Standardization of the dot enzyme-linked immunosorbent assay (Dot-ELISA) for human visceral leishmaniasis

The Dot-ELISA, a rapid, visually read micro enzyme immunoassay for visceral leishmaniasis utilizing minute volumes of antigen "dotted" on nitrocellulose filter discs and precipitable chromogenic substrate, was analyzed under a variety of experimental parameters. Raising assay incubation temperatures from 23 degrees C to 28 degrees C resulted in titer increases in three of five leishmaniasis patient sera; at 37 degrees C, all five patient sera and one of five normal human sera showed titer increases. The amount of antigen used could be reduced 50% by incubating patient serum overnight at 4 degrees C. Antigen discs stored at - 20 degrees C were optimally reactive with leishmaniasis sera over a 270-day period. Antigen discs stored at 4 degrees C and 23 degrees C showed reproducible titer decreases at 90 days. Aging either peroxidase-conjugated antibody or substrate for up to 28 days at 4 degrees C did not adversely affect titers of positive and negative control sera and reagent controls. Activated substrate stored at 23 degrees C was optimally reactive in the assay for at least 24 hours. No changes in titers of positive and negative control sera or nonspecific reactions in reagent controls occurred when using different brands of microtiter plates. The long shelf lives and stabilities of Dot-ELISA antigen and reagents indicate this test should prove useful both in the laboratory and in the field.