Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR.

Background: Introduction of microbeads into flow-cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications.

Methods: Formaldehyde-fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow-cytometric platform for various immunological and biochemical assays.

Results: We have tested these "biological microbeads" for the simultaneous titration of human alpha-fetoprotein (AFP) and human Chorionic Gonadotropin (betahCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers.

Conclusions: The use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow-cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow-cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format.