Development of an in-house serological test to detect severe acute respiratory syndrome coronavirus-2 (SARS COV-2) using recombinant nucleocapsid protein.
Date
2024
Authors
Chiyumbabeenzu, Muuba
Journal Title
Journal ISSN
Volume Title
Publisher
The University of Zambia
Abstract
Coronavirus disease-19 (COVID-19) is a major global concern for public health. This laboratorybased study was conducted at the University of Zambia, School of Veterinary Medicine (UNZAVet) laboratory. The study aimed at developing an in-house test to detect SARS-CoV-2 using the
recombinant SARS-CoV-2 nucleocapsid gene. Recombinant based protein immunoassays are frequently used in veterinary medicine to detect antibodies against different viruses. There is no in-house recombinant protein-based immunoassay (IFA/ELISA) for detection of SARS-CoV-2 antibodies in Zambia. The N gene from the full SARS-CoV-gene was obtained by PCR using N gene primers tagged with SacI and SphI restriction ends. The N gene was cloned into a vector plasmid pCAGGS-MCS. The cloned N gene was transformed into competent DH5α cells. The N gene was purified and sequenced to ensure that there were no mutations within the gene and then transfected into VERO-E6 cells for protein expression. Archived serum samples (ten samples) of individuals previously infected with COVID-19 were tested for COVID-19 using OnSite® Rapid Test. The recombinant NP expressing cells were used as an antigen for an in-house immunofluorescent antibody test (IFA). The serum samples that tested positive on the rapid test were subjected to IFA using the recombinant N antigen prepared. None of the positive testing sera showed positive results on the assay. This could have been due to the expression system used which expressed a non-reactive protein. Another possible reason could have been the serum samples having low reactivity to the recombinant protein because the time frame between infection and when the samples were collected was not known. Hence, validation of this assay could not be conducted. The techniques used in recombinant antigen development, for detection of antibodies can potentially be applied in manufacturing of serological diagnostic kits. The findings of this study indicate that there is need for further investigations into the development of serological tests for sero-surveillance of COVID-19.
Description
Thesis of Master of Science in One Health Laboratory Diagnostic Sciences.