Nonradioactive detecting methods of inflammatory myopathy HMGCR (3-hydroxy3-methylutaryl coenzyme A reductase) autoantibody and application
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A technology of autoantibodies and detection methods, applied in the direction of viruses/bacteriophages, biochemical equipment and methods, carriers, etc., can solve the problems of high cost and high false positive rate of ELISA kits, and achieve easy high expression, easy transfection, The effect of increasing sensitivity
Inactive Publication Date: 2017-04-26
CENT SOUTH UNIV
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[0007] In order to overcome the shortcomings of the above-mentioned prior art, solve the high cost, high false positive rate and radioactivity of ELISA kits and western blot membrane strips for the detection of HMGCR antibodies that are not yet commercialized in China, as well as self-coated ELISA kits for HMGCR antigens. To addres
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[0036] Example 1 Non-radiolabeled immunoprecipitation method for detecting inflammatory myopathy-specific autoantibody HMGCR
[0041] Protein G magnetic beads (Millipore, LSKMAGG10)
[0042] 2. Method
[0043] The non-radiolabeled immunoprecipitation method of HMGCR autoantibodies in inflammatory myopathy includes the following steps:
[0044] (1) Design primers HMGCR-F (5'-CCGAATTCGGGAACAAACAGAGACAGAATCTACA-3') and HMGCR-R (5'-GCGGTACCTCAGGCTGTCTTCTTGGT-3') to amplify the C-terminus of human HMGCR from the pENTER-HMGCR plasmid (Genbank: NM0008590, 1174bp- 2823bp) (as attached figure 1 (a) Shown).
[0045] (2) Recover and purify the PCR fragments, double-enzyme digestion with EcoRI and KpnI restriction enzymes, and connect with EcoRI and KpnI pCMV-myc vector that has been double-cut with EcoRI and KpnI overnight, and transform t...
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[0049] Example 2 Immunoblotting method for detecting HMGCR autoantibodies in inflammatory myopathy
[0057] The immunoblotting method for detecting HMGCR autoantibodies in inflammatory myopathy includes the following steps:
[0058] (1) Transfect HEK293 cells transiently with 3ug and 4ug pCMV-myc-HMGCRC plasmid or pCMV-myc for 48 hours, lyse HEK293 cells with SDS lysate, perform SDS-PAGE gel electrophoresis and transfer membrane, one membrane adopts c-myc The antibody was incubated, and it was found that HEK293 cells transfected with pCMV-myc-HMGCRC plasmid had specific bands of approximately 70kDa and 50kDa, while HEK293 cells transfected with pCMV-myc plasmid had no specific bands (as attached ...
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Abstract
The invention discloses nonradioactive detecting methods of an inflammatory myopathy HMGCR (3-hydroxy3-methylutaryl coenzyme A reductase) autoantibody and an application. The nonradioactive detecting methods comprise a nonradioactive labeled immunoprecipitation assay method and an immunoblotting assay method of the inflammatory myopathy HMGCR autoantibody and is used for detecting the HMGCR-autoantibody of a patient with IIM (idiopathic inflammatory myopathies), wherein the nonradioactive labeled immunoprecipitation assay method of the inflammatory myopathy HMGCR autoantibody comprises the steps as follows: Protein G magnetic beads are conjugated with 20 mu l of serum of an HMGCR antibody-positive patient with inflammatory myopathy or 20 mu l of healthy control serum respectively, the serum is then incubated with a cell lysate overexpressing HMGCR C terminal for antigen adsorption, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is performed after elution, and an c-myc antibody is used for detection; the immunoblot assay method of the inflammatory myopathy HMGCR autoantibody comprises the step that the HEK 293 cell lysate overexpressing the HMGCR C terminal is subjected to SDS-PAGE and detected by the serum of the HMGCR antibody-positive patient as a primary antibody and a rabbit-anti-human IgG H & L as secondary antibody. With the adoption of the nonradioactive detecting methods, the problems of the lack of commercial ELISA kits and immunoblotting membrane strips for detecting the HMGCR antibody at home, as well as high cost and false positive rate and low safety of radiolabeled immunoprecipitation of an ELISA kit self-coated with an HMGCR antigen are solved.
Description
technical field [0001] The application belongs to the field of biotechnology, and relates to a non-radioactive detection method and application of HMGCR autoantibodies in inflammatory myopathy, in particular to a non-radioactive labeling immunoprecipitation detection method and immunoblotting detection method and application of HMGCR autoantibodies in inflammatory myopathy. Background technique [0002] Idiopathic inflammatory myopathy (idiopathic inflammatory myopathies, IIM) is a group of systemic autoimmune diseases mainly invading skeletal muscle, the abnormality of autoimmunity is the key to the occurrence and development of IIM. According to the latest diagnostic classification criteria, IIM can be divided into polymyositis (PM), dermatomyositis (DM), immune-mediated necrotizing myopathy (IMNM), nonspecific Myositis (nonspecific myositis, NSM) and inclusion body myositis (inclusion body myositis, sIBM). The clinical features of IIM are muscle weakness in the proximal ...
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