IMMORTALIZATION OF B CELLS FROM PLASMODIUM FALCIPARUM EXPOSED INDIVIDUALS FOR THE PRODUCTION OF P ARASITE- SPECIFIC ANTIBODIES

Abstract

The continuous production of antibodies with desired properties has become very important in fields, such as biomedical research and in medicine. Methods for making such useful antibodies include the hybridoma technology and Epstein-Barr virus (EBV) transformation of antibody- secreting cells (ASCs) to make them live longer in ex vivo culture. Transformed cells are then capable of multiplying rapidly over longer periods and producing large amounts of the desired antibodies. The aim of this study is to perform an EBV transformation of ASCs in peripheral blood mononuclear cells (PBMCs) to establish immortalized ASCs and to subsequently screen culture supernatants for Plasmodium Jalciparum-specific antibodies. PBMCs were isolated from malaria exposed individuals by density gradient centrifugation using Ficoll-hypaque. Isolated PBMCs were co-cultured with EBV using the EBV-based Human Blood B Booster Kit from Dendritics™. Transformed B cells clones after day 21 were expanded and screened for total human and Plasmodium-specific antibody production using ELISA. Signs of transformation (cell aggregates, increased size and rate of growth) were seen within 3 days of commencement of the immortalization process, and after 21 days some of the polyclonal population of B cells were frozen and stored in liquid Nitrogen. Upon thawing, recovered cells were shown to be viable and underwent clonal expansion in culture. Screening of culture supernatants with chicken anti-human antibody capture ELISA showed good production of human antibodies. The quantity of human IgG produced by immortalized ASCs that had been in culture for 11 days was greater compared to yield from the same culture by day 25. Western Blot analysis confirmed human IgG bands of 160 kDa. After about 20 rounds of screening, however, Plasmodium falciparum- specific antibodies could not be isolated and this reflects a very low frequency of parasite-specific B cell clones in these exposed individuals.