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

Abstract

BackgroundIntroduction 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.MethodsFormaldehyde‐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.ResultsWe have tested these “biological microbeads” for the simultaneous titration of human α‐fetoprotein (AFP) and human Chorionic Gonadotropin (βhCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers.ConclusionsThe 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. © 2005 International Society for Analytical Cytology

Flow Cytometric and Laser Scanning Microscopic Approaches in Epigenetics Research

“…approach has been demonstrated in the case of various assays of molecular diagnostic value: immunoassays, sensitive measurement of protease or nuclease activity, detection of deletion/insertion of sequences by heteroduplex analysis, etc., that could all be adapted to a 'lab-on-beads' platform, i.e., the flow-cytometric analysis of microbead-captured macromolecules (1,18,19). Many samples can be simultaneously analyzed in a FACSarray instrument using fluorescent dyes matching its optical channels.…”

“…However, in many cases, the detection of epigenetic changes requires methods providing a high-throughput (HTP) platform. Cytometry has opened a novel approach for the quantitative measurement of molecules, including PCR products, anchored to appropriately addressed microbeads (Pataki et al 2005. Cytometry 68, 45-52).…”

Fluorescence Spectroscopy: Applications in Chemical Biology

“…Fluorescently functionalized microspheres and nanospheres are available commercially from several sources, spanning the UV-to-IR, in a variety of sizes ranging from 20 nm to 5 µm (3). Fluorescent microspheres are also commonly labeled with primary or secondary antibodies and are used as solid supports in many sandwich immunoassays, where they take full advantage of solution-based kinetics (51)(52)(53). Alternatively, microspheres can be obtained with carboxyl, amine or other surface-displayed functional groups for chemical conjugation to biologic molecules (3).…”

Multiplexed and microparticle‐based analyses: Quantitative tools for the large‐scale analysis of biological systems

“…This approach has been used to simultaneously measure activated cells and released cytokines in whole blood (86) and more examples are likely to follow. Further blurring the distinction between cell‐based assays and bead‐based assays is the notion of using cells as microparticles for multiplexed analysis (87). In this configuration, fixed bacteria or yeast cells may encode information and display receptors or other molecules, but are not themselves the object of study.…”