Yukihiro Nishikawa, Kimiko Hasegawa, Hiroki Abe, Shun Matsuzawa, Takuya Isayama, Ran Nakashima, Yoshihiko Saito, Ichizo Nishino, Masataka Kuwana
{"title":"利用非放射性生物素化重组蛋白，建立一种检测抗3-羟基-3-甲基戊二酰辅酶A还原酶自身抗体的免疫沉淀法","authors":"Yukihiro Nishikawa, Kimiko Hasegawa, Hiroki Abe, Shun Matsuzawa, Takuya Isayama, Ran Nakashima, Yoshihiko Saito, Ichizo Nishino, Masataka Kuwana","doi":"10.1111/cen3.12810","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Objectives</h3>\n \n <p>A variety of myositis-specific autoantibodies have been identified in sera from patients with idiopathic inflammatory myopathies, and they play a crucial role in tailoring personalized disease management. In particular, autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) are now recognized as a key tool for diagnosing immune-mediated necrotizing myopathy. The current gold standard for detecting anti-HMGCR autoantibodies involves immunoprecipitation (IP) using radiolabeled proteins from cell extracts or purified proteins produced by <i>in vitro</i> transcription/translation (IVTT). Unfortunately, this radioisotope labeling is technically intricate and not suitable for routine laboratory use. To address this, we developed a novel assay called “Bio-IVTT-IP” for detecting anti-HMGCR autoantibodies, which uses a nonradioactive biotinylated recombinant HMGCR protein produced by IVTT.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>We collected 14 clinical specimens containing anti-HMGCR autoantibodies from patients with immune-mediated necrotizing myopathy, which were validated using the gold standard IP assay, and a set of 35 control samples from patients with other autoimmune diseases, such as systemic lupus erythematosus.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>The Bio-IVTT-IP assay successfully identified 14 clinical samples positive for anti-HMGCR. We confirmed that the performance of the Bio-IVTT-IP assay was completely consistent with that of the gold standard IP assay using radiolabeled proteins. Notably, no immunoprecipitates were found in control samples from patients with other autoimmune diseases.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>These findings show that the Bio-IVTT-IP assay can serve as a potential alternative to the gold standard IP assay for detecting anti-HMGCR autoantibodies. It can be considered a practical immunodiagnostic tool for diagnosing immune-mediated necrotizing myopathy, particularly owing to its accessibility in routine laboratory settings.</p>\n </section>\n </div>","PeriodicalId":10193,"journal":{"name":"Clinical and Experimental Neuroimmunology","volume":"16 2","pages":"88-97"},"PeriodicalIF":0.0000,"publicationDate":"2024-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/cen3.12810","citationCount":"0","resultStr":"{\"title\":\"Development of an immunoprecipitation assay for detecting anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies using a non-radioactive biotinylated recombinant protein\",\"authors\":\"Yukihiro Nishikawa, Kimiko Hasegawa, Hiroki Abe, Shun Matsuzawa, Takuya Isayama, Ran Nakashima, Yoshihiko Saito, Ichizo Nishino, Masataka Kuwana\",\"doi\":\"10.1111/cen3.12810\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Objectives</h3>\\n \\n <p>A variety of myositis-specific autoantibodies have been identified in sera from patients with idiopathic inflammatory myopathies, and they play a crucial role in tailoring personalized disease management. In particular, autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) are now recognized as a key tool for diagnosing immune-mediated necrotizing myopathy. The current gold standard for detecting anti-HMGCR autoantibodies involves immunoprecipitation (IP) using radiolabeled proteins from cell extracts or purified proteins produced by <i>in vitro</i> transcription/translation (IVTT). Unfortunately, this radioisotope labeling is technically intricate and not suitable for routine laboratory use. To address this, we developed a novel assay called “Bio-IVTT-IP” for detecting anti-HMGCR autoantibodies, which uses a nonradioactive biotinylated recombinant HMGCR protein produced by IVTT.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>We collected 14 clinical specimens containing anti-HMGCR autoantibodies from patients with immune-mediated necrotizing myopathy, which were validated using the gold standard IP assay, and a set of 35 control samples from patients with other autoimmune diseases, such as systemic lupus erythematosus.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>The Bio-IVTT-IP assay successfully identified 14 clinical samples positive for anti-HMGCR. We confirmed that the performance of the Bio-IVTT-IP assay was completely consistent with that of the gold standard IP assay using radiolabeled proteins. Notably, no immunoprecipitates were found in control samples from patients with other autoimmune diseases.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions</h3>\\n \\n <p>These findings show that the Bio-IVTT-IP assay can serve as a potential alternative to the gold standard IP assay for detecting anti-HMGCR autoantibodies. 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Development of an immunoprecipitation assay for detecting anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies using a non-radioactive biotinylated recombinant protein
Objectives
A variety of myositis-specific autoantibodies have been identified in sera from patients with idiopathic inflammatory myopathies, and they play a crucial role in tailoring personalized disease management. In particular, autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) are now recognized as a key tool for diagnosing immune-mediated necrotizing myopathy. The current gold standard for detecting anti-HMGCR autoantibodies involves immunoprecipitation (IP) using radiolabeled proteins from cell extracts or purified proteins produced by in vitro transcription/translation (IVTT). Unfortunately, this radioisotope labeling is technically intricate and not suitable for routine laboratory use. To address this, we developed a novel assay called “Bio-IVTT-IP” for detecting anti-HMGCR autoantibodies, which uses a nonradioactive biotinylated recombinant HMGCR protein produced by IVTT.
Methods
We collected 14 clinical specimens containing anti-HMGCR autoantibodies from patients with immune-mediated necrotizing myopathy, which were validated using the gold standard IP assay, and a set of 35 control samples from patients with other autoimmune diseases, such as systemic lupus erythematosus.
Results
The Bio-IVTT-IP assay successfully identified 14 clinical samples positive for anti-HMGCR. We confirmed that the performance of the Bio-IVTT-IP assay was completely consistent with that of the gold standard IP assay using radiolabeled proteins. Notably, no immunoprecipitates were found in control samples from patients with other autoimmune diseases.
Conclusions
These findings show that the Bio-IVTT-IP assay can serve as a potential alternative to the gold standard IP assay for detecting anti-HMGCR autoantibodies. It can be considered a practical immunodiagnostic tool for diagnosing immune-mediated necrotizing myopathy, particularly owing to its accessibility in routine laboratory settings.
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