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.