Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.

A method for monitoring formation of latex particle pairs by chemiluminescence is described. Molecular oxygen is excited by a photosensitizer and an antenna dye that are dissolved in one of the particles. 1 delta gO2 diffuses to the second particle and initiates a high quantum yield chemiluminescent reaction of an olefin that is dissolved in it. The efficiency of 1 delta gO2 transfer between particles is approximately 3.5%. The technique permits real-time measurement of particle binding kinetics. Second-order rate constants increase with the number of receptor binding sites on the particles and approach diffusion control. By using antibody-coated particles, a homogeneous immunoassay capable of detecting approximately 4 amol of thyroid-stimulating hormone in 12 min was demonstrated. Single molecules of analyte produce particle heterodimers that are detected even when no larger aggregates are formed.

Action of beta-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.

Enzyme-enhancement immunoassay: a homogeneous assay for polyvalent ligands and antibodies.

A homogeneous enzyme immunoassay for proteins has been developed that avoids the need for a labeled antigen. The technique involves antibody labeled with beta-galactosidase (EC 3.2.1.23), succinylated antibody, and a macromolecular o-nitrophenyl-beta-galactoside substrate. The enzyme-labeled antibody and the succinylated antibody form an immune complex in the presence of sample antigen. An enzyme within this negatively charged microenvironment produces a product that forms a second light-scattering phase, whereas the product produced by free enzyme remains soluble. Thus the antigen modulates the rate of increase in light scattering. The technique has been applied to assays for human immunoglobulin G and C-reactive protein as well as for specific antibodies.