Efficient Conjugation of Aflatoxin M1 With Bovine Serum Albumin through Aflatoxin M(1)-(O-carboxymethyl) Oxime and Production of Anti-aflatoxin M1 Antibodies

BACKGROUND: Aflatoxins are the most extensively studied group of mycotoxins produced by molds, especially the Aspergillus group, which are highly toxic to animals and humans. OBJECTIVES: Since immunoassay is a simple and rapid method for the analysis of many toxic substances in comparison to the chromatographic methods, it is necessary to produce specific and sensitive antibodies for detection of Aflatoxin M(1) (AFM(1)). The current study was conducted to produce bioconjugate of Aflatoxin M(1) (AFM(1)) with Bovine Serum Albumin (BSA) as well as to generate specific antibodies against AFM(1) for immunoassay of the mycotoxin. MATERIALS AND METHODS: First, AFM(1) was converted to AFM(1)-(O-carboxymethyl) oxime derivative. Then, AFM(1)-oxime was coupled with BSA and the product was assessed by UV-VIS spectrophotometry. In order to generate polyclonal antibodies against AFM(1), rabbits were immunized with BSA-AFM(1) conjugate. Produced antibodies were purified using ion exchange chromatography and BSA-Sepharose 4B affinity chromatography. The titers and specificity of the produced antibodies were determined by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The results indicated that coupling of AFM(1) with O-(Carboxymethyl) hydroxylamine hemihydrochloride was suitable and 12 moles of AFM(1)-oxime were successfully coupled to each mole of BSA. In addition, the titers and specificity of the prepared antibody were considerable compared to standard anti-AFM(1) antibodies. The relative cross-reactivity of each toxin (relative to AFM(1)) with purified anti-AFM(1) antibodies, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 70, 105, 240, and 2500 ng/mL for AFB(1), AFB(2), AFG(1,) and AFG(2), respectively. CONCLUSIONS: The prepared antibody can be used for the development of an ELISA kit to assay AFM(1) in milk and other biological fluids.