CN101865913B - Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography - Google Patents

Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography Download PDF

Info

Publication number
CN101865913B
CN101865913B CN 200910049453 CN200910049453A CN101865913B CN 101865913 B CN101865913 B CN 101865913B CN 200910049453 CN200910049453 CN 200910049453 CN 200910049453 A CN200910049453 A CN 200910049453A CN 101865913 B CN101865913 B CN 101865913B
Authority
CN
China
Prior art keywords
xaa
arg
gly
cys
gln
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN 200910049453
Other languages
Chinese (zh)
Other versions
CN101865913A (en
Inventor
朱绍荣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Rongsheng Biological Pharmaceutical Co.,Ltd.
Original Assignee
Shanghai Rongsheng Biological Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Rongsheng Biological Pharmaceutical Co Ltd filed Critical Shanghai Rongsheng Biological Pharmaceutical Co Ltd
Priority to CN 200910049453 priority Critical patent/CN101865913B/en
Publication of CN101865913A publication Critical patent/CN101865913A/en
Application granted granted Critical
Publication of CN101865913B publication Critical patent/CN101865913B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Abstract

The invention relates to a composition for detecting a rheumatoid arthritis immune antibody by lateral chromatography. The composition comprises a CCP peptide, a high polymer which is covalently bonded with the CCP peptide, and a carrier medium. The composition can realize quick in-vitro detection of the rheumatoid arthritis immune antibody, is favorable for self-service diagnosis of patients, and provides reference for timely knowing the development of disease conditions. The composition has the advantages of improving the sensitivity and specificity of antibody detection, increasing detection accuracy and enlarging detection applicable people by covalently bonding the CCP peptide having different sequences with the high polymer.

Description

The composition that is used for detecting rheumatoid arthritis immune antibody by lateral chromatography
Technical field
The present invention relates to a kind of composition of detection type rheumathritis antibody, relate in particular to a kind of composition for detecting rheumatoid arthritis immune antibody by lateral chromatography, realize the fast detecting to the rheumatoid disease immune antibody of rheumatoid arthritis.
Background technology
Autoimmune disease mainly contains systemic loupus erythematosus, rheumatoid arthritis, Sjogren syndrome, dermatomyositis, polymyositis, system (multiple) property sclerosis, chorionitis etc. clinically, these diseases once were named as " connective tissue disease (CTD) ", all they were classified as rheumatic disease with domestic abroad afterwards.
Rheumatoid arthritis (rheumatoid arthritis, RA) be a kind of more common systemic autoimmune disease take chronic polyarthritis disease as main manifestations, how repeatedly its course of disease is long,, and can cause and organize irreversible damage, and cause very large misery to the patient.This sick incidence of disease worldwide is 0.5-1%, and the incidence of disease of China is about 0.36%.Patient's age of onset is many, and the women was more than the male sex at 20～50 years old, and men and women's ratio is 1: 3.Nearest epidemiology statistics, rheumatoid arthritis has year by year the trend that increases, painful swelling of joints and activity difficulty, the daystart anchylosises such as its outstanding early clinical manifestation is symmetry redness and swelling of joints heat pain, the little joint of common four limbs, refer to that a near-end arthroncus, the palm refer to, wrist, elbow and ankle, in the afternoon alleviate gradually, symptom has 20% patient subcutaneous nodule can occur approximately outside the joint; The permanent symptom in late period that does not heal then has in various degree joint deformity and tetanic, and 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 the clinical practice, the diagnostic tool commonly used of this disease is (the American College of Rheumatology of U.S. rheumatism association, ACR) rheumatoid disease criteria for classification (the Arthritis 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 mainly is to adopt the anti-inflammatory method, though this method can so that the degree of arthroncus (swelling) and erosion (erosive) is slowed down, can't make disease obtain curing.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 selectivity (Specificity) and high sensitivity (Sensitivity) is particularly necessary (the Clin Applied Immunol Rev 2004 that just seems of the early diagnosis before the bone joint is damaged for rheumatoid arthritis, 4,239-62).
Although research finds that some autoimmune antibodies interrelate with the rheumatoid arthritis disease, as: anti-calpastatin antibody (Proc Natl Acad Sci 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 both be in suffering from the patient of other autoimmune disease, in healthy individual, detect even (Clin Applied Immunol Rev 2004,4,239-62).Although some antibody wherein can be applied to the identification of rheumatic disease, and screen these antibody and also help disease surveillance and prediction, but because it lacks selectivity to rheumatoid arthritis, the overwhelming majority in these antibody seldom is applied to rheumatoid arthritis early diagnosis (Clinica Chimica Acta 2004,350,17-34).
Ideal mark (Marker) for the rheumatoid arthritis early diagnosis should satisfy 4 standards at least: (1) high sensitivity, and the patient that can detect reaches high number percent; (2) high selectivity, the as much as possible generation of limit erroneous positive findings; (3) can be applicable to early diagnosis; (4) can predict some patient can develop into aggressivity disease (erosive disease) (AutoimmunityReviews 2006,6,37-41; MEDICINE 2006,34,441-4).
Rheumatoid factor (rheumatoid factor, RF) antibody is applied in the ACR standard as Serology test, is acknowledged as the direct antibody of Fc domain on the IgG molecule.Although its detection sensitivity scope can reach 60-80%, its selectivity 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 among 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) then have higher selectivity to rheumatoid arthritis.64% Patients With Rheumatoid Arthritis can produce direct antibody for BiP, report that in addition it has the selectivity of height to this disease, but there is not 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 research 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).Other has research to disclose Sa antigen can be up to 92-99% for the selectivity of rheumatoid arthritis, but its sensitivity then seems generally, only is 30-40% (J 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 selectivity to rheumatoid arthritis after deliberation, and can indirectly record by the immunofluorescence technique take human cheek mucosa's cell as the antigen substrate (Ann Rheum Dis 1964,23,302-5).
The researcher had described a kind of rheumatoid arthritis selectivity keratoprotein antibody in 1979, it can directly suppress cuticula epithelial cell keratinization, and be anti-keratoprotein antibody (anti-keratin andtibody with the title of this antibody-like is tentative, AKA) (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 selectivity (Internal Medicine 2005,44,1122-6).Filaggrin (filamentaggregating protein) is a kind of mutually crosslinked thread body of keratin, effect is to form very firm cytoskeletal structure, its also be 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 selectivity for rheumatoid arthritis, its defective is also very outstanding.The detection sensitivity of these two kinds of antibody depends critically upon the purification process of filaggrin.In the practice, owing to be difficult to separate and obtain pure and guanidine radicals content has repeatable antigen, it is not easy to add its detection means, and the fluorescence immunoassay testing process need to expend larger workload, thereby so that be difficult to form standardization between the laboratory, 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 the rheumatoid arthritis serum.Because citrulline is not primary amino acid and can't adding in protein translation, so usual way is to take off imines enzyme (peptidyl argnine deiminine, PAD, EC3.5.3.15) catalysis arginine residues deaminizating by peptide arginine to obtain.According to these facts, the arginine side chain that some are derived from the filaggrin sequence is modified, and the polypeptide that especially contains citrulline is synthesized out, and is used for the vitro detection (US6,858,438) of rheumatoid arthritis antibody.Using Enzyme-linked Immunosorbent Assay (Enzyme-Linked immunosorbent Assay, ELISA) in the method test experience, this method is synthetic contains citrulline rectilinearity polypeptide can detect anti-citrulline peptide antibody from rheumatoid arthritis patients serum, in the situation that keeps high selectivity (greater than 90%), 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 the polypeptide is replaced then fully not reaction generation with other amino acid.These results show, citrulline partly be determine can by the antigenic determining factor of AKA and APF antibody recognition (J Clin Invest 1998,101,273-81).
Other researcher finds to contain the rectilinearity polypeptide of citrulline by the mode energy analogy β-corner structure of disulfide bond (S-S key) formation loop configuration, thereby imitate β-corner structure of initial antigenic determinant, and can increase affinity (the FASEB J 1995 of polypeptide-antibody, 9,37-42; Mol Immunol 1985,22,1255-64).Thus, a kind of cyclic peptide manually is synthesized, after it changes two serines on the main epi-position of filaggrin into halfcystine, formed by the disulfide bond cyclisation again, be first generation cyclic citrullinated peptide (the first generation cyclic citrullinatedpeptide, CCP 1) (Internal Medici ne 2005,44,1122-6).The researcher replaces with halfcystine with two serines in the citrulline peptide that is comprised of 19 amino acid residues and forms the disulfide bond that has analog structure with β-corner, is manually synthesized CCP, and the testing result of CCP1 and straight line peptide is contrasted.The result shows that employing CCP1 is that the polypeptide of antigen uses the ELISA method to detect RA patient's anti-CCP antibody, and sensitivity is that antigen is significantly increased with rectilinearity citrulline peptide, be respectively 68% and 49%, the two selectivity 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, clinically the simultaneous determination with RF and anti-CCP antibody more practical (Peking University's journal (medicine) 2005,37,894).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 aggressivity 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 changeability.Outside the pale of civilization by citrulline except the filaggrin arginine residues in the serum/plasma of rheumatoid arthritis patients, citrulline also appears in Sa antigen, collagen (I and II type), histone, myelin basic protein, fibronectin etc., and produces anti-CCP antibody.Further investigation shows, is not that all arginine deaminizatings are converted into citrulline in the autoantigen of citrulline; The citrulline of citrulline also not exclusively participates in forming epitope, namely produces anti-CCP antibody.To sum up, generation and the citrulline of anti-citrulline antibody are closely related, and the generation of the flanking sequence of citrulline antagonism citrulline antibody plays an important role, this fact is hinting that all skirt amino acids of citrulline residue have vital role for epitope (antigen episode), and anti-citrulline albumen (as: AKA and APF antibody) activity is polyclone reaction (J Clin 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).
Second generation CCP (CCP2) detection method results from 2002, obtain the second generation CCP irrelevant with filaggrin by peptide library and the rheumatoid arthritis serum screening that will contain citrulline, it has epi-position (Report on 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 that CCP2 has not only kept higher selectivity (96%) after same group of patient's test, and its sensitivity for analysis also increase significantly (Ann.Rheum.Dis.2005,64,1510-2).Many researchers studies have shown that recent five years the anti-sensitivity that contains the test of citrulline polypeptide has higher changeability, and scope is at 40%-94% (Clin.ExpRheumatol.2005,23 (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 second generation of using on the present market and third generation CCP kit (Kit) respectively, found that, in three kinds of detection methods of her Nova diagnosis (InovaDiagnostics) company exploitation, its CCP3 kit with use CCP2 only to compare as the method for antigen slightly to be improved, sensitivity separately is respectively 67% and 64%.On the contrary, the CCP3.0 that is used for anti-IgG compares rear discovery with the detection method that can detect IgG and can detect again the CCP3.1 of IgA, and both do not have a bit difference unexpectedly.As seen, the method that detects simultaneously IgG and IgA antibody does not resemble has remarkable improvement looking.Their conclusion is because the diagnostic kit sensitivity of test may be relevant with position and the quantity of guanidinated arginine residues, selectivity then may be subject to the impact of protein or assorted peptide sequence, therefore, for this two, the kind of antigen seems even more important.Both consider, and experimental data shows that the preparation of antigen is the most important variable factor that determines the assay method quality.
The people such as Lutteri L (Clinica Chimica Acta 2007,386, another value of anti-CCP has also been found in research 76-81), they point out that IgM-RF is the sensitiveest mark, can find in 77.9% rheumatoid arthritis patients.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 the diagnostic criteria.IgM-RF is considered to slightly be weak aspect the disease selectivity.Use IgM-RF to detect rheumatoid arthritis patients, wherein have the people of 13-19% negative for RF, by comparison, all positive when these patients use anti-CCP method to detect.
The inherent cause of rheumatoid arthritis can affect appearance and the generation of citrulline albumen, also can affect antibody product for these modified proteins (Clinica Chimica Acta 2004,350,17-34).A series of correlative studys show that rheumatoid arthritis and some HLA-DR allele 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 regions, these allele are also slightly different.DRB1*0401 mainly appears among the crowd of Northern Europe and north America region, evidence suggests that " especially big joint " that this gene and rheumatoid arthritis cause characterizes (extraarticular manifestations) and be related, 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 among 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 among Northern Europe and the North America crowd.Among the American Indian group then be DRB1*1402 and * 1406 (Hum Immuno 2000,61,1254-1261).Although these genes relevant with rheumatoid arthritis are had nothing in common with each other in the crowd of each department, but research finds to have shared epi-position (shared epitope in the third high degree variable region (HV3) of DRB1, SE), all be 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 allele and anti-CCP2 antibody has close inner link (Arthritis Rheum 2000,50,2113-21; Arthritis ResTher 2004,6, R303-8).Verified, this association is because the MHC molecule is shared 70-74 amino acids residue (Q/R, the K/R of epi-position β chain, R, A, A) formation the 4th grappling bag (P4), itself and electric neutrality or negative charge amino acid have high affinity (J Immuno 2003,171,538-541).When positively charged arginine after converting electroneutral citrulline under the effect of PAD enzyme, the posttranslational modification protein/polypeptide affinity that obtains is far longer than the affinity of arginine and P4, and can activate CD4 +The T cell, these " the special T cells of citrulline " can help generation (J 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 allele with shared epi-position can activate the rheumatoid arthritis patients autoimmune response.
Comprehensive present result of study, anti-CCP antibody has higher selectivity 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, and it is also longer to detect the used time, 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.
Summary of the invention
One object of the present invention is to provide a kind of composition of detection type rheumathritis antibody, comprises CCP peptide, high molecular polymer covalently bound with it and mounting medium.
Another object of the present invention is to provide a kind of composition for detecting rheumatoid arthritis immune antibody by lateral chromatography, comprise CCP peptide, high molecular polymer covalently bound with it and detect medium, realize the fast detecting to immune antibody of rheumatoid arthritis.
Another purpose of the present invention is to provide a kind of composition for detecting rheumatoid arthritis immune antibody by lateral chromatography, comprise CCP peptide, high molecular polymer covalently bound with it, sample pad, collaurum pad, detecting pad, adsorptive pads and base plate, realize the fast detecting to immune antibody of rheumatoid arthritis.
The CCP peptide is a kind ofly to be connected to each other by amido link by 5-50 amino acid, and the loop configuration organic molecule that between non-conterminous two amino acid side chains, forms by covalent bond, amino acid is selected from 20 kinds of primary amino acids, in the middle of its sequence, have at least an arginine side chain to be modified, the arginine side chain of modifying has the structure shown in the formula I
Figure G2009100494532D00081
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;
Wherein working as G 1 is NH, and G2 is NH 2The time, G3 is not NH.
Further, a kind of CCP peptide is connected to each other by amido link by 10-40 amino acid, and by forming disulfide bond between non-conterminous two cysteine side chain sulfydryls on the sequence, has a citrulline (Cit) at least in the middle of its sequence.
A kind of amino acid sequence of above-mentioned CCP peptide, specific as follows:
His-Gln-Cys-Xaa1-Xaa2-Phe-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Gly，
Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Arg; Xaa6 is Gly or His; Xaa7 is Arg; Xaa8 is Ser or Arg; Xaa9 is Arg or Leu; Xaa10 is Ala or Ile; Xaa11 is Ala or Arg, wherein has at least an arginine to be modified.
The amino acid sequence of another kind of above-mentioned CCP peptide, specific as follows:
His-Gln-Cys-Xaa 1-Xaa2-Phe-Xaa3-Xaa4-Xaa5-Xaa6-Cit-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Gly，
Wherein, Xaa1 is His or Ala; Xaa2 is Gln or Arg; Xaa3 is Arg or Gln; Xaa4 is Phe or Met; Xaa5 is Arg or Cit; Xaa6 is Gly or His; Xaa7 is Ser or Arg; Xaa8 is Arg or Leu; Xaa9 is Ala or Ile; Xaa10 is Ala or Arg.
Particularly, the amino acid sequence of above-mentioned CCP peptide such as SEQ ID No:1-261 form disulfide bond between its 3rd Cys and the sixteen bit Cys.
High molecular polymer (polymer) refers to molecular weight greater than the compound of 2,000Da, is intermolecularly interconnected via covalent bond by structural units (structural unit) or monomer.Its structure can be linear pattern (linear), ramiform (branch/multi-arm) or branch type (dendrimer).Described polymkeric substance is selected from polyglycol, polyaminoacid, polynucleotide, PLA, polylysine, poly-imines and 10 polysaccharides (polysaccharide) that above monose forms by glycosidic bond, as: starch and hydrolysate thereof, cellulose, chitin or glucosan.
Branch type polymkeric substance (dendrimer) is generally poly-amino amine (poly (amidoamine), PAMAM), can be starting material preparation (Clin.Chem., 1994,40 by ammonia or ethylenediamine respectively, 1845-1849), also have and use amino acid be polymerized (J Immuno Method, 1996,196,17-32), such as lysine and arginine.Resulting polymers has reactive group, and such 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 molecule.
Polyglycol is selected from the polyglycol that two ends are not all sealed or an end seals, and blocking groups is selected from the alkyl of C1-C30, the fatty acid of C1-C30 or glycosyl.Polyglycol is selected from molecular weight 2,000-100, and 000Da can select 2,000-50, and 000Da generally selects 5,000-50,000Da.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.
Polyaminoacid comprises the oligothiophene molecule that is polymerized by same amino acid, also comprises the poly molecule that is polymerized by different molecular, and protein or polypeptide are its natural existence forms.
Above-mentioned CCP peptide and high molecular polymer covalent bond (covalent conjugate), the covalent bond of formation is such as: amido link (amide bond), urine key (urine bond), ester bond or disulfide bond.The CCP peptide combines with high molecular polymer by its amino acid side chain, N terminal amino group or C terminal carboxyl group.This combination can by as: the reagent such as N-hydroxy-succinamide, maleic anhydride or third/aldehyde-base combine amino acid side chain, N terminal amino group or C terminal carboxyl group after activating with high molecular polymer, also can be suitable for aforementioned identical or different mode, after activating first the group of high molecular polymer, combine with the CCP peptide again.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 the carbohydrate molecule is referring to J.Pharma.Sci.87,326-332; Adv.Drug deliv.Rev., 6,103-131; Adv.Drug deliv.Rev., 13,251-267.The combination of polyglycol and above-mentioned CCP peptide is referring to Adv.Drug deliv.Rev., 28,275-299; Adv.Drug deliv.Rev., 54,453-609; Adv.Drug deliv.Rev., 60,1-88.Because the CCP peptide polymer is same or similar in conjunction with related chemical reaction, these combinations are equally applicable to the CCP peptide in the middle of reaction that other polymkeric substance is combined.
The CCP peptide connects can also pass through mode and the high molecular polymer indirect joint that connexon (space linker) connects with chemistry.This connexon is selected from the polypeptide of amino acid length 1-500 or protein, molecular weight at 100-2, the polymkeric substance of 000Da or organic molecule.As: molecular weight is 200-2, polyglycol (NOF Corporation), the NH of the two ends activation of 000Da 2(CH 2) nCOOH, SH (CH 2) nThe fatty acid of the C1-C30 of COOH or two ends activation (J.Gene.Med., 2005,7,604-612).The chemistry connection can be that CCP peptide N or C activated group terminal and on the above-mentioned connexon is connected, and also can be connected with activated group on the connexon by the some amino acid side chains of CCP peptide.Form covalent bond between connexon and the antigen, as: amido link, urine key, ester bond,, (CH=N-NH-), the oxime key (CH=N-O-) and disulfide bond for 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 bond by two monosaccharide units, the oligosaccharide (oligosaccharide) (as: oligoisomaltose, xylo-oligosaccharide or galactooligosaccharide) that is formed by glycosidic bond by 2-10 monose and molecular weight be at 100-2, the organic molecule of 000Da.
A kind of composition for detection of immune antibody of rheumatoid arthritis comprises CCP peptide, high molecular polymer and mounting medium.Wherein, CCP peptide and high molecular polymer covalent bond, high molecular polymer is incorporated on the mounting medium by form covalently or non-covalently afterwards.
Mounting medium is selected solid phase or liquid phase.Solid-phase media is selected plastics or glass.
Plastics should be understood to all or part by carbon and oxygen, hydrogen, organic and the inorganic elements chemical combination of nitrogen and other forms, become solid in the final stage of making, some stage is that (plastic material is becoming before the final products liquid in the mill, must want and to flow in some stage), thereby can heat or plus-pressure, or the mode of the two and usefulness, make it form various shapes, any in this huge and protean material same clan, such as resin, thermoset resin, cellulose derivatives etc. exist numerous repetition atom or molecule in the molecular structure of a long-chain.Plastics comprise artificial synthetic or nature organic material.Make the used plastics of mounting medium or use a kind of plastic material, or use several plastic materials physically stack or addition after compound, its concrete form is plastic plate or test strips.
Glass is appreciated that frangible amorphous material, these materials can be transparent also can be translucent, usually merged by molten silicon and silicon-carbon hydrochlorate and form.Glass can also think that a class does not have crystallization process, but is solidified and next material by molten state, substantially by Na 2O, CaO and 6SiO 2Chemical oxide forms, and has optical properties and various mechanical attributes.
Concrete, plastic plate is the straight plate of being made by polystyrene, tygon, Polyvinylchloride or polycarbonate etc. or the ELISA Plate with vesicular structure; Test strips is by independent or compounded thin slice such as polystyrene, tygon, Polyvinylchloride, polyester or cellulose and derivant thereof etc. or film.
CCP peptide and high molecular polymer covalent bond form the CCP peptide polymer, and polymer substance is incorporated into mounting medium on it, so that the CCP peptide on every square centimeter of medium can be 100ng-1000 μ g/cm greater than 20ng 2, generally select 100ng-500 μ g/cm 2, preferentially select 1 μ g-100 μ g/cm 2
Another kind of composition for detection of immune antibody of rheumatoid arthritis comprises CCP peptide, high molecular polymer and mounting medium.Wherein, the CCP peptide is selected from any one or a few combination with arbitrary proportion among the SEQ ID No:1-261, and with the high molecular polymer covalent bond, the CCP peptide polymer of formation covalently or non-covalently is incorporated on the mounting medium, the CCP peptide content of combination on the mounting medium is 1 μ g-50 μ g/cm 2
Another kind of composition for detection of immune antibody of rheumatoid arthritis comprises CCP peptide, high molecular polymer and detection medium.Wherein, the CCP peptide is selected from SEQ ID No:1,3 and 119, be covalently bonded in high molecular polymer at 1: 1: 1 with mol ratio, the CCP peptide polymer of formation covalently or non-covalently is incorporated on the mounting medium, and the CCP peptide content of combination on the mounting medium is 1 μ g-50 μ g/cm 2
With the detection of above-mentioned various compositions for detection of immune antibody of rheumatoid arthritis, its detection method can have multiple, such as: indirect enzyme-linked immunosorbent determination method, double antigens sandwich enzyme immunoassay, lateral chromatography fast detection method, immunity percolation detection method and protein chip detection method etc.
A kind of composition for detecting rheumatoid arthritis immune antibody by lateral chromatography comprises CCP peptide, high molecular polymer covalently bound with it, and the CCP peptide content that is incorporated into detecting pad is 100ng-1000 μ g/cm 2
Lateral chromatography (Lateral Flow Immunoassay, LFIA) usually adopt test strips, sample is finished one-time detection through sample pad (sample pad) loading after passing through successively mark pad (conjugate pad), detecting pad (membrane) and adsorptive pads (wick) under the effect of capillarity.The colour developing mark of testing molecule in the sample in the mark pad is combined, detection zone (test line when itself and detecting pad, the T line) molecular specific in conjunction with the time kept somewhere, the detected label of son indwelling of distinguishing is by control zone (control line, the C line) antibody combination, thereby the detection of realize target molecule.Sample pad, mark pad, detecting pad and adsorptive pads all are fixed in support baseboard (backing).
Each ingredient of lateral chromatography test strips is made by polystyrene, tygon, Polyvinylchloride, polyester or cellulose and derivant thereof etc.
Be combined with the colour developing label on the mark pad, operable label has: colloidal-carbon, collaurum, electroselenium, liposome, paramagnetic particle, quantum dot, metallic ion, organic fluorescence molecule or color micro-sphere.Usually be combined with aglucon on these labels, label on the molecular labeling can is combined and make to aglucon with the molecule in the testing sample.The organic molecule that these aglucons are selected from staphylococcal protein A, immunoglobulin (Ig), toxin, glycoprotein, enzyme, polypeptide, microbiotic, hormone and bovine serum albumin(BSA) or combine with molecule in the testing sample.
Because liposome possesses the high concentration of forgiving molecule, being applied to the lateral flow chromatography just can increase detection sensitivity 2-3 the order of magnitude.According to the actual needs that detects, visible dyes, fluorescent dye, enzyme or electric activated camplex energy direct coated are in size is the liposome of 50nm-800nm.The liposome that is used for the lateral flow chromatography is generally 200nm-400nm, and dyestuff is generally red thiocyanate.In order to adapt to the kind that detects aglucon, other molecule that just can crosslinked identification aglucon to be measured behind the functional group derivatization of surface of liposome.
Colloidal-carbon (Colloidal Carbon) is also referred to as " India Ink ", can be used as a kind of label be used for the tachysynthesis chemical detection (J.Biotechnol., 1993,30,185-195).United States Patent (USP) 5,641,689 disclose a kind of colloidal-carbon preparation method for protein adsorption, thereby have solved the unsettled problem of colloidal-carbon particle.The colloidal-carbon that is used for the lateral flow chromatography is generally 100nm-200nm.
Quantum dot (Quantum Dots) be a kind of diameter between 1-100nm, can accept the semiconductor nanoparticle of excitation light generation fluorescence.It is by CdSe, CdS, and ZnSe, InP or InAs form, or at the nuclear of CdSe one deck AnS or CdS are arranged.Prior art can be prepared into the quantum dot size extremely consistent, and has utilizing emitted light bandwidth extremely narrow in 1O-50nm.
According to needed grain size, there is various ways can in the middle of the laboratory, prepare collaurum (Colloidal Gold) (Methods for Synthesis of Colloidal Gold.In " ColloidalGold:Principles; Methods; and Applications ", Academic Press, Inc.SanDiego, Vol.1,1989).Nearly all method all is to use reductive agent gold ion to be converted into the simple substance gold in controlled scope.Used reductive agent comprises sodium borohydride, yellow phosphorus, ethanol, ascorbic acid, sodium citrate and citric acid-tannic acid.The people such as Frens (J.Microsc. (Oxford) 1981,123,201) are with gold chloride (HAuCl 4) be the most frequently used method for raw material uses sodium citrate to prepare collaurum.
Before adding reductive agent, contain 100% gold ion in the solution.Add after the reductive agent, rapid rising 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 chlorauride, 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, so that formed gold grain size all (being single decentralized) in full accord.
Gold grain has formed collaurum in the middle of being suspended in liquid.Because residual in the solution have negative ion, thereby each particle is centered on by the negative charge layer.This charge layer is referred to as Zeta electric potential, and it can make mutually exclusive between the gold grain and be suspended in the liquid.Along with the total ion concentration in the middle of the solution changes, Zeta electric potential also can compressed or expansion.
Large molecular ligand (as: protein, polypeptide or antibody) being adsorbed on the collaurum by electrostatic force (electrostatic) and hydrophobic interaction.During labelled antibody, can not there be excessive antibody to be marked to exist on the collaurum.Because excessive free antibody can with antibody and the antigen generation competitive reaction on the collaurum, cause occurring false negative, and excessive antibody protein can have influence on the stability of label.The top condition of protein and collaurum combination is near the 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: lysine) to attract each other; Albumen is by the hydrophobic suction-operated between tryptophane and the gold particle surface; The coordination valence of the sulfenyl of the halfcystine in the albumen with between gold particle is combined.
In process that collaurum is combined, the pH of control aglucon and collaurum is most important.Before combination, both pH should be adjusted to a little more than the aglucon isoelectric point.Be lower than the pKi of aglucon, the cohesion that aglucon is induced can occur, and be higher than the pKi of aglucon, then can limit adsorbance owing to the electrical charge rejection between aglucon and the collaurum.The situation of this dependence pH is general only to be occured at the protein aglucon, and the stabilizing agent that can adopt polyglycol to be used as collaurum 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 sal tartari, potassium chloride and dilution can be used for the adjusting of collaurum pH.The collaurum 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-service cut stream filter membrane.
Along with the combination of differential protein, the collaurum combination must be stablized with suitable reagent, usually uses BSA, gelatin, polyglycol or casein.Use stabilizing agent 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 antiseptic of collaurum is 0.1% triazo-compound.
When labelled antibody, particle diameter the colloid gold particle of 40-100nm can more smooth mark to IgG antibody.The colloid gold particle of 40nm provides maximum visuality and minimum sterically hindered.If be used for the molecular weight of labeled 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 collaurum has a single optical absorption peak in visible-range, the wavelength of this optical absorption peak (λ max) is in 510～550nm scope, change with the colloid gold particle size, the λ max deflection long wavelength of bulky grain collaurum, otherwise the λ max of granule collaurum then 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.(2～5nm) is orange to minimum collaurum, and (10～20nm) is claret to medium sized collaurum, and (30～80nm) then is mauve to the collaurum of larger particles.According to these characteristics, but the size of the color guestimate gold grain of the collaurum that detects by an unaided eye.
Because different its maximum absorption wavelengths at visible region of the particle diameter of collaurum (λ max) 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 mean grain size of collaurum more accurately, as: immerse in the 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 under transmission electron microscope, observe, mainly observe whether uniformity of the size of gold grain and particle.
Danscher (Histochemistry, 1984,81,331; Histochemistry, 1981,71,81) and Holgate (J.Histochem.Cytochem., 1983,31,938) in immuning tissue's dyeing, strengthen the collaurum colour developing by simple substance silver, thereby find that silver salt (as: actol or silver acetate) can strengthen gold mark signal (Mikroskopie (Vienna) 1985,42,318).After in traditional collaurum lateral flow immune detection, using silver atoms, gold mark signal can improve the 1-2 order of magnitude (J.Immunol.Meth., 140,131-134).All reactants (comprising golden combination and reinforcing agent silver salt) all are dried and only open by sample is molten on test strip.This indirectly enhancing technology is expected in the scope of pg/ml analysans be detected.The reinforcing agent silver salt places between the sample pad and colloid gold label pad on the reagent strip usually.
The material that detecting pad uses is selected from cellulose acetate, cellulose nitrate, regenerated cellulose, nylon and polyvinylidene fluoride.Wherein, cellulose nitrate is the most frequently used material.Membrane aperture is when 15-20 μ m, and the flow velocity of liquid on film can be faster.Because common fenestra is parallel to membrane plane and can't measures, the lateral flow film is classified according to the flow velocity of lateral flow usually, take " s/4cm " as unit, i.e. and chromatography time of every 4cm film water.The film of 6 kinds of models that Millipore company provides (Hi-FlowPlus 240,180,135,90,120,90 and 75), numerical value just represents the chromatography time of corresponding every 4cm film water on it, but faster its detection sensitivity of flow velocity is also lower, and is generally commonly used with 135s/4cm and two kinds of models of 180s/4cm.
The another kind of composition that is used for detecting rheumatoid arthritis immune antibody by lateral chromatography comprises CCP peptide, high molecular polymer covalently bound with it, and the CCP peptide content that is incorporated into the detection zone of detecting pad is 100ng-1000 μ g/cm 2
The another kind of composition that is used for detecting rheumatoid arthritis immune antibody by lateral chromatography comprises the CCP peptide, and high molecular polymer covalently bound with it, the CCP peptide content that is incorporated into the detection zone of detecting pad are 1 μ g-50 μ g/cm 2, described CCP peptide is selected among the SEQ ID No:1-261 any one.
The another kind of composition that is used for detecting rheumatoid arthritis immune antibody by lateral chromatography comprises the CCP peptide, and high molecular polymer covalently bound with it, the CCP peptide content that is incorporated into the detection zone of detecting pad are 1 μ g-50 μ g/cm 2, described CCP peptide is selected from arbitrarily several combinations with arbitrary proportion among the SEQ ID No:1-261.
The another kind of composition that is used for detecting rheumatoid arthritis immune antibody by lateral chromatography, comprise and be selected from SEQ ID No:1,3 and 119 CCP peptide, be covalently bonded in high molecular polymer at 1: 1: 1 with mol ratio, the CCP peptide content that is incorporated into the detection zone of detecting pad is 1 μ g-50 μ g/cm 2
Above-mentioned composition for detecting rheumatoid arthritis immune antibody by lateral chromatography, its high molecular polymer can be selected from bovine serum albumin(BSA) (bovine serum albumin, BSA), human serum albumins (Humal serum albumin, HSA), albumin rabbit serum (RSA), chicken ovalbumin (ovalbumin, OVA) keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH), dendrimer, polylysine, polyglycol or antibody.The detection zone area of detecting pad is generally 3.3mm * 0.8mm.
The beneficial effect that technical solution of the present invention realizes
The present invention is covalently bonded in the antigen that forms behind the high molecular polymer with the CCP peptide and is incorporated into the vitro detection that can realize immune antibody of rheumatoid arthritis on the detection medium.
The CCP peptide polymer is used for lateral chromatography not only can realize that the rapid in-vitro of immune antibody of rheumatoid arthritis detects, and is convenient to the self-service diagnosis of patient, provides reference in time understanding PD.
Not homotactic CCP peptide is covalently bonded in the antigen that forms behind the high molecular polymer is used for lateral chromatography, improved sensitivity and the selectivity of antibody test, increased the accuracy that detects, enlarged the applicable crowd who detects.
Term involved in the present invention is identical with its general concept.
Described " CCP peptide high molecular polymer covalently bound with it ", " CCP peptide polymer " or " CCP peptide and high molecular polymer covalent combination " refer to the CCP peptide directly or the indirect and high molecular polymer covalent bond by connexon.
Described " mounting medium " made by plastics or glass, can covalently or non-covalently be combined with the high molecular polymer covalent combination with CCP peptide or CCP peptide, for the combination of CCP peptide polymer and antibody provides support.
Described " detection medium " made by plastics or glass, can covalently or non-covalently be combined with the high molecular polymer covalent combination with CCP peptide or CCP peptide, for combination and the detection of CCP peptide polymer and antibody provides support.In the middle of lateral chromatography, detecting medium comprises sample pad, mark pad, detecting pad, adsorptive pads, support baseboard and is incorporated into essential material on the medium, such as the label on the mark pad, the aglucon that combines with label and be incorporated into antibody on the detecting pad C line etc.Wherein, aglucon is such as staphylococcal protein A, immunoglobulin (Ig), toxin, glycoprotein, enzyme, microbiotic, hormone and bovine serum albumin(BSA) etc.
Described " detection line " or " detection zone " all refer to be combined with the zone of CCP peptide in lateral chromatography.
Described " control line " or " control zone " all refer to be combined with the zone of the molecule that can combine with aglucon on the label in lateral chromatography, as: be combined with the mouse-anti human IgG on the label, this zone can be combined with sheep anti-mouse igg, combines with the mouse-anti human IgG and develops the color in this zone realizing.
Described " C1-C10 alkyl ", " C1-C20 alkyl " and " C1-C30 alkyl " refer to straight or branched alkyl, the carbon number that the numeral group is contained.
Described " fatty acid of C1-C30 " refers to saturated or undersaturated straight or branched carbochain, has a carboxyl on it at least, the carbon number that the numeral group is contained.
Described " glycosyl of C1-C30 ", the carbon number that its numeral group is contained.
Embodiment
Below describe technical scheme of the present invention in detail.The embodiment of the invention is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
If the used reagent of the present invention does not clearly indicate, then all available from Sigma-aldrich (Sigma-Aldrich).
Embodiment 1 polypeptide I's is synthetic
Polypeptide adopts the Fmoc chemical method, and is synthetic by solid phase synthesis technique.The concrete steps of the method are referring to Eur.J.Immunol.1994,24,3188-3193; J.Org.Chem.1972,37,3404-3409; Huang Weide, old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985.
The formation method of disulfide bond and step thereof can be referring to document: Huang Weide, and old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985, p85; Michael W.Pennington Peptide SynthesisProtocols (Methods in Molecular Biology), Humana Press, 1994, p91-169.
By above-mentioned steps, the concrete sequence of synthetic polypeptide is:
Polypeptide I:His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Arg-His-Cit-Arg-Leu-Il e-Arg-Cys-Gly, the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make polypeptide form ring texture, and can simulate β-corner structure.
Embodiment 2 polypeptide II's is synthetic
According to the synthetic polypeptide II:His-Gln-Cys-Ala-Arg-Phe-Gln-Met-Cit-His-Cit-Arg-Leu-I le-Arg-Cys-Gly of embodiment 1 disclosed method, the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make polypeptide form ring texture, and can simulate β-corner structure.
Embodiment 3 polypeptide III's is synthetic
According to the synthetic polypeptide III:His-Gln-Cys-His-Gln-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Ala-Ala-Cys-Gly of embodiment 1 disclosed method, the 3rd and sixteen bit Cys form disulfide bond by sulfydryl on it, and make polypeptide form ring texture, and can simulate β-corner structure.
The purifying of embodiment 4 polypeptide
With protecting group deprotection in dimethylformamide (DMF) solvent of protein or polypeptide end, neutralize first.With hydrogen fluoride polypeptide is downcut from synthetic resin again, the crude product that obtains is through reverse-phase chromatography C18 or C8 post (as: 5 μ m, 250 * 4.6mm), take mobile phase A (0.1% (v/v) trifluoroacetic acid acetonitrile solution) and Mobile phase B (0.1% (v/v) trifluoroacetic acid aqueous solution) as the gradient elution solvent.In 45 minutes, mobile phase A accounts for A, B two-phase cumulative volume 0% (v/v) changes to 100% (v/v) collection and obtains target polypeptides, remove organic solvent by desalination chromatographic column (GE Healthcare) or rotary evaporation, target polypeptides can be determined by the molecular weight that the ESI mass spectrum obtains by the mode that LC-MS is used in conjunction the polypeptide direct injected that maybe collection is obtained.(referring to: Chinese analytical chemistry 2002,30,1126-9)
Purity and the molecular weight detection of embodiment 5 polypeptide I and polypeptide II
The above-mentioned polypeptide I that synthesizes and polypeptide II measure by reverse-phase chromatography (RP-HPLC) respectively, and its concrete grammar is:
4.6 * 250mm 5 μ m C18 analytical columns (Kromasil);
Mobile phase A is that trifluoroacetic acid (trifluoroacetic, TFA) adds in 100% acetonitrile (acetonitrile, ACN), so that TFA concentration is 0.1% (v/v);
Mobile phase B is that TFA adds in 100% water, so that TFA concentration is 0.1% (v/v);
Flow velocity is 1.0ml/min;
The detection wavelength is 220nm;
Gradient: the proportional balancing method that accounts for two cumulative volumes 15% of A, B (v/v) with mobile phase A, behind the sample introduction, adopt linear gradient elution (Gradient Elution), mobile phase A accounts for two cumulative volumes of A, B and changes to the ratio of 50% (v/v) from 15% (v/v) in 25 minutes, afterwards with 100% (v/v) mobile phase A balance 5 minutes.
The retention time of polypeptide I (Retention Time) is 12.5, and its purity is greater than 95%.
The retention time of polypeptide II is 13.2, and its purity is greater than 95%.
Measure respectively the molecular weight of polypeptide I and polypeptide II by the ESI mass spectrum, its actual conditions is:
Mass spectrum (Probe) ESI
Mass spectrum voltage (Probe bias)+4.5kv
Sprayer flow velocity (Nebulizer Gas Flow) 1.5L/min
Detecting device (Detector) 1.5kv
Sample ionization gasifying device (CDL)-20.0v
Flow rate of mobile phase (T.Flow) 0.2ml/min
250 ℃ of CDL temperature (CDL Temp)
Buffer concentration (B.conc) 50%H 2O, 50%ACN
200 ℃ of heat block temperature (Block Temp)
Polypeptide I molecular weight is 2167.59Da; Polypeptide II molecular weight is 2168.58Da.
Purity and the molecular weight detection of embodiment 6 polypeptide III
The above-mentioned polypeptide III that synthesizes is by reverse-phase chromatography (RP-HPLC). and measure, its concrete grammar is:
4.6 * 250mm 5 μ m C18 analytical columns (Kromasil);
Mobile phase A is that trifluoroacetic acid (trifluoroacetic, TFA) adds in 100% acetonitrile (acetonitrile, ACN), so that TFA concentration is 0.1% (v/v);
Mobile phase B is that TFA adds in 100% water, so that TFA concentration is 0.1% (v/v);
Flow velocity is 1.0ml/min;
The detection wavelength is 220nm;
Gradient: the proportional balancing method that accounts for A, B two-phase cumulative volume 10% (v/v) with mobile phase A, behind the sample introduction, adopt linear gradient elution (Gradient Elution), mobile phase A accounts for A, B two-phase cumulative volume and changes to the ratio of 35% (v/v) from 10% (v/v) in 25 minutes, afterwards with 100% (v/v) mobile phase A balance 5 minutes.
The retention time of polypeptide III is 9.32, and its purity is greater than 95%.
Use the mass spectrum condition identical with embodiment 5, the molecular weight of mensuration polypeptide III is 2017.26Da.
The combination of embodiment 7 polypeptide and high molecular polymer
2mg carrier protein B SA is dissolved in 200 μ l deionized waters, and the above-mentioned three kinds of CCP of 2mg (mol ratio is 1: 1: 1) are dissolved in 0.5ml coupling buffer (0.1mol/L MES, 0.9mol/L NaCl, 0.2g/L NaN 3, pH4.7) in.0.5ml CCP solution is added 200 μ l carrier protein solution.Be that the 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) solution of 10mg/ml adds in the mixed liquor of CCP and BSA with 1ml concentration, mixing gently, at room temperature react 2h, with 0.01mol/L pH7.4 phosphate buffer in 4 ℃ of dialysis 3 days, after the packing 4 ℃ of preservations.
Embodiment 8IgG mark collaurum
Heating 100ml ultrapure water adds the HAuCl of 1% (w/v) to 80-90 ℃ 41.64ml, continue heating and be stirred to boiling.Fill the about 10s of boiling, the citric acid three sodium solution that rapid adding is now joined (the 0.0236g trisodium citrate is made into the concentration of about 1% (w/v)).Stir boiling reaction 6min.The room temperature cooling.
In the aurosol that is cooled to room temperature that every 100ml prepares, add the phosphate buffer of 1ml 0.1M (PH7.0), with the NaOH of 1M, transfer pH to 7.5.
Add 1mg mouse-anti human IgG (Keelung, Hangzhou Bioisystech Co., Ltd) (the 0.1M phosphate buffer is diluted to 1mg/ml), stirring at room 40min.
Add BSA solution sealing 20min above (solution of 10% (w/v) adds 1ml).The centrifugal 25min of 10,000rpm abandons supernatant.
Precipitation is the phosphate buffer cleaning (0.002M, pH7.0) of 0.01% (w/v) BSA with concentration; The centrifugal 25min of 10,000rpm abandons supernatant; The precipitation PEG20 that contains 1% (w/v) B SA, 000 dissolving, the trehalose of the sucrose and 5% (w/v) of adding 20% (w/v), filtering with microporous membrane is used in dissolving, surveys the absorption value under the 535nm wavelength.
Embodiment 9 polypeptide-BSA is incorporated into detecting pad
Get the CCP-BSA antigen of debita spissitudo, be coated on the detection zone with some film machine, the cellulose membrane drying case after being coated with is suitably dry rear for subsequent use.
Point control line on nitrocellulose filter (control line, C line) and detection line (test line, T line), discharge rate is 1.2 μ g/cm.
C line solution is for being mixed with the sheep anti-mouse igg (Keelung, Hangzhou Bioisystech Co., Ltd) of 1mg/ml with dilution 1 * PBS (3% trehalose).
T line solution is for being mixed with CCP-BSA antigen with dilution 0.02M Tris (2% trehalose, 0.5%NaCl, pH8.0), and wherein the concentration of CCP peptide is 1.8mg/ml.
The sheet material that point is good dries by the fire 2h in 37 ℃ of baking ovens.
Embodiment 10 gold medal marks detect embodiment
The anti-CCP antibody gold mark detection test paper comprises base plate, adsorptive pads, nitrocellulose filter, is combined with golden mark pad and the sample pad of marking (embodiment 8) of mouse-anti human IgG monoclonal antibody; The base plate middle part is nitrocellulose filter, be provided with a test wire (T line) and a control line (C line) on the nitrocellulose filter, base plate one end is adsorptive pads, the other end is the absorption of sample pad, the two ends of nitrocellulose filter are filled up mutual overlapping the connection with adsorptive pads with the mark of being connected respectively, adopt the anti-CCP antibody in lateral chromatography (indirectly solid-phase immunity gold chromatography) the qualitative detection sample.Be combined with the CCP-BSA bond of embodiment 7 preparations on the test wire, be combined with sheep anti-mouse igg (embodiment 9) on the control line.
Mark mouse-anti human IgG on colloid gold particle, at the coated CCP polypeptide of the detection zone (T line) of test paper, control zone (C line) is coated with dynamics.During detection, the colloid gold label mouse-anti people's of the anti-CCP antibody in the sample and Immuno gold pad IgG is combined into bond, this bond is along with the capillarity migration of film arrives detection zone, react with the CCP polypeptide on the film, a red stripes appears, no matter whether have anti-CCP antibody in the sample, when liquid level continues to migrate to when being fixed with anti-mouse IgG district and being with, a red stripes must appear in the control zone.If coloured band appears in detection zone, then point out positive findings, showing has rheumatoid arthritis antibody in detected person's body; Only have the control zone red stripes to occur, then point out negative findings, show that the measured does not have immune antibody of rheumatoid arthritis; Red stripes is simultaneously as the inner quality standard of test paper, helps to judge that sample size is whether enough or whether chromatography process is normal.Whole testing process can be finished in 15 minutes.
Sequence table
＜110〉ShangHai RongSheng Biology Pharmacy Co., Ltd
＜120〉be used for the composition of detecting rheumatoid arthritis immune antibody by lateral chromatography
<130>0911175
<160>261
<170>PatentIn version 3.3
<210>SEQ ID No 1
<211>17
<212>PRT
＜213〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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 ID No 53
<211>17
<212>PRT
＜213〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉manually close penta
＜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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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
Gly
<210>SEQ ID No 110
<211>17
<212>PRT
＜213〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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 No161
<211>17
<212>PRT
＜213〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial synthetic
＜223〉Xaa is citrulline
<400>170
His Gln Cys His Gln Phe Gln Phe Arg His Xaa Arg Leu lle Arg Cys
1 5 10 15
Gly
<210>SEQ ID No 171
<211>17
<212>PRT
＜213〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉entering the worker synthesizes
＜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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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〉artificial 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 (10)

1. composition that is used for detecting rheumatoid arthritis immune antibody by lateral chromatography, the detection medium that comprises the CCP peptide, is combined with high molecular polymer with its covalently bound high molecular polymer;
Described CCP peptide is selected from SEQ ID No:1,118 and 119 CCP peptide, is covalently bonded in high molecular polymer at 1: 1: 1 with mol ratio.
2. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1, the CCP peptide content that it is characterized in that combination on the described detection medium is 100ng-1000 μ g/cm 2
3. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1, the CCP peptide content that it is characterized in that combination on the described detection medium is 1 μ g-100 μ g/cm 2
4. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1 is characterized in that described high molecular polymer molecular weight is greater than 2,000Da.
5. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1 is characterized in that described high molecular polymer is selected from bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, chicken ovalbumin, keyhole limpet hemocyanin, branch type polymkeric substance, polyglycol or antibody.
6. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1 is characterized in that also being combined with label on the described detection medium.
7. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1 is characterized in that also being combined with the reinforcing agent silver salt on the described detection medium.
8. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 1 is characterized in that also being combined with collaurum on the described detection medium.
9. the composition for detecting rheumatoid arthritis immune antibody by lateral chromatography according to claim 8 is characterized in that described collaurum particle diameter is 40-100nm.
10. one of according to claim 1-9 described composition for detecting rheumatoid arthritis immune antibody by lateral chromatography is characterized in that the covalent bond between described CCP peptide and the high molecular polymer is selected from amido link, urine key, ester bond, thioether bond, hydrazone key, oxime key or disulfide bond.
CN 200910049453 2009-04-16 2009-04-16 Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography Active CN101865913B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200910049453 CN101865913B (en) 2009-04-16 2009-04-16 Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 200910049453 CN101865913B (en) 2009-04-16 2009-04-16 Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography

Publications (2)

Publication Number Publication Date
CN101865913A CN101865913A (en) 2010-10-20
CN101865913B true CN101865913B (en) 2013-04-03

Family

ID=42957716

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200910049453 Active CN101865913B (en) 2009-04-16 2009-04-16 Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography

Country Status (1)

Country Link
CN (1) CN101865913B (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102539786B (en) * 2011-12-31 2014-06-04 上海凯创生物技术有限公司 Microscale urinary albumin colloidal gold detection kit and preparation technology thereof
CN102539773B (en) * 2011-12-31 2014-07-16 上海凯创生物技术有限公司 KET (Ketamine) colloid gold detection kit and preparation method thereof
CN107462722B (en) * 2015-11-16 2019-10-11 孙广玉 It is a kind of for detecting the composition of tumour
CN108267576B (en) * 2017-01-03 2020-07-14 深圳市新产业生物医学工程股份有限公司 Modified CCP antigen and use thereof, anti-CCP antibody detection kit and manufacturing method thereof
CN113030456A (en) * 2021-04-02 2021-06-25 山东康华生物医疗科技股份有限公司 Detection test strip, detection card and kit for anti-cyclic citrullinated peptide antibody

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1491959A (en) * 2002-08-20 2004-04-28 昆明广博科技有限公司 Preparation of CCP polypeptide series and its use
CN1796997A (en) * 2004-12-22 2006-07-05 上海富纯中南生物技术有限公司 Detection kit for diagnosing RA, preparating kit, and method for completing standard of quality detection
CN101407541A (en) * 2008-11-26 2009-04-15 上海精臻生物科技有限公司 Modified allosteric type cyclic citrulline polypeptide, and fusion protein, antibody and reagent kit thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090117584A1 (en) * 2005-06-29 2009-05-07 Genmab A/S Non-Human mammalian Arthritis Model Featuring Human Antibodies Against Citrul-Linated Proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1491959A (en) * 2002-08-20 2004-04-28 昆明广博科技有限公司 Preparation of CCP polypeptide series and its use
CN1796997A (en) * 2004-12-22 2006-07-05 上海富纯中南生物技术有限公司 Detection kit for diagnosing RA, preparating kit, and method for completing standard of quality detection
CN101407541A (en) * 2008-11-26 2009-04-15 上海精臻生物科技有限公司 Modified allosteric type cyclic citrulline polypeptide, and fusion protein, antibody and reagent kit thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GERARD A. SCHELLEKENS,et al.THE DIAGNOSTIC PROPERTIES OF RHEUMATOID ARTHRITIS ANTIBODIES RECOGNIZING A CYCLIC CITRULLINATED PEPTIDE.《ARTHRITIS & RHEUMATISM》.2000,第43卷(第1期),155-163.
GERARD A. SCHELLEKENS,et al.THE DIAGNOSTIC PROPERTIES OF RHEUMATOID ARTHRITIS ANTIBODIES RECOGNIZING A CYCLIC CITRULLINATED PEPTIDE.《ARTHRITIS &amp *
RHEUMATISM》.2000,第43卷(第1期),155-163. *

Also Published As

Publication number Publication date
CN101865913A (en) 2010-10-20

Similar Documents

Publication Publication Date Title
CN102023219B (en) Composition for detecting rheumatoid arthritis immune antibody by colloidal gold lateral chromatography
CN101865913B (en) Composition for detecting rheumatoid arthritis immune antibody by lateral chromatography
US7514531B2 (en) Specific binding sites in collagen for integrins and use thereof
CN108700584A (en) Labeled complex and preparation method thereof, kit, application and detecting system
PL178895B1 (en) Novell cell activity controlling factor
CN109929009B (en) CCP peptide fragment, antigen, reagent, kit and application containing CCP peptide fragment
JPH11510596A (en) Quantitative measurement method of human acute phase serum amyloid A protein, recombinant protein, specific antibody
CN101995463B (en) Composition for detecting rheumatoid arthritis immune antibody by dot immuno-gold filtration assay
KR880001338B1 (en) Method and reagent for use in the assay of pivka-ii
CN102004154B (en) Composition for detecting immune antibodies of rheumatoid arthritis by dot immunogold filtration assay
CN102012431B (en) CCP (Cyclic Citrullinated Peptide) used for detecting the immune antibody of rheumatoid arthritis with dot immuno-gold filtration assay
Nexø et al. Comparison of a receptor binding assay with a radioimmunoassay for measuring human epidermal growth factor-urogastrone in urine
JPH03175360A (en) Medicament screening assay
CN101819201B (en) Polypeptide composition for detecting immune antibody of rheumatoid arthritis in vitro
JPH02253162A (en) Detection using aequorin conjugated with material having specific conjugatability
CN101726588B (en) Composition for externally detecting rheumatoid arthritis antibody and application thereof
Cuatrecasas Membrane receptors: methods for purification and characterization
CN101812119B (en) polypeptide combined with immune antibody and application thereof
AU2012309417A1 (en) Method for measuring anti-WT1 antibody
Kayser et al. Biotinylated epidermal growth factor: a useful tool for the histochemical analysis of specific binding sites
Howl et al. V1a vasopressin receptors: Selective biotinylated probes
Dürauer et al. Evaluation of a sensitive detection method for peptide arrays prepared by SPOT synthesis
CN101724024B (en) Polypeptide combined with immune antibody and application thereof
CN111748035B (en) Novel FN3 antibody mutant and application thereof
Hekimi et al. Antisera against AKHs and AKH precursors for experimental studies of an insect neurosecretory system

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CP01 Change in the name or title of a patent holder

Address after: 201108 No. 888 Xiangyang Road, Shanghai, Minhang District

Patentee after: Shanghai Rongsheng Biological Pharmaceutical Co.,Ltd.

Address before: 201108 No. 888 Xiangyang Road, Shanghai, Minhang District

Patentee before: SHANGHAI RONGSHENG BIOTECH CO.,LTD.

CP01 Change in the name or title of a patent holder