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Abstracts of Oak Ridge Posters |
1 Microsens Biotechnologies, London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0TU, England
aauthor for correspondence: fax 44-20-7691-2036, e-mail jay.patel{at}microsens.co.uk
Protein kinases play a critical role in almost every cellular regulatory process and have been identified as key players in diseases such as cancer and immune syndromes (1). For this reason, there has been substantial interest in the development of assays for protein kinases for use as both diagnostics and drug discovery tools (2). Although several assays exist for kinases, the most commonly used involve monitoring transfer of the 
-phosphoryl group from [-32P]ATP to a peptide substrate (3). These assays are cumbersome to implement because the unreacted [-32P]ATP needs to be separated from the phosphorylated peptide by use of separation techniques such as gel electrophoresis.
In drug discovery programs, fluorescence resonance energy transfer (FRET) kinase assays are becoming increasingly popular because they are homogeneous, making them relatively easy to automate (4). In typical FRET assays, the anti-phosphotyrosine antibody and the substrate peptide are labeled with "donor" and "acceptor" fluorophores. On phosphorylation of the peptide, the anti-phosphotyrosine antibody binds to the peptide, and the two fluorophores are brought in close proximity to each other. Excitation of the acceptor fluorophore leads to energy transfer to the donor molecule, which emits fluorescence. The principal disadvantage of FRET-based assays is that they are difficult to configure because the two fluorophores need to be within a closely defined distance of each other.
The Dual Phage technology is a new ultrasensitive biological amplification system of broad applicability that uses bacteriophages as biological amplification tools (5). The Dual Phage technology uses two types of bacteriophages that encode two selectable markers. The phages are labeled with an interacting pair, e.g., a receptor/agonist or enzyme/substrate combination. When the interaction takes place, the two phages become spatially linked in the complex and can then infect the same bacterial host cell (the "indicator organism"), thus conferring resistance to both selective agents. This dual infection event is markedly enhanced by the close proximity of the two phages in the complex. When the two selective agents are added to the medium, only the doubly infected indicator organisms survive, and the signal monitored is the growth of these cells.
Several other biological amplification methods have also been devised for the study of molecular interactions. For example, immuno-PCR is a sensitive technique that detects antigens by binding a DNA-tagged antibody to the antigen, amplifying the DNA by PCR, and then detecting the DNA product (6). In immunodetection amplified by T7 RNA polymerase, a double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody, and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the antibody in the antibody–antigen complex (7).
Using a version of the Dual Phage technology adapted for enzyme assays (Fig. 1A
), we have developed a highly sensitive homogeneous assay for lck kinase (p56lck). p56lck kinase is a membrane-associated nonreceptor tyrosine kinase that is found exclusively in natural killer (TK) cells and T cells (8) that play a critical role in T-cell development and activation. The p56lck kinase is localized to a site on the genome that frequently contains chromosomal abnormalities in lymphomas and neuroblastomas (9). In light of these observations, inhibitors for p56lck kinase could have important applications in the treatment of autoimmune and cancer disease.
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The kinase substrate peptide (RRLIEDAEYAARG-biotin; Pierce) was coupled to streptavidin-derivatized M13 (encoding for ampicillin resistance) by standard biotin–streptavidin conjugation techniques (10). Biotinylated anti-phosphotyrosine antibody (Sigma) was coupled to a streptavidin-derivatized M13 bacteriophage encoding for chloramphenicol resistance. These phage conjugates were purified on an affinity column containing anti-M13 antibody (Sigma) bound to agarose. Using the Sigma protein tyrosine kinase assay reagent set (nonradioactive), we determined that each phage carried 
10–100 ligands.
As illustrated in Fig. 1B
, the Dual Phage technology has been successfully applied to the development of a homogeneous microtiter plate-based assay for p56lck kinase. In the assay incubation, the optimum number of each phage was 105 virions. Previous optimization experiments had shown that use of a lower phage concentration decreased the signal and increased the detection time, whereas use of a higher concentration led to an increase in the background signal. At the optimum phage concentration, the signal-to-background ratio of the Dual Phage technology was >10:1. In replicate p56lck kinase assays, the CV was 5.1% (n = 10).
We conclude that the Dual Phage lck kinase assay has a lower detection limit (0.05 pmol/L lck kinase) lower than other homogeneous lck kinase assays. For example, the Packard HTRF lck assay has a lower detection limit of 2 pmol/L (11). The homogeneous nature of the Dual Phage assay makes it ideally suited to both automation and miniaturization. The labeled phages and the indicator organism are extremely robust (no loss of activity has been seen in phage conjugates and freeze-dried Escherichia coli stored at 4 °C over a period of 6 months) and can be readily prepared by standard techniques. The flexibility of the Dual Phage technology suggests that the kinase assay can be implemented in many formats, such as microplate, magnetic particle, or microfluidics systems. The output signal from the indicator organism can be adapted to match existing instruments such as fluorometers, colorimeters, or luminometers. Potential uses for this kinase assay include discovery of kinase inhibitors in high-throughput screening campaigns and use in clinical diagnostic assays.
References
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