title: Development of indirect competitive ELISA for quantification of mitragynine in Kratom (Mitragyna speciosa (Roxb.) Korth.).
abstract: Monoclonal antibody (MAb) against mitragynine (MG), an analgesic alkaloid from Kratom leaves (Mitragyna speciosa), was produced. MG was coupled to carrier proteins employing either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), a zero-length cross linker or a 5-carbon length glutaraldehyde cross linker. To confirm the immunogenicity, the hapten numbers were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Preparation of the MAb was accomplished by the electrofusion method. Hybridoma 1A6 that was constructed from the fusion between splenocytes of EDC/NHS conjugate immunized mice and SP2/0-Ag14 myeloma cells was selected, cloned twice and expanded. The cross-reactivities (CRs) of this MAb 1A6 with a series of indole alkaloids were 30.54%, 24.83% and 8.63% for speciogynine, paynantheine and mitraciliatine, respectively. Using this MAb, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a measurement range of 32.92-250 μg/mL. Quantitative analysis of the MG contents in plant samples by icELISA correlated well with the standard high performance liquid chromatography method (R(2)=0.994). The MAb against mitragynine provided a tool for detection of MG in Kratom preparations.
authors.affiliation: Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
authors.forename: Supattra
authors.initials: S
authors.lastname: Limsuwanchote
authors.affiliation: Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Electronic address: juraithip.w@psu.ac.th.
authors.forename: Juraithip
authors.initials: J
authors.lastname: Wungsintaweekul
authors.affiliation: Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
authors.forename: Niwat
authors.initials: N
authors.lastname: Keawpradub
authors.affiliation: Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008807:Mice, Inbred BALB C; D032065:Mitragyna / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry*; D046948:Secologanin Tryptamine Alkaloids / Q000032:analysis*
chemical_list: D000911:Antibodies, Monoclonal; D046948:Secologanin Tryptamine Alkaloids; C001801:mitragynine
keywords: Electrofusion; Kratom; Mitragynine; Monoclonal antibody; icELISA