Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure

Monoclonal antibodies (McAbs) cross-reactive With four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30 min, aflatoxin B-1, B-2, G(1) and G(2) were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to Four aflatoxins. Five McAbs were chosen for further analysis. Of the five. two antibodies had similar reaction efficiency with aflatoxin B-1, B-2. and G(1) but showed a weak cross-reaction to G(2). Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins, The concentrations of aflatoxin B-1, B-2, G(2) and G(2) at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0 mu g mL(-1) respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had Superiority and these antibodies could be used for simultaneous analysis of total aflatoxins. (C) 2009 Elsevier B.V. All rights reserved.