Development of an ELISA Test with High Diagnostic Accuracy for SARS-COV-2 Using Recombinant Nucleocapsid Protein Expressed in E. coli.

IF 2.4 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular Biotechnology Pub Date : 2025-04-07 DOI:10.1007/s12033-025-01424-6

Mahsa Darestanifarahani, Faezeh Mahmoudi, Ali Mohammadi, Ehsan Lotfi, Bahar Bahrami, Samira Shajari, Fatemeh Hamzehlooy, Fatemeh Karamali, Mahmood Barati, Davod Jafari

{"title":"Development of an ELISA Test with High Diagnostic Accuracy for SARS-COV-2 Using Recombinant Nucleocapsid Protein Expressed in E. coli.","authors":"Mahsa Darestanifarahani, Faezeh Mahmoudi, Ali Mohammadi, Ehsan Lotfi, Bahar Bahrami, Samira Shajari, Fatemeh Hamzehlooy, Fatemeh Karamali, Mahmood Barati, Davod Jafari","doi":"10.1007/s12033-025-01424-6","DOIUrl":null,"url":null,"abstract":"<p><p>This study aimed to produce a recombinant N protein to develop an enzyme-linked immunosorbent assay (ELISA). A recombinant pET28a vector was constructed, and E. coli BL 21 was transformed for recombinant protein expression. After SDS-PAGE analysis, we approved recombinant N protein expression and purification using western and dot-blotting tests. In ELISA setup tests, 2.5 µg/ml of N protein and 1:100 serum dilution was determined as optimum concentrations for anti-N IgG antibody. In validation tests, 47 out of 51 negative and 46 out of 51 positive samples were determined negative and positive, respectively, using our developed ELISA. According to the Receiver Operating Characteristic (ROC) curve analysis based on the Youden index, the sensitivity and specificity of the developed test were 92% and 90.38%, respectively. An ELISA test with the recombinant SARS-CoV-2 N protein was developed with high specificity and sensitivity for the clinical diagnosis of SARS-CoV-2 infection.</p>","PeriodicalId":18865,"journal":{"name":"Molecular Biotechnology","volume":" ","pages":""},"PeriodicalIF":2.4000,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Biotechnology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s12033-025-01424-6","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

This study aimed to produce a recombinant N protein to develop an enzyme-linked immunosorbent assay (ELISA). A recombinant pET28a vector was constructed, and E. coli BL 21 was transformed for recombinant protein expression. After SDS-PAGE analysis, we approved recombinant N protein expression and purification using western and dot-blotting tests. In ELISA setup tests, 2.5 µg/ml of N protein and 1:100 serum dilution was determined as optimum concentrations for anti-N IgG antibody. In validation tests, 47 out of 51 negative and 46 out of 51 positive samples were determined negative and positive, respectively, using our developed ELISA. According to the Receiver Operating Characteristic (ROC) curve analysis based on the Youden index, the sensitivity and specificity of the developed test were 92% and 90.38%, respectively. An ELISA test with the recombinant SARS-CoV-2 N protein was developed with high specificity and sensitivity for the clinical diagnosis of SARS-CoV-2 infection.

查看原文本刊更多论文

求助全文

约1分钟内获得全文求助全文

来源期刊

Molecular Biotechnology 医学-生化与分子生物学

CiteScore

4.10

自引率

3.80%

发文量

165

审稿时长

6 months

期刊介绍： Molecular Biotechnology publishes original research papers on the application of molecular biology to both basic and applied research in the field of biotechnology. Particular areas of interest include the following: stability and expression of cloned gene products, cell transformation, gene cloning systems and the production of recombinant proteins, protein purification and analysis, transgenic species, developmental biology, mutation analysis, the applications of DNA fingerprinting, RNA interference, and PCR technology, microarray technology, proteomics, mass spectrometry, bioinformatics, plant molecular biology, microbial genetics, gene probes and the diagnosis of disease, pharmaceutical and health care products, therapeutic agents, vaccines, gene targeting, gene therapy, stem cell technology and tissue engineering, antisense technology, protein engineering and enzyme technology, monoclonal antibodies, glycobiology and glycomics, and agricultural biotechnology.