Ian Gibbons

Homogeneous enzyme immunoassay for proteins employing β-galactosidase

Abstract A homogeneous enzyme immunoassay method for proteins is described. The assay components include a protein antigen—β-galactosidase conjugate, antiprotein antibody, and a synthetic macromolecular substrate. When antibody binds to the conjugate, enzyme activity is inhibited up to 95% because of steric exclusion of the substrate. Free-protein antigen competes for antibody, thus preventing inhibition. The extent of inhibition is related to the concentration of protein analyte. The assay is simple to perform, highly sensitive, and is generally useful for proteins with wide structural variations.

Nonseparation enzyme channeling immunometric assays.

Publisher Summary This chapter describes a set of nonseparation immunoassays in which the reagents are enzyme-labeled antibodies. These methods are designed to measure low concentrations of macromolecular antigens with excess reagents while limiting the background due to the unbound reagents. The assays provide sensitivity comparable to that of enzyme-linked immunosorbent assay (ELISA) while greatly simplifying the assay protocol. In many enzyme immunoassays, the enzyme-labeled reagent binds to a surface and the immobilized enzyme is subsequently measured. For example, in the ELISA, antigen binds to antibody that has previously been adsorbed to a solid surface. Usually, unbound antigen is removed. Enzyme-labeled antibody is then added and binds to antigen on the solid surface. After carefully washing away unbound reagent, the measurement of the bound enzyme gives a direct indication of the quantity of antigen originally present. The materials and reagents employed for the assays are largely the same and are also described in the chapter.

Action of β-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions☆

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.