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Ultrasensitive immunoassay for monocrotaline using monoclonal antibody produced by N, N’-carbonyldiimidazole mediated hapten-carrier protein conjugates

Sakamoto, Seiichi, Nagamitsu, Rika, Yusakul, Gorawit, Miyamoto, Tomofumi, Tanaka, Hiroyuki, Morimoto, Satoshi
Talanta 2017 v.168 pp. 67-72
Crotalaria, albumins, cross reaction, detection limit, enzyme-linked immunosorbent assay, food chain, humans, hydrolysates, ingestion, lysine, monoclonal antibodies, monocrotaline, poisoning, quantitative analysis, retronecine
Monocrotaline (MCT) is a naturally occurring pyrrolizidine alkaloid mainly produced in plant genus Crotalaria (Fabaceae), in which various toxicities have been reported. Recently, demand of quantitative analysis for determination/detection of MCT has increased because MCT in plants can be transferred to human by food chain, leading to intoxication. In this study, monoclonal antibody (MAb) against MCT (MAb 4B8) was produced by unique one-pot conjugation between hydrolysate of MCT, retronecine (RN) and consecutive lysine residues of bovine-serum albumin (BSA) through N,N’-carbonyldiimidazole (CDI) mediated method. The cross-reactivity test evaluated by an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that MAb 4B8 is highly specific to MCT. In addition, the developed icELISA using MAb 4B8 exhibited ultrasensitive recognition to free MCT with the limit of detection of 48.8pgmL−1. Further validation analyses suggested that the developed icELISA is sensitive, accurate, and reliable enough to be used for determining/detecting MCT before ingestion.