Immunoblotting assay method of myositis specific autoantibody NXP2 antibody and application

Abstract

The invention discloses an immunoblotting detection method and application of a myositis-specific autoantibody NXP2 antibody, and constructs a plasmid pCMV-myc-NXP2N containing a Myc tag at the N-terminal (1-500aa) or C-terminal (400-939aa) of NXP2 Or pCMV‑myc‑NXP2C, use the serum of myositis patients to be tested as the primary antibody, and rabbit anti-human IgG H&L as the secondary antibody to determine whether there are NXP2 autoantibodies in the serum of patients with myositis; solve the problem of detecting NXP2 that is not yet commercialized in China The problems of antibody ELISA kits and western blot strips, as well as the high cost and high false positive rate of self-coated NXP2 antigen ELISA kits and the safety of radiolabeled immunoprecipitation.

Description

technical field [0001] The application belongs to the field of biotechnology, in particular, it relates to the immunoblotting detection method and application of myositis-specific autoantibody NXP2 antibody. Background technique [0002] Idiopathic inflammatory myopathy (idiopathic inflammatory myopathies, IIM), also known as myositis, is a group of systemic autoimmune diseases mainly invading skeletal muscle. The abnormality of autoimmunity is the occurrence of myositis, key to development. According to the most recent diagnostic classification criteria, myositis can be divided into polymyositis (PM), dermatomyositis (DM), immune-mediated necrotizing myopathy (IMNM), nonspecific Myositis (nonspecific myositis, NSM) and sporadic inclusion body myositis (sporadic inclusion body myositis, sIBM). The clinical features of myositis are muscle weakness in the proximal extremities, characteristic rash, systemic damage, and the presence of various autoantibodies in the serum, whic...

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Problems solved by technology

[0005] The main methods for detecting autoantibodies in autoimmune diseases include enzyme-linked immunosorbent assay (ELISA), radiolabeled immunoprecipitation, and western blotting. However, there are no commercial ELISA kits and Western blot membranes for detecting NXP2 antibodies in China at present. The detection of NXP2 antibodies in most laboratories includes: (1) Purchase purified NXP2 protein-coated enzyme-labeled reaction plates, add the serum to be tested and incubate, and use enzyme-labeled anti-human immunoglobulin antibodies to detect antigen-binding The antibody, and then use the appropriate enzyme substrate for color development; (2) purchase the purified NXP2 protein and the serum to be tested for immunoprecipitation, and use western Blot to detect the precipitated product; (3) use 35 S labeled Hela cells, then immunoprecipitated with the serum to be tested, separated the precipitate by SDS-PAGE electrophoresis, and autoradiography, which is the gold standard for detecting MSA

The above methods have their own advantages and disadvantages. Enzyme-linked immunosorbent assay provides a highly sensitive and rapid method for the detection of autoantibodies. However, there is no commercial ELISA kit for the detection of NXP2 antibodies. This method requires highly purified Antigens, and the conditions for antigen coating need to be optimized, false positive results are prone to occur, and batch operations are required. For inflammatory myopathy with a low incidence, it will increase the cost; as the gold standard, radiolabeled immunoprecipitation method has High specificity and sensitivity, and this method precipitates antigens in their natural conformation and protein molecules that interact with the target antigen, but radioisotopes can only be used in well-equipped laboratories and require strict control, Not suitable for promotional use

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