Differentiation between culture-derived insect stages of T. brucei, T. vivax, T. 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 Trypanosoma brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes in suspension were applied onto NC membrane in dots and probed with unlabelled trypanosome species-specific mAbs. Bound mAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogenic substrate. The assay detected the aforementioned trypanosome species 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 the specific mAbs in the dot-ELISA which had a specificity of 100%. Some of the T. brucei, T. congolense and Nannomonas-specific mAbs could detect 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/μl/dot was eventually determined as ideal for testing in the dot-ELISA. Antigen dots stored at 4 °C under desiccated conditions did not show any loss in activity for up to 90 days. However, when stored under similar conditions at room temperature (17-26 °C), the T. congolense-specific antigen remained unaffected up to 60 days, and then showed decreased activity when tested on day 90.