Development of monoclonal antibodies against human immunodeficiency virus-1 viral protein R using hybridoma technology

Abstract

Hybridoma technology allows the development of monoclonal antibodies that are specific to a target antigen. The goal of this project was to use hybridoma technology to develop monoclonal antibodies that would be able to recognize and bind both cell bound HIV-1 Viral protein r (Vpr) and cell free HIV-1 Vpr. A mouse was immunized with Vpr antigen and the spleen from the immunized mouse was isolated and teased in order to release antibody-secreting spleen cells. Isolated spleen cells were fused with myeloma cells using polyethylene glycol and then cultured in HAT medium in order to select for hybridomas. Hybridomas secreting anti-HIV-1 Vpr were screened by indirect ELISA. About 70 % of the hybridoma cell lines were able to recognize and bind to the Vpr antigen. Antibodies from DL.VPR.D1 had the best amount of binding to the Vpr antigen when compared to the positive control. The antibodies from DL.VPR.D1 were isotyped in order to determine what class of antibody they belong to. Non-specific binding of the negative control was noticed during isotyping. The use of casein blocking buffer helped in eliminating the non-specific binding. It was found that the antibodies from DL.VPR.D1 belong to the IgG2b class. DL.VPR.D1 was further subcloned by limiting dilution in order to develop monoclonal cells. Seven subclones were derived from DL.VPR.D1. Three of the seven subclones were positive with DL.VPR.D1.G6 having the highest amount of binding to Vpr antigen. Over time DL.VPR.D1.G6 antibodies lost its binding abilities of the HIV-1 Vpr antigen. Loss of reactivity to Vpr antigen was also noticeable in some of the subclones from cell lines (EH.VPR.C5.C6, DF.VPR.C4.D12) that were initially reactive to the Vpr. Amidst the shortcomings; this study demonstrates the importance of hybridoma technology in developing antibodies against a specific target.