SPECIFICITY DETERMINATION OF A PSEUDOLENTIVIRUS-BASED NEUTRALIZATION ASSAY

• Main Author: Aissa Gunawan, Hefina
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/82726
• Institution: Institut Teknologi Bandung

A neutraliza,on assay is a method that can be used to determine COVID-19 vaccine e?cacy. Generally, neutraliza,on assays u,lize live viruses so they must be conducted in BSL-3 laboratories. Pseudoviruses are safer alterna,ves in conduc,ng neutraliza,on assays because they are unable to replicate but demonstrate tropism similar to that of wild-type viruses. This research aims to determine the specificity of a pseudolen,virus-based neutraliza,on assay using spike from wildtype SARS-CoV-2. This research was ini,ated by isola,ng plasmids from Escherichia coli DHa or TOP10. Plasmids were confirmed through circular and linear plasmid analysis. Pseudolen,virus produc,on was performed through plasmid transfec,on into HEK293T cells. To determine whether pseudolen,viruses were formed, a luciferase expression assay was performed on HEK293T-ACE2 cells. NAb produc,on was performed through plasmid co-transfec,on into HEK293T cells. The concentra,on of produced NAb was determined through indirect ELISA and a concentra,on of 36,14 ± 1,16 µg/mL was obtained. The pseudolen,virus-based neutraliza,on assay specificity was determined using monoclonal an,bodies and mouse and human serum. The specificity test using monoclonal an,bodies demonstrated that this neutraliza,on assay is able to di?eren,ate between an,bodies that recognize di?erent epitopes on SARS-CoV-2 spike. The specificity test using serum showed that this neutraliza,on assay can di?eren,ate mouse serum responses at di?erent dilu,ons, but the test serum did not exhibit neutraliza,on e?ects. The decrease in infec,on percentage is thought to have been caused by other components in the serum.