Production of a monoclonal antibody against a galactose-binding protein of Acanthamoeba castellanii and its cytotoxicity.

In this study, it was confirmed whether the galactose-binding protein (GBP) was present in Acanthamoeba castellanii, and its function on a target cell was confirmed by production of an antibody against the GBP. Since the genes for GBP have not yet been identified at all, the purification of GBP was done using galactose-beads from amoebial lysates, and monoclonal antibodies were produced using cell fusion. GBP was confirmed to have a size of about 35 kDa. After the third immunization with purified GBP in BALB/c mice, monoclonal antibody production was analyzed. The clone cultured before limiting dilution was named 2AB2 and showed the highest antibody titer in the culture supernatant of a 24-well plate. AF6 clone cultured after limiting dilution showed an antibody titer of 0.259 in a 75-T flask. Antibodies generated by collecting ascites by injecting monoclonal colonies into the abdominal cavity of mice were confirmed through gel analysis and were observed to belong to the isotype of the IgM having kappa chains. Since the cytotoxicity of A. castellanii was inhibited by about 26% by the monoclonal antibody against GBP, it was confirmed that the antibody against GBP had an inhibitory effect on cytotoxicity. This study was the first report on GBP isolated and purified from A. castellanii, and similarly to a mannose-binding protein (MBP), its involvement in contact-dependent cytotoxicity was demonstrated with monoclonal antibody production.