A HighlySensitivelmmunoenzymometric AssayInvolving“Common- Capture”Particlesand MembraneFiltration

This highly sensitive immunoenzymometnc assay method involves monoclonalantibodies, a common-capturemicrosphere, and a rapid, membrane-filtration separation step. The common-capture solid phase is monoclonal anti-fluorescain antibody covalently attached to 6.5-sm-diameter latex particles. In sandwich-type assays for large-molecule analytes, the capture antibody is conjugated with fluorescein Isothiocyanate and the probe antibody Is conjugated with beta-galactosidase (EC 3.2.1.23). in competitive assays for small analytes, the analyte-beta-galactosidase conjugate competes with the analyte in the clinical samples for the fluorescelnated capture antibody. After simultaneous incubation of the reagents for2 h, the boundand unboundreagents are separated by filtration through the bottom of each well of a 96-well plate. Substrate (4-methylumbelliferyl-beta.D-galactopyranoside) is then added to the wells, and the rate of product formation is determined kinetically for 12 mm. The rate is proportional to the concentration of analyte in the sandwich assays and inversely proportional In the competitive assays. The assay resultsfor choriogonadotropin, thyrotropin, digoxin,and thyroxinshowthe assay to be sensitive, rapid, and applicable to any size analyte. With this system, several different sandwich and (or) competitive-type assays can be performed simultaneously on the same plate.