Phosphatase probes

Figure 2 illustrates the reaction mechanisms of acridinium ester label probes and alkaline phosphatase probes using dioxetane chemiluminescent detection. Table 2 summarizes approaches for labeling DNA. 

Although these probes show potent, heat-sensitive labeling profiles with purified tyrosine phosphatases, suggestive of specific active site modification, their use in complex proteomes was not reported [109]. Zhang and coworkers introduced a more specific class of tyrosine phosphatase probe [110]. This probe consists of an a-bromobenzylphosphonate moiety that acts as a tyrosine phosphate mimic and a mechanism-based inhibitor of tyrosine phosphatases. The chemistry of the probe mode of action is shown in Scheme 7. 

Although these probes reacted specifically with tyrosine phosphatases, low water stability and limited cell permeability hindered their use in further ABPP studies [110]. The same group subsequently introduced a new class of phosphatase probes based on an aryl vinyl sulfone moiety [35]. 

Site-Directed Mutagensis of E. call Alkaline Phosphatase Probing the Active-Site Mechanism and the Signal Sequence-Mediated Transport of the Enzyme... 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

Alkaline phosphatase-labeled probes are synthesized so that 18 bases are complementary to sequences on the arms of the bDNA. Three hybridization sites are located on each branch for a total binding capacity of 45 labeled probes per bDNA molecule. The alkaline phosphatase catalyzes the dephosphorylation of chemiluminescent substrate, dioxetane (Lumi-Phos Plus, Lumigen, Detroit, MI). The intensity of the light emission is measured with a plate luminometer as relative luminescent units. 

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

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. 

Ca2+ [66], making calcium monitoring a vital tool for cellular biologists. For example, calmodulin-dependent protein phosphatase, calcineurin can be monitored using a probe based on the regulatory domain of the nuclear factor of activated T cells [69], This approach can be taken further to monitor multiple calcium-dependent events in real time [70],... 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

Jablonski, E., Moomaw, E.W., Tullis, R.H., and Ruth, J.L. (1986) Preparation of oligo-deoxynucle-otide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 14, 6115-6129. 

Kiyama, H., Emson, P.C., and Tokyama, M. (1992) In situ hybridization histochemistry using alkaline phosphatase-labeled oligodeoxynucleotide probe. In Methods in Molecular Biology, Protocols in Molecular Neurobiology, (A. Longstaff, and R Revest, eds.), Vol. 13, pp. 167-179. Humana Press, Totowa, New Jersey. 

---