Anti-mitragynine monoclonal antibody-based ELISA for determination of alkaloids in the kratom cocktail

Kratom cocktail or the fatal 4 × 100 formula is defined as a mixture of boiled kratom leaves, cola drink, and cough syrup. In the present study, we focused on application of the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using the anti-mitragynine (MG) monoclonal antibody (anti-MG mAb) to the kratom cocktail. The ic-ELISA is a rapid method for quantification of the major kratom alkaloids including MG, paynantheine, and speciogynine in kratom cocktails. Because some matrices or additives may influence the binding affinity between the alkaloids and the anti-MG mAb, a liquid–liquid extraction method using chloroform was used to clean-up samples and minimize any cross-reactivity with anti-MG mAb. The anti-MG mAb showed slight cross-reactivity to caffeine, codeine, morphine, tramadol, and dextromethorphan (