DEVELOPMENT OF A SARS-COV-2 NEUTRALIZATION ASSAY SYSTEM USING PSEUDO-LENTIVIRUS
COVID-19 is an acute respiratory disease outbreak caused by SARS-CoV-2 and has been declared a global pandemic. SARS-CoV-2 infects humans through the interaction of spike proteins that bind to the ACE2 receptor on target cells. This interaction is utilized in the development of a COVID-19 vaccine...
Saved in:
| Main Author: | |
|---|---|
| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/70723 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | COVID-19 is an acute respiratory disease outbreak caused by SARS-CoV-2 and
has been declared a global pandemic. SARS-CoV-2 infects humans through the
interaction of spike proteins that bind to the ACE2 receptor on target cells. This
interaction is utilized in the development of a COVID-19 vaccine. Therefore,
neutralizing antibodies are expected to form an adaptive immune system response.
These antibodies work by binding to the virus to inhibit the virus's formation with
the receptor. These antibodies can be detected through a neutralization assay. The
gold standard of neutralization assay is carried out by microneutralization virus
assay, which requires the wild-type virus. Therefore, using pseudovirus for the
neutralization assay of SARS-CoV-2 is a safer alternative and can be carried out in
BSL-2 laboratory facilities. This study aimed to develop a SARS-CoV-2
neutralization assay system using pseudo-lentivirus. The flow of this research
includes characterization and confirmation of the plasmid used for pseudolentivirus
production, determination of conditions for transfection and pseudolentivirus
infection, and neutralization assay using the human serum. The condition
of pseudo-lentivirus production was determined by choosing the transfection
reagent type and adding centrifugation step at the time of harvesting the pseudolentivirus.
Determining the conditions of pseudo-lentivirus infection was carried
out by determining the target cell type and the number of pseudo-lentiviruses used
for the neutralization test. Analysis of the success of pseudo-lentivirus infection was
carried out by fluorescent assay and luciferase assay. The results showed that the
plasmid used for pseudo-lentivirus production was characterized and confirmed
and showed no mutations. The optimal conditions to produce the SARS-CoV-2
pseudo-lentiviruses were obtained using lipofectamine 2000 reagent and
centrifugation during harvesting of the pseudo-lentivirus. Optimal pseudolentivirus
infection conditions were obtained using puromycin-selected HEK 293TACE2
cells as target cells. The optimal number of pseudo-lentiviruses for
neutralization assay was with an MOI of 0.075. The 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 infections.
|
|---|