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<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 <i>E. coli</i> 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&#xa0;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>

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

  • 期刊论文

  • 发表日期：

    2025年4月7日

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

摘要

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.