A monoclonal antibody-based enzyme-linked immunosorbent assay of aflatoxin B1 in peanut products.

An improved enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibody (MAb) and one-step extraction method was established for the estimation of aflatoxin B1 (AFB1) in a peanut product. AFB1 was converted to AFB1-oxime, and then conjugated with bovine serum albumin (BSA). Spleen cells from mice immunized with AFB1-BSA conjugates were fused with myeloma cells. After double selection with AFB1-ovalbumin (OVA) and carbodiimide-modified OVA, five stable hybridoma cells secreting anti-AFB1 MAbs (AF1, AF 2, AF 3, AF 4, and AF5) were cloned. Using these anti-AFB1 MAbs, we developed the indirect competitive ELISA (cELISA) with alkaline phosphatase (ALP) - labeled sheep anti - mouse IgG as marker and the direct cELISA with AFBi-oxime horseradish peroxidase (POD) as marker.The minimum detectable limits of the indirect cELISA with AF 1, 2, 3, 4, and 5 were 5, 5, 5, 5, and 50 pg of standard AFB1 per assay, respectively, and those of the direct cELISA with AF 1, 3, 4, and 5 were 2.5, 5, 25, and 100 pg of standard AFB1 per assay, respectively. The cross reactivity of each toxins with these MAbs in the indirect cELISA was as follows: (a) AF 1 and AF 2 were reactive with AFB2 as well as AFB1, weakly with AFG2 > AFG1 > aflatoxicol II (COL II) > aflatoxicol I (COL I) and less weakly with other aflatoxins; (b) AF 3 and AF 4 were reactive with COL II as well as AFB1, weakly with COLI > AFQ1 and less weakly with others; (c) AF 5 was AFQ1 as well as AFB1 weakly with COL II > AFG2 > COL I and less weakly with others.The 60% aqueous methanol extracts of oil-roasted blanched peanuts ("butter peanut"), naturally contaminated with AFB1, were assayed by the direct cELISA without further purification. The direct cELISA with the most sensitive MAb AF 1 was allowed to determine 1 ng of AFB1 per g samples.