Development of an Enzyme-linked Immunosorbent Assay Method for Detection of Tenuazonic Acid

To develop an immunoassay method for tenuazonic acid (TeA), two haptens, 2-(2-(1-(5-(sec-butyl)-2-oxo-2,5-dihydro-1H-pyrrol-3-yL)ethylidene)-hydrazinyl) acetic acid (TeAHGA) and 5-(sec-butyl)-3-(1-hydrazonoethyl-4-hydroxy-1H-pyrrol-2 (5H)-one (TeAH) derived from TeA by hydrazine hydrate and glyoxylic acid were synthesized. Then TeAHGA-BSA conjugate was used as immunogen and the specific polyclonal antibody against TeAH was prepared. Furthermore, an indirect competitive ELISA (icELISA) method for TeA with TeAH as target analyte was established. The optimized assay conditions were 0.156 mu g/L of heterologous coating antigen (TeAH-OVA, ovalbumin), with PBS as assay buffer, the incubation times for the primary antibody and the secondary antibody were 40 and 20 min, respectively. The icELISA results showed IC50 value, limit of detection (LOD) and linear range as 1.61 ng/mL, 0.08 mu g/L and 0.19-12.89 mu g/L, respectively. The average recovery rates for standard addition of TeA from tomato and flour samples were 67.2%-89.8% and 74.8%-93.7%, respectively.