Multiplex immunoassays on size-categorized individual beads using time-resolved fluorescence

Miniaturized multiplex immunoassays were studied on individual beads to det ect analytes in the same reaction mixture. With this approach hands-on time , cost, and amount of reagents as well as waste produced in the assays were reduced. Particles were categorized according to size for immunoassays of prostate specific antigen (PSA) and acute myocardial infarction (AMI) marke rs. Additionally, free and dual PSA assays were carried out on a single bea d. The analyte concentration was detected directly on the surface of the be ads using stable, intrinsically fluorescent europium and terbium chelates, and time-resolved fluorometry. Less than 0.4 ng ml(-1) PSA (corresponding t o ca. 10 amol) was detected in free, dual and multiplex PSA assays and 0.1 and 2.4 ng ml(-1) of myoglobin (Mb) and creatine kinase MB (CK-MB), respect ively, were monitored in the multiplex AMI marker assay. The effect of bead size and material on free PSA detection was also investigated. The beads w ere detected three times under different conditions; in liquid, after dryin g and after dissociating europium ions from the chelate into the DELFIA(R) fluorescence enhancement solution. The same detection sensitivity was found for the dissociative and non-dissociative methods indicating that the curr ent labeling technology for the surface detection is comparable to the comm ercial DELFIA system. (C) 2000 Elsevier Science B.V. All rights reserved.