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SARS-CoV-2 Neutralization Assay System using Pseudo-lentivirus
Indonesian Biomedical Journal ; 15(2):179-184, 2023.
Article in English | Scopus | ID: covidwho-2312649
ABSTRACT

BACKGROUND:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans' lower respiratory tracts and causes coronavirus disease-2019 (COVID-19). Neutralizing antibodies is one of the adaptive immune system responses that can reduce SARS-CoV-2 infection. This study aimed to develop a SARS-CoV-2 neutralization assay system using pseudo-lentivirus.

METHODS:

The plasmid used for pseudo-lentivirus production was characterized using restriction analysis. The gene encoding for SARS-CoV-2 spike protein was confirmed using sequencing. The transfection pseudolentivirus optimal condition was determined by choosing the transfection reagents and adding centrifugation step. Optimal pseudo-lentivirus infection was analysed using fluorescent assay and luciferase assay. The optimal condition of pseudo-lentivirus infection was determined by the target cell type and the number of pseudo-lentiviruses used for neutralization test. SARS-CoV-2 pseudo-lentivirus was used to detect neutralizing antibodies from serum samples.

RESULTS:

The plasmid used for pseudo-lentivirus production was characterized and confirmed to have no mutations. Lipofectamine 2000 reagent generated pseudolentivirus with a higher ability to infect target cells, as indicated by a percentage green fluorescent protein (GFP) of 12.68%. Pseudo-lentivirus centrifuged obtained more stable results in luciferase expression. Optimal pseudo-lentivirus infection conditions were obtained using puromycinselected HEK 293T-ACE2 cells as target cells. The number of pseudo-lentiviruses used in the neutralization assay system was multiplicity of infection (MOI) 0.075. Serum A samples with a 110 dilution had the highest neutralizing antibody activity.

CONCLUSION:

This study shows that SARS-CoV-2 neutralization assay system using pseudo-lentivirus successfully detected neutralizing antibodies in human serum, which were indicated by a decrease in the percentage of pseudo-lentivirus infections. © 2023 The Prodia Education and Research Institute
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Full text: Available Collection: Databases of international organizations Database: Scopus Language: English Journal: Indonesian Biomedical Journal Year: 2023 Document Type: Article

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Full text: Available Collection: Databases of international organizations Database: Scopus Language: English Journal: Indonesian Biomedical Journal Year: 2023 Document Type: Article