EVALUATION AND IMPROVEMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE DETECTION OF ISOMETAMIDIUM IN BOVINE SERUM

The control of bovine trypanosomiasis in Africa continues to rely heavily on the chemoprophylactic drug isometamidium (ISMM) chloride. However, despite many years of use, no methods are available that are sufficiently sensitive to measure drug levels in treated cattle. An enzyme-linked immunosorbent assay (ELISA) for the detection of ISMM in the serum of treated cattle has been developed and evaluated. Liquid-phase ISMM (sample) competes with solid-phase bound ISMM-protein conjugate for biotinylated sheep anti-ISMM IgG. The specific IgG is detected by streptavidin-peroxidase, using tetramethylbenzidine for colour development. Assay calibration is by four-parameter logistic curve-fitting. Factors contributing to absorbance variance were considered in assay optimization and improvement of precision and the lower limit of detection (approximately 0.1 ng/ml in serum). The ELISA was shown to detect serum ISMM for several months after treatment of cattle in a trypanosomiasis endemic country. The potential uses of this assay include the development of rational prophylactic drug regimens, and the indirect detection of drug-resistant trypanosomes.