An ELISA kit for detecting anti-OJ antibodies and its detection method and application

Abstract

This invention discloses an ELISA kit for detecting anti-OJ antibodies, characterized by comprising: purified dominant antigenic epitope OJ protein, wherein the amino acid sequence corresponding to the purified dominant antigenic epitope OJ protein is shown in SEQ NO.1. This invention also discloses its detection method and application. The kit of this invention can be used for anti-OJ antibody detection, filling a gap in anti-OJ antibody detection methods. It features high throughput, accurate results, high sensitivity, and simple operation, making it easier to promote and apply.

Description

Technical Field

[0001] The present invention relates to the field of antibody detection, and in particular to an ELISA kit for detecting anti-OJ antibodies and a detection method and application thereof. Background Art

[0002] Anti-synthetase syndrome (ASS) is a special type of polymyositis or dermatomyositis. Among the autoantibodies associated with inflammatory diseases, there is a class of anti-synthetase antibodies. Patients with positive anti-synthetase antibodies have more specific clinical manifestations, such as myositis interstitial lung lesions, symmetrical polyarthritis, fever, mechanic's hands, Raynaud's phenomenon, etc.

[0003] OJ is also known as isoleucyl-tRNA synthetase.

[0004] Anti-OJ antibodies can be found in 0.6%-3% of patients with anti-synthetase antibody syndrome, and 100% of patients with positive anti-OJ antibodies have interstitial pneumonia. Dynamic observation of serum anti-OJ antibody levels is helpful for diagnosing the disease, predicting the progression of the disease, and guiding clinical treatment.

[0005] However, there is no commercial anti-OJ antibody detection kit on the market yet.

[0006] Therefore, given the importance of detecting anti-OJ antibodies, it is particularly important to develop a widely applicable, efficient and sensitive anti-OJ antibody detection kit and corresponding detection methods for predicting disease progression and guiding clinical treatment. Summary of the invention

[0007] In order to overcome the above-mentioned defects of the prior art, the object of the present invention is to provide an ELISA kit for detecting anti-OJ antibodies and a detection method and application thereof.

[0008] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0009] An ELISA kit for detecting anti-OJ antibodies, comprising:

[0010] The purified dominant antigen epitope OJ protein, the amino acid sequence corresponding to the purified dominant antigen epitope OJ protein is shown in SEQ NO.1, and specifically comprises the following amino acid sequence:

[0011] .

[0012] In a preferred embodiment of the present invention, the full-length protein structure corresponding to the purified dominant antigen epitope OJ protein is shown in SEQ NO.2, which specifically comprises the following amino acid sequence:

[0013]

[0014] In a preferred embodiment of the present invention, the purified dominant antigen epitope OJ protein is amplified by the primer pair shown in SEQ NO.3 and 4, and the sequence corresponding to the primer pair is as follows:

[0015] F:ccaccgccttgctgaca;

[0016] R:ccctgctcctagtagtaaaccttagaatc.

[0017] In a preferred embodiment of the present invention, the ELISA kit further comprises:

[0018] A 96-well ELISA plate coated with the dominant antigen epitope OJ protein.

[0019] In a preferred embodiment of the present invention, the ELISA kit further comprises:

[0020] Negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate color development solution, stop solution;

[0021] The negative control serum is the negative serum of anti-OJ antibody detected by immunoblotting method in patients with anti-synthetase syndrome;

[0022] The positive control serum is the positive serum of patients with anti-synthetase syndrome detected by immunoblotting for anti-OJ antibodies;

[0023] The enzyme-labeled secondary antibody is goat anti-human IgG labeled with horseradish peroxidase.

[0024] In a preferred embodiment of the present invention, the serum diluent is a PBS solution containing BSA;

[0025] The washing solution is PBST with a concentration of 1%;

[0026] The substrate color developing solution was tetramethylbenzidine (TMB);

[0027] The stop solution is 0.5% dilute sulfuric acid solution.

[0028] A detection method of an ELISA kit for detecting anti-OJ antibodies comprises the following steps:

[0029] The 96-well ELISA plate was coated with the dominant antigen epitope OJ protein at an ambient temperature of 4° C. overnight;

[0030] The excess coating solution was removed using the PBST washing solution, and the washing step was repeated 3-5 times;

[0031] Add 300ul of 1% BSA solution to a single well of a 96-well ELISA plate, incubate at 37°C for 2 hours, then wash with the PBST, and repeat 3-5 times;

[0032] Add 100ul of the negative and positive control serum as standard and 100ul of the serum to be tested into a single well of a 96-well ELISA plate, and incubate at room temperature for 1 hour;

[0033] The residual solution after incubation was washed with PBST washing solution, and the washing steps were repeated 3-5 times;

[0034] The enzyme-labeled secondary antibody was added at a ratio of 1:60,000 and incubated at room temperature for 30 minutes;

[0035] The residual solution after incubation was washed with PBST washing solution, and the washing steps were repeated 3-5 times;

[0036] Add the substrate color developing solution (TMB) to develop color, and use the stop solution to terminate the reaction after the development is completed;

[0037] Read the results in a 450 / 630 nm microplate reader.

[0038] In a preferred embodiment of the present invention, the dilution of the positive control serum as a standard and the blank control standard are as follows:

[0039] The dilution gradient is: 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560;

[0040] The blank control used 100ul of PBS buffer with pH = 7.2

[0041] In a preferred embodiment of the present invention, the concentration of the dominant antigen epitope OJ protein is 5 ug / ml, and 100 ul is added to each reaction well.

[0042] An application of an ELISA kit for detecting anti-OJ antibodies, wherein the application is for detecting anti-OJ antibodies.

[0043] The beneficial effects of the present invention are:

[0044] Since it is difficult to express the full-length OJ protein and the expression technology requirements are high, the price of purchasing finished OJ full-length protein is even more expensive.

[0045] The present invention expresses synthetic antigens based on searching for the dominant antigen epitopes of OJ, thereby greatly reducing the cost.

[0046] The kit of the present invention can be used for anti-OJ antibody detection, fills the gap in anti-OJ antibody detection methods, has the characteristics of large detection throughput, accurate results, high sensitivity, and simple operation, and is easier to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the full-length protein structure diagram of OJ protein.

[0048] Figure 2 This is the structural diagram of the dominant antigen epitope OJ protein.

[0049] Figure 3 Schematic diagram of Western-Blot results of the dominant antigen epitope OJ protein obtained after protein recombination.

[0050] Figure 4 This is a comparative diagram of Anti-OJ antibody detection in various groups of people.

[0051] Figure 5 Schematic diagram of the changes in Anti-OJ antibody detection after treatment. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following structure, the description of the known structure and technology is omitted to avoid unnecessary confusion of the concept of the present invention.

[0053] The normal temperature referred to in the present invention is the normal temperature commonly understood in the art, ranging from 20 to 25 degrees Celsius.

[0054] 1. Since the OJ protein has a large structure, a complex spatial structure, and multiple amino acid sequences, the inventors first analyzed its full-length structure through protein dynamics to obtain its dominant antigenic structure epitope:

[0055] The full-length amino acid sequences involved are as follows:

[0056] SEQ NO.1:

[0057]

[0058] The full-length protein structure is Figure 1 shown.

[0059] The inventors of the present invention have obtained the amino acid sequence of the dominant antigen epitope through a large number of proteomic dynamics analyses as follows:

[0060] SEQ NO.2:

[0061] .

[0062] The dominant antigen structure is Figure 2 shown.

[0063] After obtaining the amino acid sequence of the dominant antigen epitope, the dominant antigen epitope protein corresponding to the amino acid sequence of the dominant antigen epitope is obtained by protein recombination. The specific protein recombination includes the following steps:

[0064] 2.1 Gene cloning;

[0065] 2.1.1 Obtaining the target gene

[0066] PCR method:

[0067] Using human cDNA containing the target gene as a template, primers were designed according to the target gene sequence as follows:

[0068] F:ccaccgccttgctgaca;

[0069] R:ccctgctcctagtagtaaaccttagaatc.

[0070] The PCR reaction conditions are as follows:

[0071] 95℃2min;

[0072] 94℃30s, 60℃30s, 65℃Xmin (3min / kb); repeat 35 cycles;

[0073] 65℃10min;

[0074] 4℃1min;

[0075] The amplified product was recovered by electrophoresis using a target fragment recovery kit (QIAGEN QIAquickGe l Extraction Kit) to obtain a purified amplified product.

[0076] 2.1.2 Construction of target cloning plasmid

[0077] 2.1.2.1 Ligation of amplified products

[0078] The connection system is constructed as follows:

[0079]

[0080] Add double distilled water to 10ul;

[0081] The ligation was carried out at 16°C overnight to obtain a ligation product. 2.1.2.2

[0083] The ligation product was heat-transformed using competent cells, and then transferred to LB culture plates with corresponding antibiotics for overnight culture to obtain colony screening plates; a single colony was picked and transferred to LB culture medium containing corresponding antibiotics for culture, and then 2 μL was taken for PCR identification; after identification, the sample containing the target fragment was inoculated into LB culture medium for overnight rotation culture. 2.1.2.3

[0085] The cultured bacterial solution was used to extract plasmids using the Axygen mini-extraction kit according to the standard steps of the kit, and the obtained plasmid was sequenced to identify whether it was the target clone plasmid.

[0086] 2.2 Cell transfection

[0087] The obtained target clone plasmid is transfected into HEK293 cells, and the cells containing the target clone plasmid after transfection are inoculated into the corresponding culture medium and cultured for 48-72 hours, and then the cultured cells are collected.

[0088] 2.3 Protein purification.

[0089] The collected cells were lysed by ultrasonic disruption, and then the lysate supernatant was collected by high-speed centrifugation. The lysate supernatant was purified by affinity purification to obtain purified protein.

[0090] 2.4 SDS-Page electrophoresis identification results refer to Figure 3 The molecular weight of the truncated protein was about 70KD after SDS-Page verification.

[0091] 3. Obtain a large amount of purified dominant antigen protein and use it as an antigen for ELISA test, including the following steps:

[0092] 3.1 Antigen concentration is 10ug / ml OJ ul coated on 96-well plate, add 100ul to each reaction well, and incubate at 4℃ overnight;

[0093] 3.2 Washing: After coating, discard the coating solution and wash with PBST, repeat 3-5 times;

[0094] 3.3 Blocking: 1% BSA, 300ul / well, incubate at 37℃ for 2h and then wash, same as 3.2;

[0095] 3.4 Add 100ul of standard sample and serum to be tested to each well and incubate at room temperature for 1 hour;

[0096] 3.5 Dilute the standard (known positive serum) as follows:

[0097] Standard dilution: blank, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560; Blank: 100ul PBS, serum to be tested was diluted 1:100, and 100ul was added respectively;

[0098] 3.6 Washing, same as 3.2;

[0099] 3.7 Add secondary antibody (horseradish peroxidase-labeled goat anti-human IgG) at a ratio of 1:60,000, incubate at room temperature for 30 minutes, wash, and repeat 3.2;

[0100] 3.8 Add substrate solution and color for 15 minutes;

[0101] 3.9 Terminate the reaction and read the result with a 450 / 630 nm microplate reader.

[0102] 4. Experimental data:

[0103] The sensitivity and specificity statistics of the Anti-OJ antibody detection kit of the present invention are shown in Table 1.

[0104] Table 1:

[0105]

[0106] As shown in Table 1, the clinical sensitivity of the detection system for anti-synthetase antibody syndrome (n=105) is 3.24%. The results of testing the sera of patients with other autoimmune diseases (n=701) and healthy blood donors (n=400) showed that its specificity was 100%.

[0107] As shown in Table 2, the correlation analysis between the detection method (ELISA) of the present invention and the immunoblotting method, 105 anti-synthetase antibody syndrome patient sera and 600 control group sera were compared, and the sensitivity of the detection method of the present invention was 31.43% and the specificity was 100%.

[0108] See Table 2 for the comparative statistics between the Anti-OJ antibody detection kit of the present invention and the traditional detection method.

[0109] Table 2:

[0110]

[0111] For comparison of Anti-OJ antibody detection in various groups of people, see Figure 4 .

[0112] The present invention confirms that anti-OJ antibodies are not positive in the sera of normal people, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and non-OJ antibody positive dermatomyositis (DM) patients, indicating that the specificity of the detection invention is high.

[0113] Schematic diagram of changes in Anti-OJ antibody detection after treatment, see Figure 5 .

[0114] The present invention detects the changes in the values ​​of anti-synthetase syndrome patients with positive anti-OJ antibodies before and after treatment, confirming that the values ​​of anti-OJ antibodies have decreased after treatment, and the corresponding clinical symptoms have been alleviated. The present invention can effectively monitor the efficacy of the test results for anti-OJ antibodies.

[0115] The basic principles and main features of the invention and the advantages of the invention are shown and described above.

[0116] Those skilled in the art should understand that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents. Sequence Listing <110> Shanghai Jiading District Central Hospital (Jiading District Central Hospital affiliated to Shanghai Health and Medical College, Jiading Branch of Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine) <120> An ELISA kit for detecting anti-OJ antibodies and its detection method and application <130> 20220509 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 653 <212> PRT <213> Homo sapiens <400> 1 Gln Gln Val Pro Glu Asn Ile Asn Phe Pro Ala Glu Glu Glu Lys Ile 1 5 10 15 Leu Glu Phe Trp Thr Glu Phe Asn Cys Phe Gln Glu Cys Leu Lys Gln 20 25 30 Ser Lys His Lys Pro Lys Phe Thr Phe Tyr Asp Gly Pro Pro Phe Ala 35 40 45 Thr Gly Leu Pro His Tyr Gly His Ile Leu Ala Gly Thr Ile Lys Asp 50 55 60 Ile Val Thr Arg Tyr Ala His Gln Ser Gly Phe His Val Asp Arg Arg 65 70 75 80 Phe Gly Trp Asp Cys His Gly Leu Pro Val Glu Tyr Glu Ile Asp Lys 85 90 95 Thr Leu Gly Ile Arg Gly Pro Glu Asp Val Ala Lys Met Gly Ile Thr 100 105 110 Glu Tyr Asn Asn Gln Cys Arg Ala Ile Val Met Arg Tyr Ser Ala Glu 115 120 125 Trp Lys Ser Thr Val Ser Arg Leu Gly Arg Trp Ile Asp Phe Asp Asn 130 135 140 Asp Tyr Lys Thr Leu Tyr Pro Gln Phe Met Glu Ser Val Trp Trp Val 145 150 155 160 Phe Lys Gln Leu Tyr Lys Gly Glu Asn Gly Met Glu Asp Cys Val Met 165 170 175 Ala Leu Glu Thr Leu Phe Ser Val Leu Leu Ser Leu Cys Arg Leu Met 180 185 190 Asp Lys Asp Thr Leu Ser Ile His Tyr Leu Met Leu Pro Arg Val Arg 195 200 205 Glu Glu Leu Ile Asp Lys Lys Thr Glu Ser Ala Val Ser Gln Met Gln 210 215 220 Ser Val Ile Glu Leu Gly Arg Val Ile Arg Asp Arg Lys Thr Ile Pro 225 230 235 240 Ile Lys Tyr Pro Leu Lys Glu Ile Val Val Ile His Gln Asp Pro Glu 245 250 255 Ala Leu Lys Asp Ile Lys Ser Leu Glu Lys Tyr Ile Ile Glu Glu Leu 260 265 270 Asn Val Arg Lys Val Thr Leu Ser Thr Asp Lys Asn Lys Tyr Gly Ile 275 280 285 Arg Leu Arg Ala Glu Pro Asp His Met Val Leu Gly Lys Arg Leu Lys 290 295 300 Gly Ala Phe Lys Ala Val Met Thr Ser Ile Lys Gln Leu Ser Ser Glu 305 310 315 320 Glu Leu Glu Gln Phe Gln Lys Thr Gly Thr Ile Val Val Glu Gly His 325 330 335 Glu Leu His Asp Glu Asp Ile Arg Leu Met Tyr Thr Phe Asp Gln Ala 340 345 350 Thr Gly Gly Thr Ala Gln Phe Glu Ala His Ser Asp Ala Gln Ala Leu 355 360 365 Val Leu Leu Asp Val Thr Pro Asp Gln Ser Met Val Asp Glu Gly Met 370 375 380 Ala Arg Glu Val Ile Asn Arg Ile Gln Lys Leu Arg Lys Lys Cys Asn 385 390 395 400 Leu Val Pro Thr Asp Glu Ile Thr Val Tyr Tyr Lys Ala Lys Ser Glu 405 410 415 Gly Thr Tyr Leu Asn Ser Val Ile Glu Ser His Thr Glu Phe Ile Phe 420 425 430 Thr Thr Ile Lys Ala Pro Leu Lys Pro Tyr Pro Val Ser Pro Ser Asp 435 440 445 Lys Val Leu Ile Gln Glu Lys Thr Gln Leu Lys Gly Ser Glu Leu Glu 450 455 460 Ile Thr Leu Thr Arg Gly Ser Ser Leu Pro Gly Pro Ala Cys Ala Tyr 465 470 475 480 Val Asn Leu Asn Ile Cys Ala Asn Gly Ser Glu Gln Gly Gly Val Leu 485 490 495 Leu Leu Glu Asn Pro Lys Gly Asp Asn Arg Leu Asp Leu Leu Lys Leu 500 505 510 Lys Ser Val Val Thr Ser Ile Phe Gly Val Lys Asn Thr Glu Leu Ala 515 520 525 Val Phe His Asp Glu Thr Glu Ile Gln Asn Gln Thr Asp Leu Leu Ser 530 535 540 Leu Ser Gly Lys Thr Leu Cys Val Thr Ala Gly Ser Ala Pro Ser Leu 545 550 555 560 Ile Asn Ser Ser Ser Thr Leu Leu Cys Gln Tyr Ile Asn Leu Gln Leu 565 570 575 Leu Asn Ala Lys Pro Gln Glu Cys Leu Met Gly Thr Val Gly Thr Leu 580 585 590 Leu Leu Glu Asn Pro Leu Gly Gln Asn Gly Leu Thr His Gln Gly Leu 595 600 605 Leu Tyr Glu Ala Ala Lys Val Phe Gly Leu Arg Ser Arg Lys Leu Lys 610 615 620 Leu Phe Leu Asn Glu Thr Gln Thr Gln Glu Ile Thr Glu Asp Ile Pro 625 630 635 640 Val Lys Thr Leu Asn Met Lys Thr Val Tyr Val Ser Val 645 650 <210> 2 <211> 1237 <212> PRT <213> Homo sapiens <400> 2 Met Leu Gln Gln Val Pro Glu Asn Ile Asn Phe Pro Ala Glu Glu Glu 1 5 10 15 Lys Ile Leu Glu Phe Trp Thr Glu Phe Asn Cys Phe Gln Glu Cys Leu 20 25 30 Lys Gln Ser Lys His Lys Pro Lys Phe Thr Phe Tyr Asp Gly Pro Pro 35 40 45 Phe Ala Thr Gly Leu Pro His Tyr Gly His Ile Leu Ala Gly Thr Ile 50 55 60 Lys Asp Ile Val Thr Arg Tyr Ala His Gln Ser Gly Phe His Val Asp 65 70 75 80 Arg Arg Phe Gly Trp Asp Cys His Gly Leu Pro Val Glu Tyr Glu Ile 85 90 95 Asp Lys Thr Leu Gly Ile Arg Gly Pro Glu Asp Val Ala Lys Met Gly 100 105 110 Ile Thr Glu Tyr Asn Asn Gln Cys Arg Ala Ile Val Met Arg Tyr Ser 115 120 125 Ala Glu Trp Lys Ser Thr Val Ser Arg Leu Gly Arg Trp Ile Asp Phe 130 135 140 Asp Asn Asp Tyr Lys Thr Leu Tyr Pro Gln Phe Met Glu Ser Val Trp 145 150 155 160 Trp Val Phe Lys Gln Leu Tyr Asp Lys Gly Leu Val Tyr Arg Gly Val 165 170 175 Lys Val Met Pro Phe Ser Thr Ala Cys Asn Thr Pro Leu Ser Asn Phe 180 185 190 Glu Ser His Gln Asn Tyr Lys Asp Val Gln Asp Pro Ser Val Phe Val 195 200 205 Thr Phe Pro Leu Glu Glu Asp Glu Thr Val Ser Leu Val Ala Trp Thr 210 215 220 Thr Thr Pro Trp Thr Leu Pro Ser Asn Leu Ala Val Cys Val Asn Pro 225 230 235 240 Glu Met Gln Tyr Val Lys Ile Lys Asp Val Ala Arg Gly Arg Leu Leu 245 250 255 Ile Leu Met Glu Ala Arg Leu Ser Ala Leu Tyr Lys Leu Glu Ser Asp 260 265 270 Tyr Glu Ile Leu Glu Arg Phe Pro Gly Ala Tyr Leu Lys Gly Lys Lys 275 280 285 Tyr Arg Pro Leu Phe Asp Tyr Phe Leu Lys Cys Lys Glu Asn Gly Ala 290 295 300 Phe Thr Val Leu Val Asp Asn Tyr Val Lys Glu Glu Glu Gly Thr Gly 305 310 315 320 Val Val His Gln Ala Pro Tyr Phe Gly Ala Glu Asp Tyr Arg Val Cys 325 330 335 Met Asp Phe Asn Ile Ile Arg Lys Asp Ser Leu Pro Val Cys Pro Val 340 345 350 Asp Ala Ser Gly Cys Phe Thr Thr Glu Val Thr Asp Phe Ala Gly Gln 355 360 365 Tyr Val Lys Asp Ala Asp Lys Ser Ile Ile Arg Thr Leu Lys Glu Gln 370 375 380 Gly Arg Leu Leu Val Ala Thr Thr Phe Thr His Ser Tyr Pro Phe Cys 385 390 395 400 Trp Arg Ser Asp Thr Pro Leu Ile Tyr Lys Ala Val Pro Ser Trp Phe 405 410 415 Val Arg Val Glu Asn Met Val Asp Gln Leu Leu Arg Asn Asn Asp Leu 420 425 430 Cys Tyr Trp Val Pro Glu Leu Val Arg Glu Lys Arg Phe Gly Asn Trp 435 440 445 Leu Lys Asp Ala Arg Asp Trp Thr Ile Ser Arg Asn Arg Tyr Trp Gly 450 455 460 Thr Pro Ile Pro Leu Trp Val Ser Asp Asp Phe Glu Glu Val Val Cys 465 470 475 480 Ile Gly Ser Val Ala Glu Leu Glu Glu Leu Ser Gly Ala Lys Ile Ser 485 490 495 Asp Leu His Arg Glu Ser Val Asp His Leu Thr Ile Pro Ser Arg Cys 500 505 510 Gly Lys Gly Ser Leu His Arg Ile Ser Glu Val Phe Asp Cys Trp Phe 515 520 525 Glu Ser Gly Ser Met Pro Tyr Ala Gln Val His Tyr Pro Phe Glu Asn 530 535 540 Lys Arg Glu Phe Glu Asp Ala Phe Pro Ala Asp Phe Ile Ala Glu Gly 545 550 555 560 Ile Asp Gln Thr Arg Gly Trp Phe Tyr Thr Leu Leu Val Leu Ala Thr 565 570 575 Ala Leu Phe Gly Gln Pro Pro Phe Lys Asn Val Ile Val Asn Gly Leu 580 585 590 Val Leu Ala Ser Asp Gly Gln Lys Met Ser Lys Arg Lys Lys Asn Tyr 595 600 605 Pro Asp Pro Val Ser Ile Ile Gln Lys Tyr Gly Ala Asp Ala Leu Arg 610 615 620 Leu Tyr Leu Ile Asn Ser Pro Val Val Arg Ala Glu Asn Leu Arg Phe 625 630 635 640 Lys Glu Glu Gly Val Arg Asp Val Leu Lys Asp Val Leu Leu Pro Trp 645 650 655 Tyr Asn Ala Tyr Arg Phe Leu Ile Gln Asn Val Leu Arg Leu Gln Lys 660 665 670 Glu Glu Glu Ile Glu Phe Leu Tyr Asn Glu Asn Thr Val Arg Glu Ser 675 680 685 Pro Asn Ile Thr Asp Arg Trp Ile Leu Ser Phe Met Gln Ser Leu Ile 690 695 700 Gly Phe Phe Glu Thr Glu Met Ala Ala Tyr Arg Leu Tyr Thr Val Val 705 710 715 720 Pro Arg Leu Val Lys Phe Val Asp Ile Leu Thr Asn Trp Tyr Val Arg 725 730 735 Met Asn Arg Arg Arg Leu Lys Gly Glu Asn Gly Met Glu Asp Cys Val 740 745 750 Met Ala Leu Glu Thr Leu Phe Ser Val Leu Leu Ser Leu Cys Arg Leu 755 760 765 Met Asp Lys Asp Thr Leu Ser Ile His Tyr Leu Met Leu Pro Arg Val 770 775 780 Arg Glu Glu Leu Ile Asp Lys Lys Thr Glu Ser Ala Val Ser Gln Met 785 790 795 800 Gln Ser Val Ile Glu Leu Gly Arg Val Ile Arg Asp Arg Lys Thr Ile 805 810 815 Pro Ile Lys Tyr Pro Leu Lys Glu Ile Val Val Ile His Gln Asp Pro 820 825 830 Glu Ala Leu Lys Asp Ile Lys Ser Leu Glu Lys Tyr Ile Ile Glu Glu 835 840 845 Leu Asn Val Arg Lys Val Thr Leu Ser Thr Asp Lys Asn Lys Tyr Gly 850 855 860 Ile Arg Leu Arg Ala Glu Pro Asp His Met Val Leu Gly Lys Arg Leu 865 870 875 880 Lys Gly Ala Phe Lys Ala Val Met Thr Ser Ile Lys Gln Leu Ser Ser 885 890 895 Glu Glu Leu Glu Gln Phe Gln Lys Thr Gly Thr Ile Val Val Glu Gly 900 905 910 His Glu Leu His Asp Glu Asp Ile Arg Leu Met Tyr Thr Phe Asp Gln 915 920 925 Ala Thr Gly Gly Thr Ala Gln Phe Glu Ala His Ser Asp Ala Gln Ala 930 935 940 Leu Val Leu Leu Asp Val Thr Pro Asp Gln Ser Met Val Asp Glu Gly 945 950 955 960 Met Ala Arg Glu Val Ile Asn Arg Ile Gln Lys Leu Arg Lys Lys Cys 965 970 975 Asn Leu Val Pro Thr Asp Glu Ile Thr Val Tyr Tyr Lys Ala Lys Ser 980 985 990 Glu Gly Thr Tyr Leu Asn Ser Val Ile Glu Ser His Thr Glu Phe Ile 995 1000 1005 Phe Thr Thr Ile Lys Ala Pro Leu Lys Pro Tyr Pro Val Ser Pro Ser 1010 1015 1020 Asp Lys Val Leu Ile Gln Glu Lys Thr Gln Leu Lys Gly Ser Glu Leu 1025 1030 1035 1040 Glu Ile Thr Leu Thr Arg Gly Ser Ser Leu Pro Gly Pro Ala Cys Ala 1045 1050 1055 Tyr Val Asn Leu Asn Ile Cys Ala Asn Gly Ser Glu Gln Gly Gly Val 1060 1065 1070 Leu Leu Leu Glu Asn Pro Lys Gly Asp Asn Arg Leu Asp Leu Leu Lys 1075 1080 1085 Leu Lys Ser Val Val Thr Ser Ile Phe Gly Val Lys Asn Thr Glu Leu 1090 1095 1100 Ala Val Phe His Asp Glu Thr Glu Ile Gln Asn Gln Thr Asp Leu Leu 1105 1110 1115 1120 Ser Leu Ser Gly Lys Thr Leu Cys Val Thr Ala Gly Ser Ala Pro Ser 1125 1130 1135 Leu Ile Asn Ser Ser Ser Thr Leu Leu Cys Gln Tyr Ile Asn Leu Gln 1140 1145 1150 Leu Leu Asn Ala Lys Pro Gln Glu Cys Leu Met Gly Thr Val Gly Thr 1155 1160 1165 Leu Leu Leu Glu Asn Pro Leu Gly Gln Asn Gly Leu Thr His Gln Gly 1170 1175 1180 Leu Leu Tyr Glu Ala Ala Lys Val Phe Gly Leu Arg Ser Arg Lys Leu 1185 1190 1195 1200 Lys Leu Phe Leu Asn Glu Thr Gln Thr Gln Glu Ile Thr Glu Asp Ile 1205 1210 1215 Pro Val Lys Thr Leu Asn Met Lys Thr Val Tyr Val Ser Val Leu Pro 1220 1225 1230 Thr Thr Ala Asp Phe 1235 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <400> 3 ccaccgcctt gctgaca 17 <210> 4 <211> 29 <212> DNA <213> Artificial sequence (Artificial sequence) <400> 4 ccctgctcct cttagac

Claims

1. An ELISA kit for detecting anti-OJ antibodies, characterized in that: include: The purified dominant antigen epitope OJ protein, the amino acid sequence corresponding to the purified dominant antigen epitope OJ protein is shown in SEQ NO.

1.

2. An ELISA kit for detecting anti-OJ antibodies according to claim 1, characterized in that: The full-length protein structure corresponding to the purified dominant antigen epitope OJ protein is shown in SEQ NO.

2.

3. An ELISA kit for detecting anti-OJ antibodies according to claim 1, characterized in that: The purified dominant antigen epitope OJ protein is amplified by the primer pair shown in SEQ NO.3 and 4, and the sequence corresponding to the primer pair is as follows: F:ccaccgccttgctgaca; R:ccctgctcctagtagtaaaccttagaatc.

4. An ELISA kit for detecting anti-OJ antibodies according to claim 1, characterized in that: The ELISA kit also includes: at least one 96-well ELISA plate coated with the dominant antigen epitope OJ protein.

5. An ELISA kit for detecting anti-OJ antibodies according to any one of claims 1 or 4, characterized in that: The ELISA kit also includes: negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate color development solution, and stop solution; The negative control serum is the negative serum of anti-OJ antibody detected by immunoblotting method in patients with anti-synthetase syndrome; The positive control serum is the positive serum of patients with anti-synthetase syndrome detected by immunoblotting for anti-OJ antibodies; The enzyme-labeled secondary antibody is goat anti-human IgG labeled with horseradish peroxidase.

6. An ELISA kit for detecting anti-OJ antibodies according to claim 5, characterized in that: The serum diluent is a PBS solution containing 0.5% BSA; the washing solution is PBST with a concentration of 1%; the substrate color developing solution is tetramethylbenzidine (TMB); and the stop solution is a 0.5% dilute sulfuric acid solution.

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