CN1403819A - Application of alpha-fodrin polypeptide resisting antibody determination in the diagnosis of xerosis syndromes - Google Patents
Application of alpha-fodrin polypeptide resisting antibody determination in the diagnosis of xerosis syndromes Download PDFInfo
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Abstract
The present invention provides one kind of alpha-fodrin polypeptide and its analog used for the detection of xerosis syndromes. This kind of polypeptide antigen may be combined specifically with the specific antibody in the body of xerosis syndromes patient and has very high sensitivity and specificity.
Description
The present invention relates to the preparation of α-fodrin polypeptide and as the application of antigen in detecting Sjogren syndrome and other disease.
Technical background
(Sj gren ' s Syndrome is a kind of to invade and exocrine glands such as lachrymal gland and salivary gland are the general autoimmune disease of feature SS) to Sjogren syndrome.The modal clinical manifestation of this disease is that eye is done, dry, and can involve organs such as lung, kidney, lymphatic system, blood vessel and liver.This sick no tangible regional distributional difference can betide any age, and with 40-60 year, particularly postclimacteric women is in the majority, and men and women's ratio is 1: 9.Morbidity rate in China is 0.77%, and the morbidity rate in elderly population is up to 4%, and there are nearly 1,000 ten thousand patients in the whole nation.Sjogren syndrome can be divided into primary Sjogren's syndrome (Primary Sjogren ' s syndrome) and secondary Sjogren syndrome (Secondary Sjogren ' s syndrome) two classes.When this disease takes place separately, be called primary Sjogren's syndrome; With other clear and definite autoimmune disease of diagnosis such as rheumatoid arthritis (rheumatoid arthritis, RA), (systemic lupus erythematosus when SLE) etc. merging exists, is called secondary Sjogren syndrome to systemic loupus erythematosus.
In recent years, people have carried out big quantity research to the cause of disease and the pathogenesis of Sjogren syndrome, but do not have breakthrough so far.At present do not find desirable serological diagnostic method clinically as yet, except that clinical symptoms, mainly lean on labial gland biopsy and parotid gland radiography etc. to have wound inspection to clarify a diagnosis, because patient's doctor is poor from property, the treatment opportunity of usually delay diagnosis, and forfeiture the best.Therefore, seek the detection method that a kind of susceptibility is strong, specificity is high and simple and easy to do, to the early diagnosis of Sjogren syndrome and treat significant.For this reason, the inventor is through having discovered the serological specificity antigen that can be used for the Sjogren syndrome early diagnosis for many years---α-fodrin polypeptide (also claiming α-fodrin polypeptide), specific antibody---the anti-α-fodrin polypeptide antibody (also claim anti-α-fodrin polypeptide antibody) of this polypeptide in can specific identification Patients with Sjogren Syndrome serum, this antibody can detect by some comparatively easy immunological detection methods (as: enzyme linked immunosorbent assay).And have very high susceptibility and specificity.
Summary of the invention
One of purpose of the present invention provides the specific antigen that can effectively detect Sjogren syndrome---α-fodrin polypeptide.
Another object of the present invention provides the purposes of described α-fodrin polypeptide in the diagnosis Sjogren syndrome.
At last, the invention provides and to contain described α-fodrin polypeptide and analog are used to detect Sjogren syndrome as antigen method.
Term used herein " α-fodrin polypeptide " refers to have common sequences RQKLEDSYRFQFFQRDAEEL amino acid or the polypeptide or the analog of (being Arg-Gln-Lys-Leu-Glu-Asp-Ser-Tyr-Arg-Phe-Gln-Phe-Phe-Gln-Arg-Asp-Ala-Glu-Glu-Leu).
Fodrin (claiming fodrin or fodrin again) is found in the endochylema of neurocyte, mainly accumulates under neurocyte and the lymphocytic serous coat, parallelly with serous coat is tabular distribution.It is the chief component of eukaryotic cell membrane skeleton, and similar cylindrical structure contains two subunits of α and β.The former molecular weight is 240KD, and the latter is 235KD.α, β subunit form heterodimer with edge-to-edge's form, link to each other with disulfide bond therebetween.This heterodimer further forms tetrad with tail to the form of tail, and links to each other with troponin, becomes the basic structure of membrane bone frame, participate in the motion of cell and keep the connection and the form of cell, may with peripheral nerve, brain, kidney, the generation of liver and general autoimmunity disease is relevant
(1)
The people such as Haneji of medical college of Japanese Tokushima university in 1997 isolate the antigen that a kind of molecular weight is 120kD from the parotid gland tissues of Sjogren syndrome model NFS/sld mouse, and find that this antigen has great expression in the labial gland tissue of Patients with Sjogren Syndrome, and in the normal person, do not detect; Find also that simultaneously this antigen can stimulate the T lymphopoiesis in the Patients with Sjogren Syndrome peripheral blood; In order to identify this antigen, people such as Haneji measure its N-terminal sequence, found that this antigen is the non-erythrocyte membrane spectrin (α-fodrin of nonerythroid α-spectrin).The personnel of this group use engineered method then, with the gst fusion protein expression system construction have expressing fusion protein system---the pGEX-2T/JS-1 of coding for alpha-fodrin amino terminal sequence cDNA, (JS-1 is the base sequence of coding for alpha-fodrin amino terminal sequence, this section sequence is totally 1784 bases), the α that this expression system gives expression to-fodrin fusion protein molecule amount is about 97.4kD.With α-fodrin fusion of being purified into as antigen, method with Western blotting (Westernblot) has detected 43 routine primary Sjogren's syndromes (pSS), 8 routine secondary Sjogren syndromes, 21 routine systemic loupus erythematosuses (SLE), 14 routine rheumatoid arthritis (RA) patients and the normal blood donor of 15 examples, the result shows that the positive rate of this albumen in primary Sjogren's syndrome is 95.3%, be 50% in the secondary Sjogren syndrome, and in SLE, RA and normal person, do not detect this antibody
(2)Yet, α-fodrin of people's such as Watanabc the 98kD that uses reorganization is as antigen, the positive rate that detects in various disease with the method for Westem blot is: primary Sjogren's syndrome: 78% (7/9), and LE secondary SS:60% (9/15), LE:7% (3/44)
(3)The method of human ELISA such as Witte, is 55% (47/85) with the α-fodrin of 93kD of reorganization as former dry dry syndrome patient's positive rate that Detection of antigen goes out, SLE secondary SS's is 40% (6/15), RA secondary SS's is 43% (3/7), RA is 41.6% (5/12), SLE is 20% (1/50), and normal person's group is 1.9% (3/160)
(4)
Based on above research, the inventor finds in α-fodrin one section amino acid is arranged through sequential analysis, the non-erythrocyte membrane spectrin of sequence and mouse sequence homology, have four conservative or conservative amino acid simultaneously in this sequence, may be relevant with the antigenic characteristic of this albumen, and in the morbidity of Sjogren syndrome, play a significant role, up to now, still have nothing to do both at home and abroad in the antigenicity of this sequence and the research of meaning in the Sjogren syndrome diagnosis thereof.This sequence is RQKLEDSYRFQFFQRDAEEL (being Arg-Gln-Lys-Leu-Glu-Asp-Ser-Tyr-Arg-Phe-Gln-Phe-Phe-Gln-Arg-Asp-Ala-Glu-Glu-Leu), is called α-fodrin polypeptide.The inventor with this polypeptide as antigen, with the α-fodrin polypeptide antibody in method detection Sjogren syndrome, rheumatoid arthritis, systemic loupus erythematosus and the normal human serum of ELISA, found that this polypeptide has higher susceptibility and specificity to the diagnosis of Sjogren syndrome, can be used for the diagnosis of Sjogren syndrome, thereby reach purpose, for the clinical diagnosis of Sjogren syndrome provides objective laboratory foundation by serological method diagnosis Sjogren syndrome.
α of the present invention-fodrin polypeptide and analog thereof can detect Patients with Sjogren Syndrome by several different methods (for example: enzyme linked immunosorbent assay, Western blot, golden labelling method etc.), and its detection method is well known to those skilled in the art.Simultaneously, the anti-α of detection-fodrin polypeptide antibody type can be the immunoglobulin (Ig) of IgG type, IgA type or other type.
α of the present invention-fodrin polypeptide can be known to such an extent that method obtains with those skilled in the art, for example: solid-phase synthesis, gene engineering expression method of purification etc.
Description of drawings 1. is at first adsorbed α-fodrin polypeptide bag on the microwell plate in enzyme linked immunological, hatches with test serum, as
Anti-α-fodrin the polypeptide antibody that exists in the fruit serum, this antibody will combine shape with the solid phase antigen of bag quilt
Become antigen-antibody complex.After unconjugated antibody and other composition rinsed well, add horseradish peroxidase
The goat anti-human igg antibody (IgG-HRP) of enzyme (HRP) mark is hatched together, this moment IgG-HRP will with
The antigen-antibody complex combination forms Ag-Ab-hrp-antibody complex, and the unconjugated composition of flush away adds
Go into substrate,, then can show specific color, and antibody drips if contain anti-α-fodrin polypeptide antibody in the serum
It is dark more to spend high more color.Under the 450nm wavelength, measure the OD value after adding stop buffer.Fig. 2. the α-positive rate of fodrin polypeptide antibody in primary and secondary Sjogren syndrome is all obviously high as seen from the figure
In SLE, RA and normal control (P all＜0.05 or＜0.01).Fig. 3. in Patients with Sjogren Syndrome, the susceptibility of anti-α-fodrin polypeptide antibody is apparently higher than SSA, SSB and anti-
Antinuclear antibodies, its specificity is a little less than anti-SSB antibody (P all＜0.01).Than SSA antibody, antinuclear antibodies is obvious
Increase (P＜0.01).Therefore, anti-α-fodrin polypeptide antibody tool in Sjogren syndrome serodiagnosis is important
Be worth.
The design of EXAMPLE Example 1 α-fodrin polypeptide and synthetic
Aforesaid research work (1-4) is verified, and the α-fodrin fusion that utilizes engineered method to be purified into can be specific in conjunction with the α in the Patients with Sjogren Syndrome body-fodrin antibody, it can be detected by specific method then.At first utilize Flechsler etc. in the test of the present invention
(5)The solid-phase synthesis of report has synthesized totally 20 amino acid whose α-fodrin polypeptide, and its sequence is as follows:
RQKLEDSYRFQFFQRDAEEL
This sequence of Arg-Gln-Lys-Leu-Glu-Asp-Ser-Tyr-Arg-Phe-Gln-Phe-Phe-Gln-Arg-Asp-Ala-Glu-Glu-Leu is non-erythrocyte membrane spectrin (the amino section sequence of the α-fodrin of nonerythroid α-spectrin).
Studies show that: above-mentioned α-fodrin polypeptide has the effect of the specific antibody in can specific identification Patients with Sjogren Syndrome serum, and the diagnosis of Sjogren syndrome is had great importance.Embodiment 2 α-fodrin polypeptide is to detection of antibodies in the Patients with Sjogren Syndrome serum
This research adopts enzyme linked immunosorbent assay (being the ELISA method) that the anti-α-fodrin polypeptide antibody among Sjogren syndrome, rheumatoid arthritis (RA), systemic loupus erythematosus (SLE) patients serum is detected, to estimate the distribution of this polypeptide antibody in various diseases.
At first α-fodrin polypeptide bag is adsorbed on the microwell plate in enzyme linked immunological, hatch with test serum, if the anti-α-fodrin polypeptide antibody that exists in the serum, this antibody will combine with the solid phase antigen of bag quilt, forms antigen-antibody complex.After unconjugated antibody and other composition rinsed well, the goat anti-human igg antibody (IgG-HRP) who adds horseradish peroxidase (HRP) mark is hatched together, this moment, IgG-HRP will combine with antigen-antibody complex, form Ag-Ab-hrp-antibody complex, the unconjugated composition of flush away adds substrate, if contain anti-α-fodrin polypeptide antibody in the serum, then can show specific color, and the high more color of antibody titer is dark more.Under the 450nm wavelength, measure the OD value after adding stop buffer.Detect principle schematic and see Fig. 1.
This research detects Patients with Sjogren Syndrome 39 examples altogether, Patients with SLE 25 examples, rheumatoid arthritis patients 20 examples, normal person's 59 examples.Adopt sample OD value to be measured (A value)＞average OD value of normal person (X) ± positive threshold value of 2 * standard deviation (2s).
Present embodiment is mainly illustrated the meaning of polypeptide of the present invention in the Patients with Sjogren Syndrome diagnosis by the following aspects: 1, α-fodrin polypeptide antibody positive rate is in Sjogren syndrome, rheumatoid arthritis, systemic loupus erythematosus and normal person's comparison.
This research with the ELISA method detected α-fodrin polypeptide antibody Sjogren syndrome, rheumatoid arthritis, systemic loupus erythematosus and normal person's positive rate (table 1, Fig. 2).Use u-test and respectively organize data, the result shows that α-fodrin polypeptide antibody is significantly higher than rheumatoid arthritis, systemic loupus erythematosus and normal person at former the positive rate with secondary Sjogren syndrome, and the p value all＜0.05.Former the positive rate with the α-fodrin polypeptide antibody of secondary Sjogren syndrome is close, no difference of science of statistics.And α-fodrin polypeptide antibody is significantly higher than normal person (p＜0.05) at the positive rate of systemic loupus erythematosus and rheumatoid arthritis.
Table 1. α-fodrin polypeptide antibody is at the positive rate of various disease
Anti-α-fodrin polypeptide antibody is diagnosed routine number u value p value
Positive routine number positive rate (%) Sjogren syndrome 39 30 76.9
Primary 31 24 77.4 0.13＞0.05
*Secondary cases 86 75 0.12＞0.05 systemic loupus erythematosus 25 6 24 4.23＜0.01 rheumatoid arthritis 20 8 40 3.04＜0.05 normal person 59 2 3.8 6.43＜0.01
*Comprise SLE secondary SS patient 1 example, RA secondary SS person 5 examples, dermatomyositis secondary SS, each 1 example of the secondary SS patient of UCTD.2, anti-α-fodrin polypeptide antibody is in the comparison of Sjogren syndrome, systemic loupus erythematosus, rheumatoid arthritis and normal person's titre.
By the visible α of table 2-fodrin polypeptide antibody in the titre of Sjogren syndrome apparently higher than SLE patient and normal person.But, compare the titre no significant difference of α-fodrin polypeptide antibody and SS with RA patient.The RA patient's age of considering this 8 routine α-fodrin polypeptide antibody positive is big, the course of disease is longer, may develop into secondary Sjogren syndrome, and traceable investigation is with clearly.In this research, 2 examples significantly are lower than Patients with Sjogren Syndrome with α-fodrin polypeptide reaction normal person's serum titer.
The comparison of the anti-α of table 2. SS, SLE, RA and normal person-Fordin antibody titer
Detect positive positive rate OD value t value P value
△
Example numerical example number (%) (former SS 31 24 77.4 0.29 ± 0.08 secondary SS 86 75 0.34 ± 0.09 1.322 0.194SLE 25 6 24 0.23 ± 0.09 2.933 0.005 of M ± SD)
* RA 20 8 40 0.28 ± 0.09 0.761 0.450 normal person 59 2 3.8 0.17 ± 0.04 7.415 0.000
*3, α-fodrin polypeptide antibody and primary SS, SLE and RA patient's course of disease and relationship with age.
By table 3 as seen, among the primary Sjogren's syndrome patient of 31 routine α-fodrin polypeptide antibody positive, the course of disease is seemingly irrelevant with the course of disease at the positive rate of the 8 years α with interior person-fodrin polypeptide antibodies.But α-fodrin polypeptide antibody of the 8 routine patients of the course of disease above 8 years is all positive, and prompting α-fodrin polypeptide antibody may be relevant with severity extent, and prolonging positive rate with the course of disease increases.
The positive rate and the primary Sjogren's syndrome patient's age of α-fodrin polypeptide antibody do not have obvious relation between persistence.
The course of disease (year) 12345678＞8 that concerns of the table 3. α-fodrin polypeptide antibody and the Patients with Sjogren Syndrome course of disease adds up to number positive 632523118 31 total several 834623328 39 percent of example (%) 75 100 50 83 100 100 33 50 100 77.4
Analysis to α-fodrin polypeptide antibody and rheumatoid arthritis patients age and course of disease relation is found, older and the course of disease of the rheumatoid arthritis patients of α-fodrin polypeptide antibody positive is longer, with α-fodrin polypeptide antibody negative RA patient notable difference (table 4) is arranged relatively.As if but the α of systemic loupus erythematosus-fodrin polypeptide antibody positive rate is irrelevant with the patient's age and the course of disease.
The relation of α-fodrin polypeptide antibody and the patient's age and the course of disease among table 4.RA and the SLE
The positive α of α-fodrin polypeptide antibody-fodrin polypeptide antibody feminine gender
Example age several years (year) course of disease (year) routine age several years (year) course of disease (year) RA 8 70 ± 5.2
a9.3 ± 3.7
b12 52 ± 13.6 3.5 ± 3.0SLE 6 31 ± 5.6 6 ± 4.4 19 30 ± 4.3 5 ± 6.4
aCompare t=2.48, p＜0.05 with the negative group of α-fodrin polypeptide antibody.
bCompare t=1.99, p＜0.05 with the negative group of α-fodrin polypeptide antibody.4, α-fodrin polypeptide antibody and the primary Sjogren's syndrome patient internal organs relation of getting involved.
By table 5 as seen, primary Sjogren's syndrome patient's internal organ such as the pulmonary fibrosis rate of getting involved is relevant with α-fodrin polypeptide antibody, and the significant difference on the statistics shows that α-fodrin polypeptide antibody positive patient internal organs rate of getting involved is higher than α-fodrin polypeptide antibody negative patient.
The relation that table 5. α-fodrin polypeptide antibody and primary Sjogren's syndrome patient internal organs are got involved
α-fodrin polypeptide antibody α-fodrin polypeptide antibody
The P value
Several 13 41.9 2 25 pulmonary fibrosis, 10 32.2 1 13＜0.05 vasculitis, 10 32.2 1 13＜0.05 cirrhosis, 3 9.7 00＜0.05 renal insufficiency, 3 9.7 00＜0.05 hypersplenias 2 6.4 1 135 of example, α-autoantibodies such as fodrin polypeptide antibody sensitiveness and the specific comparison at Sjogren syndrome of getting involved of example number % example number % internal organs.
Table 6 has shown α-fodrin polypeptide antibody, SSA antibody, SSB antibody and ANA susceptibility and the specificity in Sjogren syndrome.The specificity of SSB antibody is apparently higher than α-fodrin polypeptide antibody, SSA antibody and ANA, but susceptibility only is 17.9%, significantly is lower than α-antibody such as fodrin polypeptide.Therefore, in conjunction with susceptibility and specificity, the α-meaning of fodrin polypeptide antibody in diagnosis may be better than other autoantibodies.
Table 6 autoantibody is at the susceptibility and positive routine number susceptibility (%) specificity (%) α of specific antibody classification-fodrin polypeptide antibody 31 79.5 87.4SSA antibody 17 43.6 84.7SSB antibody 7 17.9 100ANA 17 43.6 72.6 of Sjogren syndrome
△Primary SS compares with Secondary cases SS, SLE, RA and normal person
* P＜0.01, the difference highly significant.
List of references 1.Cheney R, Hirokawa N, Levine J, Willard M, Intracellular movement of Fodrin.Cell Motil 1983; 3 (5-6): 649-55.53232.Haneji, N., Nakamura T, Takio K, et al.Identification of α-Fodrin as a candidate autoantigen in primary Sjogren ' s Syndrome.Science 1997; 276:604-7.3.Watanabc T, Tsuchida T, Kanda N, et al.Anti-α-Fodrin antibodies in Sjogren ' s Syndrome and lupus erythematosus.Arch Dermatol 1999; 135:535-539.4.Witte T, Matthias T, Arnett C F, et al.IgA and IgG autoantibodies against α-Fodrin as marker for Sjogren ' s Syndrome.J Rheumatol 2000; 27:2617-20.5.Anderson EC, et al:Proc, Natl.Acad.USA 95:7574-79,1998.
Sequence table＜110〉The People's Hospital of Peking University＜120〉anti-α-fodrin polypeptide antibody is determined at application＜140 in the Sjogren syndrome diagnosis〉＜141〉2002-08-06＜160〉1＜170〉PatentIn version 3.1＜210〉1＜211〉20 (amino acid)＜213〉artificial sequence＜223〉synthetic＜400〉1Arg Gln Lys Leu Glu Asp Ser Tyr Arg Phe Gln Phe Phe Gln ArgAsp Ala Glu Glu Leu
Claims (3)
1. has α-fodrin (α-Fodrin) amino acid, polypeptide analog and the albumen of sequence RQKLEDSYRFQFFQRDAEEL (being Arg-Gln-Lys-Leu-Glu-Asp-Ser-Tyr-Arg-Phe-Gln-Phe-Phe-Gln-Arg-Asp-Ala-Glu-Glu-Leu).
2. contain the α-fodrin polypeptide of right requirement 1 or the diagnostic kit of its analog.
3. α-fodrin the polypeptide or its analog that contain right requirement 1 are used for the ELISA detection kit.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100572391C (en) * | 2006-08-30 | 2009-12-23 | 中国科学院广州生物医药与健康研究院 | Beta-fodrin antigen epitope polypeptide and screening method thereof and application |
| CN1979165B (en) * | 2006-12-12 | 2011-09-21 | 中国科学院广州生物医药与健康研究院 | Alpha-fodrin antigen epi-position polypeptide mixture and its use |
| 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 |
| CN104655850A (en) * | 2013-11-18 | 2015-05-27 | 李捷 | Detection method and detection kit of early-stage irreversible damage degree of cells |
| CN114280304A (en) * | 2021-04-30 | 2022-04-05 | 北京纳么美科技有限公司 | Poly antigen and application thereof in detection of autoimmune disease |
-
2002
- 2002-09-23 CN CN 02130999 patent/CN1403819A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100572391C (en) * | 2006-08-30 | 2009-12-23 | 中国科学院广州生物医药与健康研究院 | Beta-fodrin antigen epitope polypeptide and screening method thereof and application |
| CN1979165B (en) * | 2006-12-12 | 2011-09-21 | 中国科学院广州生物医药与健康研究院 | Alpha-fodrin antigen epi-position polypeptide mixture and its use |
| 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 |
| CN104655850A (en) * | 2013-11-18 | 2015-05-27 | 李捷 | Detection method and detection kit of early-stage irreversible damage degree of cells |
| CN114280304A (en) * | 2021-04-30 | 2022-04-05 | 北京纳么美科技有限公司 | Poly antigen and application thereof in detection of autoimmune disease |
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