WO2012100599A1

WO2012100599A1 - Use of peroxiredoxin iv in preparing in vitro diagnostic reagents for rheumatoid arthritis

Info

Publication number: WO2012100599A1
Application number: PCT/CN2011/083637
Authority: WO
Inventors: 吴乔; 吴玉章
Original assignee: 中国人民解放军第三军医大学
Priority date: 2011-01-24
Filing date: 2011-12-07
Publication date: 2012-08-02
Also published as: CN102346186B; CN102346186A

Abstract

Provided is an ELISA reagents kit for detecting peroxiredoxin IV antibody in biological samples, comprising an antibody capture agent, an enzyme-labeled secondary antibody and a substrate. Also provided are applications for the kit in detecting volumes of peroxiredoxin IV antibody and rheumatoid arthritis.

Description

Application of Peroxidase Reductase IV in Preparation of Rheumatoid Arthritis In Vitro Diagnostic Reagents

The present invention relates to the field of medical testing, and in particular to an indirect ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen, and a method and use thereof.

Background technique

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovitis as a basic pathological change characterized by chronic destructive joint disease. Most of the conditions are progressive, eventually leading to joint fibrosis or bony rigidity. Severe disability, seriously affecting the quality of life of patients. Rheumatoid arthritis without proper treatment can be delayed and even cause joint deformities. As a self-immune disease, pathogenic antigen-driven autoreactive CD4+ T cell activation is a central pathway in the pathogenesis of rheumatoid arthritis.

For the diagnosis of rheumatoid arthritis, the current mainstream still uses the classification criteria of the American College of Rheumatology 1987 RA, which mainly relies on clinical manifestations and excludes other arthritis to judge RA. In addition, X-ray changes and rheumatoid factors are also common criteria for determining RA. However, the above methods are cumbersome and non-specific, and are not suitable for early diagnosis.

It is well known that the pathological changes of RA gradually progress from cartilage tissue to cartilage to bone. If RA is not effectively treated in the early stage, it will cause joint dysfunction and even labor loss, and cause multiple organs in the body to be involved and endangered. If you can diagnose the condition of RA as soon as possible and treat it early to prevent or delay its development, it will effectively improve the treatment effect of RA and reduce its complications. The detection of high sensitivity and specificity in the early stage of RA is a prerequisite for the early diagnosis and treatment of RA.

In early diagnostic indicators, IL-8 and IL-6 levels were associated with elevated cytomegalovirus DNA-positive rheumatoid arthritis patients and CMV genomes, but with low specificity and sensitivity. In terms of RA autoantibodies as indicators: Rheumatoid factor (RF) has the disadvantage of low sensitivity; 53.3% of patients with early RF-negative rheumatoid arthritis are positive for anti-peripheral factor (APF), but APF antigen The storage time of the tablets is short (only 1~2 weeks), and the detection stability is low; the positive rate of Sa antibody in rheumatoid arthritis is 40%, the specificity is 98.9%, but the early stage of rheumatoid arthritis is only about 23%. Correlation studies between peroxide reductase IV and rheumatoid arthritis have not been reported.

Summary of the invention

In view of the above, it is an object of the present invention to provide a kit capable of rapidly detecting the content of a peroxide reductase IV antibody, which is simple in operation and low in cost.

To achieve the above object, the technical solution of the present invention is: An ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen, the ELISA kit comprising an antibody capture reagent, an enzyme-labeled secondary antibody, and a substrate.

2. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 1, wherein the antibody capture reagent is a dilution of not less than 1 μg/mL of the peroxide reductase IV.

3. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 1, wherein the antibody capture reagent is attached to the solid phase support.

4. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 3, wherein the solid phase support is an ELISA plate or a biochip.

5. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 1, wherein the biological specimen is serum, urine, cells, tissue or plasma.

6. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 5, wherein the biological specimen is serum, and the serum is a serum dilution of not more than 200 volume dilutions.

7. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 1, wherein the enzyme-labeled secondary antibody is a horseradish-labeled goat anti-human IgG as a dilution of no more than 20,000 dilutions. .

8. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 1, wherein the ratio of the antibody capture agent to the enzyme-labeled secondary antibody is 1:1.

A second object of the present invention is to provide a use of the above kit, which can make up for the technical blank in the field of detection of a peroxide reductase IV antibody, and provides a diagnostic idea for rheumatoid arthritis and other immune diseases, and is particularly suitable for use in Detection of biological specimens.

To achieve the above object, the technical solution of the present invention is:

9. The use of an ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen as described in 1, 1-8 for detecting an antibody content of an oxidoreductase IV antibody.

10. The use of an ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 9, the use of the ELISA kit in the preparation of a kit for detecting rheumatoid arthritis.

11. The use of an ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 9, the use of the ELISA kit in the preparation of an early rheumatoid arthritis kit.

12. The use of an ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 9, wherein the ELISA kit detects the peroxide reductase IV antibody of the serum to be tested and normal human serum. The content, when the OD _45Qnm value of the serum to be tested is greater than 0.5610 is positive for the peroxide reductase IV antibody, and less than or equal to 0.5610 is negative for the peroxide reductase IV antibody. A third object of the present invention is to provide a method for the content of a peroxide reductase IV antibody which is highly sensitive and simple to handle.

13. The method for detecting the content of the peroxide reductase IV antibody by using the ELISA kit of any of 1-8, a, contacting and incubating the biological specimen with the antibody capture agent in the ELISA kit; b, unbound The biological specimen is isolated to obtain an antigen-antibody complex; c. The antigen-antibody complex obtained in step b is added to the enzyme-labeled secondary antibody in the ELISA kit to obtain a double-antibody complex; d. detecting binding to the antibody-trapping agent The content of the oxide reductase IV antibody.

14. The method for detecting a content of a peroxide reductase IV antibody using an ELISA kit according to 13, the method further comprising the content of the peroxide reductase IV antibody obtained in step d and normal human peroxidase reductase The content of the IV antibody was compared and judged.

15. The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to 13, in step a, coating the antibody capture agent with an enzyme label, contacting the biological sample to be tested with the antibody capture agent, and keeping warm and using The blocking solution is blocked; the biological specimen is a serum dilution of no more than 200 volume dilutions, and the antibody capture agent is a dilution of less than lg/ml of the peroxide reductase IV.

16. The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to 13, in step b, separating the unbound biological specimen by washing the enzyme plate sealed in step a and blocked with a blocking solution, Antigen-antibody complex.

17. The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to 13, wherein in step c, the antigen-antibody complex obtained in step b is added to the enzyme-labeled secondary antibody in the ELISA kit for incubation reaction. The double-anti-sandwich complex is obtained; the enzyme-labeled secondary antibody is a horseradish-labeled goat anti-human IgG as a dilution of no more than 20,000-fold dilution.

18. The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to 13, wherein in step d, the substrate is added to the double-antibody complex obtained in step c, and the substrate is protected from light and then terminated. The colorant sample solution is reacted, and the content of the peroxide reductase IV antibody bound to the antibody capture agent is detected by measuring the OD _45Qnm value of the color developing sample solution.

19. The method for detecting a content of a peroxide reductase IV antibody using an ELISA kit according to 13, the method further comprising a negative control group and a blank control group, wherein the negative control group is a biological specimen derived from a normal person. .

20. The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to 19, wherein the OD _45Qnm value corresponding to the maximum value of the sensitivity/specificity of the biological sample to be tested is a critical value. When the OD _45Qnm value of the biological specimen to be tested is greater than the critical value, it is determined that the peroxide reductase IV antibody is positive, and the OD _45Qnm value of the biological specimen to be tested is less than or equal to the critical value, and the peroxide reductase IV antibody is determined to be negative.

The beneficial effects of the invention are: the kit can specifically detect the content of the peroxide reductase IV antibody, Low, sensitive; use this kit to detect the content of peroxide reductase IV antibody, and compare with the normal human peroxide reductase IV antibody level, can specifically teach high diagnostic rheumatoid arthritis Therefore, the kit can be prepared into a rheumatoid arthritis diagnostic kit, which is especially suitable for early diagnosis.

DRAWINGS

In order to make the objects, technical solutions and advantages of the present invention more comprehensible, the present invention will be further described in detail with reference to the accompanying drawings in which:

Figure 1 shows the analysis of the expression of peroxide reductase IV antibody in early RA, late RA and normal human serum by indirect ELISA.

Figure 2 is a standard curve of indirect ELISA for peroxide reductase IV. The abscissa is the dilution factor of mouse anti-human monoclonal antibody, and the ordinate is the OD ₄₅₍₎ value corresponding to different dilution factor antibodies.

Figure 3 is a graph showing the specificity and sensitivity of the ELISA kit in the analysis of the analysis curve of subjects with early rheumatoid arthritis, where the abscissa is sensitivity and the ordinate is (100-specific).

Figure 4 is a graph showing the specificity and sensitivity of the ELISA kit in the analysis of the analysis curve of subjects with advanced rheumatoid arthritis, where the abscissa is sensitivity and the ordinate is (100-specific).

detailed description

In order to make the objects, technical solutions and advantages of the present invention more comprehensible, the preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. In the present embodiment, the antigen-coated human peroxidase reductase IV is obtained by constructing a prokaryotic expression plasmid, transforming the expression bacteria, and fermenting the culture, and collecting a large amount of the bacterial cell precipitate containing the peroxide reductase IV, which is spicy. Root peroxide» (HRP)-labeled goat anti-human G, as a secondary antibody for indirect ELISA; further determined the concentration of human peroxidase reductase IV coating and the optimal concentration range of serum and the positive-positive threshold It was confirmed that a specific and sensitive method for detecting rheumatoid arthritis peroxide reductive IV antibody capable of capturing 7.8 ng/mL of peroxide reductase IV antibody was developed.

EXAMPLES Peroxide Reductase IV Antibody Indirect Method ELISA Kit and Method and Application

I. Preparation of coated human peroxide reduction KIV preparation

A human peroxidase reductase IV expression plasmid was constructed, and a recombinant antigen TRX-peroxide reductase IV fusion protein was prepared using the pET prokaryotic expression system.

1 According to the human peroxidase reductase IV mature protein sequence retrieved in enBank, the DNA encoding the peroxidase reductase IV protein was designed and synthesized;

2 EcoR I and Hhidlll restriction sites were designed at the 5' and 3' ends of DNA, and the recombinant plasmid pET peroxide reductase IV was constructed by polymerase chain reaction (PCR).

3 induced expression by BLTG in BL21 (DE3);

Correction page (Article 91) ISA/CN 4 Ni-NTA affinity chromatography to prepare TRX-peroxide reductase IV;

5 The correctness of the TRX-peroxide reductase IV plasmid and the expressed peroxidase IV antigen was identified by DNA sequencing, relative molecular mass analysis of the fusion protein and Western blotting.

Second, the indirect method of peroxide reductase IV antibody ELISA kit and its method and application

1. Experimental materials

1.1, reagent

Horseradish-labeled goat anti-human IgM was purchased from Lifescience; horseradish-labeled goat anti-mouse IgG (H+L) was purchased from Beijing Zhongshan Biotechnology Co., Ltd.; mouse anti-human peroxide reductase IV monoclonal antibody was purchased from Abeam Company (potency 1:12800);

1.2, instrument

UV spectrophotometer Backmen.

1.3, the main reagent preparation

1% blocking solution: Add 10mL blocking solution to 90ml TBS and store at -15~-25°C. 0.5% blocking solution: Add 5 mL of blocking solution to 95 mL of TBS and store at -15~-25 °C. Serum dilution: The primary antibody was diluted with 0.5% blocking solution. HRP-labeled anti-mouse or human IgG was diluted 1:0.5 times with 0.5% blocking solution to obtain a secondary antibody working solution. Coating solution: 0.85M (pH 9.6) Carbonate buffer: 1.59g Na ₂ C0 ₃ plus 2.93g NaHC0 ₃ , three distilled water to a constant volume of 1000mL. Washing solution: 0.01 M pH 7.4 PBS Ten Tween-20 (T) The final mass fraction was 0.05%. Substrate solution: Phosphate-citrate buffer (pH 5.0~5.5): 0.1M Citric acid 24.3mL Add 25.7ml 0.2M Na ₂ HP0 ₄ · 12H ₂ 0 Add o-phenylenediamine 40.0mg, dilute to 50mL , before use, add 0.15mL volume fraction of 30% 3⁄40 ₂ . Terminator: 22.2 mL of sulfuric acid was added to 177.8 mL of distilled water.

1.4. Samples of RA The 5 cases of RA synovial tissue were provided by Professor Li Zhanguo from the Department of Rheumatology and Immunology, Peking University Affiliated People's Hospital; 534 cases of RA (67 cases in early stage, 467 cases in advanced stage), 65 cases of OA, 120 cases of SLE, 10 cases of crust disease

(scleroderma), 15 cases of dermatomyositis and 120 normal controls (NC) blood samples were obtained from Xi'an Hospital.

The collected procoagulant samples were centrifuged at 3,000 rpm for 30 minutes at room temperature overnight, and the supernatant was taken, lOOul was dispensed, -70

°C save.

2. ELISA kit for peroxide reductase IV antibody, method and application thereof

2.1, Determination of optimal peroxide reductase IV coating concentration and detection of serum dilution factor

The square matrix titration method was used as follows: (1) The human peroxidase IV containing recombinant expression of 10 g/ul was diluted at the following concentration (5 g/ml, 10 μg/mK 20 g/mU 50 μ). g/mU lOO g/ml), 100 μ Ι 7 wells added to the enzyme In the standard plate, incubate in a wet box at 4 °C for 12 hours, wash the plate 3 times, 5 min/time, and pat dry; (2) Add 100 μΐ/well blocking solution, block at 4 °C wet overnight, wash plate Machine washing 3 times, 5min / time, patted dry; (3) serum to be tested according to 1: 20, 1: 50, 1: 100, 1: 200 times dilution, 100 μ ΐ / hole, 37 ° C wet box effect lh Wash the plate 10 times, 3 min / time, pat dry; (4) Dilute HRP-labeled goat anti-human IgG according to 1:4000, ΙΟΟ μ Ι / L, add to the plate, incubate in a 37 ° humid box 30 minutes, washing machine washing 10 times, 3min / time, patted; enzyme standard secondary antibody according to the determined ratio of 1: 4 000; (5) adding TMB substrate 100 μ 1 / hole, 37 °C protected from light After 5 min, the reaction was terminated by adding 100 μl/well of 2 mol/L sulfuric acid; (6) Detection: The OD _{450 nm} value was measured by a microplate reader. At the same time, 1% BSA/PBS solution was used as a blank control, and the concentration of human peroxide reductase IV coating and the serum dilution to be tested with the highest P/N value were the optimal working concentrations.

The human peroxide reductase IV coating and the serum to be tested were serially diluted, and the square matrix test was carried out. The results are shown in Table 1. The results of square array titration showed that the concentration of human peroxidase reductase IV was 50 g/ml, and the serum to be tested was diluted 1:200, the serum OD _{45 () nm} value of rheumatoid arthritis was greater than 1.0, negative positive serum OD _{45 () nm} ratio (PIN) maximum of 10.45; human peroxide reductase IV coating concentration of 20 g / ml, serum 1: 200 dilution, rheumatoid arthritis serum OD _{45 () nm} value is greater than 1.0, the ratio of OD _{45 () nm} in the positive-positive serum (P / N) was 10.08, which was only 0.37 less than the ratio of OD _{450nm in the} largest negative-positive serum. Considering the cost of antigen coating, the concentration of human peroxidase IV coating was selected. 20 g/ml is the optimal coating concentration; the serum to be tested is diluted 1:200 to the optimal dilution.

Table 1 Determination of the optimal working concentration of human peroxidase reductase IV and human test serum. Dilution factor of human test serum. Human peroxide reductase IV coating concentration (μg/ml)

100 50 20 10 5

Serum OD _45Qnm value in patients with rheumatoid arthritis 10 3.013 2.845 2.644 2.442 2.009

(P value) 50 2.866 2.342 2.201 2.002 1.867

100 2.620 2.177 2.009 1.911 1.766

200 2.221 2.102 1.977 1.800 1.633 Normal human serum OD _45Qnn i 10 0.623 0.587 0.545 0.496 0.477

(N value) 50 0.532 0.388 0.335 0.321 0.302

100 0.371 0.221 0.215 0.208 0.199

200 0.321 0.201 0.196 0.195 0.188

P/N value 10 4.84 4.85 4.85 4.92 6.65

50 5.39 6.04 6.57 6.24 6.18

100 7.06 9.85 9.34 9.19 8.87

200 6.89 10.45 10.08 9.23 8.69

2.2. Determination of the positive value of negative Yin Table 2 30 normal human serum indirect ELISA peroxide reductase IV antibody detection result number 1 2 3 4 5 6 7 8

OD ₄ 50nm 0.250 0.310 0.380 0.390 0.450 0.510 0.520 0.390 No. 9 10 11 12 13 14 15 16

OD450 0.370 0.340 0.330 0.660 0.520 0.490 0.460 0.530 No. 17 18 19 20 21 22 23 24

OD ₄ 50nm 0.520 0.510 0.550 0.370 0.360 0.310 0.420 0.440 No. 25 26 27 28 29 30

OD ₄ 50nm 0.380 0.480 0.420 0.390 0.300 0.410

Table 3 30 indirect early RA patients with serum indirect ELISA peroxidase IV antibody detection U results No. 1 2 3 4 5 6 7 8

OD450 0.970 1.890 1.200 1.930 1.470 1.870 1.900 1.470 No. 9 10 11 12 13 14 15 16

OD450 1.210 2.120 0.790 0.360 0.950 0.810 0.300 0.280 No. 17 18 19 20 21 22 23 24

OD ₄ 50nm 0.790 0.910 0.300 0.950 1.550 1.860 1.600 2.000 No. 25 26 27 28 29 30

OD450 0.570 0.410 0.440 0.480 0.880 1.780

Table 4 Serum indirect ELISA peroxidase IV antibody in 17 patients with advanced RA U results No. 1 2 3 4 5 6 7 8

OD450 1.860 1.890 1.800 1.990 0.620 1.300 1.200 1.100 No. 9 10 11 12 13 14 15 16

OD ₄ 50nm 0.700 0.870 0.750 1.370 1.870 1.900 2.100 1.200 No. 17

OD _450nm 0.780

Table 5 Results of the receiver operating curve critical value analysis

OD value Sensitivity % 95% CI Specificity% 95%CI Likelihood ratio

> 0.2650 100.0 92.45% to 100.0% 3.333 0.08436% to 17.22% 1.03

> 0.2900 97.87 88.71 % to 99.95% 3.333 0.08436% to 17.22% 1.01

> 0.3050 93.62 82.46% to 98.66% 10.00 2.1 12% to 26.53% 1.04

> 0.3200 93.62 82.46% to 98.66% 13.33 3.755% to 30.72% 1.08

> 0.3450 93.62 82.46% to 98.66% 16.67 5.642% to 34.72% 1.12

> 0.3650 91.49 79.62% to 97.63% 20.00 7.713% to 38.57% 1.14

> 0.3720 91.49 79.62% to 97.63% 26.67 12.28% to 45.89% 1.25

> 0.3770 91.49 79.62% to 97.63% 30.00 14.73% to 49.40% 1.31

> 0.3850 91.49 79.62% to 97.63% 36.67 19.93% to 56.14% 1.44

> 0.4000 91.49 79.62% to 97.63% 46.67 28.34% to 65.67% 1.72

> 0.4150 89.36 76.90% to 96.45% 50.00 31.30% to 68.70% 1.79

> 0.4300 89.36 76.90% to 96.45% 56.67 37.43% to 74.54% 2.06

> 0.4450 87.23 74.26% to 95.17% 60.00 40.60% to 77.34% 2.18

> 0.4550 87.23 74.26% to 95.17% 63.33 43.86% to 80.07% 2.38

> 0.4700 87.23 74.26% to 95.17% 66.67 47.19% to 82.71 % 2.62 > 0.4850 85.1 1 71 .69% to 93.80% 70.00 50.60% to 85.27% 2.84

> 0.5000 85.1 1 71 .69% to 93.80% 73.33 54.1 1 % to 87.72% 3.19

> 0.5150 85.1 1 71 .69% to 93.80% 80.00 61 .43% to 92.29% 4.26

> 0.5250 85.1 1 71 .69% to 93.80% 90.00 73.47% to 97.89% 8.51

> 0.5400 85.1 1 71 .69% to 93.80% 93.33 77.93% to 99.18% 12.77

> 0.5610 85.1 1 71 .69% to 93.80% 96.67 82.78% to 99.92% 25.53

> 0.5960 82.98 69.19% to 92.35% 96.67 82.78% to 99.92% 24.89

> 0.6400 80.85 66.74% to 90.85% 96.67 82.78% to 99.92% 24.26

30 normal human serum, 30 early RA, and 17 advanced RA patients' serum were detected by indirect ELISA using the above-described peroxide reductase IV antibody. The OD450 values measured by each sample were grouped into GraphPad Prism software for subject working curve (R0C) analysis, and the specific value of 0D450 corresponding to the maximum value (specificity%+sensitivity%) -1 was selected, such as As shown in Table 5, 0. 5610 is a negative-positive cut-off value, and the OD450nm value of the sample to be tested is greater than 0. 5610 is positive for peroxide reductase IV, and negative for less than or equal to.

2.3 Specificity test

Osteoarthritis (0A), systemic lupus erythematosus (SLE), and other autoimmune diseases (AD), including atherosclerosis, scleroderma, dermatomyositis, rheumatoid arthritis (RA), and normal people The control (NC) serum was tested according to the method in the present example, and the results were observed.

Peroxidase reductase IV antibody indirect method ELISA specific test results

Disease or normal human serum OA AD SLE RA NC

The average value of OD _45Qmn value is 0.2146 0.2152 0.2336 2.4231 0.1667

The 2 g/ul mouse anti-human peroxide reductase IV monoclonal antibody was diluted according to the following concentration gradient (2000 ng/ml, 1000 ng/mK 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ Ml, 31.25 ng/mU 15.625 ng/mK 7.8125 ng/ml), 100 μL/well was added to the ELISA plate, and each dilution was repeated 3 times. The indirect ELISA method was established according to the established peroxide reductase IV antibody. The results are shown in Table 3 and Figure 2. When the mouse anti-human peroxide reductase IV monoclonal antibody was diluted to 7.8125 ng/ml, the OD450nm value was 1.082, which was slightly larger than the blank control value of 0.895, since lOO L was added to each enzyme standard well, therefore, in serum The minimum content of the peroxide-containing reductase IV antibody was determined to be 0.78 ng.

Table 7 Indirect method of peroxide reductase IV antibody ELISA sensitivity test results

Peroxide reductase IV ratio dilution (ng/ml)

2000 1000 500 250 125 62. 5 31. 25 15. 625 7. 8125 0

OD450nm

Average 2. 823 1. 920 1. 501 1. 277 1. 167 1. 129 1. 094 1. 087 1. 082 0. 895 Linear regression formula: Y=0.0009X+1.0391 2.5 Repeatability test results

(1) Intra-assay repeatability test: Three rheumatoid arthritis sera collected at different times were diluted at the optimal dilution and added to the antigen-coated wells. Three replicate ELISAs were performed for each sample. The average OD _{45 () nn} (X) and standard deviation (SD) were calculated from the OD _{45 () nm} values of each sample, and the intra-assay coefficient of variation for each sample was calculated [CV = (SD / X) X 100 ] _; inter-assay reproducibility (2) test batch: under otherwise identical conditions, with 3 parts of purified mouse anti-human prepare different batches of Peroxiredoxin IV antibody-coated microtiter plates, parts of three different The rheumatoid arthritis serum collected at the time was tested. The average OD _{45 () nn} (X) and SD were calculated from the OD _{45 () nm} values of each sample, and the inter-assay coefficient of variation for each sample was calculated.

According to statistical analysis, the coefficient of variation (CV) of OD _45ftm ^ between batches of rheumatoid arthritis serum collected at different times was 2.34%~4.87%, less than 5%; 3 collected at different times The CV of the OD _45Qnm value of the rheumatoid arthritis serum 3 batches was 1.27%~3.79%, less than 5% (Table 4). It is indicated that the established indirect ELISA method for peroxide reductase IV antibody has good intra- and inter-assay repeatability.

Table 8 Repeatability test results

Item Serum number of times Times of test mean standard deviation coefficient of variation

Item Sera 1st 2nd 3rd X SD /% cv

Inboard 1 2.133 2.147 2.326 2.202 0.1076 4.87%

Within 2 2.536 2.521 2.632 2.563 0.060 2.34%

Plate 3 1.955 1.823 1.949 1.909 0.745 3.90%

Item Serum determination 4 times Times of test Mean Standard deviation Coefficient of variation

Item Sera 1st 2nd 3rd X SD /% cv

Board room 1 2.823 2.970 2.857 2.883 0.077 2.67%

Among 2 1.723 1.745 1.701 1.723 0.022 1.27%

Plates 3 1.643 1.649 1.540 1.611 0.061 3.79%

2.6. Determination of test methods

Conclusion: The titer of peroxide reductase IV in serum of RA patients is significantly higher than that of normal human serum (P<0.01). In particular, the titer of peroxide reductase IV in serum of late RA was significantly higher than that of normal human serum (P < 0.001); the positive rate was determined by cutoff value (0.5610), and the peroxide was reduced in serum of patients with advanced RA. The specificity of the enzyme IV antibody was 96.67%, and the sensitivity was 94.12%. The specificity of the peroxide reductase IV antibody in the serum of early RA patients was 96.67%, and the sensitivity was 76.67%.

3. Application of ELISA kit containing peroxide reductase IV antibody in RA diagnosis

The early rheumatoid arthritis described in the present application is defined as a patient who finds the clinical symptom within 2 months. 30 patients with early RA and 17 patients with advanced RA were tested for OD _{45Q by} the kit and method described in Example 2, as shown in Table 3, 4 7 .

The serum OD _45Q values of the above-mentioned early RA patients and normal subjects were _analyzed by the GraphPad Prism software for the receiver operating curve (R0C), as shown in Fig. 3, the specific sensitivities were _{76.67%, 96.67} %, respectively. .

The OD _45Q value of the above-mentioned patients with advanced RA and normal subjects was _analyzed by the GraphPad Prism software for the receiver operating curve (R0C), as shown in Fig. 4, the specific sensitivities were _{94.12%, 96.67} %, respectively. .

It is to be understood that the above embodiments are merely illustrative of the embodiments of the present invention, and are not intended to be The various changes and modifications of the invention are intended to be

Claims

Claim

An ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen, characterized in that the ELISA kit comprises an antibody capture reagent, an enzyme-labeled secondary antibody, and a substrate.

The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 1, wherein the antibody capture agent is a peroxide reductase IV at a final concentration of not less than 1 A dilution of μ g/mL.

3. The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 1, wherein the antibody capture agent is attached to the solid phase support.

The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 3, wherein the solid phase support is an ELISA plate or a biochip.

The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 1, wherein the biological specimen is serum, urine, cells, tissue or plasma.

The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 5, wherein the biological specimen is serum, and the serum is not more than 200 times volume dilution. Serum dilution.

The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 1, wherein the enzyme-labeled secondary antibody is horseradish-labeled goat anti-human IgG. More than 20,000 times diluted dilution.

The ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claim 1, wherein the antibody capture agent and the enzyme-labeled secondary antibody are used in a volume ratio of 1:1.

9. Use of an ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to claims 1-8 for detecting a content of a peroxide reductase IV antibody.

10. The use of an ELISA kit according to claim 9, wherein: the ELISA kit is used in the preparation of a kit for detecting rheumatoid arthritis.

11. The use of an ELISA kit according to claim 9, wherein: the ELISA kit is used in the preparation of an early rheumatoid arthritis kit.

12. The use of the ELISA kit according to claim 9, wherein: the ELISA kit detects the content of a peroxide reductase IV antibody in the serum to be tested and normal human serum, and the OD _{45Qnm of} the serum to be tested. A value greater than 0.5610 is positive for the peroxide reductase IV antibody, and less than or equal to 0.5610 is negative for the peroxide reductase IV antibody.

13. A method for detecting a content of a peroxide reductase IV antibody using the ELISA kit according to any one of claims 1-8, a, contacting and incubating the biological specimen with the antibody capture agent in the ELISA kit; b. The bound biological specimen is isolated to obtain an antigen-antibody complex; c. The antigen-antibody complex obtained in step b is added to the enzyme-labeled secondary antibody in the ELISA kit to obtain a double-antibody complex; d. detecting binding to the antibody capturing agent The content of the peroxide reductase IV antibody.

The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to claim 13, wherein the method further comprises: the content of the peroxide reductase IV antibody obtained in the step d is normal. The content of human peroxide reductase IV antibody was compared and judged.

The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to claim 13, wherein: in step a, the antibody capture agent is coated with an enzyme label, and the biological sample and the antibody to be tested are used. The capture agent is contacted, insulated and blocked with a blocking solution; the biological specimen is a serum dilution of no more than 200 volume dilutions, and the antibody capture agent is a dilution of not less than 1 μg/ml of the peroxide reductase IV.

The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to claim 13, wherein: in step b, the step of washing in the step a and the enzyme plate sealed with the blocking solution are unbound. The biological specimen is isolated to obtain an antigen-antibody complex.

The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to claim 13, wherein: in step c, the antigen-antibody complex obtained in step b is added to the enzyme in the ELISA kit. The secondary antibody is subjected to an incubation reaction to obtain a double-antibody sandwich complex; the enzyme-labeled secondary antibody is a horseradish-labeled goat anti-human IgG as a dilution of no more than 20,000-fold dilution.

The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to claim 13, wherein in the step d, the substrate is added to the double-antibody complex obtained in the step c to protect the light from color. _Thereafter , the reaction solution was terminated by adding a stop solution, and the content of the peroxide reductase IV antibody bound to the antibody capture agent was detected by measuring the OD _45Qnm value of the chromogenic sample solution.

19. The method for detecting a content of a peroxide reductase IV antibody using an ELISA kit according to claim 13, wherein: the method further comprises a negative control group and a blank control group, wherein the negative control group is derived from Biological specimens of normal people.

The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to claim 19, wherein: the OD _45Qnm value of the biological sample to be tested corresponds to (sensitivity + specificity)-1 When it is the maximum value, it is set as the critical value. When the OD _45Qnm value of the specimen is larger than the critical value, it is judged that the peroxide reductase IV antibody is positive, and the sample OD _450nm value is less than or equal to the critical value.