Fig. 3.

The rabbit serum against Leishmania 40S ribosomal protein S12 antigen (1357) displayed the highest sensitivity and specificity in a direct ELISA. To determine whether these rabbit antisera can detect the corresponding antigen in L. donovani cell lysate in traditional ELISA, ELISA plates were directly coated with L. donovani promastigote lysate ranging from 10 to 1 million promastigotes per well as indicated in a. Each rabbit antiserum in 1:2000 dilution was added to the plate, followed by adding the HRP-linked anti-rabbit antibody and HRP substrate TMB. The color change proportional to the level of target antigen detected was measured at A450nm. b and c To determine whether these antisera are able to detect Leishmania antigen in L. donovani-infected macrophages, the lysate of L. donovani-infected B10R-mouse macrophages were mixed with non-infected B10R macrophage lysate (b) or human H1299 cell lysate (c) in different ratios (denoted as B or H in the figure legends, respectively) and coated on the plate so that each well contained the same amount of cell lysate from a total 3000 (infected plus non-infected) mammalian cells where the total number of amastigotes ranged from 100 to 6400 per well, as indicated; the plate was then hybridized with these rabbit antisera as a