Preparation of Horseradish Peroxidase-Carbamide and its Use in Hapten Immunoassays

Preparation of horseradish peroxidase (HRP) carbamide that is HRP linked to urea (HRP-carbamide/HRP-U) is demonstrated and its potential application in the development of enzyme immunoassays (EIAs) for haptens is described. In this new strategy, the lysine residues of HRP were acylated and then acylated HRP was activated to create highly reactive functional groups by periodate oxidation of its carbohydrate moiety and, subsequently, forming a peptide bond with one of the amino groups of urea. The resulting HRP-carbamide was then coupled to carboxylic derivatives of cortisol-21-hemisuccinate (F-21-HS), 17α-OH progesterone-3-O-carboxymethyl-oxime (17α-OHP-3-O-CMO) and nandrolone-3-O-carboxymethyl-oxime (N-3-O-CMO) to prepare enzyme conjugates adopting N-hydroxysuccinimide-carbodiimide method. The F-21-HS-U-HRP, 17α-OHP-3-O-CMO-U-HRP, and N-3-O-CMO-U-HRP enzyme conjugates thus prepared were used for the development of an enzyme linked immunosorbent assays (ELISAs) for the estimation of cortisol, 17α-OHP and nandrolone. The sensitivity of cortisol, 17α-OHP and nandrolone assays were 0.4 ng/ml, 0.05 ng/ml, and 0.12 ng/ml, respectively, and the analytical recovery ranged from 93.3% to 100%, 94.3% to 98.7%, and 93.5 % to 104%, respectively. In the present study, the strategy adopted for preparing HRP-carbamide and its subsequent use in preparing enzyme conjugate has been shown to result in an increase in thermostability of enzyme conjugate, amino group's availability in enzyme for conjugation with increase in bridge length between analyte and enzyme to reduce steric hindrance.