Quantum Dot-Based Luminescent Oxygen Channeling Assay for Potential Application in Homogeneous Bioassays

Abstract

Luminescent oxygen channeling assay (LOCITM) is a homogeneous immunoassay method capable of rapid, quantitative determination of a wide range of analytes-including high and very low concentrations of large and small molecules , free (unbound) drugs, DNA, and specific 1gM. Assays have been carried out in serum and in lysed blood. Reliable detection of 1.25 tUfL thyrotropin (TSH) and 5 ngfL hepatitis B surface antigen (HBsAg) corresponds to detection limits-3-and-20-fold lower, respectively, than those of the best commercially available assays. An assay of chorionic gonadotropin is capable of quantification over a 106-fold range of concentrations without a biphasic response. Latex particle pairs are formed in the assay through specific binding interactions by sequentially combining the sample and two reagents. One particle contains a photosen-sitizer, the other a chemiluminescer. Irradiation causes photosensitized formation of singlet oxygen, which migrates to a bound particle and activates the chemilumi-nescer, thereby initiating a delayed luminescence emission. Assay times range from 1 to 25 mm. INDEXING TERMS: latexagglutination,singletoxygen #{149} chemi-luminescence. photosensitizers. biotin-streptavidin interaction #{149} digoxin #{149} cyclosporine#{149} thyrotropin#{149} hepatitis #{149} chorionic gonadotropin Development of a sensitive homogeneous immunoassay methodology has been the goal of numerous researchefforts [1, 2]. A method thatwould eliminatethe need to separatebound from free labeled analyte(and the associatedwash steps)would be particularly useful if it were suitable for both haptens and proteins and sufficiently sensitive to permit quantification of all common analytes. Chemiluminescence-based homogeneous im-munoassays provide the required sensitivity [3, 4], but the complex chemistry of chemical excitation and emission renders them highly susceptible to matrix effects. Latex agglutination techniques [5], in which detection is based on low-angle light scattering, also have the theoretical ability to provide exceptionally high sensitivity, but the measurements require rigorous exclusion of adventitious particles [6-8]. 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 chro-mogenic 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 channel-ing assay (LOCIm'M) principle, which has been described previously [10], uses two different ligand-or receptor-coated polystyrene p...

Establishment of a sandwich light‐initiated chemiluminescence assay with double antigen for detecting human cytomegalovirus IgG antibody

“…Moreover, this LICA analysis system of HCMV IgG is a homogeneous immunoassay, providing a liquid three-dimensional reaction environment, in which steric hindrance effect does not exist. 33,34 This system exhibited good precision and sensitivity.…”