Institute for Research in Molecular Medicine (INFORMM), Main Campus, Universiti Sains Malaysia, 11800, Penang, Malaysia.
Centre for Drug Research (CDR), Main Campus, Universiti Sains Malaysia, 11800, Penang, Malaysia.
Anal Biochem. 2020 Jun 15;599:113733. doi: 10.1016/j.ab.2020.113733. Epub 2020 Apr 14.
An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.
建立了一种酶联免疫吸附测定法（ELISA），用于检测 mitragynine、其他密切相关的 kratom 生物碱和代谢物，该方法使用多克隆抗体。通过曼尼希反应将 mitragynine 与载体蛋白阳离子化牛血清白蛋白偶联。合成的抗原被注入兔子体内，以引发针对 mitragynine 的特异性多克隆抗体。合成了酶缀合物来评估其与产生的抗体的性能。该测定法对 mitragynine 的 IC 为 7.3ng/mL，检测限为 15ng/mL。产生的抗体对 mitragynine（100%）、其他密切相关的 kratom 生物碱如 paynantheine（54%）、specioctiline（63%）、7α-羟基-7H- mitragynine（83%）具有高亲和力，并且与代谢物 9-O-去甲米曲肼（79%）、16-羧基米曲肼（103%）、9-O-去甲米曲肼硫酸盐（263%）、9-O-去甲米曲肼葡萄糖醛酸苷（60%）、16-羧基米曲肼葡萄糖醛酸苷（60%）、9-O-去甲-16-羧基米曲肼硫酸盐（270%）和 17-O-去甲-16,17-二氢米曲肼葡萄糖醛酸苷（34%）发生交叉反应。它对利血平、可待因、吗啡、咖啡因、美沙酮、安非他命和可卡因的交叉反应小于 0.01%。在测定中使用 10 倍稀释的尿液来减少基质效应。回收率在 83%至 112%之间，日内和日间变异系数分别小于 8%和 6%。该 ELISA 被证明是一种方便的工具，可用于诊断人尿液中的 mitragynine、其他密切相关的 kratom 生物碱和代谢物。
