LAUSR

To establish cholyglycine (CG) detection via enzyme‐multiplied immunoassay technique (EMIT), glucose‐6‐phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten–enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine, and CG‐G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG‐G6PD conjugates were investigated. Consequently, CG amount, nicotinamide adenine dinucleotide, d‐glucose‐6‐phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG‐G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG‐G6PD conjugate as test kit, the cholyglycine–EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten–G6PD conjugates.