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, however , 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-PЈ-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 difference in amino group availability in different batches of commercial preparations of HRP makes it difficult to establish standard reaction conditions that could be applicable 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 conjugate 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. To 10 mg of HRP (HRP type VI, lot no. 28H7848; Sigma Chemical Company; HRP of this lot failed to conjugate by the direct method) per 1 mL of water, 10 ␮L of freshly prepared 0.1 m aqueous solution of sodium meta-perio-date was added to oxidize the vicinal hydroxy group of carbohydrate moieties of HRP to generate HRP-aldehyde (Fig. 1A). The reaction mixture was vortex-mixed and kept at room temperature for 30 min in the dark. Activated HRP (HRP-aldehyde) was passed through a Seph-adex G-25 column previously equilibrated with 10 mm ammonium carbonate, pH 9.3. The brown colored fraction of activated HRP was directly …