Production of monoclonal antibody for lumpy skin disease, sheep pox, and goat pox viruses to develop Enzyme-linked immunosorbent assay
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Date
2024
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Addis Abeba University
Abstract
Lumpy skin disease (LSD), sheeppox (SPP), and goatpox (GTP) are economically significant pox disease of ruminants, caused by lumpy skin disease virus (LSDV),
sheeppox virus (SPPV), goatpox virus (GTPV), respectively. The new emergence of disease caused by capripoxviruses and spreading outside of their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. This experimental study was conducted to produce Monoclonal antibodies (mAbs) against LSDV, SPPV, and GTPV to development ELISA assay. The study was conducted in African Union Pan African Veterinary Vaccine Centre (AU-PANVAC), from October 2023 to May 2024. The mAbs were produced through immunization of BALB/C mice with purified antigen of LSD, SPP, and GTP, with consecutive booster injection. A hybridoma technology was used to produce the hybridoma clones (LC 5.14 and LC 5.4) which were subsequently mass-produced and tested. These LC 5.14 and LC 5.4 mAbs were precipitated and then quantified through the Bicinchoninic Acid protein assay kit. The isotype for the two mAbs were determined through Pierceā¢ Rapid Antibody Isotyping Kit, and both mAbs were IgG1. The cross-reaction of the two mAbs with GTPV and SPPV Ag were studied and those two mAbs were cross-reacted with the GTPV Ag but not with the SPPV Ag LC 5.14 and LC 5.4 mAbs were then conjugated with horseradish peroxidase enzyme; hence enzyme linked mAbs were used for ELISA development. The dot blot test was conducted by using of the LSDV, SPPV, and GTPV Ags with the two conjugated mAbs on the nitrocellulose membrane. Finally, the conjugated mAbs (LC 5.14 and LC 5.4) were
titrated to determine the concentration which is required for ELISA test, and the titer of 1/10 for LC 5.14 and titer of 1/5 for LC 5.4 were determined to use for ELISA assay. Further repeated tests with the positive serum from immunized cattle, sheep, and goat and the negative serum from the same species of animals are required to produce the validate kit which can be used as a diagnostic purpose for the capripoxviruses.
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Keywords
ELISA, GTPV, LSDV, mAbs, SPPV