Homogeneous immunoassays

Abstract

Luminescent [6][7][8]. Enzyme channeling immunoassays are applicable to both small and large molecules but have limited sensitivity because of their susceptibility to matrix effects. In these assays, an immune reaction brings two enzymes into proximity on a surface; at that surface, one enzyme produces a product that serves as a chromogenic substrate for the second enzyme [9]. The product reaches higher concentrations near the surface than in the bulk solution, which leads to an increase in the rate of reaction with the proximate second enzyme.We report here on the development of an unusually robust and sensitive homogeneous chemiluminescent immunoassay that is based on a combination of the principles of latex agglutination and channeling. The luminescent oxygen channeling assay (LOCIm'M) principle, which has been described previously [10], uses two different ligand-or receptor-coated polystyrene particles at much lower concentrations than ordinarily used in latex agglutination. ' Typically, particle pairs are formed rather than large aggregates, and photochemically triggered chemiluminescence replaces detection by nephelometry or turbidity. One particle has dissolved in it a photosensitizer that Nonstandard abbreviations: }lBsAg, hepatitis B surfaceantigen;biotin-NIIS ester, biotinyl-6-aminohexanoic acid N-hvdroxvsuccinimide ester; I-lAy, hepatitis A virus;TSH, thyroid-stimulating hormone (thvrotropin);and hCG, human chorionicgonadotropin.

Production of Immunoassay Reagents

“…Immunoassays have been divided into homogeneous assays, which require no physical separation (Rubenstein et al, 1972;Boguslaski and Li, 1982;Looney, 1984), and heterogeneous assays, which require physical separation in order to determine degree of displacement or binding by monitoring the bound/free ratio of labeled ligand or receptor (see Chapter 1). An overview of separation methods most commonly employed in heterogeneous immunoassays is presented in Table IV. A. Liquid-Phase Methods Separation of free and bound labeled ligand or receptor was accomplished in many early immunoassays by differential adsorption of the labeled reagent to a solid material.…”

A Review of Immunoassays and Their Application to Salmonellae Detection in Foods

“…Such labels have included enzymes, co-enzymes, fluorescent dyes, fluorogenic substrates, chemiluminescence precursors, stable free radicals, a luminol reactive heme-containing compound, bacteriophages and metal atoms. This subject has been reviewed by Oellerich (114), Schall and Tenoso (134,135) Chang (29), Visor and Schulman (158), and Boguslaski and Li (23).…”

“…However, the use of enzymes as tracer in immunoassays was first introduced by van Weeman and Schuurs (154) and Engvall and Perlmann (43). Since that time, a number of different concepts have been introduced and reviews dealing with methodology principles, characteristics, application, automation and semi-automation (23,134,136,139,159,162), application in diagnostic medicine (160), microbiology (42) and clinical chemistry (114) have been published. In addition, studies on the adsorptive characteristics (immobilization) of antigens on plastics for use in EIA (27,87,141), the optimization of EIA (25,48,153,167), factors affecting their sensitivity (73,74,132), and interpretation of data (56,127) have been reported.…”