Synthesis and evaluation of luminescent tracers and hapten-protein conjugates for use in luminescence immunoassays with immobilised antibodies and antigens. A critical study of macro solid phases for use in immunoassay systems, Part II

Abstract

This article describes the synthesis of labels and hapten-protein conjugates for use in bio- and chemiluminescent immunoassay systems, together with the problems encountered. The effects of maleimide upon acetate-, adenylate- and pyruvate kinase activity have been studied, as well as upon the luciferin-luciferase monitoring system. Maleimide inhibited both acetate and adenylate kinase but showed no inhibition of pyruvate kinase and the monitoring reagent. Four heterobifunctional reagents were tested for their capability in forming pyruvate kinase-donkey-anti-rabbit IgG conjugates which retained enzyme and antibody activity. The best results were obtained with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and succinimidyl-6-(p-maleimidophenyl)-hexanoate. The relationship between the amounts of succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and IgG was studied with respect to enzymic activity of the conjugate. The Michaelis-Menten constants for both conjugated and non-conjugated pyruvate kinase were calculated and compared. It was found that the maximal velocity (Vmax) of the conjugated enzyme was lower than that of the non-conjugated enzyme although the "apparent" Km value was the same for both conjugated and non-conjugated pyruvate kinase. The pyruvate kinase-anti rabbit IgG conjugate was tested for its ability to bind to rabbit-IgG coated polystyrene balls. In addition to bioluminescent labels, the synthesis of chemiluminescent markers was undertaken and optimised. The three substances used for labelling were diazoluminol, diazoisoluminol and N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide the latter being used as an N-hydroxysuccinamide "active" ester. The ratio of label to IgG was studied for diazoluminol and N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide active ester after it had been discovered that diazoisoluminol was not suitable for coupling to antibodies. The optimal molar ratios label: IgG were for diazoluminol 40:1 and for N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide active ester 60:1. Increasing the substitution rate led to a lessening of the dynamic range, shown by an increase in the ratio between unspecific binding (noise) to maximal binding (signal) in an assay. The synthesis of hapten-protein conjugates for covalent coupling to polystyrene balls was undertaken as this formed part of the preparation for the assays described in Part III. The optimal production of gentamicin-bovine serum albumin and thyroxine-transferrin conjugates has been described in detail.