Development and Validation of an ELISA to Detect Antibody to Onchocerca volvulus Infection Using Mammalian Expressed...

Programs designed to eliminate Onchocerca volvulus transmission rely on measurement of Ov16-specific antibodies in children <10 years old and molecular detection of parasites in blackfly vectors. Antibody testing can be conducted using rapid diagnostic tests or an ELISA, depending on location of testing and sample throughput needs. In previous efforts to establish anti-Ov16 testing in endemic country laboratories using existing tests, we encountered difficulties with assay reproducibility, reagent availability and shipping, and user friendliness. To address these challenges, we developed and validated an ELISA using Ov16 recombinant antigen expressed in a mammalian protein expression system that can be completed within 2 hours and does not require refrigeration during shipment. The assay was developed and validated using two distinct sets of dried blood spot samples that included defined positive, negative, and samples from infections with potential cross-reactivity. During development, the ELISA had 92.98% (95% CI = 88.28-97.68%) sensitivity and a specificity of 98.50% (95% CI = 93.80-100%). The cutoff point established during development was then applied to the validation set of specimens, which demonstrated a sensitivity of 89.47% (95% CI = 84.77-94.17%) and 98.32% (95% CI = 93.62-100%) specificity. When repeated over 4 days by 2 different operators, all replicates yielded reproducible results within a coefficient of variance of 20%. In addition, we performed stability testing on assay reagents to evaluate their suitability for shipping at ambient temperatures. The resulting assay will be useful for monitoring anti-Ov16 prevalence in endemic country laboratories.