DIFFERENTIATION BETWEEN CULTURE-DERIVED INSECT STAGES OF TRYPANOSOMA-BRUCEI, T-VIVAX, TRYPANOSOMA-CONGOLENSE AND T-SIMIAE USING A MONOCLONAL ANTIBODY-BASED DOT-ELISA

A sensitive and specific nitrocellulose (NC) membrane-based dot-ELISA, utilizing a panel of monoclonal antibodies (mAbs), was developed for differentiation between in vitro-derived procyclic forms of Trypanosom a brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes in suspension were applied onto NC membrane in dots and p robed with unlabelled trypanosome species-specific mAbs. Bound mAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogen ic substrate. The assay detected the aforementioned trypanosome specie s in both single and artificially mixed preparations. Ten T. brucei, 4 T. vivax, 7 T. congolense and 3 T. simiae procyclic stocks and clones from different geographical areas were tested and identified using th e specific mAbs in the dot-ELISA which had a specificity of 100%. Some of the T. brucei, T. congolense and Nannomonas-specific mAbs could de tect as few as 10 trypanosomes/dot, whilst 1 T. vivax mAb was able to detect a minimum of 100 trypanosomes/dot in monospecies preparations. A concentration of 1 x 10(4) trypanosomes/mu l/dot was eventually dete rmined as ideal for testing in the dot-ELISA. Antigen dots stored at 4 degrees C under desiccated conditions did not show any loss in activi ty for up to 90 days. However, when stored under similar conditions at room temperature (17-26 degrees C), the T. congolense-specific antige n remained unaffected up to 60 days, and then showed decreased activit y when tested on day 90.