NUCLEAR-MAGNETIC-RESONANCE CONTROLLED METHOD FOR COUPLING OF FENOTEROL TO A CARRIER AND ENZYME

Fenoterol is a phenethanolamine with beta-adrenergic agonist activity. The development of an enzyme immunoassay for fenoterol requires coupl ing to a carrier (bovine serum albumin, BSA) and an enzyme (horseradis h peroxidase, HRP). 1,4-Butanediol diglycidyl ether was used as the co upling agent, providing for a 12-atom spacer. During the coupling proc edure of the spacer to the protein and fenoterol to the spacer the cou pling yield was monitored by nuclear- magnetic resonance (NMR). This e nsured a coupling at desired hapten: protein ratios. The average coupl ing ratios obtained using this method were 30 moles of fenoterol ol to 1 mole of BSA and 1 mole of fenoterol to 1 mole of HRP. In practice, this method can be used for the coupling of compounds containing pheno lic, anilinic, primary amine and thiol groups to proteins. Its major a dvantage is the real-time NMR quantification of coupling efficiency an d the option to interrupt the coupling reaction to obtain the desired hapten:protein ratio.