Research paperElimination of falsely reactive results in a commercially-available West Nile virus IgM capture enzyme-linked immunosorbent assay by heterophilic antibody blocking reagents

All sera initially reactive in the Focus Diagnostics West Nile virus IgM capture enzyme-linked immunosorbent assay (WNV IgM ELISA) must be retested with background subtraction to identify falsely-reactive (FR) samples due to antibodies that bind to immunoglobulins of other animal species (heterophilic antibodies). In some settings, such as pre-transplant testing of organ donors, the reporting delay associated with retesting can have an adverse impact on donor procurement and organ placement. We sought to determine if inclusion of heterophilic antibody blockers in assay conjugate could eliminate the nonspecific reactivity of FR samples. Of 6 blocking reagents evaluated using a well-characterized FR sample, immunoglobulin inhibiting reagent from Bioreclamation (IIR) and blocker from Fitzgerald Industries (BFI) were superior in their ability to inhibit false reactivity; these 2 blockers were then used to evaluate 20 additional FR and 21 truly-reactive (TR) samples. Both blockers eliminated the reactivity of 20/21 FR samples, whereas all 21 TR samples remained reactive; further, all 13 truly non-reactive (NR) samples evaluated remained non-reactive when using blocker-containing conjugate. A subset of 22 samples were tested in parallel using the initial lot and a second lot of IIR and BFI; with one exception, all samples showed the same qualitative result using both lots of a given blocker. These findings demonstrate that modification of the Focus WNV IgM screening ELISA to include heterophilic antibody blocker IIR or BFI in assay conjugate eliminates the reactivity of most FR samples, markedly reducing the number of samples requiring further testing by background subtraction.