Development of surface plasmon resonance-based immunoassay for aflatoxin B(1).

Aflatoxins are a group of highly toxic fungal secondary metabolites that occur in Aspergillus species and may contaminate foodstuffs and feeds. Two different anti-aflatoxin B(1) antibodies were examined to develop a surface plasmon resonance (SPR)-based immunoassay to aflatoxin B(1). A conjugate consisting of aflatoxin B(1)-bovine serum albumin (BSA) was immobilized on the dextran gel surface. Competition between immobilized aflatoxin B(1) conjugate and free aflatoxin B(1) in solution for binding to antibody injected over the surface formed the basis for the assay. Regeneration of the antibody from the immobilized conjugate surface is essential for the development of such an inhibitive immunoassay. Problems were encountered with the regeneration of the sensor surface, due to the high-affinity binding of the antibodies. Conventional regeneration solutions consisting of low concentrations of NaOH and HCl worked to a degree, but regeneration was at the expense of the integrity of the immobilized conjugate. A polyclonal anti-aflatoxin B(1) antibody was produced and was found to be regenerable using an organic solution consisting of 1 M ethanolamine with 20% (v/v) acetonitrile, pH 12.0. This combined high ionic strength and extreme pH, as well as chaotrophic properties and allowed the development of an inhibitive immunoassay. The assay had a linear range of 3.0-98.0 ng mL(-1) with good reproducibility.

Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody.

Phosphonic acid (trans-4-phosphono-2-butenic acid; TPB) was used as a generic hapten in order to generate broad specificity antibodies against a group of organophosphorus pesticides. The polyclonal antiserum showed, in an indirect enzyme-linked immunosorbent assay (ELISA) format, preferential binding toward pesticides containing unsaturated diethyl-phosphate functionalities rather than the equivalent thiophosphate or dimethyl structures. The level of detection in the ELISA using a heterologous system was investigated and showed a 20-fold improvement when a conjugate for which the antibody had lower affinity was immobilized on the plate. Biosensor assays using parathion as a standard indicated that the antibody had a relatively high dissociation rate, and reproducible cycles of regeneration were achieved. The potential for using TPB as a generic hapten is discussed.