An ELISA kit for detecting anti-NXP2 antibodies and its detection method and application

Abstract

The present invention discloses an ELISA kit for detecting anti-NXP2 antibodies, characterized in that it comprises: a purified dominant antigen epitope NXP2 protein, wherein the amino acid sequence corresponding to the purified dominant antigen epitope NXP2 protein is shown in SEQ NO.1. The present invention also discloses a detection method and application thereof. The kit of the present invention can be used for anti-NXP2 antibody detection, fills the gap in the anti-NXP2 antibody detection method, has the characteristics of large detection flux, accurate results, high sensitivity, and simple operation, and is 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-NXP2 antibodies and a detection method thereof. Background Art

[0002] Dermatomyositis (DM) is a common life-threatening inflammatory myopathy in clinical practice, mainly affecting the skin and muscles.

[0003] It is currently considered an autoimmune disease. The annual incidence of dermatomyositis is around 0.5-1 / 100,000 people, and is on the rise.

[0004] NXP2 is called nuclear matrix protein 2, also known as MJ or MORC3. Anti-NXP2 antibodies can be found in 20%-25% of juvenile dermatomyositis patients, and the incidence in adult dermatomyositis patients is about 1.6%-30%.

[0005] The most typical rash in patients with NXP2 antibody positive is calcicutaneous positivity and peripheral edema, and they often present with frequent and severe myalgias and weakness in the neck and distal limbs (in contrast to the typical proximal weakness).

[0006] Anti-NXP2 antibodies are specific antibodies for dermatomyositis, especially juvenile dermatomyositis. Dynamic observation of anti-NXP2 antibody levels in serum can help predict the evolution of the disease and guide clinical treatment.

[0007] However, there is no commercial anti-NXP2 antibody detection kit on the market. Therefore, given the importance of detecting anti-NXP2 antibodies, it is particularly important to develop a widely applicable, efficient and sensitive anti-NXP2 antibody detection kit and corresponding detection methods for predicting disease progression and guiding clinical treatment. Summary of the invention

[0008] 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-NXP2 antibodies and a detection method and application thereof.

[0009] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0010] An ELISA kit for detecting anti-NXP2 antibodies, comprising:

[0011] The purified dominant antigen epitope NXP2 protein, the amino acid sequence corresponding to the purified dominant antigen epitope NXP2 protein is shown in SEQ NO.1, and specifically comprises the following amino acid sequence:

[0012] MDYKDDDDKAAQPPRGIRLSALCPKFLHTNSTSHTWPFSAVAELIDNAYDPDVNAKQIWIDKTVINDHICLTFTDNGNGMTSDKLHKMLSFGFSDKVTMNGHVPVGLKWYCSNNPDPQFRNCEVPEEPEDEDLVHPTYEKTYKKTNKEKFRIRQPEMIPRINAELLFRPTALSTPSFS SPKESVPRRHLSEGTNSYATRLLNNHQVPPQSEPESNSLKRRLSTRSSILNAKNRRLSSQFENSVYKGDDDDEDVIILEENSTPKPAVDHDIDMKSEQSHVEQGGVQVEFVGDSEPCGQTGSTSTSSSRCDQGNTAATQTEVPSLVVKKEETVEDEIDVRNDAVILPSCVEAEAKIHE.

[0013] In a preferred embodiment of the present invention, the full-length structure corresponding to the purified dominant antigen epitope NXP2 protein is shown in SEQ NO.2, which specifically comprises the following amino acid sequence:

[0014] MDYKDDDDKAAQPPRGIRLSALCPKFLHTNSTSHTWPFSAVAELIDNAYDPDVNAKQIWIDKTVINDHICLTFTDNGNGMTSDKLHKMLSFGFSDKVTMNGHVPVGLYGNGFKSGSMRLGKDAIVFTKNGESMSVGLLSQTYLEVIKAEHVVVPIVAFNKHRQMINLAESKASLAAILEHSLFSTEQKLLAELDAIIGKKGTRIIIWNLRSYKNATEFDFEKDKYDIRIPEDLDEITGKKGYKKQERMDQIAPESDYSLRAYCSILYLKPRMQIILRGQKVKTQLVSKSLAYIERDVYRPKFLSKTVRITFGFNCRNKDHYGIMMYHRNRLIKAYEKVGCQLRANNMGVGVVGIIECNFLKPTHNKQDFDYTNEYRLTITALGEKLNDYWNEMKVKKNTEYPLNLPVEDIQKRPDQTWVQCDACLKWRKLPDGMDQLPEKWYCSNNPDPQFRNCEVPEEPEDEDLVHPTYEKTYKKTNKEKFRIRQPEMIPRINAELLFRPTALSTPSFSSPKESVPRRHLSEGTNSYATRLLNNHQVPPQSEPESNSLKRRLSTRSSILNAKNRRLSSQFENSVYKGDDDDEDVIILEENSTPKPAVDHDIDMKSEQSHVEQGGVQVEFVGDSEPCGQTGSTSTSSSRCDQGNTAATQTEVPSLVVKKEETVEDEIDVRNDAVILPSCVEAEAKIHETQETTDKSADDAGCQLQELRNQLLLVTEEKENYKRQCHMFTDQIKVLQQRILEMNDKYVKKETCHQSTETDAVFLLESINGKSESPDHMVSQYQQALEEIERLKKQCSALQHVKAECSQCSNNESKSEMDEMAVQLDDVFRQLDKCSIERDQYKSEVELLEMEKSQIRSQCEELKTEVEQLKSTNQQTATDVSTSSNIEESVNHMDGESLKLRSLRVNVGQLLAMIVPDLDLQQVNYDVDVVDEILGQVVEQMSEISSTD。

[0015] In a preferred embodiment of the present invention, the purified dominant antigen epitope NXP2 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:

[0016] F:acagtcgttccgccacc;

[0017] R: ctaagattccaaatgatgatcctcgtccc.

[0018] In a preferred embodiment of the present invention, the ELISA kit further comprises:

[0019] A 96-well ELISA plate coated with the dominant antigen epitope NXP2 protein.

[0020] In a preferred embodiment of the present invention, the ELISA kit further comprises:

[0021] Negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate color development solution, stop solution;

[0022] The negative control serum is the negative serum of CADM patients detected by immunoblotting for anti-NXP2 antibodies;

[0023] The positive control serum is the anti-NXP2 antibody positive serum detected by immunoblotting of CADM patients;

[0024] The enzyme-labeled secondary antibody is goat anti-human IgG labeled with horseradish peroxidase.

[0025] In a preferred embodiment of the present invention, the serum diluent is a PBS solution containing 0.5% BSA;

[0026] The washing solution is PBST with a concentration of 1%;

[0027] The substrate color developing solution is tetramethylbenzidine (TMB);

[0028] The stop solution is a 0.5% dilute sulfuric acid solution.

[0029] A detection method of an ELISA kit for detecting anti-NXP2 antibodies comprises the following steps:

[0030] The 96-well ELISA plate was coated with the dominant antigen epitope NXP2 protein at an ambient temperature of 4° C. overnight;

[0031] The excess coating solution was removed using the PBST washing solution, and the washing step was repeated 3-5 times;

[0032] 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;

[0033] Add 100ul of the negative and / or positive control serum as a 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;

[0034] The residual solution after incubation was washed with PBST washing solution, and the washing steps were repeated 3-5 times;

[0035] The enzyme-labeled secondary antibody was added at a ratio of 1:60,000 and incubated at room temperature for 30 minutes;

[0036] The residual solution after incubation was washed with PBST washing solution, and the washing steps were repeated 3-5 times;

[0037] Add the substrate color developing solution to develop color, and use the stop solution to terminate the reaction after the development is completed;

[0038] Read the results in a 450 / 630 nm microplate reader.

[0039] 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:

[0040] The dilution gradient is: 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560;

[0041] The blank control used 100ul of PBS buffer with pH=7.2.

[0042] In a preferred embodiment of the present invention, the concentration of the dominant antigen epitope NXP2 protein is 5 ug / ml, and 100 ul is added to each reaction well.

[0043] An application of an ELISA kit for detecting anti-NXP2 antibodies, wherein the application is for detecting anti-NXP2 antibodies.

[0044] The beneficial effects of the present invention are:

[0045] Since it is difficult to express the full-length NXP2 protein and the expression technology is demanding, it is even more expensive to purchase the finished product of the full-length NXP2 protein.

[0046] The present invention expresses synthetic antigens based on searching for NXP2 dominant antigen epitopes, thereby greatly reducing costs.

[0047] The kit of the present invention can be used for anti-NXP2 antibody detection, fills the gap in anti-NXP2 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

[0048] Figure 1 This is a diagram of the NXP2 protein structure.

[0049] Figure 2 This is the structural diagram of the dominant antigen epitope NXP2 protein.

[0050] Figure 3 Schematic diagram of Western-Blot results of the dominant antigen epitope NXP2 protein obtained after protein recombination.

[0051] Figure 4 This is a comparative diagram of Anti-NXP2 antibody detection in various groups of people.

[0052] Figure 5 Schematic diagram of the changes in Anti-NXP2 antibody detection after treatment. DETAILED DESCRIPTION

[0053] 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.

[0054] 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.

[0055] 1. Since the NXP2 protein has a large structure and multiple amino acid sequences, the inventors first analyzed its full-length structure through protein dynamics to obtain its dominant antigenic structure epitope:

[0056] The full-length amino acid sequences involved are as follows:

[0057] SEQ NO.1:

[0058] MDYKDDDDKAAQPPRGIRLSALCPKFLHTNSTSHTWPFSAVAELIDNAYDPDVNAKQIWIDKTVINDHICLTFTDNGNGMTSDKLHKMLSFGFSDKVTMNGHVPVGLYGNGFKSGSMRLGKDAIVFTKNGESMSVGLLSQTYLEVIKAEHVVVPIVAFNKHRQMINLAESKASLAAILEHSLFSTEQKLLAELDAIIGKKGTRIIIWNLRSYKNATEFDFEKDKYDIRIPEDLDEITGKKGYKKQERMDQIAPESDYSLRAYCSILYLKPRMQIILRGQKVKTQLVSKSLAYIERDVYRPKFLSKTVRITFGFNCRNKDHYGIMMYHRNRLIKAYEKVGCQLRANNMGVGVVGIIECNFLKPTHNKQDFDYTNEYRLTITALGEKLNDYWNEMKVKKNTEYPLNLPVEDIQKRPDQTWVQCDACLKWRKLPDGMDQLPEKWYCSNNPDPQFRNCEVPEEPEDEDLVHPTYEKTYKKTNKEKFRIRQPEMIPRINAELLFRPTALSTPSFSSPKESVPRRHLSEGTNSYATRLLNNHQVPPQSEPESNSLKRRLSTRSSILNAKNRRLSSQFENSVYKGDDDDEDVIILEENSTPKPAVDHDIDMKSEQSHVEQGGVQVEFVGDSEPCGQTGSTSTSSSRCDQGNTAATQTEVPSLVVKKEETVEDEIDVRNDAVILPSCVEAEAKIHETQETTDKSADDAGCQLQELRNQLLLVTEEKENYKRQCHMFTDQIKVLQQRILEMNDKYVKKETCHQSTETDAVFLLESINGKSESPDHMVSQYQQALEEIERLKKQCSALQHVKAECSQCSNNESKSEMDEMAVQLDDVFRQLDKCSIERDQYKSEVELLEMEKSQIRSQCEELKTEVEQLKSTNQQTATDVSTSSNIEESVNHMDGESLKLRSLRVNVGQLLAMIVPDLDLQQVNYDVDVVDEILGQVVEQMSEISSTD。

[0059] The full-length protein structure is as Figure 1 shown.

[0060] 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:

[0061] SEQ NO.2:

[0062] MDYKDDDDKAAQPPRGIRLSALCPKFLHTNSTSHTWPFSAVAELIDNAYDPDVNAKQIWIDKTVINDHICLTFTDNGNGMTSDKLHKMLSFGFSDKVTMNGHVPVGLKWYCSNNPDPQFRNCEVPEEPEDEDLVHPTYEKTYKKTNKEKFRIRQPEMIPRINAELLFRPTALSTPSFS SPKESVPRRHLSEGTNSYATRLLNNHQVPPQSEPESNSLKRRLSTRSSILNAKNRRLSSQFENSVYKGDDDDEDVIILEENSTPKPAVDHDIDMKSEQSHVEQGGVQVEFVGDSEPCGQTGSTSTSSSRCDQGNTAATQTEVPSLVVKKEETVEDEIDVRNDAVILPSCVEAEAKIH.

[0063] The dominant antigen structure is Figure 2 shown.

[0064] 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:

[0065] 2.1 Gene cloning;

[0066] 2.1.1 Obtaining the target gene

[0067] PCR method:

[0068] Using human cDNA containing the target gene as a template, primers were designed according to the target gene sequence as follows:

[0069] F:acagtcgttccgccacc,

[0070] R: ctaagattccaaatgatgatcctcgtccc.

[0071] The PCR reaction conditions are as follows:

[0072] 95℃ 2min;

[0073] 94℃ 30s, 60℃ 30s, 65℃ Xmin (3min / kb); repeat 35 cycles;

[0074] 65℃ 10min;

[0075] 4℃ 1min;

[0076] The amplified product was recovered by electrophoresis using a target fragment recovery kit (QIAGEN QIAquickGe l Extraction Kit) to obtain a purified amplified product.

[0077] 2.1.2 Construction of target cloning plasmid

[0078] 2.1.2.1 Ligation of amplified products

[0079] The connection system is constructed as follows:

[0080]

[0081] Add double distilled water to 10ul;

[0082] The ligation was carried out at 16°C overnight to obtain a ligation product. 2.1.2.2

[0084] 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

[0086] 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.

[0087] 2.2 Cell transfection

[0088] 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.

[0089] 2.3 Protein purification.

[0090] 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.

[0091] 2.4 Western-Blot identification results see Figure 3 ,like Figure 3 As shown, the molecular weight of the truncated protein was 75KD after Western-Blot verification.

[0092] 3. Obtain a large amount of purified dominant antigen protein and use it as an antigen for ELISA test, including the following steps:

[0093] 3.1 Antigen concentration is 5ug / ml NXP2 ul coated on a 96-well plate, add 100ul to each reaction well, and incubate at 4°C overnight;

[0094] 3.2 Washing: After coating, discard the coating solution and wash with PBST, repeat 3-5 times;

[0095] 3.3 Blocking: 1% BSA, 300ul / well, incubate at 37℃ for 2h and then wash, same as 3.2;

[0096] 3.4 Add 100ul of standard sample and serum to be tested to each well and incubate at room temperature for 1 hour;

[0097] 3.5 Dilute the standard (known positive serum) as follows:

[0098] 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;

[0099] 3.6 Washing, same as 3.2;

[0100] 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;

[0101] 3.8 Add substrate solution and color for 15 minutes;

[0102] 3.9 Terminate the reaction and read the result with a 450 / 630 nm microplate reader.

[0103] 4. Experimental data:

[0104] The sensitivity and specificity statistics of the Anti-NXP2 antibody detection kit in the present invention are shown in Table 1.

[0105] Table 1

[0106]

[0107] As shown in Table 1, the clinical sensitivity of the detection system of the present invention for dermatomyositis (n=500) is 21.4%. The results of testing the serum of patients with other autoimmune diseases (n=545) and healthy blood donors (n=400) show that its specificity is 100%.

[0108] The correlation analysis between the detection method (ELISA) of the present invention and the immunoblotting method was conducted by comparing 500 sera of dermatomyositis patients and 600 sera of the control group. The sensitivity of the detection method of the present invention was 21.6% and the specificity was 100%.

[0109] The comparative statistics of the Anti-NXP2 antibody detection kit in the present invention and the traditional detection method are shown in Table 2.

[0110] Table 2

[0111]

[0112]

[0113] For comparison of Anti-NXP2 antibody detection in various groups of people, see Figure 4 The present invention confirms that anti-NXP2 antibodies are not positive in the serum of normal people, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and non-NXP2 antibody positive dermatomyositis (DM) patients, indicating that the specificity of the present detection invention is high.

[0114] Schematic diagram of changes in Anti-NXP2 antibody detection after treatment, see Figure 5 The present invention detects the changes in the values ​​of DM patients with positive anti-NXP2 antibodies before and after treatment, confirming that the values ​​of anti-NXP2 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 of anti-NXP2 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-NXP2 antibodies and a detection method thereof <130> 20180815 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1025 <212> PRT <213> Homo sapiens <400> 1 Met Ser Asn Gly Tyr Ser Thr Asp Glu Asn Phe Arg Tyr Leu Ile Ser 1 5 10 15 Cys Phe Arg Ala Arg Val Lys Met Tyr Ile Gln Val Glu Pro Val Leu 20 25 30 Asp Tyr Leu Thr Phe Leu Pro Ala Glu Val Lys Glu Gln Ile Gln Arg 35 40 45 Thr Val Ala Thr Ser Gly Asn Met Gln Ala Val Glu Leu Leu Leu Ser 50 55 60 Thr Leu Glu Lys Gly Val Trp His Leu Gly Trp Thr Arg Glu Phe Val 65 70 75 80 Glu Ala Leu Arg Arg Thr Gly Ser Pro Leu Ala Ala Arg Tyr Met Asn 85 90 95 Pro Glu Leu Thr Asp Leu Pro Ser Pro Ser Phe Glu Asn Ala His Asp 100 105 110 Glu Tyr Leu Gln Leu Leu Asn Leu Leu Gln Pro Thr Leu Val Asp Lys 115 120 125 Leu Leu Val Arg Asp Val Leu Asp Lys Cys Met Glu Glu Glu Leu Leu 130 135 140 Thr Ile Glu Asp Arg Asn Arg Ile Ala Ala Ala Glu Asn Asn Gly Asn 145 150 155 160 Glu Ser Gly Val Arg Glu Leu Leu Lys Arg Ile Val Gln Lys Glu Asn 165 170 175 Trp Phe Ser Ala Phe Leu Asn Val Leu Arg Gln Thr Gly Asn Asn Glu 180 185 190 Leu Val Gln Glu Leu Thr Gly Ser Asp Cys Ser Glu Ser Asn Ala Glu 195 200 205 Ile Glu Asn Leu Ser Gln Val Asp Gly Pro Gln Val Glu Glu Gln Leu 210 215 220 Leu Ser Thr Thr Val Gln Pro Asn Leu Glu Lys Glu Val Trp Gly Met 225 230 235 240 Glu Asn Asn Ser Ser Glu Ser Ser Phe Ala Asp Ser Ser Val Val Ser 245 250 255 Glu Ser Asp Thr Ser Leu Ala Glu Gly Ser Val Ser Cys Leu Asp Glu 260 265 270 Ser Leu Gly His Asn Ser Asn Met Gly Ser Asp Ser Gly Thr Met Gly 275 280 285 Ser Asp Ser Asp Glu Glu Asn Val Ala Ala Arg Ala Ser Pro Glu Pro 290 295 300 Glu Leu Gln Leu Arg Pro Tyr Gln Met Glu Val Ala Gln Pro Ala Leu 305 310 315 320 Glu Gly Lys Asn Ile Ile Ile Cys Leu Pro Thr Gly Ser Gly Lys Thr 325 330 335 Arg Val Ala Val Tyr Ile Ala Lys Asp His Leu Asp Lys Lys Lys Lys 340 345 350 Ala Ser Glu Pro Gly Lys Val Ile Val Leu Val Asn Lys Val Leu Leu 355 360 365 Val Glu Gln Leu Phe Arg Lys Glu Phe Gln Pro Phe Leu Lys Lys Trp 370 375 380 Tyr Arg Val Ile Gly Leu Ser Gly Asp Thr Gln Leu Lys Ile Ser Phe 385 390 395 400 Pro Glu Val Val Lys Ser Cys Asp Ile Ile Ile Ser Thr Ala Gln Ile 405 410 415 Leu Glu Asn Ser Leu Leu Asn Leu Glu Asn Gly Glu Asp Ala Gly Val 420 425 430 Gln Leu Ser Asp Phe Ser Leu Ile Ile Ile Asp Glu Cys His His Thr 435 440 445 Asn Lys Glu Ala Val Tyr Asn Asn Ile Met Arg His Tyr Leu Met Gln 450 455 460 Lys Leu Lys Asn Asn Arg Leu Lys Lys Glu Asn Lys Pro Val Ile Pro 465 470 475 480 Leu Pro Gln Ile Leu Gly Leu Thr Ala Ser Pro Gly Val Gly Gly Ala 485 490 495 Thr Lys Gln Ala Lys Ala Glu Glu His Ile Leu Lys Leu Cys Ala Asn 500 505 510 Leu Asp Ala Phe Thr Ile Lys Thr Val Lys Glu Asn Leu Asp Gln Leu 515 520 525 Lys Asn Gln Ile Gln Glu Pro Cys Lys Lys Phe Ala Ile Ala Asp Ala 530 535 540 Thr Arg Glu Asp Pro Phe Lys Glu Lys Leu Leu Glu Ile Met Thr Arg 545 550 555 560 Ile Gln Thr Tyr Cys Gln Met Ser Pro Met Ser Asp Phe Gly Thr Gln 565 570 575 Pro Tyr Glu Gln Trp Ala Ile Gln Met Glu Lys Lys Ala Ala Lys Lys 580 585 590 Gly Asn Arg Lys Glu Arg Val Cys Ala Glu His Leu Arg Lys Tyr Asn 595 600 605 Glu Ala Leu Gln Ile Asn Asp Thr Ile Arg Met Ile Asp Ala Tyr Thr 610 615 620 His Leu Glu Thr Phe Tyr Asn Glu Glu Lys Asp Lys Lys Phe Ala Val 625 630 635 640 Ile Glu Asp Asp Ser Asp Glu Gly Gly Asp Asp Glu Tyr Cys Asp Gly 645 650 655 Asp Glu Asp Glu Asp Asp Leu Lys Lys Pro Leu Lys Leu Asp Glu Thr 660 665 670 Asp Arg Phe Leu Met Thr Leu Phe Phe Glu Asn Asn Lys Met Leu Lys 675 680 685 Arg Leu Ala Glu Asn Pro Glu Tyr Glu Asn Glu Lys Leu Thr Lys Leu 690 695 700 Arg Asn Thr Ile Met Glu Gln Tyr Thr Arg Thr Glu Glu Ser Ala Arg 705 710 715 720 Gly Ile Ile Phe Thr Lys Thr Arg Gln Ser Ala Tyr Ala Leu Ser Gln 725 730 735 Trp Ile Thr Glu Asn Glu Lys Phe Ala Glu Val Gly Val Lys Ala His 740 745 750 His Leu Ile Gly Ala Gly His Ser Ser Glu Phe Lys Pro Met Thr Gln 755 760 765 Asn Glu Gln Lys Glu Val Ile Ser Lys Phe Arg Thr Gly Lys Ile Asn 770 775 780 Leu Leu Ile Ala Thr Thr Val Ala Glu Glu Gly Leu Asp Ile Lys Glu 785 790 795 800 Cys Asn Ile Val Ile Arg Tyr Gly Leu Val Thr Asn Glu Ile Ala Met 805 810 815 Val Gln Ala Arg Gly Arg Ala Arg Ala Asp Glu Ser Thr Tyr Val Leu 820 825 830 Val Ala His Ser Gly Ser Gly Val Ile Glu His Glu Thr Val Asn Asp 835 840 845 Phe Arg Glu Lys Met Met Tyr Lys Ala Ile His Cys Val Gln Asn Met 850 855 860 Lys Pro Glu Glu Tyr Ala His Lys Ile Leu Glu Leu Gln Met Gln Ser 865 870 875 880 Ile Met Glu Lys Lys Met Lys Thr Lys Arg Asn Ile Ala Lys His Tyr 885 890 895 Lys Asn Asn Pro Ser Leu Ile Thr Phe Leu Cys Lys Asn Cys Ser Val 900 905 910 Leu Ala Cys Ser Gly Glu Asp Ile His Val Ile Glu Lys Met His His 915 920 925 Val Asn Met Thr Pro Glu Phe Lys Glu Leu Tyr Ile Val Arg Glu Asn 930 935 940 Lys Ala Leu Gln Lys Lys Cys Ala Asp Tyr Gln Ile Asn Gly Glu Ile 945 950 955 960 Ile Cys Lys Cys Gly Gln Ala Trp Gly Thr Met Met Val His Lys Gly 965 970 975 Leu Asp Leu Pro Cys Leu Lys Ile Arg Asn Phe Val Val Val Phe Lys 980 985 990 Asn Asn Ser Thr Lys Lys Gln Tyr Lys Lys Trp Val Glu Leu Pro Ile 995 1000 1005 Thr Phe Pro Asn Leu Asp Tyr Ser Glu Cys Cys Leu Phe Ser Asp Glu 1010 1015 1020 Asp 1025 <210> 2 <211> 86 <212> PRT <213> Homo sapiens <400> 2 Asn Ser Asn Met Gly Ser Asp Ser Gly Thr Met Gly Ser Asp Ser Asp 1 5 10 15 Glu Glu Asn Val Ala Ala Arg Ala Ser Pro Glu Pro Glu Leu Gln Leu 20 25 30 Leu Leu Asn Leu Glu Asn Gly Glu Asp Ala Gly Val Gln Leu Ser Asp 35 40 45 Phe Ser Leu Ile Ile Ile Asp Glu Cys His His Thr Asn Lys Glu Ala 50 55 60 Val Tyr Asn Asn Ile Met Arg His Tyr Leu Met Gln Lys Leu Lys Asn 65 70 75 80 Leu Gly Leu Thr Ala Ser 85 <210> 3 <211> 56 <212> DNA <213> Artificial sequence <400> 3 tcgaatgggt attccacaga cgagaatttr tcttgctgcc acttctcttc tatgaa 56

Claims

1. An ELISA kit for detecting anti-NXP2 antibodies, characterized in that: include: The purified dominant antigen epitope NXP2 protein, the amino acid sequence corresponding to the purified dominant antigen epitope NXP2 protein is shown in SEQ NO.1, and specifically comprises the following amino acid sequence: MDYKDDDDKAAQPPRGIRLSALCPKFLHTNSTSHTWPFSAVAELIDNAYDPDVNAKQIWIDKTVINDHICLTFTDNGNGMTSDKLHKMLSFGFSDKVTMNGHVPVGLKWYCSNNPDPQFRNCEVPEEPEDEDLVHPTYEKTYKKTNKEKFRIRQPEMIPRINAELLFRPTALSTPSFS SPKESVPRRHLSEGTNSYATRLLNNHQVPPQSEPESNSLKRRLSTRSSILNAKNRRLSSQFENSVYKGDDDDEDVIILEENSTPKPAVDHDIDMKSEQSHVEQGGVQVEFVGDSEPCGQTGSTSTSSSRCDQGNTAATQTEVPSLVVKKEETVEDEIDVRNDAVILPSCVEAEAKIHE; The full-length structure of the purified dominant antigen epitope NXP2 protein is shown in SEQ NO.2, which specifically comprises the following amino acid sequence: MDYKDDDDKAAQPPRGIRLSALCPKFLHTNSTSHTWPFSAVAELIDNAYDPDVNAKQIWIDKTVINDHICLTFTDNGNGMTSDKLHKMLSFGFSDKVTMNGHVPVGLYGNGFKSGSMRLGKDAIVFTKNGESMSVGLLSQTYLEVIKAEHVVVPIVAFNKHRQMINLAESKASLAAILEHSLFSTEQKLLAELDAIIGKKGTRIIIWNLRSYKNATEFDFEKDKYDIRIPEDLDEITGKKGYKKQERMDQIAPESDYSLRAYCSILYLKPRMQIILRGQKVKTQLVSKSLAYIERDVYRPKFLSKTVRITFGFNCRNKDHYGIMMYHRNRLIKAYEKVGCQLRANNMGVGVVGIIECNFLKPTHNKQDFDYTNEYRLTITALGEKLNDYWNEMKVKKNTEYPLNLPVEDIQKRPDQTWVQCDACLKWRKLPDGMDQLPEKWYCSNNPDPQFRNCEVPEEPEDEDLVHPTYEKTYKKTNKEKFRIRQPEMIPRINAELLFRPTALSTPSFSSPKESVPRRHLSEGTNSYATRLLNNHQVPPQSEPESNSLKRRLSTRSSILNAKNRRLSSQFENSVYKGDDDDEDVIILEENSTPKPAVDHDIDMKSEQSHVEQGGVQVEFVGDSEPCGQTGSTSTSSSRCDQGNTAATQTEVPSLVVKKEETVEDEIDVRNDAVILPSCVEAEAKIHETQETTDKSADDAGCQLQELRNQLLLVTEEKENYKRQCHMFTDQIKVLQQRILEMNDKYVKKETCHQSTETDAVFLLESINGKSESPDHMVSQYQQALEEIERLKKQCSALQHVKAECSQCSNNESKSEMDEMAVQLDDVFRQLDKCSIERDQYKSEVELLEMEKSQIRSQCEELKTEVEQLKSTNQQTATDVSTSSNIEESVNHMDGESLKLRSLRVNVGQLLAMIVPDLDLQQVNYDVDVVDEILGQVVEQMSEISSTD; The purified dominant antigen epitope NXP2 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:acagtcgttccgccacc; R: ctaagattccaaatgatgatcctcgtccc.

2. An ELISA kit for detecting anti-NXP2 antibodies according to claim 1, characterized in that, The ELISA kit also includes: A 96-well ELISA plate coated with the dominant antigen epitope NXP2 protein.

3. An ELISA kit for detecting anti-NXP2 antibodies according to claim 1, 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, stop solution; The negative control serum is the negative serum of CADM patients detected by immunoblotting for anti-NXP2 antibodies; The positive control serum is the anti-NXP2 antibody positive serum detected by immunoblotting of CADM patients; The enzyme-labeled secondary antibody is goat anti-human IgG labeled with horseradish peroxidase; 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); The stop solution is a 0.5% dilute sulfuric acid solution.

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