CN1979165B - Alpha-fodrin antigen epi-position polypeptide mixture and its use - Google Patents
Alpha-fodrin antigen epi-position polypeptide mixture and its use Download PDFInfo
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Abstract
The invention relates to screen for antigen epitope polypeptide, and building up of immunology detection method and the manufacturing and application for reagent for clinical diagnosis, especially for alpha-cyst albumen antigen epitope polypeptide.
Description
Technical field
The invention belongs to field of immunology, relate to the screening of antigen epitope polypeptide, the foundation of immunological detection method and the preparation of reagent for clinical diagnosis and application, especially relating to the screening of alpha-fodrin antigen epi-position polypeptide and the design of alpha-fodrin antigen epi-position polypeptide mixture forms, obtain having two best alpha-fodrin antigen epi-position polypeptide corresponding amino acid sequence of diagnostic significance, and the immunological method that set up to detect α-total antibody of fodrin polypeptide IgG/IgA in patient's serum, and with two best alpha-fodrin antigen epi-position polypeptides of synthetic as the application of hybrid antigen in the medicine of preparation diagnosis sjogren syndrome.
Exactly, the present invention relates to a kind of new epitope polypeptide and corresponding amino acid sequence thereof of people's sjogren syndrome antigen protein that adopts modern information biology, immunological technique screening to obtain, these two antigen epitope polypeptides can be distinguished anti-IgG, the IgA antibody in the specific detection Patients with Sjogren Syndrome serum.The invention still further relates to two antigen epitope polypeptide optimal conditions combinations " mixing ELISA " combination " antibody repertoire ELISA " method of carrying out and detect the immunological method of anti-α-total antibody of fodrin polypeptide IgG/IgA.In addition, the invention provides the application of this antigen epitope polypeptide mixture in the clinical diagnosis of people's sjogren syndrome.
Technical background
Sjogren syndrome (
Syndrome is a kind of based on mouth, xerophthalmia scheorma SS), and the general autoimmune disease of involving a plurality of systems of whole body and organ, is divided into primary (pSS) and secondary Sjogren syndrome (sSS).Epidemiology survey is found, the morbidity of China's sjogren syndrome is 0.77%, and the morbidity in crowd more than 50 years old reaches 5%, even apparently higher than the morbidity of diabetes (0.67%), rheumatic heart disease (0.19%) and cor pulmonale (0.47%), total number of patients has exceeded 8,000,000.Yet, clinically, the Patients with Sjogren Syndrome of exhausted most (97%) is diagnosed timely and is treated, and 52% patient is interstitial pulmonary fibrosis, autoallergic and ephritis etc. by mistaken diagnosis, and fail to obtain regular treatment or miss to control, even the organ injury of appearance appears controlling because of mistake.This disease is chronic general autoimmune disease, engenders that without the correct patient who treats organ dysfunction is unusual, depleted more, even dead.Malignant tumour such as lymphoma, myelomatosis can be up to 40 times of normal people in the incidence of sjogren syndrome.At present, this sick early diagnosis is a puzzlement clinicist greatest problem for many years.Clinical diagnosis is mainly by indexs such as symptom, sign, labial gland biopsy and non-specific antinuclear antibody.Wherein the labial gland biopsy belongs to traumatic inspection, and susceptibility is poor, and Most patients can not be accepted.Then specificity is relatively poor for antinuclear antibody, SSA antibody, SSB antibody, and susceptibility is also undesirable.During most of patient has belonged to when making a definite diagnosis, late period, often be in a bad way, and the impaired or complication of a plurality of internal organs arranged, very difficult in the treatment.
Hayashi equals to find in 1997 a kind of organ specific antigen at Patients with Sjogren Syndrome sialisterium ductal epithelial cell, further be tested and appraised amino-acid residue and determine that this antigen is human skelemin α-fodrin, relevant (the Haneji N with sjogren syndrome of antigen of this 120KD is proposed first, Nakamura T, Yakiok K et al, Identification of α-Fodrin as a candidate autoantigen in primary
Syndrome.Science, 1997,276 (25): 604-606.).In recent years, foreign scholar and the inventor continued the relevant α-fodrin antibody of sjogren syndrome is studied, and be concentrated in the following areas about the pathogenesis of this antibody and sjogren syndrome and diagnosis correlative study and existing problems:
(1) positive rate of α-fodrin antibody:, can use its purifying antigen, or the α of lysis-fodrin product detects the antibody in patient's serum because α-fodrin antigen is more special to Patients with Sjogren Syndrome.But present result of study is not the same.Hayashi research group uses α-fodrin fusion rotein-JS-1, utilizes Western blot method, detects primary Sjogren's syndrome 43 examples, secondary Sjogren syndrome 8 examples, systemic lupus erythematous (SLE) 21 examples, rheumatoid arthritis (RA) 14 examples, normal control 15 examples.The result shows, the positive rate of α-fodrin antibody is 95.35% in the primary Sjogren's syndrome patient, the secondary Sjogren syndrome patient is 50%, and at RA, do not detect α-fodrin antibody (Janicke RU in SLE and the normal population, Ng P, Sprengart ML, Porter AG, Caspase-3 is required for alpha-Fodrin cleavage but dispensable for cleavage of other deathsubstrates in apoptosis.J Biol Chem, 1998,273 (25): 15540-5.).Yet, Masataka etc. discover, α-fodrin antibody only is 50% (42/82) at the positive rate of Patients with Sjogren Syndrome, susceptibility is 52%, specificity is 96% (Masataka K, Tetsuroh O, Yoko O, Junichi D, and Yutaka K, Autoantibodies tothe Amino-Terminal Fragment of β-Fodrin expressed in Glandular Epithelial Cells in Patientswith Sjogren ' s syndrome.The Journal of Immunology, 2001,167:5449-5456).
(2) to the detection of children's sjogren syndrome: application Western blot methods such as Maeno detect the α-fodrin antibody in the 15 routine sjogren syndrome infant serum, 15 routine infant serum α-fodrin antibody are all positive, and all negative (Haneji.N of normal child, Nakamura.T, Takio.K, et al, Identification of alpha-Fodrin antibodiex inSjogren ' s syndrome in children.J Rheumatol, 2001,28:860.).This shows that the effect of α-fodrin in adult's primary Sjogren's syndrome pathogenesis may have identical meaning in children's sjogren syndrome, but its precise mechanism still need to further study clear and definite.
(3) IgA of α-fodrin and IgG antibody subtype: it is generally acknowledged that mostly unusual immunoglobulin (Ig) is the IgG hypotype in the Patients with Sjogren Syndrome body.Witte etc. use IgA and IgG antibody test primary Sjogren's syndrome (85 example) at first respectively, are secondary to the sjogren syndrome (15 example) of SLE and are secondary to α-fodrin antibody among sjogren syndrome (7 example) patients serum of RA.The result shows, 64% primary Sjogren's syndrome, and 47% sjogren syndrome and 86% that is secondary to SLE is secondary to the IgA antibody positive of the Patients with Sjogren Syndrome of RA.In control group, healthy blood donor (1/160), IgA antibody positive among the SLE (1/50), RA (2/12).It is relatively low to measure the IgG antibody positive rate with same method, and at primary Sjogren's syndrome, the positive rate in the sjogren syndrome of the sjogren syndrome of secondary SLE and secondary RA is respectively 55%, 40% and 43%.The healthy blood donor, SLE, the IgG antibody positive rate is respectively 3/160,1/50 and 5/12 among the RA.Therefore, IgA antibody and IgG antibody are compared, positive rate and the specificity of IgA are higher, and to secondary Sjogren syndrome more special (Witte T, Matthias T, Arnett FC, Deter HH, etal, IgA and IgG autoantibodies against alpha-fodrin as markers for Sjogren ' s syndrome.JRheumatol, 2000,27 (11): 2617-20.).Subsequently, many IgA and the researchs of IgG antibody subtype in dry syndrome detects about α-fodrin are arranged respectively, majority thinks that the IgA of α-fodrin is relevant with dry syndrome with the IgG antibody subtype, be well to diagnose antibody, but the result is not quite similar, mainly with the detection antigen-like material relevant (WitteT in source, Matthias T, Oppermann M, et al, Prevalence of antibodies against alpha-fodrin in Sjogren ' ssyndrome:comparison of 2sets of classification criteria.J Rheumatol, 2003,30 (10): 2157-9.Ruffatti A, Ostuni P, Grypiotis P, et al, Sensitivity and specificity for primary Sjogren ' ssyndrome of IgA and IgG anti-alpha-fodrrin antibodies detected by ELISA.J Rheumatol, 2004,31 (3): 504-7.Kahaly GJ, Bang H, Berg W, et al, Alpha-fodrin as a putative autoantigen inGraves ' ophthalmopathy.Clin Exp Immunol, 2005,140 (1): 166-72.Turkcapar N, Olmez U, Tutkak H, et al, The importance of alpha-fodrin antibodies in the diagnosis of Sjogren ' ssyndrome.Rheumatol Int, 2006,26 (4): 354-9.Yavuz S, Toker E, Bicakcigil M, et al, Comparative analysis of autoantibodies against a-fodrin in serum, tear fluid, and saliva frompatients with Sjogren ' s syndrome.J Rheumatol, 2006,33 (7): 1289-92.).
(4) research of alpha-fodrin antigen epi-position polypeptide: above research is carried out antibody test with the albumen of expression and purification, detected result can be subjected to the restriction of the character and the stability of purifying protein, thereby very big to true value and the stabilization result influence of α-fodrin in antibody test.People such as the inventor and Shiari R use overlapping protein fragments of expression and immunoassay screening to obtain the relevant alpha-fodrin antigen epi-position polypeptide (or albumen segment) of some sjogren syndromes, its susceptibility, specificity is compared antinuclear antibody, SSA is anti-, SSB antibody all increases, the result is also very stable, but finally because of the overlapping recombinant protein binding immunoassay of utilization prokaryotic expression check and analysis in the screening method, still do not meet with the independent detection value of antigen epitope polypeptide, add in the external a lot of research that the case sample is little etc., the specificity of this antibody test in the sjogren syndrome diagnosis, susceptibility is still not fully up to expectations, the more important thing is the research (He Jing that does not also have corresponding epitope polypeptide at more significant IgA antibody test, Li Jing, Li Zhanguo, Chen Qiaolin. the meaning of anti-α-fodrin polypeptide antibody in the sjogren syndrome diagnosis, China's rheumatology magazine, 2003,7 (10): 600-603.He Jing, Chen Qiaolin, Li Zhanguo, Antibodies to α-fodrin-derived peptide in Sjogren ' s Syndrome.Annals of Rheumatic Diseases, 2006,65 (4): 549-550. Chen Qiao woods, Piao Haiyan, He Jing, Li Jing, Li Zhanguo. the screening and the evaluation of sjogren syndrome antigen alpha-fodrin antigen epi-position, China's Journal of Immunology, 2005,11 (21): 864～872.Shiari R, Kobayashi I, Toita N, et al, Epitope mapping of anti-alpha-fodrin autoantibody in juvenile Sjogren ' ssyndrome:difference in major epitopes between primary and secondary cases.J Rheumatol, 2006,33 (7): 1395-400.Fox RI, Konttinen Yj, Fisher A, Use of Muscarinic Agonists in theTreatment of Sjogren ' s syndrome.Clin Immunol.2001,101 (3): 249-263.).
In sum, in the present known sjogren syndrome diagnostic antigen (albumen or polypeptide), still lack a kind of at people's sjogren syndrome specificity height, and the also high mark of positive rate; Lacking specific diagnostic method also is the very corn of a subject.At the dry syndrome related antigen---the exploratory development of Serum Antibody Detection and antigen epitope polypeptide screening has been carried out comprising in α-fodrin field, but the problem that still has many aspects is still needed clearing and is solved, as: the antigen epitope polypeptide that high specific, strong sensitivity α-fodrin IgG, the antibody test of IgA type are used still lacks further investigation; Still lack at α-fodrin IgG/IgA type antibody and detect the method for usefulness simultaneously.Therefore, screen and identify new α-fodrin related antigen epitope polypeptide, seek a kind of susceptibility height, high specificity and the clinical antibody that is easy to detect is set up better diagnostic method, the clinical diagnosis of sjogren syndrome is had important and urgent meaning.
Summary of the invention
At aforementioned present Research and sjogren syndrome clinical diagnosis demand, first purpose of the present invention is the alpha-fodrin antigen epi-position polypeptide (comprising its derivative) that provides new, with and corresponding amino acid sequence.Described two alpha-fodrin antigen epi-position polypeptides (comprising its derivative) can be distinguished anti-α-fodrin polypeptide IgG, the IgA antibody that detects specifically in the Patients with Sjogren Syndrome serum, article two, the alpha-fodrin antigen epi-position polypeptide mixture result that is used for detecting the total antibody of IgG/IgA is presented at the positive rate height of Patients with Sjogren Syndrome (former, secondary) serum, and positive rate is very low in other contrast diseases (rheumatoid arthritis, systemic lupus erythematous) and the normal population serum, meets the needs of clinical diagnosis fully.Thereby described two alpha-fodrin antigen epi-position polypeptides (comprising its derivative) mixture can be provided as people's sjogren syndrome clinical diagnosis hybrid antigen.
Second purpose of the present invention is to provide the screening method of described two alpha-fodrin antigen epi-position polypeptides and derivative thereof.The present invention adopts bioinformatics method binding immunoassay check and analysis method, and screening obtains can be used as the alpha-fodrin antigen epi-position polypeptide and the derivative thereof of people's sjogren syndrome hybrid antigen.
The 3rd purpose of the present invention is to provide a kind of and will contains described alpha-fodrin antigen epi-position polypeptide or derivatives thereof and be used to detect the method for the anti-α of Patients with Sjogren Syndrome serum-total antibody of fodrin polypeptide IgG/IgA as hybrid antigen, combination " antibody repertoire ELISA " method of promptly " mixing ELISA ".The present invention all optimizes the optimum mixture ratio example of two antigen epitope polypeptides and the various conditions of described immunological detection method, and obtains experimental verification.
The 4th purpose of the present invention is to provide described alpha-fodrin antigen epi-position polypeptide mixture to be used for diagnosing the application of the medicine of sjogren syndrome in preparation.This application is based on described alpha-fodrin antigen epi-position polypeptide mixture and measures the value evaluation result of anti-α-total antibody of fodrin polypeptide IgG/IgA in the sjogren syndrome diagnosis.Alpha-fodrin antigen epi-position polypeptide mixture of the present invention can be used to prepare the detection kit of sjogren syndrome.
According to first purpose of the present invention, the invention provides alpha-fodrin antigen epi-position polypeptide, comprising:
(a), the polypeptide of forming by the aminoacid sequence shown in the SEQ ID NO.1;
(b), the process of the aminoacid sequence in (a) replaces, lacking or add one or several amino acid and have can be active by (a) polypeptides derived with the epitope of anti-α-fodrin polypeptide IgG antibody response;
(c), the polypeptide of forming by the aminoacid sequence shown in the SEQ ID NO.2;
(d), the process of the aminoacid sequence in (c) replaces, lacking or add one or several amino acid and have can be active by (c) polypeptides derived with the epitope of anti-α-fodrin polypeptide IgA antibody response.
SEQ?ID?NO.1
NH3-Gln-Asp-Leu-Glu-His-Val-Glu-Val-Leu-Gln-COOH
SEQ?ID?NO.2
NH3-Arg-Asp-Leu-Ala-Ser-Val-Gln-Ala-Leu-Leu-Arg-Lys-COOH
Polypeptide is the partial amino-acid residue in α-fodrin sequence shown in SEQ ID NO.1, the SEQ ID NO.2.
According to second purpose of the present invention, the inventor chooses that 1-1680 nucleotide sequence corresponding amino acid sequence is an object among people α-fodrin ORF, by adopting Standard, Karplus, Emini, Amphiphi, 5 kinds of worlds such as Pellequer are about epitope forecast method (Odorico M, Pellequer JL, BEPITOPE:predicting thelocation of continuous epitopes and patterns in proteins.J Mol Recognit.2003, Jan-Feb; 16 (1): 20-22.), forecast analysis mainly comprises the secondary structure relevant with protein antigenicity, accessibility, wetting ability, β-corner, signal peptide cutting site, wears film spiral, N-glycosylation site etc., by analyzing antigen trend index, tentatively determined the distribution of antigenic determinant amino-acid residue of the α-fodrin of above-mentioned 560aa length.Further by synthetic polypeptide and immunology detection analysis, two corresponding separately antibody susceptibility and all very high antigen epitope polypeptides of specificity of detecting have been obtained, article one, can be fine anti-α-fodrin polypeptide IgG type antibody in the detection patient serum, the anti-α-fodrin polypeptide IgA type antibody in can fine detection patient serum.Simultaneously, through replacing, lack or adding one or several amino acid and have and to derive and next a series of polypeptide by these two peptide sequences with the epitope of anti-α-fodrin polypeptide IgG, IgA antibody response is active, also show good antigen-antibody binding reaction, these two polypeptide and corresponding series derivates (polypeptide) thereof are alpha-fodrin antigen epi-position polypeptide of the present invention.Alpha-fodrin antigen epi-position polypeptide in this invention separately all can the anti-α of fine detection-fodrin polypeptide IgG or IgA type antibody, and its mixture then can well detect anti-α-total antibody of fodrin polypeptide IgG/IgA.Up to now, still have nothing to do both at home and abroad in the antigenicity of these two antigen epitope polypeptides of α-fodrin (comprising two antigen epitope polypeptides and the corresponding polypeptide of deriving that screen) sequence, method that this antigen epitope polypeptide mixture detects the total antibody of corresponding IgG/IgA in serum and as the research of hybrid antigen meaning etc. in the sjogren syndrome diagnosis.
Alpha-fodrin antigen epi-position polypeptide of the present invention can obtain with the known method of those skilled in the art, for example: solid-phase synthesis etc.
Among above-mentioned selected people α-fodrin ORF the 1-1680nt gene order derive from NCBI (＞gi|4507190|ref|NM_003127.1|[4507190] spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) (SPTAN1), mRNA[Homo sapiens]).
According to the 3rd purpose of invention, the present invention has set up and has contained described alpha-fodrin antigen epi-position polypeptide mixture (SEQ ID NO.1 and SEQ ID NO.2) is used to detect the anti-α of Patients with Sjogren Syndrome serum-total antibody of fodrin polypeptide IgG/IgA as hybrid antigen method (" mixing ELISA " combination " antibody repertoire ELISA " method).
Enzyme-linked immunosorbent assay (ELISA) testing product roughly can be divided into three kinds: " monospecific ELISA " test kit (being coated with monospecific antigen) can provide the quantitative vitro detection to a kind of antibody; " antibody repertoire ELISA " test kit can carry out the sxemiquantitative vitro detection respectively to multiple different antibodies on a microwell plate; The solid-phase coating of " mixing ELISA " test kit has hybrid antigen, but the multiple antibody of half-quantitative detection." mixing ELISA " combination " antibody repertoire ELISA " method of the used scope of having widened specifically refers among the present invention: the utilization hybrid antigen, simultaneously different subtype antibody is detected, and the present invention's utilization is to carry out the different subtype detection of antibodies in the same micropore.
During " mixing ELISA " combination " antibody repertoire ELISA " method detects, at first the most important thing is the blending ratio between the envelope antigen (mixture), the inventor is by repeatedly groping, select the synthetic polypeptide of SEQ ID NO.1 sequence to be optimized experiment with the different matched proportion densities of the synthetic polypeptide of SEQID NO.2 sequence: 1: 2,6: 7,9: 14,5: 7,11: 14,6: 7,13: 14.Final definite optimum mixture ratio example is 11: 14, mix alpha-fodrin antigen epi-position polypeptide (SEQ ID NO.1 and SEQ ID NO.2) with this blending ratio, be coated on the polystyrene micropore plate of solid phase, add positive Patients with Sjogren Syndrome serum then, the final mixture (equal proportion) that adds two kinds of antibody of anti-human IgG/IgA of horseradish peroxidase (HRP) mark, reaction is best.
Secondly, because antigen antibody reaction needs necessary in conjunction with condition (pH value, ionic concn and iso-electric point etc.), especially in " mixing ELISA " combination " antibody repertoire ELISA " method detects, condition to hybrid antigen and each antibody response is more accurate, the inventor is in the experiment starting stage, weak a lot of after the addition when finding hybrid antigen with the independent reaction of different antibodies antibody response summation ratio, later stage (comprising: pH6.01 * PBS through a series of pH values and reactive ion concentration, pH6.51 * PBS, pH7.01 * PBS, pH7.51 * PBS, pH8.01 * PBS, pH8.51 * PBS, pH9.01 * PBS, pH9.51 * PBS etc.) after the adjustment, determined that stable the best bag is cushioned liquid (pH8.01 * PBS); The excessive concentration of envelope antigen all can influence specificity and the susceptibility that ELISA detects with crossing to hang down in addition, and especially under the unbalanced situation of clinical patients serum antibody titer, the best bag of selecting antigenic peptide is the key element of ELISA detection by concentration.This inventor has been carried out corresponding experiment, with described alpha-fodrin antigen epi-position polypeptide (SEQID NO.1 and SEQ ID NO.2) by 11: 14 mixed after repeatedly react after the dilution of the concentration gradient of different range, in the concrete concentration gradient dilution test, total package amount is respectively: 1ng, 10ng, 100ng, 1000ng; 10ng, 40ng, 60ng, 80ng, 100ng; 60ng, 65ng, 70ng, 75ng, 80ng.Finally determined the best package amount (70ng) of detection polypeptide (SEQ ID NO.1 and SEQ ID NO.2 polypeptide mixture), carried out Serum Antibody Detection with this package amount, effect is best; Having, about the prevention problem to antigen-antibody nonspecific reaction in the serum, also is another key factor that any clinical ELISA detection reagent can obtain fine application again.The inventor is by place research group experience in the past, adopt 1%OVA/PBS respectively, 1%BCA/PBS, 1% skim-milk/PBS and sealing are with just using rabbit anteserum etc. always, finally by clinical serum sample detection reaction background comparison, (be called: BNRS) (specific procedure is the unified in the industry sealing of definite utilization: will seal with 62 ℃ of deactivations of normal rabbit serum elder generation 20 minutes as confining liquid in the reaction after particular procedure with normal rabbit serum, use in the sealing of 25ml deactivation then and add 0.3275g Tris in the normal rabbit serum, 0.21915g NaCl, transfer to pH8.0 with HCl again), like this can be so that the background (non-specific binding) that anti-α-fodrin polypeptide antibody detects drops to minimum.
By above condition optimizing, the inventor will be contained described alpha-fodrin antigen epi-position polypeptide (SEQ ID NO.1 and SEQ ID NO.2) by optimum mixture ratio example (11: 14), by specific concentrations (total amount is 70ng), be coated on the polystyrene micropore plate of solid phase, the sealing that utilization is handled well is sealed with normal rabbit serum, add Patients with Sjogren Syndrome serum then, the final anti-human IgG/IgA mixtures of antibodies (equal proportion) that adds the HRP mark, merge outside the above optimal conditions, other links are identical with conventional mixing ELISA.The present invention widen scope through what optimize to set up " mixing ELISA " combination " antibody repertoire ELISA " method can well detect the total antibody of anti-IgG/IgA of clinical Patients with Sjogren Syndrome serum, experimental result shows, good, the high specificity of susceptibility of the anti-α of the Patients with Sjogren Syndrome serum that this method obtains-total antibody of fodrin polypeptide IgG/IgA meets the requirement of disease clinical diagnosis fully.
Compared with prior art, alpha-fodrin antigen epi-position polypeptide of the present invention is measured the value evaluation of anti-α-total antibody of fodrin polypeptide IgG/IgA in the sjogren syndrome diagnosis very remarkable advantages, and details are as follows:
At present, though launched big quantity research both at home and abroad at the IgA of α-fodrin and the detection of IgG antibody subtype, but the albumen with expression and purification carries out antibody test, the result can be subjected to the restriction of the character and batch stability of purifying protein, thereby very big to true effect and the interpretation of result influence of α-fodrin in antibody test.Research to antigen epitope polypeptide (or segment), the needs of clinical prods have been catered to, improved the stability that detects, but former studies is finally because of the overlapping recombinant protein binding immunoassay of utilization prokaryotic expression check and analysis in the screening method, the independent detection of having limited to antigen epitope polypeptide is worth, add in the external a lot of research that the case sample is little etc., specificity, the susceptibility of this antibody in the sjogren syndrome diagnosis is not fully up to expectations, but also does not have the correlative study of the epitope polypeptide of IgA antibody test correspondence.
Based on above present Research, the inventor adopts Standard, Karplus, Emini, Amphiphi, 5 kinds of worlds such as Pellequer are about the epitope forecast method, analyzed the antigenic determinant of the α-fodrin of 560aa length, further by synthetic polypeptide and immunodetection, two corresponding separately antibody susceptibility and all very high antigenic peptides of specificity of detecting have been obtained, article one, can the anti-α of fine detection-fodrin polypeptide IgG type antibody, one can be detected anti-α-fodrin polypeptide IgA type antibody.As detecting hybrid antigen, " mix ELISA " combination " antibody repertoire ELISA " method good with above-mentioned foundation detects the anti-α-total antibody of fodrin polypeptide IgG/IgA in sjogren syndrome, rheumatoid arthritis, systemic lupus erythematous and the normal human serum to the inventor with these two polypeptide.The present invention studies common detection Patients with Sjogren Syndrome 182 examples, Patients with SLE 113 examples, rheumatoid arthritis patients 136 examples, normal people's 212 examples.Anti-α-total the antibody of fodrin polypeptide IgG/IgA that found that the detected sjogren syndrome of this polypeptide mixture has higher susceptibility and specificity, can reach purpose, for the clinical diagnosis of sjogren syndrome provides objective laboratory foundation by serological method diagnosis sjogren syndrome.
According to the 4th purpose of the present invention, the present invention also provides described alpha-fodrin antigen epi-position polypeptide mixture to be used for diagnosing the application of the medicine of sjogren syndrome in preparation.
Measure the requirement that value evaluation result, the alpha-fodrin antigen epi-position polypeptide mixture of anti-α-total antibody of fodrin polypeptide IgG/IgA in the sjogren syndrome diagnosis measured anti-α-preliminary immunological method of the total antibody of fodrin polypeptide IgG/IgA (" mixing ELISA " combination " antibody repertoire ELISA " method) and sjogren syndrome the detection kit according to above-mentioned about alpha-fodrin antigen epi-position polypeptide mixture, the inventor has prepared the α-total antibody clinical diagnosis of fodrin polypeptide IgG/IgA test kit.What specifically adopt is to mix the multiple antibody act of direct solid-phase enzyme-linked immune adsorption analysis (" mixing ELISA " combination " antibody repertoire ELISA " method), is used to detect anti-α-total antibody of fodrin polypeptide IgG/IgA; Described alpha-fodrin antigen epi-position polypeptide (SEQ ID NO.1 and SEQ ID NO.2) mixes coated on microwell plate and through sealing by above-mentioned condition, detachable microwell plate (1 * 8) is totally 12 laths, can finish the detection of 96 person-portions.
This test kit is through 3 different step of reaction and form mixed complex, produces enzyme connection color reaction at last, and the depth of color becomes certain proportionlity with the concentration of antibody in the sample.Step 1: sample, standard substance and quality controlled serum add in the reacting hole of microwell plate (wrapped in the hole by above-mentioned hybrid antigen and finish sealing), the antibodies of any existence is at the micropore internal surface, hatches after scouring in 30 minutes to remove non-reacted constituent.Step 2: add horseradish peroxidase (HRP) anti-human IgG of mark goat and the anti-people IgA of HRP mark goat equal proportion mixtures of antibodies, hatched after scouring in 30 minutes to remove unconjugated unnecessary enzyme labelled antibody.Step 3: adding 3,3 ', 5,5 '-tetramethyl benzidine (being called for short TMB) substrate, hatched back reagent color and become blueness, adding stop buffer (containing 1mol/L HCl) termination reaction, reagent color yellowing in 15 minutes.
The concentration proportion relation of the total antibody of IgG/IgA in the depth of color and the sample.The total anti-body contg of the IgG/IgA of sample can be inferred out by typical curve according to its OD value.In microplate reader or spectrophotometer, read the result.450nm is as initial wavelength, and 600 to 650nm (being preferably 620nm) conduct is with reference to wavelength.
Description of drawings
Fig. 1 is the alpha-fodrin antigen epi-position antigen trend index analysis figure as a result of 560aa length.X-coordinate is represented the position of α-fodrin aminoacid sequence, and ordinate zou is represented antigen trend exponential size, and＞1.012 is possible epitope, and antigen trend index is big more, and the possibility of epitope is big more.This method accuracy is 75%.
Fig. 2 detects the total antibody principle schematic of anti-α-fodrin polypeptide IgG/IgA for mixing the multiple antibody act of direct solid-phase enzyme-linked immune adsorption analysis (" mixing ELISA " combination " antibody repertoire ELISA " method).Among the figure, by microwell plate, make solid phase carrier, and finish sealing with hybrid antigen (alpha-fodrin antigen epi-position polypeptide mixture) bag.Add patient's (sjogren syndrome and disease control group etc.) serum in plate hole, its contained antibody (anti-α-fodrin polypeptide IgG, IgA antibody) combines with the antigen that exists in the solid phase carrier specifically, forms the mixed immunity mixture.After removing unnecessary material, add the anti-human IgG of goat and the IgA mixtures of antibodies of horseradish peroxidase-labeled, make it and above-mentioned immunocomplex reaction.Wash plate, remove unnecessary binding substances, add substrate (TMB).This reaction produces color product, and colour intensity is directly proportional with specific antibody content.
Fig. 3 is the trace routine synoptic diagram of alpha-fodrin antigen epi-position polypeptide mixture foundation of the present invention " mixing ELISA " combination " antibody repertoire ELISA " method.
Fig. 4 shows the positive rate of α-total antibody of fodrin polypeptide IgG/IgA in various disease.The anti-as seen from the figure α-positive rate of the total antibody of fodrin polypeptide IgG/IgA in primary and secondary Sjogren syndrome is apparently higher than SLE, RA and normal control (P all＜0.01).
Fig. 5 shows susceptibility and the specificity that autoantibody detects in the Patients with Sjogren Syndrome serum sample.In Patients with Sjogren Syndrome, the susceptibility of anti-α-total antibody of fodrin polypeptide IgG/IgA and specificity are apparently higher than SSA, SSB antinuclear antibody (P all＜0.01) and anti-α-fodrin polypeptide IgG antibody, susceptibility is than anti-α-the independent detection of fodrin polypeptide IgA antibody also improves a lot, but specificity is lower slightly than it.Comprehensive, the anti-α-total antibody of fodrin polypeptide tool important value in sjogren syndrome serodiagnosis.
Embodiment
Term used herein:
Alpha-fodrin antigen epi-position polypeptide: be meant polypeptide, and have the polypeptide of SEQ ID NO.2 sequence RDLASVQALLRK (being NH3-Arg-Asp-Leu-Ala-Ser-Val-Gln-Ala-Leu-Leu-Arg-Lys-COOH) or above-mentioned two amino acid sequence of polypeptide are passed through replacement with SEQ ID NO.1 sequence QDLEHVEVLQ (being NH3-Gln-Asp-Leu-Glu-His-Val-Glu-Val-Leu-Gln-COOH), the disappearance or add one or several amino acid and have can with anti-α-fodrin polypeptide IgG, active the deriving of epitope of IgA antibody response and the polypeptide that comes.
Alpha-fodrin antigen epi-position polypeptide mixture: be meant to be optimized the mixture that forms after the mixed with having derive polypeptide and polypeptide or the above-mentioned corresponding polypeptide of deriving of the polypeptide of SEQ ID NO.1 common sequences or above-mentioned correspondence with SEQ ID NO.2 common sequences.
The screening and the evaluation of embodiment 1 alpha-fodrin antigen epi-position polypeptide
Aforesaid research work is verified, α-fodrin the fusion rotein that utilizes engineered method to be purified into can be specific in conjunction with the anti-α-fodrin antibody (IgA or IgG) in the Patients with Sjogren Syndrome serum, it can be detected (Cheney R by specific method then, Hirokawa N, Levine J, Willard M, Intracellular movementof Fodrin.Cell Motil, 1983,3 (5-6): 649-55.Haneji, N., Nakamura T, Takio K, et al, Identification of α-Fodrin as a candidate autoantigen in primary Sjogren ' s Syndrome.Science, 1997,276:604-7.Watanabc T, Tsuchida T, Kanda N, et al, Anti-α-Fodrin antibodies inSjogren ' s Syndrome and lupus erythematosus.Arch Dermatol, 1999,135:535-539.Witte T, Matthias T, Arnett C F, et al, IgA and IgG autoantibodies against α-Fodrin as marker forSjogren ' s Syndrome.J Rheumatol, 2000,27:2617-20.).In the experiment of the present invention, the contriver chooses that 1-1680 nucleotide sequence corresponding amino acid sequence is an object among α-fodrin ORF, adopt Standard, Karplus, Emini, Amphiphi, 5 kinds of worlds such as Pellequer are about the standard method of epitope prediction, multianalysis the antigenic determinant of α-fodrin of above-mentioned 560aa length, press the arrangement of antigen trend index, the results are shown in Figure 1, synthesized 23 polypeptide altogether according to the result.The corresponding sequence of the α of the 560aa length of choosing-fodrin and 23 polypeptid acid sequences of synthetic are respectively:
The 560aa sequence of 1-1680 nucleotide sequence correspondence is (holding the ordering of COOH end from NH3) among the α that chooses-fodrin ORF:
MDPSGVKVLETAEDIQERRQQVLDRYHRFKELSTLRRQKLEDSYRFQFFQRDAEELE
KWIQEKLQIASDENYKDPTNLQGKLQKHQAFEAEVQANSGAIVKLDETGNLMISEGH
FASETIRTRLMELHRQWELLLEKMREKGIKLLQAQKLVQYLRECEDVMDWINDKEAI
VTSEELGQDLEHVEVLQKKFEEFQTDMAAHEERVNEVNQFAAKLIQEQHPEEELIKT
KQDEVNAAWQRLKGLALQRQGKLFGAAEVQRFNRDVDETISWIKEKEQLMASDDF
GRDLASVQALLRKHEGLERDLAALEDKVKALCAEADRLQQSHPLSATQIQVKREELI
TNWEQIRTLAAERHARLNDSYRLQRFLADFRDLTSWVTEMKALINADELASDVAGAE
ALLDRHQEHKGEIDAHEDSFKSADESGQALLAAGHYASDEVREKLTVLSEERAALLE
LWELRRQQYEQCMDLQLFYRDTEQVDNWMSKQEAFLLNEDLGDSLDSVEALLKKH
EDFEKSLSAQEEKITALDEFATKLIQNNHYAMEDVATRRDALLSRRNALHE
23 peptide sequences of synthetic are (be from NH3 and hold the ordering of COOH end):
SGVKVLE?RRQQVLDR?IQEKLQI?LQGKLQKHQAFEAEV?SGAIVKL
RQWELLL?GIKLLQAQKLVQYLRECED?KEAIVTS?QDLEHVEVLQ
NQFAAKLIQE?RLKGLALQRQ?KLFGAAEV?RDLASVQALIRK
ERDLAALEDKVKALCAE?RLQQSHPLSATQIQVKR?YRLQRFLAD
LASDVAGAEALLDR?GQALLAAGH?VREKLTVLS?RAALLEL
QYEQCMDLQLFY?QEAFLLN?LGDFLDSVEALLKK
Adopt monospecific ELISA method to detect clinical 10 routine α-fodrin IgG antibody test male sjogren syndrome patient diagnosed's serum antibody and 10 routine α-fodrin IgA antibody test male sjogren syndrome patient diagnosed serum antibody respectively above-mentioned 23 synthetic α-fodrin polypeptide.
Concrete steps are: 23 synthetic α-fodrin polypeptide are cushioned liquid with bag respectively, and (1 * PBS pH8.0) is diluted to 0.5 μ g/ml, coated elisa plate (100 μ l/ hole), 37 ℃ of incubations 4 hours.Wash plate 5 times with 0.1%PBS-Tween, every hole adds sealing normal rabbit serum damping fluid (BNRS) (through handling, see before and state with preceding) 150 μ l, and 4 ℃ of overnight incubation are washed plate 5 times.Each adds (100 μ l/ hole) respectively after diluting 50 times with PBS the positive SS patients serum of anti-α-fodrin sample (comprising positive 10 examples of IgG, positive 10 examples of IgA), hatches 30min for 37 ℃.Wash the anti-human IgG of goat, the IgA two anti-(SantaCruz product) of the horseradish peroxidase-labeled of the corresponding respectively PBS of adding dilution in 1: 1000 behind the plate 5 times, hatch 30min for 37 ℃.Add TMB colour developing liquid colour developing 15min after washing plate 5 times, the HCL that adds 1mol/L stops the back and measures the absorption value (A value) of sample at the 450nm place.Every plate is established blank, positive control and negative control (all from the German BL alpha-Fodrin of company IgG antibody, the independent quantitative ELISA detection kit of IgA) simultaneously.Adopt the mean value of reaction OD value, the reaction power of 23 synthetic polypeptide of comparison and SS patients serum IgG, IgA, the polypeptide of the strongest reaction that obtains is regarded as SS (sjogren syndrome) associated antibodies optimum detection antigen---alpha-fodrin antigen epi-position polypeptide.
By above immunodetection analysis, two the strongest antigen epitope polypeptides of reaction have been obtained: the 9th, QDLEHVEVLQ (NH3-Gln-Asp-Leu-Glu-His-Val-Glu-Val-Leu-Gln-COOH), can the anti-α of fine detection-fodrin polypeptide IgG type antibody, the strongest with sjogren syndrome positive serum IgG antibody response in 23 synthetic polypeptide, OD mean value is: 0.68; Article 13,, RDLASVQALLRK (NH3-Arg-Asp-Leu-Ala-Ser-Val-Gln-Ala-Leu-Leu-Arg-Lys-COOH), can detect anti-α-fodrin polypeptide IgA type antibody, the strongest with sjogren syndrome positive serum IgA antibody response in 23 synthetic polypeptide, OD mean value is: 0.75.The results are shown in Table 1.
The sequence and the monospecific ELISA detected result of the synthetic polypeptide of 23 α-fodrins of table 1
| No | Starting point | Aminoacid sequence | Terminal point | IgG reaction OD mean value | IgA reaction OD mean value |
| 1 | 4 | SGVKVLE | 10 | 0.11 | 0.09 |
| 2 | 18 | RRQQVLDR | 25 | 0.14 | 0.18 |
| 3 | 60 | IQEKLQI | 66 | 0.08 | 0.06 |
| 4 | 78 | LQGKLQKHQAFEAEV | 92 | 0.25 | 0.16 |
| 5 | 96 | SGAIVKL | 102 | 0.09 | 0.07 |
| 6 | 129 | RQWELLL | 135 | 0.10 | 0.07 |
| 7 | 142 | GIKLLQAQKLVQYLRECED | 160 | 0.34 | 0.12 |
| 8 | 168 | KEAIVTS | 174 | 0.12 | 0.09 |
| 9 | 179 | QDLEHVEVLQ | 188 | 0.68 | 0.14 |
| 10 | 209 | NQFAAKLIQE | 218 | 0.21 | 0.31 |
| 11 | 239 | RLKGLALQRQ | 248 | 0.31 | 0.38 |
| 12 | 250 | KLFGAAEV | 257 | 0.24 | 0.21 |
| 13 | 285 | RDLASVQALLRK | 296 | 0.23 | 0.75 |
| 14 | 301 | ERDLAALEDKVKALCAE | 317 | 0.28 | 0.54 |
| 15 | 320 | RLQQSHPLSATQIQVKR | 336 | 0.44 | 0.15 |
| 16 | 361 | YRLQRFLAD | 369 | 0.17 | 0.22 |
| 17 | 389 | LASDVAGAEALLDR | 402 | 0.36 | 0.14 |
| 18 | 424 | GQALLAAGH | 432 | 0.23 | 0.11 |
| 19 | 438 | VREKLTVLS | 446 | 0.12 | 0.13 |
| 20 | 449 | RAALLEL | 455 | 0.07 | 0.07 |
| 21 | 462 | QYEQCMDLQLFY | 473 | 0.14 | 0.36 |
| 22 | 486 | QEAFLLN | 492 | 0.06 | 0.10 |
| 23 | 495 | LGDFLDSVEALLKK | 508 | 0.31 | 0.26 |
Be SEQ ID NO.1 and SEQ ID NO.2 with the 9th polypeptide and the 13rd polypeptide marker respectively, in order to analyze the SEQ ID NO.1 polypeptide that above-mentioned screening obtains and the general stability of SEQ ID NO.2 polypeptide antigen, to SEQ IDNO.1 and the process replacement respectively of SEQ ID NO.2 sequence, disappearance or add one or several amino acid but guarantee and anti-α-fodrin polypeptide IgG, the epitope of IgA antibody response is active to derive a series of polypeptide thus, again synthetic this corresponding series polypeptide, utilize above immunologic detection method to detect, finding respectively can the anti-α of fine detection-fodrin polypeptide IgG antibody, IgA antibody, show and sjogren syndrome positive serum IgG respectively, IgA antibody well reacts, and OD mean value the results are shown in Table 2 and table 3.
The sequence and the monospecific ELISA detected result thereof of 10 α of table 2-fodrin SEQ ID NO.1 polypeptide derivative
| No | Aminoacid sequence | IgG reaction OD mean value |
| 1 | GQDLEHVEVLQ | 0.65 |
| 2 | QDLEHVEVLQ K | 0.57 |
| 3 | ELGQDLEHVEVLQ | 0.63 |
| 4 | QDLEHVEVLQ KKF | 0.65 |
| 5 | ELGQDLEHVEVLQ KKF | 0.79 |
| 6 | QDLE PVEVLQ | 0.56 |
| 7 | QDLE PVEVL P | 0.53 |
| 8 | QDLEHVEVL | 0.54 |
| 9 | DLEHVEVLQ | 0.51 |
| 10 | DLEHVEVL | 0.45 |
In the last table, sequence number is that the sequence of 1-5 is the sequence of adding different quantities amino acid (marking with underscore) at the first and last end of SEQ ID NO.1 sequence respectively; Sequence number is that 6 and 7 sequence is to replace one or more amino acid whose sequences (marking with underscore) in SEQ ID NO.1 sequence; Sequence number is that the sequence of 8-10 is to lack one or more amino acid whose sequences at SEQ ID NO.1 sequence first and last end.
The sequence and the monospecific ELISA detected result of 10 α of table 3-fodrin SEQ ID NO.2 polypeptide derivative
| No | Aminoacid sequence | IgA reaction OD mean value |
| 1 | GRDLASVQALLRK | 0.72 |
| 2 | RDLASVQALLRK H | 0.67 |
| 3 | DFGRDLASVQALLRK | 0.72 |
| 4 | RDLASVQALLRK HEG | 0.65 |
| 5 | DFGRDLASVQALLRK HEG | 0.82 |
| 6 | RDLASV PALLRK | 0.64 |
| 7 | RDLASV PALLR P | 0.61 |
| 8 | RDLASVQALLR | 0.67 |
| 9 | DLASVQALLRK | 0.61 |
| 10 | DLASVQALLR | 0.60 |
In the last table, sequence number is that the sequence of 1-5 is the sequence of adding different quantities amino acid (marking with underscore) at the first and last end of SEQ ID NO.2 sequence; Sequence number is that 6 and 7 sequence is to replace one or more amino acid whose sequences (marking with underscore) in SEQ ID NO.2 sequence; Sequence number is that the sequence of 8-10 is to lack one or more amino acid whose sequences at SEQ ID NO.2 sequence first and last end.
The contriver utilizes in the test of the present invention is solid-phase synthesis (the Anderson EC of report such as Flechsler, et al, Proc, Natl.Acad.USA 1998,95:7574-79.) synthesized these two totally 22 amino acid whose α-fodrin polypeptide (SEQ ID NO.1 and SEQ ID NO.2) and corresponding polypeptide derivatives thereof.Up to now, still have nothing to do both at home and abroad in the antigenicity of this peptide sequence (SEQ ID NO.1 and SEQ ID NO.2 and corresponding polypeptide derivative), method that this antigen epitope polypeptide mixture detects the total antibody of corresponding IgG/IgA in serum and as the research of hybrid antigen meaning in the sjogren syndrome diagnosis.
The melting concn design of embodiment 2 alpha-fodrin antigen epi-position polypeptide mixtures
In order to satisfy the needs that " mixing ELISA " combination " antibody repertoire ELISA " method detects, among the present invention according to alpha-fodrin antigen epi-position polypeptide (SEQ ID NO.1 and SEQ ID NO.2) respectively as detecting separately antigen, adopt monospecific ELISA method to detect the result of the positive and 10 routine α-fodrin IgA antibody test male sjogren syndrome patient diagnosed serum antibody of clinical 10 routine α-fodrin IgG antibody test respectively.As hybrid antigen, adjust the different matched proportion densities of two polypeptide with these two polypeptide, the total antibody of this 20 routine clinical definite Patients with Sjogren Syndrome serum IgG/IgA " mixing ELISA " combination " antibody repertoire ELISA " method of carrying out is detected.By tentatively groping comparative result, further select the different matched proportion densities (1: 2,6: 7,9: 14,5: 7,11: 14,6: 7,13: 14) of the synthetic polypeptide of SEQ ID NO.1 and the synthetic polypeptide of SEQ ID NO.2 to carry out finally determining to test.
Combination " antibody repertoire ELISA " method detection concrete steps are " to mix ELISA ": at first select above-mentioned two alpha-fodrin antigen epi-position polypeptides that screen (SEQ ID NO.1 and SEQ ID NO.2), respectively with above-mentioned different matched proportion density mixed, every group of mixed polypeptide is cushioned liquid (1 * PBS with bag, pH8.0, this gropes the result for test before, specifically see embodiment 3) be diluted to 0.7 μ g/ml, coated elisa plate (100 μ l/ hole), 37 ℃ of incubations 4 hours.Wash plate 5 times with 0.1%PBS-Tween, every hole adds sealing normal rabbit serum damping fluid (BNRS) (through handling, package amount and confining liquid are test and grope the result, specifically see embodiment 3 and aforementioned with preceding) 150 μ l, and 4 ℃ of overnight incubation are washed plate 5 times.Add each bag by (100 μ l/ hole) in the hole after the positive SS patients serum of the 20 routine anti-α-fodrin sample of front experiment usefulness (comprising positive 10 examples of IgG, positive 10 examples of IgA) dilutes 50 times with 1 * PBS respectively, hatch 30min for 37 ℃.Wash grouping behind the plate 5 times and add anti-people IgA two anti-(being Santa Cruz product) mixture (equal proportions of anti-human IgG two goats anti-and horseradish peroxidase-labeled of goat of the horseradish peroxidase-labeled of 1 * PBS dilution in 1: 1000,200 μ l/ holes), hatch 30min for 37 ℃.Add TMB colour developing liquid colour developing 15min after washing plate, the HCL that adds 1mol/L stops the back and measures sample in the A at 450nm place value.Every plate is established blank, positive control and negative control (all from the German BL alpha-Fodrin of company IgG antibody, the independent quantitative ELISA detection kit of IgA) simultaneously.Adopt sample A value to be measured＞positive thresholding of normal specimen mean+2 * standard deviation, analyze the response situation of two synthetic polypeptide mixture and SS patients serum IgG/IgA.
Analyze " mixing ELISA " combination " antibody repertoire ELISA " method detected result and compare, select the matched proportion density of the best with result that front monospecific ELISA detects.Two polypeptide optimum mixture ratio examples that requirement obtains meet the demands: mix two polypeptide as hybrid antigen with this concentration ratio, detecting the total antibody of Patients with Sjogren Syndrome serum IgG/IgA, to have background low and positive reaction is strong.The results are shown in Table 4:
The OD mean value of table 4SEQ ID NO.1 and SEQ ID NO.2 different ratios mixed polypeptide antibody response
Finally pass through optimal conditions in the experiment of the present invention, determined that the best mixing match that these two polypeptide (SEQ ID NO.1 and SEQ IDNO.2) " mix ELISA " combination " antibody repertoire ELISA " method of carrying out detects anti-α-total antibody of fodrin polypeptide IgG/IgA is 11: 14, mixed by this best proportioning and detect antigen (polypeptide) the mixture thing that α-total antibody of fodrin polypeptide IgG/IgA uses and be called alpha-fodrin antigen epi-position polypeptide mixture.
Experimental study among the present invention shows: above-mentioned alpha-fodrin antigen epi-position polypeptide mixture has the effect of the total specific antibody of IgG/IgA in the good identification Patients with Sjogren Syndrome serum, and is significant to the diagnosis of sjogren syndrome.
Embodiment 3 alpha-fodrin antigen epi-position polypeptide mixtures are to anti-α among the patients serum-total detection of antibodies of fodrin polypeptide IgG/IgA and be worth evaluation
At the aminoacids characteristic of synthetic polypeptide and the situation of hybrid antigen and antibody response, after the adjustment through a series of pH values and reactive ion concentration, we have determined that the autogamy 1 * PBS with pH8.0 serves as that bag is cushioned liquid in the present invention's experiment; Also carried out corresponding experiment simultaneously at the polypeptide mixture blending ratio and the optimum concn of bag quilt, react after this α-fodrin polypeptide SEQ ID NO.1 and SEQ ID NO.2 mixed by different ratios respectively, the result shows, being cushioned liquid with autogamy 1 * PBS of above pH8.0 bag is 0.7 μ g/ml with two antigenic peptides by mixed dilution in 11: 14, every hole adds 100 μ l, 37 ℃ of incubations carried out Serum Antibody Detection after 4 hours, and effect is best; In addition, about prevention problem to nonspecific reaction, by contriver's former experiments experience, the unified in the industry sealing of utilization first with normal rabbit serum after particular procedure as confining liquid, it is best that the specific performance that makes anti-α-fodrin polypeptide antibody detect reaches.Concrete operations are as follows:
Sealing: every hole adds treated sealing normal rabbit serum damping fluid (BNRS) 150 μ l, and 4 ℃ are incubated overnight.
The preparation of BNRS: normal rabbit serum 25ml (62 ℃ of deactivations of elder generation 20 minutes) adds 0.3275g Tris again, and 0.21915g NaCl transfers pH to 8.0 with HCl at last.
SEQ ID NO.1 that this research and utilization is above-mentioned and SEQ ID NO.2 polypeptide adopt the above-mentioned good enzyme-linked immunosorbent assay (" mixing ELISA " combination " antibody repertoire ELISA " method) of setting up through optimizing that the anti-α in sjogren syndrome (SS), rheumatoid arthritis (RA), systemic lupus erythematous (SLE) patient and the normal human serum-total antibody of fodrin polypeptide IgG/IgA is detected, to estimate the distribution of this polypeptide antibody in various diseases.
Concrete steps are: at first select the above-mentioned alpha-fodrin antigen epi-position polypeptide that screens (SEQ ID NO.1 and SEQID NO.2) to synthesize dry powder, mix the back in ratio weighing in 11: 14 and be cushioned liquid (above-mentioned self-control 1 * PBS damping fluid with bag, pH8.0) be diluted to 0.7 μ g/ml, coated elisa plate (100 μ l/ hole), 37 ℃ of incubations 4 hours.Wash plate 5 times with 0.1%PBS-Tween, every hole adds treated sealing rabbit anteserum damping fluid (BNRS) (through handling, seeing above-mentioned with preceding) 150 μ l, and 4 ℃ of overnight incubation are washed plate 5 times.Add bag respectively by (100 μ l/ hole) in the hole after patients serum's sample (comprising Patients with Sjogren Syndrome, Patients with SLE, rheumatoid arthritis patients and normal people) dilutes 50 times with pH7.2PBS, hatch 30min for 37 ℃.Wash the anti-human IgG two anti-people IgA two anti-mixtures of goat anti-and horseradish peroxidase-labeled of goat that grouping behind the plate 5 times adds the horseradish peroxidase-labeled of pH7.2PBS dilution in 1: 1000 and (be Santa Cruz product, equal proportion is mixed, 200 μ l/ holes), hatch 30min for 37 ℃.Add TMB colour developing liquid colour developing 15min after washing plate, the HCL that adds 1mol/L stops the back and measures sample in the A at 450nm place value.Every plate is established blank, positive control and negative control (all from the German BL alpha-Fodrin of company IgG antibody, the independent quantitative ELISA detection kit of IgA) simultaneously.Adopt sample A value to be measured＞positive thresholding of normal specimen mean+2 * standard deviation, analyze the response situation of alpha-fodrin antigen epi-position polypeptide (SEQ ID NO.1 and SEQ ID NO.2) mixture and SS patient and the total antibody of control group serum IgG/IgA.
Combination " antibody repertoire ELISA " method detection principle schematic is seen Fig. 2 " to mix ELISA ".
The present invention studies common detection Patients with Sjogren Syndrome 182 examples, Patients with SLE 113 examples, rheumatoid arthritis patients 136 examples, normal people's 212 examples.
1. trace routine summary: as shown in Figure 3, wrap quilt behind the polypeptide mixed diluting, the 70ng/ hole, 37 ℃ of incubations 4 hours, washing adds sealing with normal rabbit serum damping fluid (BNRS) 150 μ l, and 4 ℃ are spent the night, washing.Sample diluting liquid 1: 50 dilution clinical patient serum with the sample of dilution and available control serum immediately, is added to respectively (100 μ l) in micro-the hole in, hatches 30 minutes/37 ℃ in the box that wets.Washing again adds the enzyme labelling IgG/IgA equal proportion mixing solutions 200 μ l that diluted, hatches 30 minutes/37 ℃ again in wet box, washing adds substrate solution 100 μ l, hatches 15 minutes/37 ℃ again in wet box, add stop buffer (100 μ l), at 450nm place reading.
2. testing process
2.1 will detect requisite number purpose capillary strip is put on microwell plate (wrapped by good polypeptide SEQ ID NO.1 and SEQ ID NO.2 mixture, and sealed and the finished) frame and is ready to one and initial.
2.2 in micro-hole, add the diluted sample of 100 μ l or available control serum immediately respectively.Stay a hole to be that substrate is blank and use, for example:
2.3 with sample 37 ℃ (± 1 ℃) in wet box hatch 30min (± 5min).
Wash plate hole (using automatic washer or the manual plate of washing) 2.4 hatch the back with the washing lotion damping fluid:
-inhale and go or get rid of washing lotion
Add 300 μ l washing lotions in-every hole
-inhale and go or get rid of washing lotion
-repeated washing process 4 times (totally 5 times! )
-microwell plate turned on paper handkerchief pat, make and no longer contain liquid in the micropore
2.5 adding enzymic-labelled antibody.
(except the substrate blank) adds 200 μ l IgG enzymic-labelled antibodies and IgA enzymic-labelled antibody equal proportion mixture in appropriate well
2.6 in the wet box 37 ℃ (± 1 ℃) hatch 30min (± 1min).
2.7 after hatching, clean plate hole (washing see on) with washing lotion
2.8 adding substrate
In every hole, add 100 μ l substrate solutions (comprising the substrate blank well)
2.9 in the wet box 37 ℃ (± 1 ℃) hatch 15min (± 1min).
2.10 termination reaction
Add 100 μ l stop buffers in every hole, the microwell plate that slightly vibrates is with mixing solutions.
2.11 read dullness
With the substrate blank is blank liquid, reads the OD value of 450nm in the 60min, and the reference wavelength scope is 620nm-690nm (for example 650nm).
Present embodiment is mainly illustrated the meaning of alpha-fodrin antigen epi-position polypeptide of the present invention (SEQ IDNO.1 and SEQ ID NO.2) in the Patients with Sjogren Syndrome diagnosis by the following aspects:
1, the α-comparison of the total antibody positive rate of fodrin polypeptide IgG/IgA between sjogren syndrome, rheumatoid arthritis, systemic lupus erythematous and normal people.
In the research experiment of the present invention, the contriver with " mix ELISA " combination " antibody repertoire ELISA " method detected the positive rate of α-total antibody of fodrin polypeptide IgG/IgA in sjogren syndrome, rheumatoid arthritis, systemic lupus erythematous and the normal human serum detection (table 5, Fig. 4).Statistics Application is respectively organized data, the result shows, the positive rate of α-total antibody of fodrin polypeptide IgG/IgA in former and secondary Sjogren syndrome patients serum detect is significantly higher than rheumatoid arthritis, systemic lupus erythematous and normal people, and p value is equal＜and 0.01.Former the positive rate with the α-total antibody of fodrin polypeptide IgG/IgA of secondary Sjogren syndrome is close, no difference of science of statistics.
Table 5. α-total antibody of fodrin polypeptide IgG/IgA is at the positive rate of various disease
*Comprise SLE secondary SS patient 13 examples, RA secondary SS person 19 examples, dermatomyositis secondary SS, each 2 example of undifferentiated connective tissue disease secondary SS patient.
2, anti-α-total antibody of fodrin polypeptide IgG/IgA is in the comparison of sjogren syndrome, systemic lupus erythematous, rheumatoid arthritis and normal human serum titre.
By the visible α of table 6-total antibody of fodrin polypeptide IgG/IgA at the average titer of sjogren syndrome apparently higher than RA, SLE patient and normal people.
The comparison of table 6.SS, SLE, RA and the anti-α of normal people-total antibody titers of fodrin polypeptide IgG/IgA
3, anti-α-total antibody of fodrin polypeptide IgG/IgA and other autoantibody are in the susceptibility and the specific comparison of sjogren syndrome.
Table 7, Fig. 5 have shown anti-α-total antibody of fodrin polypeptide IgG/IgA, α-fodrin polypeptide IgG, α-fodrin polypeptide IgA, SSA antibody, SSB antibody and susceptibility and the specificity of ANA (antinuclear antibody) in sjogren syndrome.Wherein monospecific ELISA method detects anti-α-fodrin IgG or IgA antibody: corresponding detection kit and method are provided by German BL company; The Ou Meng spotting method detects anti-SSA, SSB antibody: corresponding detection kit and method are provided by German Europe traditional Mongolian medicine laboratory diagnosis company limited; Indirect immunofluorescence detects antinuclear antibody (ANA): corresponding detection kit and method are provided by German Europe traditional Mongolian medicine laboratory diagnosis company limited.Experimental result of the present invention shows anti-α-total antibodies specific of fodrin polypeptide IgG/IgA and susceptibility all apparently higher than SSB antibody, SSA antibody, ANA and α-fodrin polypeptide IgG, than α-fodrin polypeptide IgA monospecific antibody susceptibility obviously improves but specificity is low slightly.Therefore, in conjunction with susceptibility and specificity, the anti-α-meaning of the total antibody of fodrin polypeptide IgG/IgA in diagnosis is better than other autoantibodies.
Table 7 autoantibody is in the susceptibility and the specificity of sjogren syndrome
Embodiment 3 alpha-fodrin antigen epi-position polypeptide mixtures are as the application of hybrid antigen in the clinical sjogren syndrome diagnostic kit of preparation
The inventor sets up optimization " mixing ELISA " combination " antibody repertoire ELISA " method, with described alpha-fodrin antigen epi-position polypeptide mixture bag by on microwell plate, prepared the α-total antibody clinical diagnosis of fodrin polypeptide IgG/IgA test kit, be used to detect anti-α-total antibody of fodrin polypeptide IgG/IgA, can finish the detection of 96 person-portions altogether.Particular content is as follows:
1. test kit composition material
Bag is by 1 of detachable microwell plate (1 * 8) 12 lath of highly purified alpha-fodrin antigen epi-position polypeptide mixture; Each 1 bottle of α-fodrin polypeptide IgG and IgA standard substance: 0/6.3/12.5/25/50/100U/ml, every bottle of 1.5ml;
Each 1 bottle of quality controlled serum (containing IgG, IgA and negative Quality Control), 1.5ml;
1 bottle of spissated sample buffer, 20ml;
1 bottle of the anti-human IgG of HRP peroxidase labelling goat, 15ml;
1 bottle of the anti-people IgA of HRP peroxidase labelling goat, 15ml;
1 bottle of tmb substrate, 15ml;
1 bottle of stop buffer (containing 1M HCl), 15ml;
1 bottle of thickening and washing damping fluid, 20ml
2. technical parameter
(1) sample: serum
(2) sample size: at every turn test the undiluted sample of 100 μ l
(3) always hatch: (18-28 ℃) 30min under the room temperature
(4) Quality Control scope: 0-100U/ml
(5) sensitivity: 1U/ml
(6) store: 2-8 ℃
(7) validity period: produced back 9 months or indicate validity period
(8) packing specifications: 96 person-portions
3. test kit performance
(1) the highly purified α of specificity-the fodrin polypeptide mixture is coated on microwell plate, and the alpha-fodrin antigen epi-position polypeptide mixture test kit of preparation only is used for the total antibody of IgG/IgA of specific detection.
(2) proofread and correct the α-total antibody test of fodrin polypeptide IgG/IgA system and do not have the international standard correction.
4. immunoassay program
(1) sample collection is handled and is preserved
Use serum sample to detect α-total antibody of fodrin polypeptide IgG/IgA, serum is with the dilution proportion (20 μ l sample+1ml damping fluid) of diluted sample damping fluid with 1: 50, and patient need not fasting, also need not prepare especially, the blood sampling is got in venipuncture, forms the centrifugal absorption supernatant in back that condenses.
If sample does not directly use, can preserve under 5 days or-20 ℃ under 2-8 ℃ and can preserve 6 months, it is divided into small portion avoids repeatedly freezing.
Bilirubin and hemolysin do not have influence to the result.
(2) preparation/storage of reagent
Except that sample buffer and lavation buffer solution, all reagent is instant liquid; 2-8 ℃ of the reagent in back of uncapping can preserve at least 30 days down or label on validity period.
No lath is put back in the packing of band siccative immediately in the experiment, and 2-8 ℃ of lower seal preserved, in order to avoid rotten.
The preparation of lavation buffer solution: install to the 1000ml container after 50 times of dilutions of distilled water.Damping fluid after the dilution can preserved at least under 2-8 ℃ 30 days or label on validity period.
The preparation of dilution buffer liquid: install to the 100ml container after 5 times of dilutions of distilled water.Damping fluid after the dilution can preserved at least under 2-8 ℃ 30 days or label on validity period.
(3) technical essential
Quality controlled serum or sample must be analyzed as unknown at every turn, with the reliability of test experience.
Application of sample and sample process: use the micro sample adding appliance sampling of band disposable tip; Directly add the micropore bottom.For avoiding crossed contamination, change the suction nozzle sampling at every turn.Should further dilute the patient's sample of suspecting high density, and when the result calculates, adjust.
(4) process of the test
Different lot numbers are not used with.All reagent and sample return to room temperature before using.
All patient's sample mix with the dilution proportion (20,1 sample+1ml damping fluid) of diluted sample damping fluid with 1: 50.Standard substance and quality controlled serum are instant, needn't dilute.
Quantity adds standard substance and the required lath of Quality Control decision per sample, each sample, and standard substance and Quality Control all should be done double.The testing program of using:
| 1 | 2 | 3 | 4 | 5 | 6 |
| A | Mark 1 | Mark 1 | Sample 1 | Sample 1 | ||
| B | Mark 2 | Mark 2 | Sample 2 | Sample 2 | ||
| C | Mark 3 | Mark 3 | Sample 3 | Sample 3 | ||
| D | Mark 4 | Mark 4 | Sample 4 | Sample 4 | ||
| E | Mark 5 | Mark 5 | Sample 5 | Sample 5 | ||
| F | Mark 6 | Mark 6 | … | …. | ||
| G | Sun | Sun | ||||
| H | Cloudy | Cloudy |
Every hole adds 100ul serum dilution, standard substance and quality controlled serum, hatches 1 hour (18-28 ℃) under the room temperature; The turned letter microwell plate, every hole is washed 3 times with the 300ul lavation buffer solution; Every hole adds 100ul enzyme mark liquid, hatches under the room temperature 60 minutes; The turned letter microwell plate, every hole is washed 3 times with the 300ul lavation buffer solution; Every hole adds 100ul substrate TMB, hatches in the dark under the room temperature 10 minutes; Every hole adds the stop buffer of 100ul, static 5 minutes; Behind the stopped reaction in 30 minutes, measurement result, 450nm is as initial wavelength, and 620nm (can with 600 to 690nm) conduct is with reference to wavelength.
(5) interpretation of result
Make typical curve (straight line, semilog or logarithmic paper) with absorbancy and standard substance concentration.
Can directly determine the antibody concentration of serum by typical curve.
(6) experiment parameter
Precision:
Sensitivity: 1U/ml
Corresponding: in dilution experiment, the sample of high antibody concentration dilutes with sample buffer and detects.The result is linear in whole test specifications.
SEQUENCE LISTING (sequence table)
＜110〉Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
＜120〉alpha-fodrin antigen epi-position polypeptide mixture and application thereof
<130>
<141>2006-11-22
<160>2
<170>PatentIn?version?3.3
<210>1
<211>10
<212>PRT
＜213〉artificial sequence
<400>1
Gln?Asp?Leu?Glu?His?Val?Glu?Val?Leu?Gln
1 5 10
<210>2
<211>12
<212>PRT
＜213〉artificial sequence
<400>2
Arg?Asp?Leu?Ala?Ser?Val?Gln?Ala?Leu?Leu?Arg?Lys
1 5 10
Claims (3)
1.a-the fodrin antigen epitope polypeptide mixture is characterized in that, comprises
(a), the polypeptide of forming by the aminoacid sequence shown in the SEQ ID NO.1;
(b), the aminoacid sequence in (a) is through replacing, lack or adding one or several amino acid and have with the epitope of anti-α-fodrin polypeptide IgG antibody response active by (a) polypeptides derived;
(c), the polypeptide of forming by the aminoacid sequence shown in the SEQ ID NO.2;
(d), the aminoacid sequence in (c) is through replacing, lack or adding one or several amino acid and have with the epitope of anti-α-fodrin polypeptide IgA antibody response active by (c) polypeptides derived.
2. the screening method of alpha-fodrin antigen epi-position polypeptide mixture as claimed in claim 1 is characterized in that, may further comprise the steps:
A) choose that 1-1680 nucleotide sequence corresponding amino acid sequence is an object among people α-fodrin ORF, by adopting Standard, Karplus, Emini, Amphiphi, five kinds of Pellequer are in the world about the epitope forecast method, forecast analysis mainly comprises the secondary structure relevant with protein antigenicity, accessibility, wetting ability, signal peptide cutting site, wears film spiral, N-glycosylation site, by analyzing antigen trend index, tentatively determined the distribution of antigenic determinant amino-acid residue of the α-fodrin of 560aa length;
B) further by synthetic polypeptide and immunology detection analysis, obtain as described two corresponding separately detection antibody susceptibility of (a) in the claim 1 and (c) and all very high antigen epitope polypeptide of specificity.
3. the test kit that contains the diagnosis sjogren syndrome of a-fodrin antigen epitope polypeptide mixture as claimed in claim 1.
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| CN1979165B true CN1979165B (en) | 2011-09-21 |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102236016A (en) * | 2010-04-20 | 2011-11-09 | 中国科学院广州生物医药与健康研究院 | Colloidal gold testing card for testing sjogren's syndrome alpha-fodrin antibody |
| CN104098686B (en) * | 2013-04-03 | 2016-03-23 | 苏州偲聚生物材料有限公司 | Polypeptide, the detection means comprising this polypeptide and detection kit |
| CN103880941B (en) * | 2012-12-21 | 2016-02-10 | 苏州偲聚生物材料有限公司 | Polypeptide, the detection means comprising this polypeptide and detection kit |
| CN103880924B (en) * | 2012-12-21 | 2016-03-23 | 苏州偲聚生物材料有限公司 | Polypeptide, the detection means comprising this polypeptide and detection kit |
| CN104098671A (en) * | 2013-04-03 | 2014-10-15 | 苏州偲聚生物材料有限公司 | Polypeptide, and detection member and detection kit both containing same |
| CN104098685B (en) * | 2013-04-03 | 2016-03-23 | 苏州偲聚生物材料有限公司 | Polypeptide, the detection means comprising this polypeptide and detection kit |
| CN104098681B (en) * | 2013-04-03 | 2016-08-10 | 苏州偲聚生物材料有限公司 | Polypeptide, the detection device comprising this polypeptide and detection kit |
| CN104655850B (en) * | 2013-11-18 | 2016-04-20 | 谱天（天津）生物科技有限公司 | The detection method of the early stage irreversible degree of injury of a kind of cell and detection kit |
Citations (3)
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|---|---|---|---|---|
| US6121057A (en) * | 1996-04-23 | 2000-09-19 | Takeda Chemical Industries, Ltd. | Methods of detecting antibodies to α-Fodrin and fragments thereof in diagnosing sjogrens' |
| CN1393694A (en) * | 2001-06-21 | 2003-01-29 | 昆明广博科技有限公司 | Diagnosing reagent for treating xerosis syndrome and its preparing process |
| CN1403819A (en) * | 2002-09-23 | 2003-03-19 | 北京大学人民医院 | Application of alpha-fodrin polypeptide resisting antibody determination in the diagnosis of xerosis syndromes |
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- 2006-12-12 CN CN2006101241689A patent/CN1979165B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6121057A (en) * | 1996-04-23 | 2000-09-19 | Takeda Chemical Industries, Ltd. | Methods of detecting antibodies to α-Fodrin and fragments thereof in diagnosing sjogrens' |
| US6703021B2 (en) * | 1996-04-23 | 2004-03-09 | Yoshio Hayashi | Composition containing α-fodrin or α-fodrin fragment protein |
| CN1393694A (en) * | 2001-06-21 | 2003-01-29 | 昆明广博科技有限公司 | Diagnosing reagent for treating xerosis syndrome and its preparing process |
| CN1403819A (en) * | 2002-09-23 | 2003-03-19 | 北京大学人民医院 | Application of alpha-fodrin polypeptide resisting antibody determination in the diagnosis of xerosis syndromes |
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| Title |
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| Witte T,et al.IgA and IgG autoantibodies against alpha-fodrin as markers for Sjogren"s syndrome.《JOURNAL OF RHEUMATOLOGY》.2000,第27卷(第11期),2617-2620. * |
| 陈巧林，等.干燥综合征特异性α-胞衬蛋白的抗原表位筛选及鉴定.《中国免疫学杂志》.2005,第21卷(第11期),864-867. * |
| 高明，等.抗α-fodrin抗体在干燥综合征诊断中的意义.《中华内科杂志》.2005,第44卷(第6期),456-457. * |
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