The PROBE Assay

Incubate the GN cultures for 6 h at 35 C (for other foods, see Note 4) and then remove 0.25 mL of each GN culture to perform the probe assay. 

The PROBE assay has been further developed into a homogeneous assay (now termed homogeneous MassEXTEND hME) using the same purification principle, whereby all sequential enzymatic steps are performed by a simple addition of the reagents to the reaction well. No washing steps are required, and the ion-... 

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

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. 

Purification of the radioactive tracer was modified to include a fractional sublimation before a single extraction—recrystallization cycle to conserve the tracer material. Microgram samples were prepared in melting point capillaries for assay by mass spectroscopic analysis (Table III), made by direct probe injection of the sample into the ion source (18). The probe was heated rapidly to 200°C, and mass spectra were obtained during vaporization of the sample. Tri-, tetra-, and pentachlorodibenzo-p-dioxins vaporized simultaneously with no observed fractionation. 

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. 

As far as we know, this is the first molecular probe that includes two different types of reporter units activated upon on a specific stimulus. The other option to achieve dual detection would be to use two separate probes. However, in this case there could be a problem of competitive catalysis (circumstances in which the Km of the two substrate is not identical). In our probe, 6-aminoquinoline and 4-nitrophenol, detected by fluorescence and absorbance spectroscopy, respectively, were used as reporter units. Due to the synthetic flexibility of our approach, other reporter molecules with different types of functional groups, like amine or hydroxyl, can be linked to our molecular probe. The two assays must be orthogonal to each other, in order to prevent disturbances in the detection measurement. Another advantage of the probe is the aqueous solubility... 

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

The standard RNAs were used to test the ability of the bDNA assay to quantify accurately target RNAs regardless of size or slight sequence variation. Standard RNA preparations of 1.3,2.2, and 3.2 kb showed no detectable effect on quantitation. The quantitation of standard transcripts prepared from clones of HCV sub-type la and 3a differed by a factor of 1.6-fold with one probe design and were indistinguishable with another probe design. These two 475-mer transcripts differed at 30 positions. 

The level of HBV DNA in serum or plasma probably better reflects the replicative activity of HBV. Several assays for the quantitation of HBV DNA are commercially available. In the Genostics assay (Abbott Laboratories), an, 25I-labeled probe binds to single-stranded HBV DNA in solution, followed by separation of free probe and hybrids using Sepharose chromatography (Kuhn et al., 1988). The... 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

Figure 7.22b is a similar plot for the other two lipids considered olive oil (unfilled symbols) and octanol (filled symbols). Both lipids can be described by a bilinear relationship, patterned after the case in Fig. 7.19d [Eq. (7.44)]. Octanol shows a declining log Pe relationship for very lipophilic molecules (log Kd > 2). The probe set of 32 molecules does not have examples of very hydrophilic molecules, with log Kd < —2, so the expected hydrophilic ascending part of the solid curve in Fig. 7.22b is not fully shown. Nevertheless, the shape of the plot is very similar to that reported by Camenisch et al. [546], shown in Fig. 7.8c. The UWL in the latter study (stirred solutions) is estimated to be 460 pm (Fig. 7.8b), whereas the corresponding value in unstirred 96-well microtiter late assay is about 2300 pm. For this reason, the high point in Fig. 7.22b is 16 x 10-6 cm/s, whereas it is 70 x 10 6 cm/s in Fig. 7.8c.

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Three factors have to be considered during the probe immobilization first, it is very crucial for the immobilization chemistry to be stable during the subsequent assay steps second, the probes have to remain functional after the attachment and last, to prevent base pairing restrain, biomolecules have to be immobilized with an appropriate orientation and configuration. Signals are obtained from the arrays caused by the... 

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