Production of monoclonal antibodies to guanine imidazole ring-opened aflatoxin B1DNA, the persistent DNA adduct in vivo

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Abstract
Monoclonal antibodies were produced following immunisation of mice with guanine imidazole ring-opened aflatoxin B1 DNA (iro AFB1 DNA), coupled electrostatically to methylated keyhole limpet haemocyanin. Three monoclonal hybridoma lines producing antibodies specific for iro AFB1 DNA were grown as ascites tumours and suitable dilutions of the ascitic fluid (1:8000–1:50 000) used in a competitive enzyme linked immunosorbent assay (ELISA) to measure reactivity of the antibodies to a variety of aflatoxin and nucleic acid-related compounds. These antibodies recognise AFB1 bound to DNA at levels 104–105 times lower concentration than unmodified calf thymus DNA or 8,9-dihydro-8,9-dihydroxy-aflatoxin B1; and show 2–5 times the affinity to iro AFB1 DNA compared to AFB1 DNA. The concentration of AFB1 in iro AFB1 DNA producing 50% inhibition in a competitive ELISA was 1.8 × 10−7 molar. Using the most sensitive hybridoma line, levels of 1 adduct in 300 000 nudeotides would be detectable, which is the level of binding found in the rat and hamster in vivo. These monoclonal antibodies should therefore prove useful in detecting these lesions in animal and human tissue samples exposed to aflatoxins.