title: Ultrasensitive immunoassay for monocrotaline using monoclonal antibody produced by N, N'-carbonyldiimidazole mediated hapten-carrier protein conjugates.
abstract: 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.
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: s.sakamoto@phar.kyushu-u.ac.jp.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sakamoto
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Rika
authors.initials: R
authors.lastname: Nagamitsu
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Gorawit
authors.initials: G
authors.lastname: Yusakul
authors.affiliation: Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: htanaka@phar.kyushu-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D007093:Imidazoles / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D016686:Monocrotaline / Q000032:analysis* / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D006241:Haptens; D007093:Imidazoles; D012710:Serum Albumin, Bovine; C006900:N,N-carbonyldiimidazole; D016686:Monocrotaline
keywords: Crotalaria; Enzyme-linked immunosorbent assay (ELISA); Monoclonal antibody (MAb); Monocrotaline (MCT); N; N’-carbonyldiimidazole (CDI)