Improvement of Leishmania major Amastigote ELISA Test Using Various Antigen Antibody Dissociating Agents and Pepsin Digestion.

Amastigotes arc main pathogenic stage of I.eishmania parasite in mammalian host. During the course of leishmania infection the amastigotc surface would be covered by host proteins like immunoglobulins and complement components. These proteins produce false positive reaction and low signal/noise ratio in evaluation of antibody raising against lesion-dcrived amastigotc by indirect ELISA. 1'0 overcome this problcm we were treated lesion-derived amastigotes with several known antigen-antibody dissociating agents like glycine, potassium thiosufate, chloroquine, guanidine Hel, urea, elc. Our results indieated that thcse agents were ineftèctive in ELISA results and could not produce a clear discrimination between positive and negativc controls. We were also studied the eftèct of pepsin digestion on amastigotes and coatcd these pepsin-treated amastigotes to check the ELISA results. Our results revealed combination of tests including pepsin treated amastigotes coated in alkaline pH and protein A-peroxidase conjugates as secondary antibody in an indirect ELISA design, possess high signal/noise ratio i.e. low background color.