Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR.
ABSTRACT 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.
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.
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.
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.
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ABSTRACT: Our understanding of epigenetics has been transformed in recent years by the advance of technological possibilities based primarily on a powerful tool, chromatin immunoprecipitation (ChIP). 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). Here we show selected examples for the utility of two different cytometry-based platforms of epigenetic analysis: ChIP-on-beads, a flow-cytometric test of local histone modifications (Szekvolgyi et al. 2006. Cytometry 69, 1086-1091), and the laser scanning cytometry-based measurement of global epigenetic modifications that might help predict clinical behavior in different pathological conditions. We anticipate that such alternative tools may shortly become indispensable in clinical practice, translating the systematic screening of epigenetic tags from basic research into routine diagnostics of HTP demand.Methods in molecular biology (Clifton, N.J.) 02/2009; 567:99-111. · 1.29 Impact Factor
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ABSTRACT: We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin- and dye labeled reaction products are immobilized on microbeads and the homo- and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero- from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli.Cytometry Part A 04/2008; 73(3):238-45. · 3.71 Impact Factor
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ABSTRACT: Exploring the possibilities offered by flow cytometric microbead analyses for the detection of genetic alterations, an assay based on the dependence of the melting point of double-stranded DNA molecules on their length has been developed, making use of PCR products carrying biotin and fluorescent moiety on their two ends. The samples of different length PCR products immobilized on streptavidine coated microbeads are heat-treated in the presence of formamide at temperatures between the melting point of the longer and that of the shorter PCR product, when the mean fluorescence intensity of the beads carrying the shorter molecules decreases as a result of denaturation, as opposed to the sample containing the longer product. The efficacy and sensitivity of the method is demonstrated in the case of the assessment of the degree of triplet expansion in Huntington's disease. Its utility for the detection of point mutations in heterozygous clinical samples is shown in the case of the BRCA1 gene. The assay is simple and may be offered for the purposes of clinical diagnostics of a number of genetic conditions. These include screening of samples for triplet expansions and SNPs predisposing for particular pathological or pharmacogenomic conditions. In general, the method described herein is offered for the diagnosis of any pathological condition where the length of a genomic or cDNA sequence is expected to be different from that of the normal allele.Cytometry Part A 09/2011; 79(9):720-6. · 3.71 Impact Factor