Serum Free Replacement Media as an Alternative Method for Ascites Production: A Step-Forward for Large Scale Production of Anti-Schistosomal Monoclonal Antibody
This research was carried out to study the diagnostic potential of monoclonal antibody (MAb) produced by in vitro hybridoma cell culture in serum low or free medium, and their potentials, versus those raised in ascitic fluid. Revival of hybridoma cell lines raised against Schistosoma (S) mansoni soluble egg antigens (SEA) was done. IgM MAb secreting hybridoma cell line (6D/6F) highly reactive and specific to S. mansoni SEA was selected by indirect ELISA and cultured in conventional medium. This cell line was injected intraperitoneally into BALB/c mice for ascites production. After propagation in conventional medium, the cells were passaged in three different media; conventional, serum low replacement medium (SLRM) and serum free replacement medium (SFRM). Hybridoma cell count and viability were comparable when using conventional medium and SLRM. They were better than those cultured in SFRM. Reactivity of antischistosomal MAbs produced in ascitic fluid and in culture supernate of SLRM were comparable when tested against S. mansoni SEA by indirect ELISA. Supernatants of SLRM and ascitic fluid were collected and purification of IgM MAbs was done by euglobulin precipitation method. Culturing in SFRM was excluded due to decrease in cell count and viability as well as having the lowest Schistosoma MAb reactivity compared to other media. MAb (6D/6F) was employed in sandwich ELISA for detection of circulating schistosome antigen (CSA). The lower detection limit of the assay in both standard curves by using MAb (6D/6F) of ascitic fluid and SLRM culture was 1.1 ng/ml of S. mansoni SEA. The assay was performed on sera of 34 individuals (24 stool positive for S. mansoni eggs and 10 stool negative); besides non-infected healthy controls to determine the cut-off level of the assay. Using MAb (6D/6F) produced either by cells cultured in ascitic fluid or SLRM, sensitivity and specificity of the assay for each were 95% & 92% respectively and specificity 90% for both (ascitic fluid & SLRM). The diagnostic efficacy of the assay was 94% and 91% respectively with no statistical differences. In conclusion, these data showed that the diagnostic efficacy of antischistosomal MAbs was comparable on usage of either in vitro cell culture supernate in SLRM or in vivo ascitic fluid production. It is recommended to use SLRM for large scale production of MAbs as an alternative method to ascitic fluid production in BALB/c mice which may result in less cost and higher production yield.
Keywords- Monoclonal antibody; Replacement media; Schistosomiasis; Hybridoma; Soluble egg antigen; Ascitic fluid.