High performance thin layer chromatography ELISAGRAM. Application of a multi-hapten immunoassay to analysis of the zearalenone and aflatoxin mycotoxin families.

A new immunoblot approach called ELISAGRAM was devised that combines the sensitivity and selectivity of competitive ELISA with the capacity of high performance thin layer chromatography (HPTLC) to separate structurally related haptens. The procedure involved (1) separation of haptens by monoclonal antibody, (3) incubation of NC with hapten-enzyme conjugate to identify unreacted antibody binding sites. (4) detection of bound enzyme conjugate with a precipitating substrate and (5) visual or densitometric assessment of inhibition bands indicative of a cross-reacting hapten. The technique was applied to two major mycotoxin families, the zearalenones and aflatoxins (AFs), which are to toxicological significance. Detection limit for zearalenone and alpha-zearalenol in the method was 300 pg/assay. AFB1, AFB2, and AFG1 were detectable at 380 pg/assay and AFG2 was detectable at 1500 pg/assay. Multiple standard curves for the zearalenones and AFs could be constructed using scanning densitometry. Cross-reactivity in ELISAGRAM curves was analogous to that found in competitive ELISA. This procedure should be widely applicable to the simultaneous quantitation and confirmation of multiple haptens with a single cross-reactive antibody.