 2006,6,37-41; MEDICINE 2006,34,441-4).

Rheumatoid factors, polyclonal (rheumatoid factor, RF) antibody is applied in the ACR standard as Serology test, is acknowledged as the direct antibody of Fc structural domain on the IgG molecule.Although its detection sensitivity scope can reach 60-80%, its specificity for rheumatoid arthritis is also more general, is 60%.In addition, RF equally can be in other suffers from the patient of autoimmune disease, suffer from the patient of infectious diseases and in the 3-5% healthy population (the wherein the elderly of 10-30%) detect (Ann Rheum Dis 2003,62,261-3).

Anti-BiP (p68) antibody, anti-Sa antibody and antioxidant cyclic guanidine propylhomoserin protein antibodies (APF, AKA, anti-filaggrin and anti-CCP) have higher specificity to rheumatoid arthritis.64% Patients With Rheumatoid Arthritis can produce direct antibody for BiP, report that in addition it has the specificity of height to this disease, but do not have at present Data support BiP having effect aspect the prediction rheumatoid arthritis, and the BiP antibody of these reports also needs products for further to pass through independently clinical study to be confirmed (Clinica Chimica Acta 2004,350,17-34).Anti-Sa antibody is to stem from the middle of the extract of people's spleen and placenta, molecular weight be 50kDa albumen (Internal Medicine 2005,44,1122-6).Separately having research to disclose Sa antigen can be up to 92-99% for the specificity of rheumatoid arthritis, but its sensitivity seems generally, is only 30-40% (J Rheumatol 1994,21,1027-33; J Rheumatol 1999,26,7-13).

Antiperinuclear factor (antiperinuclear factor, APF) is disclosed in 1964 first.These autoimmune antibodies have specificity 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 Dis 1964,23,302-5).

The investigator had described a kind of rheumatoid arthritis specificity keratoprotein antibody in 1979, it can directly suppress stratum corneum epithelial cell keratinization, and be anti-keratoprotein antibody (anti-keratin andtibody with the title of this antibody-like is tentative, AKA) (BMJ 1979,2,97-9).Studies confirm that afterwards, although the discovery of AKA and APF is mutually independently, both directly corresponding antigen be that consistent (J.Clin.Invest 1995,95,2672-9).Many researchs show that further AKA and APF have many common characteristics, they all for RA patient show the height specificity (Internal Medicine 2005,44,1122-6).Filaggrin (filamentaggregating protein) is a kind of crosslinked thread body of Keratin sulfate mutually, effect is to form very firm cytoskeletal structure, its be also AKA and APF public antigen (Clin AppliedImmunol Rev 2004,4,239-62).Wherein, the target antigen of APF is profilaggrin, and the antigen of AKA be filaggrin (J Clin Invest 1995,95,2672-9).

Although AKA and APF have specificity 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 practice, obtain pure and guanidine radicals content has repeatable antigen owing to being difficult to separate, add its detection means not easy, and the fluorescence immunoassay testing process need to expend larger workload, be difficult to form stdn between the laboratory thereby make, the main stream approach that causes AKA and APF not to become rheumatoid arthritis detecting (Clin Applied Immunol Rev 2004,4,239-62).

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 rheumatoid arthritis serum.Because citrulline is not primary amino acid and can't adding in protein translation, so usual method 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 keep high specificity (greater than 90%), also greatly improved 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 polypeptide is replaced not reaction generation fully with other amino acid.These results show, citrulline be partly determine can by the antigenic determining factor of AKA and APF antibody recognition (J Clin Invest 1998,101,273-81).

Other investigator finds that the rectilinearity polypeptide that will contain citrulline forms the mode energy analogy β-corner structure of ring structure by disulfide linkage (S-S key), thereby imitate β-corner structure of initial antigenic determinant, and can increase avidity (the FASEB J 1995 of polypeptide-antibody, 9,37-42; Mol Immunol 1985,22,1255-64).Thus, a kind of cyclic peptide by synthetic out, after it changes two Serines on the main epi-position of filaggrin into halfcystine, formed by the disulfide linkage cyclisation again, be first-generation cyclic citrullinated peptide (the first generation cyclic citrullinatedpeptide, CCP1) (Internal Medicine 2005,44,1122-6).In the citrulline peptide that the investigator is comprised of 19 amino-acid residues, two Serines replace with halfcystine and form the disulfide linkage that has analog structure with β-corner, obtain synthetic CCP, and the detected result of CCP1 and straight line peptide is contrasted.The result demonstration, employing CCP1 is that the polypeptide of antigen detects RA patient's anti-CCP antibody with the ELISA method, sensitivity is that antigen is significantly increased with rectilinearity citrulline peptide, be respectively 68% and 49%, both specificity similar (Arthritis Rheum 2000,43,155-63).The people such as Mu Rong detect the mode of RF and AKA antibody, APF and anti-CCP antibody in 266 routine RA patients and 186 routine collator's serum, assessed the meaning of antifilaggrin antibody group (anti-filaggrin antibodies, AFAs) with the RF joint-detection.Because the susceptibility of AKA and APF is too low, the simultaneous determination with RF and anti-CCP antibody more practical (Peking University's journal (medicine) 2005,37,894) clinically.Research is also found, can also be as important indicator of the early stage patient of rheumatoid arthritis by detecting anti-CCP antibody, for this class crowd's early prevention and treatment provides with reference to (J India Rheumatol Assoc 2004,12,143-6), its can also predict the aggressiveness disease (Clin Applied Immunol Rev 2004,4,239-62).

In containing the reaction pattern of different citrulline polypeptides, the rheumatoid arthritis serum detected representation goes out huge mutability.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 protein (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 Invest 1998,101,273-81).Based on the attribute of filaggrin, it be considered to simulate the citrulline epitope natural stock (Clin Applied Immunol Rev 2004,4,239-62).

S-generation CCP (CCP2) detection method results from 2002, obtain the s-generation CCP irrelevant with filaggrin by peptide library and the rheumatoid arthritis serum screening that will contain citrulline, it has epi-position (Report on the 5th 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 after same group of patient's test, and CCP2 has not only kept higher specificity (96%), and its sensitivity for analysis also increase significantly (Ann.Rheum.Dis.2005,64,1510-2).Many investigators studies have shown that recent five years, resist the sensitivity of the test that contains citrulline polypeptide to have higher mutability, and scope is at 40%-94% (Clin.ExpRheumatol.2005,23 (Suppl39), 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 Chem 2007 such as Bizzaro N, 53,1527-33) and people (the Clinica Chimica Acta 2007 such as Lutteri L, 386,76-81) compare for the s-generation of using on present market and third generation CCP test kit (Kit) respectively, found that, her Nova is diagnosed in three kinds of detection methods of (InovaDiagnostics) company exploitation, its CCP3 test 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, specificity may be subject to the impact of protein or assorted peptide sequence, therefore, to this both, 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 measuring method quality.

The people such as Lutteri L (Clinica Chimica Acta 2007,386, another value of anti-CCP has also been found in research 76-81), and they point out, and IgM-RF is the sensitiveest mark, can find in 77.9% rheumatoid arthritis patients.Must be noted that as 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 Case definition.IgM-RF is considered to slightly be weak aspect the disease specificity.Use IgM-RF to detect rheumatoid arthritis patients, wherein have the people of 13-19% negative for RF, by comparison, all positive when these patients use anti-CCP method to detect.

The inherited genetic factors of rheumatoid arthritis can affect appearance and the generation of citrulline albumen, also can affect antibody product for these modified proteins (Clinica Chimica Acta 2004,350,17-34).A series of correlative studys show, rheumatoid arthritis and some HLA-DR allelotrope have close ties, and especially (Cell 1996,85,307-10) for HLA-DRB1*0401 and HLA-DRB1*0404.Compare with containing accordingly arginic polypeptide, the polypeptide citrullineization just can be combined better with HLA-DRB1*0401 and two kinds of " bag shape " antigens of HLA-DRB1*0404 (J India Rheumatol Assoc 2004,12,143-6).In the crowd of different areas, these allelotrope are also slightly different.DRB1*0401 mainly appears in 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, particularly outstanding in DRB1*0401/DRB1*0404 heterozygous genes type.The development that can increase the rheumatoid arthritis state of an illness after DRB1*0403, DRB1*0406 and DRB1*0407 and DRB1*0401, * 0404 or * 0101 assortment of genes may.As long as DRB1*0404 and DRB1*0408 are present in the white race.DRB1*0405 is seldom in the crowd of Northern Europe, and is but extremely general in the crowd of Asia and ring Mediterranean country.DRB1*0101 mainly is present in Northern Europe and North America crowd.In American Indian group be DRB1*1402 and * 1406 (Hum Immuno 2000,61,1254-1261).Although these genes relevant to rheumatoid arthritis are had nothing in common with each other in the crowd of each department, but research finds to have shared epi-position (shared epitope in the third high degree variable region (HV3) of DRB1, SE), be all QKRAA, QRRAA or RRRAA (ClinicaChimica Acta 2004 at 70-74 amino acids residue, 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 allelotrope and anti-CCP2 antibody has close inner link (Arthritis Rheum 2000,50,2113-21; Arthritis ResTher 2004,6, R303-8).Verified, this association is to share 70-74 amino acids residue (Q/R, the K/R of epi-position β chain due to the MHC molecule, R, A, A) formation the 4th grappling bag (P4), itself and electric neutrality or negative charge amino acid have high affinity (J Immuno 2003,171,538-541).After converting electroneutral citrulline under the effect of PAD enzyme, the posttranslational modification protein/polypeptide avidity that obtains is far longer than the avidity of arginine and P4, and can activate CD4 when positively charged arginine ⁺The T cell, these " the special T cells of citrulline " can help generation (J Immuno 2003,171, the 538-541 of anti-citrulline albumen (polypeptide) antibody; Arthritis Rheum1987,30,1205-13; Rheum Dis Clin North Am 1992,18,741-59; ArthritisRheum 2005,52,1063-68).These results show that the DBR1 allelotrope with shared epi-position can activate the rheumatoid arthritis patients autoimmune response.

Comprehensive present result of study, anti-CCP antibody has higher specificity to RA, RA can be distinguished mutually to other disease similar to RA, this antibody-like is present in most patient bodies, just can detect at the early stage of disease, help the prediction to disease, can be detected by simple mode (J India Rheumatol Assoc 2004,12,143-6).

The external ELISA detection that the CCP peptide is used for rheumatoid arthritis has obtained broad research, and many business-like detection kit are also arranged.Single ELISA detects needs larger sample number usually, detects the time used also longer, and its testing process also needs the personnel of specialty to operate, and is larger to the dependency degree of medical institutions, thereby is unfavorable for that common sufferer is to the self-service monitoring of the course of disease.

Radioactive colloidal gold gold mark lateral chromatography method also has application in immune antibody of rheumatoid arthritis detects, the method utilizes capillarity to make test sample flow through successively marker, detection zone and control region.Due in whole testing process, the combination of antibody molecule to be measured and traget antibody/antigen is in the middle of the process of adsorption and desorption all the time, and in the middle of practical application, its detection sensitivity is subject to the impact of detection film used, is unfavorable for the extensive clinical application of product.

Summary of the invention

The object of the present invention is to provide a kind of composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay, comprise the CCP peptide combinations, with the covalently bound high molecular polymer of CCP peptide, millipore filtration and water sucting medium, realize to immune antibody of rheumatoid arthritis fast, accurately detect.

The CCP peptide is a kind of interconnection by amido linkage by 5-50 amino acid, and the ring structure organic molecule that forms by covalent linkage between non-conterminous two amino acid side chains.Amino acid is the organic compound that contains simultaneously amino and carboxyl in molecular structure, is selected from glycine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, phenylalanine, tryptophane, tyrosine, aspartic acid, Histidine, l-asparagine, L-glutamic acid, Methionin, glutamine, methionine(Met), arginine, Serine, Threonine, halfcystine and proline(Pro).The sequence of CCP peptide is as: U.S. Patent application 2006/0039924A1, Chinese patent application 02133648.2,02824647.0,200410093354.1,200510044046.4 and document J.Clin.Invest.1998, the polypeptide of putting down in writing in 273-281.Have at least an arginine side chain to be modified in the middle of described CCP peptide sequence, the arginine side chain of modification has the structure shown in 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 ₃Or NHCH ₃N is 2,3 or 4; When G1 is NH, G2 is NH ₂The time, G3 is O, NHCH ₃Or NHCH ₃

CCP peptide combinations of the present invention comprises CCP peptide I and CCP peptide II, and the mol ratio ratio of CCP peptide I and CCP peptide combinations is 0-1;

CCP peptide I holds to hold to C from N and comprises following aminoacid 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 side chains to be modified;

CCP peptide II holds to hold to C from N and comprises following aminoacid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Xaa5-Xaa6-Xaa7-X aa8-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 side chain to be modified.

Another kind of CCP peptide combinations of the present invention, comprise CCP peptide I and CCP peptide II, described CCP peptide I holds to hold to C from N and comprises following aminoacid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-Gly-Arg-Xaa5-Xaa 6-Xaa7-Xaa8-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 Ser or Arg; Xaa6 is Arg or Leu; Xaa7 is Ala or Ile; Xaa8 is Ala or Arg, wherein has at least two arginine side chains to be modified;

CCP peptide II holds to hold to C from N and comprises following aminoacid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Arg-His-Arg-Xaa5-Xaa 6-Xaa7-Xaa8-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 Ser or Arg; Xaa6 is Arg or Leu; Xaa7 is Ala or Ile; Xaa8 is Ala or Arg, wherein has at least two arginine side chains to be modified.

Another kind of CCP peptide combinations of the present invention, comprise CCP peptide I and CCP peptide II, described CCP peptide I holds to hold to C from N and comprises following aminoacid sequence: His-Gln-Cys-His-Gln-Phe-Xaa1-Xaa2-Xaa3-Gly-Xaa4-Xaa5-Xaa 6-Xaa7-Xaa8-Cys-Gly

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

CCP peptide II holds to hold to C from N and comprises following aminoacid sequence: His-Gln-Cys-Ala-Arg-Phe-Xaa1-Xaa2-Xaa3-His-Xaa4-Xaa5-Xaa 6-Xaa7-Xaa8-Cys-Gly,

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

Another kind of CCP peptide combinations of the present invention comprises CCP peptide I and CCP peptide II, and described CCP peptide I holds to hold to C from N and comprises following aminoacid 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 CCP peptide II holds to hold to C from N and comprises following aminoacid sequence: His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Xaa5-His-Xaa6-Xaa7-X aa8-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; The Arg of Xaa5 for modifying; 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.

The cyclisation of CCP peptide of the present invention can realize by the mode that non-conterminous two amino acid side chains on sequence form covalent linkage.Have at least an arginine residues to be modified in the middle of its sequence, as: be modified into citrulline residue (Cit).

Can adopt between two non-conterminous cysteine side chain sulfydryls the mode that forms disulfide linkage to realize the cyclisation of polypeptide in the CCP peptide sequence, as: form disulfide linkage between the 3rd and sixteen bit halfcystine, and obtain final CCP peptide.For the polypeptide with same acid sequence, its formed annular polypeptide also has larger difference for the binding ability of rheumatoid arthritis antibody.As: the relative position of two non-conterminous halfcystines in peptide sequence plays an important role to sensitivity and the specificity of formed CCP peptide and rheumatoid arthritis antibodies, and has influence on the result of detection.Those skilled in the art can be by after forming in CCP peptide sequence of the present invention halfcystine and other one or more amino acid and exchanging, carry out high flux screening in conjunction with combinatorial chemistry and proteomics, searching out other two non-conterminous halfcystine positions in peptide sequence, and then make itself and rheumatoid arthritis antibodies also have relatively high sensitivity (〉=60%) and specificity (〉=90%).

CCP peptide combinations of the present invention, the aminoacid sequence that described CCP peptide I comprises is selected from one of SEQID No:1-118; The aminoacid sequence that described CCP peptide II comprises is selected from one of SEQ IDNo:119-261, as: the CCP peptide combinations that is formed by SEQ ID No:1 and SEQ ID No:119.

CCP peptide combinations of the present invention, the aminoacid sequence that described CCP peptide I comprises is selected from one of SEQID No:1-118; The aminoacid sequence that described CCP peptide II comprises is selected from one of SEQ ID No:1-118, and CCP peptide I is not identical with the sequence of CCP peptide II.As: the CCP peptide combinations that is formed by SEQ ID No:1 and SEQ ID No:118.

CCP peptide combinations of the present invention, the mol ratio ratio of CCP peptide I and CCP peptide combinations is 0.1-0.9; Can select 0.2-0.8, as: 0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.55,0.60,0.65,0.70,0.75 or 0.80, preferentially select 0.5, i.e. CCP peptide I: CCP peptide II=1: 1.

High molecular polymer (polymer) refers to molecular weight greater than the compound of 2,000Da, is intermolecularly interconnected via covalent linkage by structure unit (structural unit) or monomer.It can be the different monomer of a plurality of structures or by the polymer of the formed structure unit of different structure molecule, it can be also the oligomer of same monomer, can also be directly or indirectly to be combined or polymerization by plural polymkeric substance, as: segmented copolymer PEG-PLA or PLA-PEG-PLA that polyoxyethylene glycol (PEG) and poly(lactic acid) (PLA) form.The morphological structure of polymkeric substance can be linear pattern (linear), ramiform (branch/multi-arm) or branch type (dendrimer).Described polymkeric substance is selected from polyoxyethylene glycol, polyamino acid, polynucleotide, poly(lactic acid), polylysine, poly-imines and 10 saccharans (polysaccharide) that above monose forms by glycosidic link, as: starch and hydrolysate thereof, Mierocrystalline cellulose, chitin or dextran.

Branch type polymkeric substance (dendrimer) is generally poly-amino amine (poly (amidoamine), PAMAM), can be initiator preparation (Clin.Chem., 1994,40 by ammonia or quadrol respectively, 1845-1849), also have and use amino acid be polymerized (J Immuno Method, 1996,196,17-32), as Methionin and arginine.Resulting polymers has active group, and as amino, hydroxyl or carboxyl, aggregation number is more, and its molecular weight is larger, and functional group is also more, energy in conjunction with also corresponding increase of molecular amounts.

Polyoxyethylene glycol is selected from the polyoxyethylene glycol that two ends are not all sealed or an end seals, and blocking groups is selected from the alkyl of C1-C30, the lipid acid of C1-C30 or glycosyl.Polyoxyethylene glycol is selected from molecular weight 2,000Da-100, and 000Da can select 2,000Da-50, and 000Da generally selects 5,000Da-50,000Da.Further, described blocking groups is selected from the alkyl of C1-C20, usually selects the alkyl of C1-C10, preferentially selects methyl, ethyl or propyl group.

Polyamino acid comprises the oligothiophene molecule that is polymerized by same amino acid, also comprises the poly molecule that is polymerized by differing molecular, and protein or polypeptide are its natural existence forms.

CCP peptide of the present invention and high molecular polymer covalent attachment (covalent conjugate), the covalent linkage of formation is as: amido linkage (amide bond), urine key (urine bond), ester bond or disulfide linkage.The CCP peptide combines with high molecular polymer by its amino acid side chain, N-terminal amino or C-terminal carboxyl.This combination can by as: the reagent such as N-hydroxy-succinamide, maleic anhydride or third/aldehyde-base are combining amino acid side chain, N-terminal amino or C-terminal carboxyl after activating with high molecular polymer, also can be suitable for aforementioned identical or different mode, after first activating the group of high molecular polymer, then combine with the CCP peptide.Other also have by the modes such as hydrazone (hydrazone), oxime (oxime), sulfydryl or thiazolidine (thiazolidine) in conjunction with (J.Immuno.Method, 1996,196,17-32).

Combination between above-mentioned CCP peptide and carbohydrate molecule can be referring to document: J.Pharma.Sci.87,326-332; Adv.Drug deliv.Rev., 6,103-131; Adv.Drug deliv.Rev., 13,251-267.The combination of polyoxyethylene glycol and above-mentioned CCP peptide is referring to document: Adv.Drugdeliv.Rev., 28,275-299; Adv.Drug deliv.Rev., 54,453-609; Adv.Drugdeliv.Rev., 60,1-88.Because the CCP peptide polymer is same or similar in conjunction with related chemical reaction, those skilled in the art can be applied to these combinations the CCP peptide in the middle of reaction that other polymkeric substance is combined.

The CCP peptide connects can also pass through connexon (space linker) with the mode (as: organic synthesis) and high molecular polymer indirect joint of chemical reaction, as: in chemosynthesis CCP peptide process, connexon first is connected in the CCP peptide, and then is connected with high molecular polymer by this connexon.This class connexon is selected from the polypeptide of amino acid length 1-500 or protein, molecular weight at 100-2, the macromole of 000Da or organic molecule.As: molecular weight is 200-2, polyoxyethylene glycol (NOF Corporation), the NH of the two ends activation of 000Da ₂(CH ₂) _nCOOH, SH (CH ₂) _nThe lipid acid of the C1-C30 of COOH or two ends activation (J.Gene.Med., 2005,7,604-612).Can to be CCP peptide N-terminal or C-terminal be connected with activating group on above-mentioned connexon in the chemistry connection, also can be connected with activating group on connexon by the some amino acid side chains of CCP peptide.Form covalent linkage between connexon and antigen, (CH=N-NH-), the oxime key (CH=N-O-) and disulfide linkage as: amido linkage, urine key, ester bond, thioether bond, hydrazone key.

The disaccharides (disaccharide) (as: sucrose, lactose or maltose) that organic molecule is selected from each amino acid, all kinds of monose, each biostearin (Vitamin), formed by glycosidic link by two monosaccharide units, the oligosaccharide (oligosaccharide) (as: oligomeric isomaltose, xylo-oligosaccharide or oligomeric galactose) that is formed by glycosidic link by 2-10 monose and molecular weight are at 100-2, the organic molecule of 000Da, as: vitamin H.

CCP peptide of the present invention is combined with non covalent bond with high molecular polymer, as: will be combined with CCP peptide and the non-covalent combination of avidin of vitamin H on sequence.Avidin (Avidin) is that a kind of molecular mass is 68,000Da, and pI is a kind of alkaline glycoprotein of 10.5, claims again avidin, and content is abundanter in Ovum Gallus domesticus album, therefore generally extracts from albumin.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 vitamin H binding sites, has high-affinity with vitamin H.

A kind of composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay comprises CCP peptide combinations, high molecular polymer, millipore filtration, water sucting medium and mounting medium.Wherein, the CCP peptide combinations comprises CCP peptide I and CCP peptide II, and CCP peptide combinations and high molecular polymer covalently or non-covalently are combined the CCP peptide polymer that forms and covalently or non-covalently are incorporated on millipore filtration.Millipore filtration is placed or is fixed on water sucting medium, then is positioned in mounting medium, and a side of described mounting medium offers the through hole relative with millipore filtration, so that point sample detects.

Millipore filtration is nitrocellulose filter.Membrane pore size is at 0.1-15 μ m, as: 0.1 μ m, 0.2 μ m, 0.45 μ m and 0.5 μ m etc., preferentially select 0.45 μ m.Millipore filtration (bottomless lining form) by pure nitrocellulose consists of has very large surface-area and very high protein adsorption quantity.Its advantage as: be that the longitudinal stream test makes, eliminate the problem that rises and cause by capillary; Less aperture, result is accurate, lower non-specific adsorption, higher sensitivity; The surface-area that increases, high binding ability, highly sensitive obtains extraordinary signal to noise ratio; 100% pure nitrocellulose has guaranteed best adsorptive power.In numerous nitrocellulose filters, with the BA series of Whatman company, as: BA79, BA83, BA85, BA-S83 and BA-S85 are the most commonly used.

Thieving paper is the paper take Mierocrystalline cellulose as main component.

Mounting medium provides support for detecting, as: comprise base plate and the lid that is buckled on base plate, offer the through hole over against millipore filtration on lid, so that point sample.The material of carrier is selected plastics or glass.

plastics should be understood to all or part by carbon and oxygen, hydrogen, nitrogen and other organic and inorganic elements chemical combination form, become solid in the final stage of making, some stage is that (plastic material is becoming before the finished product liquid in the mill, must want and to flow in some stage), thereby can heat or plus-pressure, or both and the mode of use, make it form different shape, any in this huge and protean material same clan, as resin, thermosetting resin, derivatived cellulose etc., there are numerous repetition atom or molecule in the molecular structure of a long-chain.Plastics comprise synthetic or nature organic materials.Make the plastics used of mounting medium or use a kind of plastic material, or use several plastic materials physically stack or addition after compound.

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

Concrete, mounting medium is made by polystyrene, polyethylene, polyvinyl chloride or polycarbonate etc.

CCP peptide combinations and high molecular polymer covalent attachment form the CCP peptide polymer, and on it, polymer substance is incorporated into millipore filtration, make CCP peptide polymer on every cubic centimetre of filter membrane greater than 1 μ g, can be 2 μ g-400 μ g/cm ³, generally select 50 μ g-300 μ g/cm ³, preferentially select 50 μ g-150 μ g/cm ³, as: 50 μ g/cm ³, 80 μ g/cm ³, 100 μ g/cm ³, 110 μ g/cm ³, 130 μ g/cm ³With 150 μ g/cm ³Wherein, the weight in average molecular weight of high molecular polymer is preferentially selected 20kD-100kD greater than 2,000Da.

The composition of another kind of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay comprises CCP peptide combinations, high molecular polymer, millipore filtration, water sucting medium and mounting medium.Wherein, the CCP peptide combinations comprises CCP peptide I (as: SEQ ID No:1) and CCP peptide II (as: SEQID No:118), the CCP peptide polymer that CCP peptide combinations and high molecular polymer covalent attachment form covalently or non-covalently is incorporated on millipore filtration, and the CCP peptide polymer content of combination on millipore filtration is 50 μ g-150 μ g/cm ³, as: 50 μ g/cm ³, 80 μ g/cm ³, 100 μ g/cm ³, 110 μ g/cm ³, 130 μ g/cm ³With 150 μ g/cm ³

The composition of another kind of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay comprises CCP peptide combinations, high molecular polymer, millipore filtration, water sucting medium and mounting medium.Wherein, the CCP peptide combinations comprises CCP peptide I and CCP peptide II, the non-covalent CCP peptide polymer in conjunction with forming of CCP peptide combinations and high molecular polymer covalently or non-covalently is incorporated on millipore filtration, and the CCP peptide polymer content of combination on millipore filtration is 50 μ g-150 μ g/cm ³, as: 50 μ g/cm ³, 80 μ g/cm ³, 100 μ g/cm ³, 110 μ g/cm ³, 130 μ g/cm ³With 150 μ g/cm ³

The composition of another kind of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay comprises CCP peptide combinations, high molecular polymer, millipore filtration, water sucting medium and mounting medium.Wherein, the CCP peptide combinations comprises SEQ ID No:1 and SEQ ID No:118, the CCP peptide combinations covalently or non-covalently is incorporated on millipore filtration by vitamin H and the non-covalent CCP peptide polymer in conjunction with forming of avidin that connects on it, and the CCP peptide polymer content of combination on millipore filtration is 50 μ g-150 μ g/cm ³, as: 50 μ g/cm ³, 80 μ g/cm ³, 100 μ g/cm ³, 110 μ g/cm ³, 130 μ g/cm ³With 150 μ g/cm ³

The composition that is used for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay of the present invention, its high molecular polymer preferentially is selected from bovine serum albumin (bovine serum albumin, BSA), human serum albumin (Humal serum albumin, HSA), albumin rabbit serum (RSA), chicken ovalbumin (ovalbumin, OVA) keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH), dendrimer, polylysine, polyoxyethylene glycol, avidin or antibody.

Dot immuno gold filtration assay method (Dot-Immunogold Filtration Assay, DIGFA) detect, first antibody/antigen is fixed in detection zone (the test line on millipore filtration, the T line), to check antibody to be fixed in control region (control line, the C line), then drip successively sample to be tested on film, through reagent such as the Radioactive colloidal gold of mark and washingss, realize the detection to target molecule in sample.

According to needed granular size, there is various ways can prepare Radioactive colloidal gold (Colloidal Gold) (Methods for Synthesis of Colloidal Gold.In " ColloidalGold:Principles; Methods; and Applications " in the middle of the laboratory, Academic Press, Inc.SanDiego, Vol.1,1989).Nearly all method is all to use reductive agent gold ion to be converted into the simple substance gold in controlled scope.Reductive agent used comprises sodium borohydride, yellow phosphorus, ethanol, xitix, Trisodium Citrate and citric acid-Weibull.The people such as Frens (J.Microsc. (Oxford) 1981,123,201) are with hydrochloro-auric acid (HAuCl ₄) be the most frequently used method for raw material uses Trisodium Citrate to prepare Radioactive colloidal gold.

Before adding reductive agent, contain 100% gold ion in solution.After adding reductive agent, rising sharply appears in the content of GOLD FROM PLATING SOLUTION atom immediately, until it reaches supersaturation.And then in a process that is known as coring, aggegation occurs, form the icosahedron gold core of the central authorities that are made of 11 gold atoms in the coring site.Reductive agent is more, and the nucleus of generation is more, thereby the gold grain that produces is also more.In a solution that contains the prearranged number gold trichloride, the number in the coring site of formation is more, and the size of final each gold grain that forms is just less.Thereby the amount of the large I of the particulate reductive agent that passes through to add is come finely regulating.If working condition is through optimizing, all coring sites can occur simultaneously in moment, made formed gold grain size all (being single decentralized) in full accord.

Gold grain has formed Radioactive colloidal gold in the middle of being suspended in liquid.Due to residual in solution, negative ion is arranged, thus make each particle by negative charge layer institute around.This charge layer is referred to as Zeta electric potential, and it can make mutually exclusive between gold grain and be suspended in liquid.Along with the total ion concentration in the middle of solution changes, Zeta electric potential also can compressed or expansion.

Macromole aglucon (as: protein, polypeptide or antibody) being adsorbed on Radioactive colloidal gold by electrostatic force (electrostatic) and hydrophobic interaction.During traget antibody, can not there be excessive antibody to be marked to exist on Radioactive colloidal gold.Because excessive free antibody can with antibody and the antigen generation competing reaction on Radioactive colloidal gold, cause occurring false negative, and excessive antibody protein can have influence on the stability of marker.The top condition of protein and Radioactive colloidal gold combination is near isoelectric point of protein.Albumen was adsorbed in gold surface by following mechanism within several seconds: gold particle institute electronegative with albumen in be with positive electric charge amino acid (as: Methionin) to attract each other; Albumen is by the hydrophobic adsorption between tryptophane and gold particle surface; The coordination valence of the sulfenyl of the halfcystine in albumen with between gold particle is combined.

In process that Radioactive colloidal gold is combined, the pH that controls aglucon and Radioactive colloidal gold is most important.Before combination, should be with both pH regulator to a little more than the aglucon iso-electric point.Lower than the pKi of aglucon, the cohesion that aglucon is induced can occur, and higher than the pKi of aglucon, can limit adsorptive capacity due to the electrical charge rejection between aglucon and Radioactive colloidal gold.The situation of this dependence pH is general only to be occured on the protein aglucon, and the stablizer that can adopt polyoxyethylene glycol to be used as Radioactive colloidal gold solves.The best pH value of association colloid gold can be determined (Experimentia 1975,31,1147) with a kind of simple method.For most of antibody, pH7.5 is one and has blanket value.The hydrochloric acid of salt of wormwood, Repone K and dilution can be used for the adjusting of Radioactive colloidal gold pH.The Radioactive colloidal gold combination for preparing (colloidalgold conjugate) can be collected by low-speed centrifugal or cut stream filter membrane, for fear of a large amount of losses of combination, and the most handy BSA solution pre-treatment cut stream filter membrane.

Along with the combination of differential protein, the Radioactive colloidal gold combination must be stablized with suitable reagent, usually uses BSA, gelatin, polyoxyethylene glycol or casein.Use stablizer that dual-use function is arranged, the one, it can not reduce nonspecific reaction with the site of specific proteins combination by the sealing colloid surface; The 2nd, it can help the formation of more stable suspension.The most frequently used sanitas of Radioactive colloidal gold is 0.1% triazo-compound.

When traget antibody, particle diameter the colloid gold particle of 40-100nm can more smooth mark to IgG antibody.After antibody labeling, can be with the protein solution absorbancy of ultraviolet spectrophotometer before and after 280nm place measures colloid gold label IgG, with the calculating mark rate, when mark rate greater than 60% the time, this colloid gold label thing could for detection of.The colloid gold particle of 40nm provides maximum visuality and minimum sterically hindered.If be used for the molecular weight of tagged molecule less than 160kDa, the 20nm colloid gold particle is more suitable for.The 20nm colloid gold particle is generally used for labelled streptavidin, A albumen and antigen, and these molecular weight that are labeled albumen are less than 60kDa.

The light absorption Radioactive colloidal gold has a single optical absorption peak in visible-range, the wavelength of this optical absorption peak (λ max) is in the 510-550nm scope, change with the colloid gold particle size, the λ max deflection long wavelength of macrobead Radioactive colloidal gold, otherwise the λ max of small-particle Radioactive colloidal gold is partial to the short wavelength.Colour generation molecule colloid takes on a red color, but the colloid colour generation of different sizes has certain difference.Minimum Radioactive colloidal gold (2-5nm) is orange, and medium sized Radioactive colloidal gold (10-20nm) is burgundy, and the Radioactive colloidal gold of larger particles (30-80nm) is mauve.According to these characteristics, but the size of the color guestimate gold grain of the Radioactive colloidal gold that detects by an unaided eye.

Because different its maximum absorption wavelengths at visible region of particle diameter (λ max) of Radioactive colloidal gold are also had any different, can adopt spectrophotometer scanning λ max to estimate the particle diameter of colloid gold particle.Electron microscopic observation can be measured the median size of Radioactive colloidal gold more accurately, as: immerse in colloidal gold solution with the nickel screen that is covered with the Formvar film of anticipating, enchashment is placed on air drying or 37 ℃ of baking boxs are dried, then observe under transmission electron microscope, mainly observe whether uniformity of the size of gold grain and particle.

In the present invention, the marker that is used for the mark Radioactive colloidal gold is selected from SP, streptococcus protein G, streptococcal protein L and can identifies the antibody of immune antibody of rheumatoid arthritis, as: the mouse-anti human IgG.

The beneficial effect that technical solution of the present invention realizes

With other detection method, as: ELISA compares, and the present invention can realize the vitro detection of immune antibody of rheumatoid arthritis.It is basically identical that the sensitivity of its antibody test and specificity are compared with ELISA, enlarged the applicable crowd who detects.

Dot immuno gold filtration assay method of the present invention not only can realize that the rapid in-vitro of immune antibody of rheumatoid arthritis detects, and has improved the accuracy that detects, and is convenient to the self-service diagnosis of patient, provides reference in time understanding PD.

Term involved in the present invention is identical with its general concept.

The arginine that described " Arg of modification " modifies for side chain, its structure is suc as formula shown in I.

Described " CCP peptide " refers to that with described " high molecular polymer " covalent attachment CCP peptide and high molecular polymer are directly in conjunction with forming covalent linkage, or between CCP peptide and high molecular polymer by the indirect combination of connexon, between its CCP peptide and connexon, between connexon and high molecular polymer, formed key is covalent linkage.

Described " CCP peptide " refers to that with described " high molecular polymer " non-covalent combination CCP peptide and high molecular polymer are directly in conjunction with forming non covalent bond, or between CCP peptide and high molecular polymer by the indirect combination of connexon, between its CCP peptide and connexon, between connexon and high molecular polymer, formed key has one at least for non covalent bond, as being connected between: avidin (high molecular polymer) and vitamin H (connexon).Non covalent bond is as hydrophobic interaction, ionic linkage, hydrogen bond or Van der Waals force etc.

Described " C1-C10 alkyl ", " C1-C20 alkyl " and " C1-C30 alkyl " refer to the straight or branched alkyl, as: methyl, ethyl, propyl group, sec.-propyl, butyl or isobutyl-etc.Wherein, letter C represents carbon atom, and numeral is positive integer thereafter, as: 1,2,3,4 or 5 etc., the contained carbon atom number of expression group.

Described " lipid acid of C1-C30 " refers to saturated or undersaturated straight or branched carbochain, has a carboxyl on it at least, as: formic acid, acetic acid, propionic acid, oleic acid, linolic acid, Palmiticacid, therapic acid, succsinic acid and oxysuccinic acid etc.Wherein, letter C represents carbon atom, and numeral is positive integer thereafter, as: 1,2,3,4 or 5 etc., the contained carbon atom number of expression group.

Described " glycosyl of C3-C30 ", as: triose base, tetrose base, pentose base and hexose-based etc.Wherein, letter C represents carbon atom, and numeral is positive integer thereafter, as: 3,4 or 5 etc., the contained carbon atom number of expression group.

Described " sensitivity " and " susceptibility " all refers in the middle of the detection of antigen antagonist, and because of the detected result difference that the change of test sample produces, its account form is: true positives/(true positives+false negative).

Description of drawings

Fig. 1 is for being used for the structural representation of dot immuno gold filtration assay method one embodiment of the present invention.

Embodiment

Below describe technical scheme of the present invention in detail.The embodiment of the present 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 modify 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 claim scope of the present invention.

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

Embodiment 1CCP peptide I and CCP peptide II's is synthetic

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

The formation method of disulfide linkage 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 Synthesis Protocols (Methods in Molecular Biology), Humana Press, 1994, p91-169.

In the present embodiment, various CCP peptides are synthetic by gill biochemical (Shanghai) Co., Ltd., and concrete sequence is as follows:

CCP peptide I:His-Gln-Cys-His-Gln-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Al a-Ala-Cys-Gly, on it, the 3rd and sixteen bit Cys form disulfide linkage by sulfydryl, and make polypeptide form ring texture, and can simulate β-corner structure.

CCP peptide II:His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Cit-His-Cit-Arg-Leu-I le-Arg-Cys-Gly, on it, the 3rd and sixteen bit Cys form disulfide linkage by sulfydryl, and make polypeptide form ring texture, and can simulate β-corner structure.

The combination of embodiment 2CCP peptide I and CCP peptide II and high molecular polymer

2mg carrier proteins BSA is dissolved in 200 μ l deionized waters, and 2mgCCP peptide I and CCP peptide II (mol ratio is 1: 1) are dissolved in 0.5ml coupling buffer (0.1mol/L MES, 0.9mol/LNaCl, 0.2g/L NaN ₃, pH4.7) in.0.5ml CCP solution is added 200 μ l carrier proteins solution.The 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) solution that is 10mg/ml with 1ml concentration adds in the mixed solution of CCP and BSA, mixing gently, at room temperature react 2h, with 0.01mol/L pH7.4 phosphoric acid buffer in 4 ℃ of dialysis 3 days, after packing 4 ℃ of preservations.

Embodiment 3IgG mark Radioactive colloidal gold

Heating 99ml ultrapure water adds the HAuCl of 1% (w/v) to 80-90 ℃ ₄1ml continues heated and stirred to boiling.Fill approximately 10s of boiling, add rapidly the citric acid three sodium solution (the 0.0236g trisodium citrate is made into the approximately concentration of 1% (w/v)) of now joining, stir boiling reaction 10min.Add ultrapure water after room temperature is cooling and complement to 100ml.The Radioactive colloidal gold that obtains is carried out full wavelength scanner at 400-700nm, require the absorption value under the 525-535nm wavelength to reach 0.92-0.98.

In the gold sol that is cooled to room temperature that 100ml prepares, with 1% (w/v) K ₂CO ₃Accent pH to 8.2.

Add 1.5mg mouse-anti human IgG (Keelung, Hangzhou Bioisystech Co., Ltd) (the 0.1M phosphoric acid buffer is diluted to 1mg/ml), stirring at room 30min.

Add BSA solution sealing 30min (solution of 5% (w/v) adds 25ml).The centrifugal 25min of 10,000rpm abandons supernatant.With the protein solution absorbancy of ultraviolet spectrophotometer before and after the 280nm mensuration colloid gold label IgG of place, after calculating, mark rate is 70%.Show and be marked with IgG on Radioactive colloidal gold.

Precipitation is the Tris-HCl buffer solution for cleaning (0.02M, pH8.0) of 1% (w/v) BSA with concentration; The centrifugal 25min of 10,000rpm abandons supernatant; Precipitation is with the Tris-HCl dissolving that contains 1% (w/v) BSA, adds the trehalose of sucrose and 5% (w/v) of 20% (w/v) resuspended, at last by antibody activity after the immune response certification mark.

Embodiment 4CCP peptide combinations-BSA is incorporated into millipore filtration

Get the CCP peptide combinations of proper concn-BSA antigen, at the upper point of nitrocellulose filter (BA85, a Whatman) detection line (test line, T line), making the CCP peptide polymer content on millipore filtration is 115 μ g/cm ³Then point control line (control line, C line).

C line solution is for being mixed with the sheep anti-mouse igg (Keelung, Hangzhou Bioisystech Co., Ltd) of 1mg/ml with diluent 1 * PBS (3% trehalose).

T line solution is for being mixed with CCP peptide combinations-BSA antigen with diluent 0.02M Tris (2% trehalose, 0.5%NaCl, pH8.0), and wherein the concentration of CCP peptide combinations is 1.8mg/ml.

The sheet material that point is good dries by the fire 2h in 37 ℃ of baking ovens.

Embodiment 5 dot immunogold marks detect immune antibody of rheumatoid arthritis

Fig. 1 is the structural representation for dot immuno gold filtration assay method one embodiment of the present invention.This device comprises cover plate 1, base plate 2, millipore filtration 3 and thieving paper 4, and millipore filtration 3 is placed on thieving paper 4, then is positioned in base plate, covers at last the cover plate 1 that offers through hole 11 (reacting hole).Be combined with CCP peptide antigen on described millipore filtration 3, and relative with described through hole 11.

Afterwards, drip 1 washings (the 10mM pH7.4 phosphate buffered saline(PBS) of 0.5%Tween20) with the balance detection film in reacting hole.

Get 40 μ l samples to be tested and add reacting hole, so that the antibody in sample to be tested is combined with CCP peptide antigen, and waits for the downward diafiltration of liquid and absorbed by thieving paper.

Then, drip 4 above-mentioned washingss in reacting hole, with clear film, and wait for the downward diafiltration of liquid and absorbed by thieving paper.

Afterwards, drip the 1 immune colloid gold solution through mark in reacting hole, make the marker identification antibody on it, and wait for the downward diafiltration of liquid and absorbed by thieving paper.

At last, drip 4 above-mentioned washingss in reacting hole, with clear film, and wait for the downward diafiltration of liquid and absorbed by thieving paper.Read test-results by range estimation or gold mark detector (Beijing Bo Maida bio tech ltd).

Whole testing process can be completed in 5 minutes.

Embodiment 6 dot immunogold marks detect the comparison that detects with ELISA

Use the serum of 250 Patients With Rheumatoid Arthritis and the serum of 250 Healthy Peoples.

According to US Patent No. 6,858, the mode of putting down in writing in 438 prepares the ELISA check-out console of the embodiment of the present invention 4 gained CCP peptide combinations-BSA.According to the dot immunogold mark diafiltration check-out console of this specification sheets Preparation Example 4 gained CCP peptide combinations-BSA, the CCP peptide polymer content on millipore filtration is 115 μ g/cm ³Use two kinds of detection methods to detect respectively above-mentioned serum, specificity and susceptibility result see Table respectively 1 and table 2.

The specificity contrast of table 1CCP peptide combinations detection type rheumatic sufferers

The susceptibility contrast of table 2CCP peptide combinations detection type rheumatic sufferers

From Table 1 and Table 2, the vitro detection that the worry method can realize immune antibody of rheumatoid arthritis is oozed in the immunity of spot gold, and it is basically identical that the sensitivity of its antibody test and specificity are compared with ELISA, thereby enlarged the applicable crowd who detects.

Embodiment 7 dot immunogold marks detect the comparison that detects with ELISA

According to document Reviews in Molecular Biotechnology 2002; 90; 195-229; and in the reference of quoting, disclosed synthesized polymer Methionin method obtains three polylysine pLys, and the chemosynthesis C-terminal is connected with CCP peptide I-NHS and the CCP peptide II-NHS (CCP peptide side chain and N-terminal have protecting group) of N-hydroxy-succinamide (NHS).In the pH7.4 phosphate buffered saline(PBS), CCP peptide I-NHS and CCP peptide II-NHS were attached to pLys in 1: 1 in molar ratio, then obtained respectively being combined with on pLys the pLys-CCP polymkeric substance of 2 CCP peptide I and CCP peptide II through RP-HPLC (acetonitrile-trifluoroacetic acid system) purifying.

Take the pLys-CCP polymkeric substance as detecting the antigen of immune antibody of rheumatoid arthritis, and be respectively used to ELISA and dot immunogold mark and detect that (the CCP peptide polymer content on millipore filtration is 115 μ g/cm ³), the results are shown in Table 3.

The comparison of table 3ELISA and dot immunogold mark method detection type rheumatic sufferers

As seen from Table 3, the vitro detection that the worry method can realize immune antibody of rheumatoid arthritis is oozed in the immunity of spot gold, and it is basically identical that the sensitivity of its antibody test and specificity are compared with ELISA, thereby enlarged the applicable crowd who detects.

Sequence table

＜110〉ShangHai RongSheng Biology Pharmacy Co., Ltd

＜120〉composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay

<130>BRS.HF.9002

<160>261

<170>PatentIn version 3.3

<210>SEQ ID No 1

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>1

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 2

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>2

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 3

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>3

His Gln Cys His Arg Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 4

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>4

His Gln Cys His Gln Phe Gln Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 5

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>5

His Gln Cys His Gln Phe Arg Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 6

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>6

His Gln Cys His Gln Phe Arg Phe Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 7

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>7

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 8

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>8

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 9

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>9

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 10

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>10

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 11

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>11

His Gln Cys Ala Arg Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 12

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>12

His Gln Cys His Gln Phe Gln Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 13

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>13

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 14

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>14

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 15

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>15

His Gln Cys His Gln Phe Arg Phe Xaa His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 16

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>16

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Ser Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 17

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>17

His Gln Cys Ala Gln Phe Gln Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 18

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>18

His Gln Cys Ala Gln Phe Arg Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 19

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>19

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 20

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>20

His Gln Cys Ala Gln Phe Arg Phe Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 21

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>21

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 22

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>22

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 23

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>23

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 24

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>24

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 25

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>25

His Gln Cys His Arg Phe Glu Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 26

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>26

His Gln Cys His Arg Phe Arg Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 27

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>27

His Gln Cys His Arg Phe Arg Phe Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 28

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>28

His Gln Cys His Arg Phe Arg Phe Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 29

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>29

His Gln Cys His Arg Phe Arg Phe Xaa Gly Xaa Ser Arg Leu Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 30

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>30

His Gln Cys His Arg Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Ile Cys

1 5 10 15

Gly

<210>SEQ ID No 31

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>31

His Gln Cys His Gln Phe Gln Phe Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 32

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>32

His Gln Cys His Gln Phe Gln Phe Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 33

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>33

His Gln Cys His Gln Phe Gln Phe Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 34

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>34

His Gln Cys His Gln Phe Gln Phe Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 35

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>35

His Gln Cys His Gln Phe Gln Phe Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 36

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>36

His Gln Cys His Gln Phe Arg Met Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 37

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>37

His Gln Cys His Gln Phe Arg Met Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 38

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>38

His Gln Cys His Gln Phe Arg Met Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 39

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>39

His Gln Cys His Gln Phe Arg Met Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 40

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>40

His Gln Cys His Gln Phe Arg Met Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 41

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>41

His Gln Cys Ala Arg Phe Gln Phe Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 42

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>42

His Gln Cys Ala Arg Phe Arg Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 43

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>43

His Gln Cys Ala Arg Phe Arg Phe Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 44

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>44

His Gln Cys Ala Arg Phe Arg Phe Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 45

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>45

His Gln Cys Ala Arg Phe Arg Phe Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 46

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>46

His Gln Cys Ala Arg Phe Arg Phe Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 47

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>47

His Gln Cys Ala Arg Phe Arg Phe Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 48

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>48

His Gln Cys Ala Gln Phe Gln Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 49

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>49

His Gln Cys His Arg Phe Gln Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 50

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>50

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 51

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>51

His Gln Cys His Gln Phe Gln Met Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 52

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>52

His Gln Cys His Gln Phe Gln Met Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ IDNo 53

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>53

His Gln Cys His Gln Phe Gln Met Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 54

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>54

His Gln Cys His Gln Phe Gln Met Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 55

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>55

His Gln Cys Ala Gln Phe Arg Phe Xaa Gly Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 56

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>56

His Gln Cys His Arg Phe Arg Phe Xaa Gly Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 57

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>57

His Gln Cys His Gln Phe Gln Phe Xaa Gly Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 58

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>58

His Gln Cys His Gln Phe Arg Met Xaa Gly Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 59

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>59

His Gln Cys His Gln Phe Arg Phe Xaa His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 60

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>60

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 61

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>61

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 62

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>62

His Gln Cys Ala Arg Phe Gln Met Xaa Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 63

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>63

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 64

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>64

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 65

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>65

His Gln Cys His Gln Phe Arg Met Xaa His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 66

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>66

His Gln Cys His Gln Phe Arg Phe Xaa His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 67

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>67

His Gln Cys His Gln Phe Arg Phe Xaa Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 68

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>68

His Gln Cys Ala Arg Phe Gln Phe Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 69

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>69

His Gln Cys Ala Arg Phe Gln Phe Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 70

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>70

His Gln Cys Ala Arg Phe Gln Phe Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 71

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>71

His Gln Cys Ala Arg Phe Gln Phe Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 72

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>72

His Gln Cys Ala Arg Phe Gln Phe Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 73

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>73

His Gln Cys His Arg Phe Gln Met Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 74

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>74

His Gln Cys His Arg Phe Gln Met Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 75

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>75

His Gln Cys His Arg Phe Gln Met Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 76

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>76

His Gln Cys His Arg Phe Gln Met Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 77

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>77

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 78

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>78

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 79

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>79

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 80

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>80

His Gln Cys His Gln Phe Arg Met Xaa His Xaa Arg Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 81

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>81

His Gln Cys His Gln Phe Arg Met Xaa His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 82

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>82

His Gln Cys His Gln Phe Arg Phe Xaa His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 83

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>83

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 84

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>84

His Gln Cys Ala Arg Phe Gln Met Xaa Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 85

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>85

His Gln Cys Ala Arg Phe Gln Met Xaa Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 86

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>86

His Gln Cys Ala Arg Phe Gln Met Xaa Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 87

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>87

His Gln Cys Ala Arg Phe Gln Met Xaa Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 88

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>88

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 89

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>89

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 90

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>90

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 91

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>91

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 92

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>92

His Gln Cys Ala Gln Phe Gln Met Xaa His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 93

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>93

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 94

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>94

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 95

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>95

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 96

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>96

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 97

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>97

His Gln Cys His Gln Phe Arg Met Xaa His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 98

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>98

His Gln Cys His Gln Phe Arg Met Xaa His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 99

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>99

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 100

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>100

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 101

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>101

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 102

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>102

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 103

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>103

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 104

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>1

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 105

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>105

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 106

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>106

His Gln Cys Ala Gln Phe Gln Met Xaa His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 107

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>107

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 108

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>108

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 109

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>109

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Arg Leu Ala Ala Cys

1 5 10 15

G1y

<210>SEQ ID No 110

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>110

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Arg Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 111

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>111

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 112

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>112

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 113

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>113

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 114

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>114

His Gln Cys Ala Gln Phe Gln Met Xaa His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 115

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>115

His Gln Cys His Gln Phe Gln Met Xaa His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 116

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>116

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 117

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>117

His Gln Cys His Arg Phe Gln Met Xaa His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 118

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>118

His Gln Cys Ala Arg Phe Gln Met Xaa His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 119

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>119

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 120

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>120

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 121

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>121

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 122

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>122

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 123

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>35

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 124

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>36

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 125

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>125

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 126

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>126

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 127

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>127

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 128

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>128

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 129

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>129

His Gln Cys His Gln Phe Gln Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 130

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>131

His Gln Cys Ala Arg Phe Arg Phe Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 132

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>132

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 133

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>133

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 134

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>134

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 135

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>135

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 136

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>136

His Gln Cys His Gln Phe Arg Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 137

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>137

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 138

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>138

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 139

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>139

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 140

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>140

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 141

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>141

His Gln Cys His Arg Phe Arg Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 142

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>142

His Gln Cys His Arg Phe Gln Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 143

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>143

His Gln Cys His Arg Phe Gln Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 144

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>144

His Gln Cys His Arg Phe Gln Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 145

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>145

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 146

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>146

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 147

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>147

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 148

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>148

His Gln Cys Ala Gln Phe Arg Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 149

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>149

His Gln Cys Ala Gln Phe Gln Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 150

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>150

His Gln Cys Ala Gln Phe Gln Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 151

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>151

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 152

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>152

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 153

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>153

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 154

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>154

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 155

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>155

His Gln Cys Ala Arg Phe Arg Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 156

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>156

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 157

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>157

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 158

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>158

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 159

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>159

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 160

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>160

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 161

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>161

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 162

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>162

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 163

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>163

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 164

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>164

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 165

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>165

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 166

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>166

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 167

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>167

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 168

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>168

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 169

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>169

His Gln Cys His Gln Phe Arg Met Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 170

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>170

His Gln Cys His Gln Phe Gln Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 171

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>171

His Gln Cys His Gln Phe Gln Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 172

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>172

His Gln Cys His Gln Phe Gln Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 173

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>173

His Gln Cys His Gln Phe Gln Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 174

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>174

His Gln Cys His Gln Phe Gln Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 175

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>175

His Gln Cys His Gln Phe Gln Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 176

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>176

His Gln Cys Ala Gln Phe Arg Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 177

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>177

His Gln Cys Ala Gln Phe Arg Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 178

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>178

His Gln Cys Ala Gln Phe Arg Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 179

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>179

His Gln Cys Ala Gln Phe Arg Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 180

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>180

His Gln Cys Ala Gln Phe Arg Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 181

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>181

His Gln Cys Ala Gln Phe Arg Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 182

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>182

His Gln Cys His Arg Phe Arg Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 183

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>183

His Gln Cys Ala Arg Phe Arg Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 184

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>184

His Gln Cys Ala Arg Phe Arg Phe Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 185

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>185

His Gln Cys Ala Arg Phe Arg Phe Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 186

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>186

His Gln Cys Ala Arg Phe Arg Phe Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 187

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>187

His Gln Cys Ala Arg Phe Arg Phe Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 188

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>188

His Gln Cys His Arg Phe Gln Phe Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 189

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>189

His Gln Cys Ala Gln Phe Gln Phe Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 190

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>190

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 191

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>191

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 192

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>192

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 193

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>193

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 194

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>1

His Gln Cys His Arg Phe Gln Met Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 195

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>195

His Gln Cys Ala Gln Phe Gln Met Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 196

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>196

His Gln Cys Ala Arg Phe Arg Met Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 197

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>197

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 198

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>198

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 199

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>199

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 200

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>200

His Gln Cys His Arg Phe Gln Met Arg His Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 201

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>201

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 202

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>202

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 203

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>203

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 204

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>204

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 205

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>205

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 206

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>206

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 207

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>207

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 208

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>208

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 209

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>209

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 210

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>210

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 211

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>211

His Gln Cys Ala Arg Phe Gln Met Arg His Ser Arg Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 212

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>212

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 213

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>213

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 214

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>214

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 215

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>215

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 216

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>216

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Arg Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 217

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>217

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 218

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>218

His Gln Cys His Gln Phe Arg Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 219

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>219

His Gln Cys His Gln Phe Arg Met Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 220

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>220

His Gln Cys His Gln Phe Arg Met Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 221

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>221

His Gln Cys His Gln Phe Arg Met Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 222

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>222

His Gln Cys His Gln Phe Arg Met Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 223

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>223

His Gln Cys His Gln Phe Arg Met Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 224

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>224

His Gln Cys Ala Gln Phe Arg Phe Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 225

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>225

His Gln Cys Ala Gln Phe Arg Phe Arg His Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 226

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>226

His Gln Cys Ala Gln Phe Arg Phe Arg His Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 227

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>227

His Gln Cys Ala Gln Phe Arg Phe Arg His Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 228

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>228

His Gln Cys Ala Gln Phe Arg Phe Arg His Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 229

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>229

His Gln Cys Ala Arg Phe Arg Phe Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 230

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>230

His Gln Cys Ala Arg Phe Arg Phe Arg Gly Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 231

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>231

His Gln Cys Ala Arg Phe Arg Phe Arg Gly Xaa Arg Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 232

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>232

His Gln Cys Ala Arg Phe Arg Phe Arg Gly Xaa Arg Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 233

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>233

His Gln Cys His Arg Phe Arg Phe Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 234

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>234

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 235

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>235

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Ser Leu Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 236

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>236

His Gln Cys Ala Arg Phe Gln Phe Arg Gly Xaa Ser Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 237

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>237

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 238

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>238

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Ser Arg Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 239

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>239

His Gln Cys Ala Arg Phe Arg Met Arg Gly Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 240

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>240

His Gln Cys Ala Gln Phe Gln Met Arg Gly Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 241

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>241

His Gln Cys His Arg Phe Gln Met Arg Gly Xaa Ser Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 242

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>242

His Gln Cys Ala Arg Phe Gln Met Arg His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 243

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>243

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 244

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>244

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 245

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>245

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 246

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>246

His Gln Cys His Arg Phe Gln Met Arg His Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 247

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>247

His Gln Cys Ala Arg Phe Gln Met Arg Gly Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 248

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>248

His Gln Cys Ala Arg Phe Gln Phe Arg His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 249

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>249

His Gln Cys Ala Arg Phe Arg Met Arg His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 250

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>250

His Gln Cys Ala Gln Phe Gln Met Arg His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 251

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>251

His Gln Cys His Arg Phe Gln Met Arg His Xaa Arg Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 252

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>252

His Gln Cys His Gln Phe Arg Phe Arg His Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 253

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>253

His Gln Cys His Gln Phe Arg Phe Arg Gly Xaa Arg Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 254

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>47

His Gln Cys His Gln Phe Arg Phe Arg Gly Xaa Ser Leu Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 255

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>255

His Gln Cys His Gln Phe Arg Phe Arg Gly Xaa Arg Arg Ile Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 256

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>256

His Gln Cys His Gln Phe Arg Phe Arg Gly Xaa Ser Arg Ala Arg Cys

1 5 10 15

Gly

<210>SEQ ID No 257

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>257

His Gln Cys His Gln Phe Arg Phe Arg Gly Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 258

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>258

His Gln Cys His Gln Phe Gln Met Arg Gly Xaa Ser Leu Ile Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 259

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>259

His Gln Cys His Gln Phe Gln Phe Arg Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 260

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>260

His Gln Cys His Gln Phe Arg Met Arg His Xaa Ser Arg Ala Ala Cys

1 5 10 15

Gly

<210>SEQ ID No 261

<211>17

<212>PRT

＜213〉synthetic

＜223〉Xaa is citrulline

<400>261

His Gln Cys His Gln Phe Arg Met Arg Gly Xaa Ser Leu Ala Ala Cys

1 5 10 15

Gly

Claims (7)

1. the composition of a detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay, comprise CCP peptide combinations, high molecular polymer, millipore filtration, water sucting medium and mounting medium, high molecular polymer is incorporated on millipore filtration by covalent linkage or non covalent bond;

Wherein, the CCP peptide combinations comprises CCP peptide I and CCP peptide II, and CCP peptide I and CCP peptide II all with the non-covalent combination of high molecular polymer; Described CCP peptide I is SEQ ID No:1, and described CCP peptide II is SEQ ID No:118; The mol ratio ratio of described CCP peptide I and described CCP peptide II is 1: 1.

2. the composition of a detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay, comprise CCP peptide combinations, high molecular polymer, millipore filtration, water sucting medium and mounting medium, high molecular polymer is incorporated on millipore filtration by covalent linkage or non covalent bond;

Wherein, the CCP peptide combinations comprises CCP peptide I and CCP peptide II, and CCP peptide I and CCP peptide II all with the high molecular polymer covalent attachment; Described CCP peptide I is SEQ ID No:1, and described CCP peptide II is SEQ ID No:118; The mol ratio of described CCP peptide I and described CCP peptide II is 1: 1.

3. the composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay according to claim 1 and 2, the CCP peptide polymer content that it is characterized in that combination on described millipore filtration is 50 μ g-300 μ g/cm ³

4. the composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay according to claim 1 and 2, the CCP peptide polymer content that it is characterized in that combination on described millipore filtration is 50 μ g-150 μ g/cm ³

5. the composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay according to claim 1 and 2, is characterized in that described high molecular polymer molecular weight is greater than 2,000Da.

6. the composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay according to claim 1 and 2, is characterized in that described high molecular polymer is selected from bovine serum albumin, human serum albumin, albumin rabbit serum, chicken ovalbumin, keyhole limpet hemocyanin, branch type polymkeric substance, polylysine, polyoxyethylene glycol, avidin or antibody.

7. the composition of detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay according to claim 1 and 2, is characterized in that the covalent linkage between described CCP peptide and high molecular polymer is selected from amido linkage, urine key, ester bond, thioether bond, hydrazone key, oxime key or disulfide linkage.

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