PROBE assay

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

Electrogenerated chemiluminescence (ECL) has proved to be useful for analytical applications including organic analysis, ECL-based immunosensors, DNA probe assays, and enzymatic biosensors. In the last few years, the electrochemistry and ECL of compound semiconductor nanocrystallites have attracted much attention due to their potential applications in analytical chemistry (ECL sensors). 

The three main categories of hybridization probes for real-time PCR are (1) cleavage based assays such as TaqMan, (2) displaceable probe assays such as Molecular Beacons and (3) probes which are incorporated directly into primers such as Scorpions. 

Herzberg, M. (1984) Molecular genetic probe, assay technique, and a kit using this molecular genetic probe. European Patent Application, 0128018. 

Nucleation sites, in ferrosilicon, 22 516 Nucleation track membranes, 15 802 Nucleic acid bases, recognition of, 16 794 Nucleic Acid Database (NDB), 17 606 Nucleic acid probe assays, 16 380. See also DNA analysis Nucleic acid probes, 14 153 Nucleic acids, 17 602-643 20 444-447. See also Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA)... 

Isoform P450S Antibodies cDNA probe Assays 1... 

Table 3.2.5 Disorders detectable by the in vitro probe assay. ETF Electron transfer flavoprotein, MADD multiple acyl-CoA dehydrogenase deficiency...

Fig. 3.2.8a-c Profiles of acylcarnitines as their methyl esters in cell culture medium (precursor of m/z 99 scan) following the in vitro probe assay in fibroblast cultures of a normal control (a) and a patient with the milder (b) and the more severe variant (c) of VLCAD deficiency. Note the more prominent elevation of dodecanoyl- (Ci2 m/z 358 peak 1) and myristoylcarnitine (Ci4 m/z 386 peak 2) compared to a relatively normal accumulation of palmitoylcarnitine ( , m/z 416 peak 3) in the milder VLCAD variant compared to the severe variant, where palmitoylcarnitine is markedly elevated. The asterisks represent the internal standards (from left to right) d3-acetyl-carnitine (C2 m/z 221), d9-isovalerylcarnitine (C5 m/z 269), d3-octanoylcarnitine (C8 m/z 305), d3-dodecanoylcarnitine (Ci2 m/z 361), and d3-palmitoylcarnitine ( m/z 419)... 

W.E. Lee, H.G. Thompson, J.G. Hall and D.E. Bader, Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor, Biosens. Bioelectron., 14(10-11) (2000) 795-804. 

The use of ECL processes for the detection of biological compounds is a rapidly growing area of interest, both for quantitation of analytes and to measure biomolecular interactions. By using ECL active chromophores as labels for biological compounds, a variety of applications is possible, including assays for enzymatic activity, binding assays, immunoassays, and nucleic acid probe assays. 

Since Blackburn et al. reported the first use of ECL detection for the development of immunoassays and DNA probe assays [54], several automated systems have become commercially available. An activated species that readily labels proteins and nucleic acids is obtained by chemically modifying one of the bipyridyl ligands of [(bpy)3Ru]2+ with N-hydroxysuccinimide (Fig. 11a). The... 

Schutz E, von Ahsen N, Oellerich M. Genotyping of eight thiopurine methyltransferase mutations three-color multiplexing, two-color/shared anchor, and fluorescence-quenching hybridization probe assays based on thermodynamic nearest-neighbor probe design. Clin Chem 2000 46 1728-1737. 

Sueda et al. (2000, 2002) reported a dual FRET probe assay, where two combinations, Eu-BHHCT-Cy 5 and Tb-BPTA-Cy 3, were tested for the donor-acceptor combination. They used a pair of 15-mer oligonucleotide probes to detect 31-34 synthetic oligonucleotide targets. As noted above, the emission intensities from Eu3+ and Tb3+ are negligibly weak at the Cy5 and Cy3 emission wavelengths (669 and 565 nm, respectively). Additionally, the initial emission intensities from Cy3 and Cy5 were quite weak when excited at the excitation wavelengths for the Eu3+ and Tb3+ chelates (340 and 325 nm, respectively). These properties enabled sensitive detection without quenchers. The detection limits were reported to be 200 pM and 30 pM for the Eu3+-Cy5 and Tb3+-Cy3 systems, respectively. 

The application of in situ hybridization (ISH) has advanced from short lived, non-specific isotopic methods, to very specific, long lived, multiple color fluorescent-ISH probe assays (FISH). Improvements in the optics, filter technology, microscopes, cameras, and data handling by software, have allowed for a cost effective FISH setup to be within reach of most researchers. The application of mFISH (multiplex-FISH), coupled to the advances in digital imaging microscopy, have vastly improved the capabilities for non-isotopic detection and analysis of multiple nucleic acid sequences in chromosomes and genes (1). 

Christel LA, Petersen K, McWilliam W, Northrup MA. Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration. J Biomech Eng 1999 121 272-279. 

Nomanbhoy TK, Rosenblum J, Aban A, Burbaum JJ. Inhibitor focusing direct selection of drug targets from pro-teomes using activity-based probes. Assay Drug Dev. Technol. 2003 1 137-146. 

The magnetic bead / ECL approach has also been used in DNA-probe assays. Perkin Elmer Corporation has developed an ECL-based assay for post-PCR product detection [63]. (Figure 11). The method allows PCR product identification following fewer PCR cycles than other available analytical methods. 

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