Preparation of Enzyme Conjugate through Adipic Acid Dihydrazide as Linker and Its Use in Immunoassays

The direct coupling of the carboxylic derivative of steroids to the amino group of enzymes is a well-established method in steroid enzyme immunoassays for making enzyme conjugates (1). Horseradish peroxidase (HRP), containing six lysine residues in the sequence, is a widely used enzyme in enzyme immunoassays; in practice, how- ever, only one or two of these are generally available for reaction (2). This variation in amino group content is caused by changes in the extraction conditions used for the isolation of HRP from the roots of the horseradish plant (2, 3). The low yield of HRP coupled to the IgG by the use of bifunctional reagents, namely glutaraldehyde, carbodiimide, cyanuric chloride, bis-diazotized O-dianisi- dine, and P-P-difluoro-m, m-dinitro-diphenyl sulfone (FNPS), and so forth, prompted Nakane and Kawaoi to investigate another method (periodate method) for the conjugation of HRP to IgG (4). Comparative coupling efficiency studies were carried out with the use of glutar- aldehyde, periodate, and N-succinimidyl 3-(2-pyridyl- dithio) propionate (SPDP) as cross-linking reagents (5, 6) for the preparation of HRP-IgG conjugates. These studies revealed that the most efficient HRP-IgG conjugate was obtained by the periodate method. In practice, the differ- ence in amino group availability in different batches of commercial preparations of HRP makes it difficult to establish standard reaction conditions that could be ap- plicable for more than one batch. Therefore, adipic acid dihydrazide (ADH) has been coupled to HRP with the use of the periodate method reaction to provide the necessary amino group for the preparation of hapten enzyme con- jugate with the use of the carbodiimide method. We describe for the first time the use of ADH as a linking reagent between glycoenzyme (HRP) and a ste- roid carboxylic derivative to prepare enzyme conjugate for ELISA. The conjugation of HRP to cortisol through ADH as the link is described below.