CN104820098B - Application of the anti-TUBA antibody in Behcet disease diagnosis - Google Patents
Application of the anti-TUBA antibody in Behcet disease diagnosis Download PDFInfo
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- 208000009137 Behcet syndrome Diseases 0.000 title claims abstract description 57
- 208000027496 Behcet disease Diseases 0.000 title claims abstract description 56
- 238000003745 diagnosis Methods 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 239000000427 antigen Substances 0.000 claims abstract description 6
- 102000036639 antigens Human genes 0.000 claims abstract description 6
- 108091007433 antigens Proteins 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract 8
- 101150050146 DNMBP gene Proteins 0.000 claims description 28
- 102100024821 Dynamin-binding protein Human genes 0.000 claims description 28
- 101150103035 tubA gene Proteins 0.000 claims description 28
- 210000002966 serum Anatomy 0.000 claims description 19
- 238000005406 washing Methods 0.000 claims description 15
- 208000027866 inflammatory disease Diseases 0.000 claims description 5
- 230000002792 vascular Effects 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 241000283707 Capra Species 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000003759 clinical diagnosis Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000013642 negative control Substances 0.000 claims description 2
- -1 nitrite ions Chemical class 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 239000013641 positive control Substances 0.000 claims description 2
- 235000011149 sulphuric acid Nutrition 0.000 claims description 2
- 239000001117 sulphuric acid Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 4
- 210000004369 blood Anatomy 0.000 claims 3
- 239000008280 blood Substances 0.000 claims 3
- 239000007853 buffer solution Substances 0.000 claims 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 102000004243 Tubulin Human genes 0.000 abstract description 2
- 108090000704 Tubulin Proteins 0.000 abstract description 2
- 210000005259 peripheral blood Anatomy 0.000 abstract description 2
- 239000011886 peripheral blood Substances 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract 1
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- 201000000596 systemic lupus erythematosus Diseases 0.000 description 8
- 208000001106 Takayasu Arteritis Diseases 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 108010074051 C-Reactive Protein Proteins 0.000 description 6
- 102100032752 C-reactive protein Human genes 0.000 description 6
- 206010047115 Vasculitis Diseases 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 208000025865 Ulcer Diseases 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 231100000397 ulcer Toxicity 0.000 description 5
- 206010015150 Erythema Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 231100000321 erythema Toxicity 0.000 description 4
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- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 206010047249 Venous thrombosis Diseases 0.000 description 3
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- 238000010219 correlation analysis Methods 0.000 description 3
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- 238000001962 electrophoresis Methods 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000000306 recurrent effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 101100332088 Homo sapiens DNMBP gene Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 206010028034 Mouth ulceration Diseases 0.000 description 2
- 208000025747 Rheumatic disease Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
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- 210000003743 erythrocyte Anatomy 0.000 description 2
- 102000046525 human DNMBP Human genes 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 208000014644 Brain disease Diseases 0.000 description 1
- 206010051055 Deep vein thrombosis Diseases 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 208000007117 Oral Ulcer Diseases 0.000 description 1
- 208000034189 Sclerosis Diseases 0.000 description 1
- 238000012352 Spearman correlation analysis Methods 0.000 description 1
- 208000004732 Systemic Vasculitis Diseases 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 208000002399 aphthous stomatitis Diseases 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
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- 238000010369 molecular cloning Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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Abstract
The present invention relates to using albumen TUBA as antigen, the antibody for Tubulin in Behcet disease peripheral blood in patients is detected, so as to realize the diagnosis of dialogue plug patient.Specially express TUBA albumen in conventional manner, set up qualitative or quantitative method and the matched reagent box of the anti-TUBA antibody of detection.
Description
1. technical field
The present invention relates to using albumen TUBA as antigen, detect in Behcet disease peripheral blood in patients for the anti-of Tubulin
Body, so that realize the diagnosis of dialogue plug patient.
2. background technology
Behcet disease (Behcet's disease, BD) is a kind of general, chronic, vascular inflammatory disease.Clinically with mouth
Chamber ulcer, genital ulcer, ophthalmia and skin lesion are outstanding behaviours.The disease often involve nervous system, digestive tract, lung, kidney with
And the organ such as epididymis.BD once has internal organs to be involved, normal recurrent exerbation, and can cause blind, fills in encephalopathy in vain, and Alimentary Tract Perforation etc. is tight
Weight complication, or even cause death.As preferably specific diagnosis antibody is there is no in current serum, cause early stage not
The diagnosis of typical Behcet disease and early treatment become a difficult problem for rheumatism scientific circles.Because of the present circumstance, Behcet disease is found in an urgent demand
Biomarker special in early days.
3. content of the invention
For the problems referred to above, this research is by Orbitrap analytical technique of mass spectrum from BD, RA, SSc patient and normal control
Find 300 multiple proteins in immune complex in person's serum altogether, remove immunoglobulin, keratin and high-abundance proteins,
Find that TUBA is one of specific autoantigens of BD in BD circulating immune complex.
An object of the present invention is to provide the specific antigen TUBA for being capable of effective detection BD.
It is a further object of the present invention to provide the method that detection is used for detecting BD for the antibody of TUBA.
The aminoacid sequence of term used herein " TUBA " is
·1 mrecisihvgqagvqignacwelyclehgiqpdgqmpsdktigggddsfntffsetgagk
·61 hvpravfvdleptvidevrtgtyrqlfhpeqlitgkedaannyarghytigkeiidlvld
·121 rirkladqctglqgflvfhsfgggtgsgftsllmerlsvdygkksklefsiypapqvsta
·181 vvepynsiltthttlehsdcafmvdneaiydicrmldierptytnlnrlisqivssita
·241 slrfdgalnvdltefqtnlvpyprihfplatyapvisaekayheqltvaeitnacfepan
·301 qmvkcdprhgkymaccllyrgdvvpkdvnaaiatiktkrtiqfvdwcptgfkvginyqpp
·361 tvvpggdlakvqravcmlsnttavaeawarldhkfdlmyakrafvhwyvgegmeegefse
·421aredmaalekdyeevgadsadgedegeey(SEQ ID NO.1)
Or any amino acid sequence segments that is forgiven by above-mentioned sequence.
We are found that in the research to BD autoantigens TUBA is the specificity autoantigen of BD.Further research is sent out
Existing, there is the autoantibody for the sequence of high titre in most BD patient's bodies, be named as anti-TUBA antibody, the antibody can lead to
Cross some relatively simple immunological detection methods (such as：Indirect enzyme-linked immunosorbent assay) detect, and the diagnosis to BD
There is good Sensitivity and Specificity.
Mesh of the present invention is to provide the detection method of effective detection BD specific autoantibody, and is applied to the diagnosis of BD.Base
In above research, the present inventor by recombined human TUBA as antigen, with the method detection BD of ELISA, RA, SLE, SSc, RAU, big
Anti- TUBA antibody in arteritis, ANCA relevant blood vessels inflammation patient and normal healthy controls serum, so as to reach by serological
Method diagnoses the purpose of BD, provides objective laboratory foundation for BD clinical diagnosises.
Recombiant protein of the present invention and the like can detect to BD patient by enzyme linked immunosorbent assay which detects
Method is well known to those skilled in the art.Meanwhile, the TUBA Antibody types of detection can be IgG types, IgA types, IgM types or its
The immunoglobulin of its type.
Recombined human TUBA albumen of the present invention can use the prokaryotic protein expression or eucaryon known to those skilled in the art
Protein expression is obtained, or is obtained using business-like method synthesizing and purifying.
4. illustrate
Fig. 1 TUBA SDS-PAGE electrophoresis result figures.M：Albumen Marker：Lane1: 100mM imidazoles eluting result；
Lane2,3: 100mM imidazoles result；Lane4: 60mM imidazoles eluting result, it is seen that have obvious protein band at 58kD.
Level of the anti-TUBA antibody of Fig. 2 in disease group and healthy control group, wherein systemic lupus erythematosus (sle) (SLE), be
System property sclerosiss (SSc), rheumatoid arthritis (RA), recurrent oral ulceration (RAU), Takayasu arteritiss (TA), Behcet disease (BD),
Normal healthy controls (HC), AASV (ANCA (ANCA) correlation system vasculitises).
The anti-TUBA antibody horizontals of Fig. 3 and Behcet disease patients with clinical manifestations and the relation of disease-activity.Anti- TUBA antibody
The erythrocyte sedimentation rate (ESR) of level and Behcet disease patient, c reactive protein (CRP), Birmingham vasculitises activity score (BVAS)
It is in notable positive correlation.
Specific embodiment
In order to be more clearly understood that the present invention, the present invention is further described referring now to lower example embodiment and accompanying drawing.Embodiment
It is only used for explaining and limiting the present invention never in any form.In embodiment, the experimental technique of not marked actual conditions is affiliated neck
Conventional method known to domain and normal condition, or according to the condition proposed by test kit and apparatus manufacturer.
Embodiment 1：The detection of anti-TUBA antibody and its dependency of the diagnosis to Behcet disease and Behcet disease disease feature
Research
First, case and check sample inclusion criteria
BD patient and other Patients with Rheumatic Diseases are in January, 2009 in December, 2012 in The People's Hospital of Peking University
The patient that rheumatism immunity section is in hospital, selected patient meet the international diagnosis of respective disease or criteria for classification.Wherein BD patient 44
Example, including male 28, women 16,40.49 ± 15.2 years old mean age, average course of disease 11.47 ± 12.27 years；Wherein, close
And oral cavity ulcers 41, vulval ulcer 28, intestinal involvement 2, merge ophthalmia 14, phlebothrombosises 1, erythema nodosa
10, arthritis 11.Other Patients with Rheumatic Diseases 220, male 46, women 154,45 ± 18.7 years old mean age,
Including systemic lupus erythematosus (sle) (SLE) 51, systemic sclerosiss (SSc) patient 51, rheumatoid arthritis (RA) are suffered from
Person 40, recurrent oral ulceration (RAU) 32, Takayasu arteritiss (TA) patient 13, ANCA relevant blood vessels are scorching 25, and health is right
According to 59.Normal healthy controls are all from our hospital Physical Examination person.Each group serum derives from the remaining specimen of Clinical Laboratory.BD patient
Clinical data collection as follows：
1) clinical data：Including sex, the age, the course of disease, whether there is oral ulcer, vulval ulcer, erythema nodosa, eye
Involvement, venous thrombosis etc..
2) lab index：
1. erythrocyte sedimentation rate (ESR)：Wei Shifa detections, normal value≤20mm/h；
2. c reactive protein (CRP)：Immunoturbidimetry detects that ＞ 10mg/L think abnormal；
3. IgM rheumatoid factor (RF)：Immunoturbidimetry detects that ＞ 20IU/mL think abnormal；
4. antinuclear antibody：Indirect immunofluorescence (German Ou Meng) half-quantitative detection, titre ＞ 1: 40 are judged to the positive；
5. immunoglobulin (IgG, IgM, IgA) is determined using U.S. Bake Man protein analyzer.
3) activeness of Behcet disease is evaluated using Birmingham vasculitises activity score (BVAS), as shown in table 1.
1 Birmingham systemic vasculitiss activity scores (BVAS) of table
Explanation：Above in surrounding recently, performance recently caused by vasculitises increases.
63 points of each item rating total score highest, more than 15 points of expression state of an illness activities.
2nd, experimental program
TUBA prokaryotic expression SDS-PAGE electrophoresis results：
To TUBA(SEQ ID NO.1)Prokaryotic expression, purification, expressing protein SDS-PAGE colloid electrophoresis result such as Fig. 1 institutes
Show.(correlation technique referring to：《Molecular Cloning:A Laboratory guide (third edition)》(volume two, J. Pehanorm Brookers, D.W. Russells write,
Huang Peitang etc. is translated) the 15th chapter, page 1217 to page 1265)
The level of anti-TUBA antibody in ELISA method detection BD serum
1) wrapper sheet：TUBA, 10 μ g/ml of concentration are diluted with carbonate containing buffer (PH 9.6), coated 96 orifice plates, 100 μ l/
Hole, 4 DEG C of wrapper sheets are overnight；
2) board-washing：PBS (PBS-T) board-washing containing 0.05%Tween 20 4 times, every time 5 minutes；
3) close：Addition 3%BSA-PBS, 300 μ l/ holes, 37 DEG C are closed 3.5 hours；
4) board-washing：With 0.05%PBS-T board-washings 4 times, every time 5 minutes；
5) an anti-incubation：Addition patient and normal healthy controls serum, 100 μ l/ holes, serum is with the PBS-T 1 containing 1%BSA:
400 dilutions, 37 DEG C are incubated 1 hour；
6) board-washing：PBS-T board-washings 6 times, 5 minutes every time；
7) two anti-incubation：Add the goat of the horseradish peroxidase-labeled diluted with the PBS-T 1: 5000 containing 1%BSA
Anti-human igg (company of Zhong Shan Golden Bridge), 37 DEG C are incubated 30 minutes, board-washing 4 times；
8) develop the color：OPD nitrite ions, 100 μ l/ holes, lucifuge is added to react 10 minutes；
9) terminate：With 2mol/L sulphuric acid terminating reactions, 450nm reads OD values；
10) control and result are calculated：All samples do multiple holes, are all provided with positive control, negative control per plate；Respectively with strong
Anti-+2 standard deviation AU values of TUBA antibody average of health people are set to positive threshold value.
AU values=[ODPeptide-ODNon-specific background]Test serum/ [OD peptide-OD non-specific backgrounds] standard serum × 100, standard serum are sun
Property standard serum.
3rd, analysis of experimental data：
Using 13.0 statistical softwares of SPSS, between group, rate relatively adopts chi-square criterion, and the dosage information of normal distribution is with mean
± standard deviation is represented, compares and checked using t between group.Between two variables, correlation analysiss adopt Spearman correlation analysiss.p
＜ 0.05 represents that difference has statistical significance.
4th, experimental result：
Anti- TUBA antibody horizontals compare in the level of BD and other connective tissue diseases：
Application ELISA method detection TUBA antibody is scorching in BD, RA, SLE, SSc, RAU, Takayasu arteritiss, ANCA relevant blood vessels
The level (expression of AU values) of patient and normal healthy controls, the t assays of independent sample point out BD patient anti-TUBA antibody horizontals
Apparently higher than healthy control group, (72.80 ± 37.5vs35.13 ± 17.67, t=6.114, p ＜ is 0.001).SLE, SSc patient resists
TUBA antibody horizontals are substantially also higher than healthy control group (50.54 ± 29.7vs35.13 ± 17.67, t=3.095, p=
0.003)；(73.68 ± 40.95vs 35.13 ± 17.67, t=6395, p ＜ is 0001).RA, RAU, Takayasu arteritiss, ANCA are related
The anti-TUBA antibody of vasculitises patients serum does not draw significant difference with normal healthy controls.As shown in Figure 2.
Anti- TUBA antibody horizontals compare in BD and other connective tissue disease positive rates：
Positive threshold value (AU is set to the anti-TUBA antibody average ≠ 2 standard deviation AU values of Healthy People：70.67), thus calculate
The positive rate of BD patient's Tubulin- α -1 antibody be 56.8%, SLE be 27.5%, SSc be 27.5%, RA be 7.5%, RAU
For 3.11%, Takayasu arteritiss be 23.1%, AASV be 4.0%, healthy control group be 3.39%.Anti- TUBA antibody is for diagnosis BD
Sensitivity be 56.8%, specificity be 82.7% (as shown in table 2).
Positive rate of the 2. anti-TUBA antibody of table in various disease
Anti- TUBA antibody horizontals are in BD clinical manifestations and disease-activity correlation analysiss；
There is the positive rate of deep venous thrombosis and skin noduless erythema apparently higher than anti-in anti-TUBA antibody positives patient
TUBA negative antibody groups, have therebetween significant sex differernce (p ＜ 0.05) as shown in Figure 3.Correlation analysiss are pointed out, and anti-TUBA resists
Positive correlation in body level and ESR, CRP, BVAS.As shown in table 3.It can be seen that there is deep vein in anti-TUBA antibody positives patient
The positive rate of bolt and skin noduless erythema is apparently higher than anti-TUBA negative antibodies group.
3 anti-TUBA of table is positive and negative, and group BD patient system's involvements are compared
In sum, by detecting BD and the anti-TUBA antibody water in HC and a series of other relevant disease control serums
Flat, it is found that anti-TUBA antibody horizontals in BD patient apparently higher than other each matched groups, according to anti-TUBA in normal healthy controls
After antibody horizontal determines positive threshold value, it can be found that anti-TUBA antibody is 56.8% for the sensitivity of diagnosis BD, specificity is
82.7%, the clinical efficiency diagnosed by BD can be improved, is a kind of preferable BD in-vitro diagnosis biomarker.Meanwhile, in BD
In anti-TUBA antibody horizontals and BD patient disease activity degree (BVAS) and degree of inflammation (ESR and CRP) be all in notable positive
Close, the anti-TUBA antibody horizontals of prompting are higher, and BD conditions of patients is heavier, and on the other hand, in BD patient, anti-TUBA antibody horizontals also show
It is higher than other each matched groups to write, and these two aspects illustrates that anti-TUBA antibody has good dependency with BD.And in other matched groups
In, anti-TUBA antibody positive rate is very low, while also failing to find there is significant correlation with the state of an illness, more highlights anti-TUBA antibody
Importances and value for specific diagnosises BD.
Claims (3)
- Application of the 1.TUBA antigens in the reagent for clinical diagnosis or detectable substance for preparing diagnosis vascular inflammatory disease, the TUBA's Sequence is as shown in SEQ ID NO.1；The vascular inflammatory disease is selected from Behcet disease.
- 2. application according to claim 1, it is characterised in that：Prepare TUBA antigens in conventional manner, set up detection anti- The quantitative approach of TUBA antibody and matched reagent box；The quantitative approach is：1) wrapper sheet：TUBA, 10 μ g/ml of concentration are diluted with the carbonate buffer solution of pH9.6, coated 96 orifice plates, 100 μ l/ holes, 4 DEG C Wrapper sheet is overnight；2) board-washing：PBS containing 0.05%Tween20, i.e. PBS-T, board-washing 4 times, 5 minutes every time；3) close：Addition 3%BSA-PBS, 300 μ l/ holes, 37 DEG C are closed 3.5 hours；4) board-washing：With 0.05%PBS-T board-washings 4 times, every time 5 minutes；5) an anti-incubation：Patient and normal healthy controls serum is added, 100 μ l/ holes, serum are dilute with PBS-T1: 400 containing 1%BSA Release, 37 DEG C are incubated 1 hour；6) board-washing：PBS-T board-washings 6 times, 5 minutes every time；7) two anti-incubation：Add with the Goat anti human of the horseradish peroxidase-labeled of PBS-T1: 5000 dilutions containing 1%BSA IgG, 37 DEG C are incubated 30 minutes, board-washing 4 times；8) develop the color：OPD nitrite ions, 100 μ l/ holes, lucifuge is added to react 10 minutes；9) terminate：With 2mol/L sulphuric acid terminating reactions, 450nm reads OD values；10) control and result are calculated：All samples do multiple holes, are all provided with positive control, negative control per plate：Respectively with Healthy People + 2 standard deviation AU values of anti-TUBA antibody average are set to positive threshold value,AU values=[OD peptide-OD non-specific backgrounds]Test serum/ [OD peptide-OD non-specific backgrounds]Standard serum× 100, standard serum is the positive Standard serum.
- 3. a kind of diagnosis vascular inflammatory disease Blood diagnosis reagent, it is characterised in that：Including TUBA albumen, HRP-IgG antibody, And the conventional reagent in Blood diagnosis reagent, the sequence of the TUBA is as shown in SEQ ID NO.1；The vascular inflammatory disease It is selected from Behcet disease；Conventional reagent in the Blood diagnosis reagent includes carbonate buffer solution, PBST washing liquids, confining liquid, colour developing Liquid and terminate liquid.
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