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University of Wyoming

UW Technologies Available for Licensing


Technology Disclosure: 05-099 A Bacteriophage Linked Immunosorbent Assay (BALISA) for Rapid, Sensitive, Detection of Multiple Analytes


We have developed a standardized platform that enables more sensitive and less expensive testing for a variety of biomolecules, including bacteria, viruses, toxins, bioterrorist agents such as anthrax spores, and prions. The assay combines two proven methods, reporter bacteriophage technology, and Enzyme linked Immunosorbent Assay (ELISA) into one integrated method. The integrated technology, known as the Bacteriophage Linked Immunosorbent Assay (BALISA) harnesses the signal amplification produced by bacteriophage amplification, and enzymatic cleavage of a substrate to produce a very sensitive assay, capable of rapid detection of the target biomolecule. See the US patent application UA20070054292A1, patent issued under 7,276,332.

All immunoassays, regardless of their format or usage, require a reliable and sensitive detection system. t\The most sensitive immunoassays are radioimmunoassays. However, the ELISA is the most widely used. ELISA assays rely on enzyme catalyzed detection, and typically employ colorimetric substrates that result in color formation. The enhanced sensitivity enabled by the BALISA will greatly improve the quality of immunologically based tests. The sensitive detection afforded by the BALISA offers other advantages over existing technologies. If one chooses to trade off sensitivity for a shorter assay time period, use of ultra-sensitive detection technology enables faster measurement of analytes. Finally, ultra-sensitive detection technology enables one to further dilute difficult samples such as meat. This improved sensitivity is especially critical in the detection of biomolecules which may be present in low concentrations, such as prions or microorganisms that have a very slow generation time, such as Mycobacterium tuberculosis. The BALISA consists of a reporter bacteriophage that has been genetically modified to carry the lacZ reporter gene, which encodes for β-galactosidase. The availability of the different classes of substrates (colorimetric, fluorescent, or luminescent)for β-galactosidase allows the BALISA to become extremely versatile. For example, if the BALISA was used in a field setting where it would be difficult to employ the use of instrumentation, the colorimetric substrate could be employed, enabling visual detection of the test result. If the reporter assay was conducted in a laboratory, where the use of instrumentation is more practical, the fluorescent or luminescent substrates could be utilized, allowing for more sensitive detection of the biomolecule in question. The reporter bacteriophage is further modified, such that any biomolecule binding moiety, such as an antibody or aptamer, is specifically attached to the capsid of the bacteriophage. The reporter bacteriophage remains infectious, because the ligand is specifically attached to the capsid (head of the bacteriophage), allowing the tail fibers to remain free. The technology has been developed using bacteriophage T4, but any bacteriophage could be utilized in the BALISA.

Like an ELISA, a BALISA is an antibody sandwich capture assay in which one antibody is immobilized and serves to capture a ligand while a second antibody, which binds to a different epitope on the ligand, is used for quantitation. In ELISA, the second antibody is labeled with biotin so that a streptavidin/enzyme conjugate can be used to produce the signal. In BALISA, however, the second antibody is attached to the capsid of the reporter bacteriophage (to produce a bacteriophage linked antibody (BLA)). Therefore, when the secondary antibody binds to the target ligand, the reporter bacteriophage is also bound to the ligand via its capsid. After several wash steps to remove any unbound BLA, a helper bacteria (E. coli JM109 or similar lacZ- strain), and a β-galactosidase substrate is added. The reporter bacteriophage will infect the helper bacteria and produce copies of itself, and at the same time, produce multiple copies of β-galactosidase, which is detected by cleavage of the substrate. The BALISA is outlined in Figure 1 below.

The advantages of this system over other systems are numerous. The major advantage is sensitivity. Since the detection aspect of the assay is based on bacteriophage replication and enzymatic cleavage of a substrate, the assay has two built in signal amplification steps. It is clear that many bacteriophages can bind to a single target ligand. Therefore, the sensitivity of this assay should be vastly superior to other methods because as few as 10-100 bacteriophages would be able to produce a detectable signal. In practical terms, this means that the system should be able to directly detect 10-100 particles of the target ligand, and the actual detection number is expected to be lower than that, since more than one bacteriophage will bind to a single ligand. In addition, the method described here, in which multiple assays can be produced from a single bacteriophage is advantageous, and cost effective, because the assays can be produced based on a standardized platform. Also, the choice of different substrates imparts a versatility on the BALISA not observed with other tests. The use of colorimetric substrates would allow for the test to be performed in the field without instrumentation. While the colorimetric substrates are the least sensitive, the bacteriophage amplification and enzymatic cleavage steps will still allow for sensitive detection of the target ligand. The luminescent substrates allow for extremely sensitive detection of the target ligand. We are currently developing the BALSIA so that it can be read using handheld luminometers, thereby allowing for very sensitive detection of the target ligand in the field. Handheld luminometers are already widely used in the pharmaceutical and food industries for hygiene monitoring.

If your company would like to learn more about this technology and how your company may apply it in commercial or industrial situations or even sponsor a research project to help reduce it to practice, please contact the director of the University of Wyoming Research Products Center, Davona Douglass.