Preparation of multiplex immunofluorescent platform for serology
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Date
2023
Authors
Maswanganyi, Nyiko Given
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Arboviruses are transmitted by blood-feeding insects such as mosquitoes and ticks, and pose significant global health challenges. In South Africa there are arboviruses that are known to occur causing outbreaks annually after heavy rainfall and there are arboviruses that have been detected historically but their medical and veterinary importance is unknown. Due to the unavailability of commercial assays for many arboviruses, the development of in-house assays becomes crucial. This study aimed to develop a cost-effective multiplexing technique using immunofluorescent polyvalent antigen slides for simultaneous screening of IgG antibodies against multiple arboviruses.
The nucleoprotein (NP), envelope domain III (EDIII), and capsid (C) genes for Crimean-Congo hemorrhagic fever virus (CCHFV), West Nile virus (WNV), and Sindbis virus (SINV) respectively were codon optimized and cloned into pcDNA 3.1+ plasmids. The plasmids were transfected into human embryonic kidney (HEK) 293 mammalian cells, and the expression of these genes was confirmed through immunofluorescent assay (IFA). The expressed proteins were further validated by SDS and Western blot analyses. In-house enzyme-linked immunosorbent assays (ELISAs) were developed and optimized to screen human and cattle serum samples for IgG antibodies against SINV and CCHFV. A total of 386 human serum samples and 97 cattle serum samples were screened. Polyvalent IFA slides were developed and used to screen all positive serum samples, some negative and borderline from inhouse ELISAs.
The results demonstrated successful expression of CCHFV NP, confirmed through positive IFA reactions with anti-CCHF IgG. SDS and Western blot analyses further validated the size of the expressed CCHFV. WNV showed low IFA reactivity with antihis antibody and SINV showed no expression. Hence it was decided to continue using SINV-infected cells to replace SINV transfected cells, confirmed using IFA with antiSINV sera. WNV was omitted going forward due to low yield. Among the samples positive for SINV IgG ELISA, 39 exhibited concordant positive results with the polyvalent slides. Similarly, three samples positive for CCHFV IgG antibodies with CCHFV ELISA were concordant with polyvalent slides, while eight negative serum samples tested negative using the ELISAs and IFA. Three ELISA borderline samples for SINV ELISA tested positive for SINV suggesting increased sensitivity for IFA. Furthermore, specificity testing of IFA revealed an accuracy rate of 83.3%.
Multiplex assays offer a low-cost and fast method for simultaneous detection of IgG antibodies against multiple arboviruses on a single platform. The IFA results were concordant with the ELISA, suggesting the reliability of the developed technique. The positive reactors among the borderline samples for SINV antibody ELISA indicate that additional screening of negative samples could improve the cut-off value. Importantly, all samples tested negative with ELISAs were also negative with the multiplex IFA. This approach enables efficient serosurveillance, particularly in resource-limited regions. The accuracy and sensitivity of the multiplex assay were evident through the confirmation of IgG presence by monovalent IFA.
In conclusion, the developed cost-effective multiplexing technique using immunofluorescent polyvalent antigen slides provides a valuable tool for simultaneous screening of IgG antibodies against multiple arboviruses. The accuracy and sensitivity of the multiplex assay were validated through monovalent IFA confirmation. Subsequent research and refinement of this technique hold the potential to broaden its platform, enabling the screening of a more extensive array of viruses.
Description
Dissertation (M.Sc.(Virology))--University of the Free State, 2023
Keywords
Arboviruses, multiplexing, immunofluorescent polyvalent antigen slides, IgG antibodies, In-house assays, ELISA, CCHFV, WNV, SINV, resource-limited