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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 microorganisms (bacteria, viruses, toxins, bioterrorist agents such as anthrax spores), prions, and nucleic acids. 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.

The BALISA consists of a T4 reporter bacteriophage that has been genetically modified to carry the lacZ reporter gene, which encodes for β-galactosidase. 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 ligand (which could be an antibody, aptamer, single stranded DNA molecule, etc.), which binds to a different epitope on the ligand, is used for detection. In an ELISA, the second antibody is labeled with an enzyme so that an enzyme substrate can be used to produce the signal. In BALISA, however, the second ligand is attached to the capsid of the reporter bacteriophage. Therefore, when the secondary ligand binds to the target, the reporter bacteriophage is also bound to the target via its capsid, allowing its tail fibers to remain free. After several wash steps to remove any unbound bacteriophage a helper bacteria (E. coli JM109 or similar lacZ- strain), and a β-galactosidase substrate is added.  The reporter bacteriophage will attach to the helper bacteria via its tail fibers, infect the 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.

Figure 1.  The Bacteriophage Linked Immunosorbent Assay (BALISA).  (1) A sample containing the target (as well as background flora is placed into a tube, or microtiter well.  (2) Immunomagnetic particles, specific for the target, are added (alternatively, specific monoclonal or polyclonal antibodies could be attached to the walls and bottom of the tube/well).  The immunomagnetic particles will specifically bind to the target.  (3) A magnet is applied, and the immunomagnetic particles (with the bound target) are attracted to the magnet.  (4)  The background flora is removed, and a wash step is performed to remove any non-specifically bound background flora.  (5) The bacteriophage linked ligand (which could be an antibody) (BLA) is added.  The BLA will bind capsid first (via the monoclonal antibody) to the target.  (6) The BLA –target  – immunomagnetic particle complex is captured by the magnet, allowing any unbound BLAs to be removed.   Several wash steps are performed to remove any unbound BLAs.  (7)  The helper bacteria is added.  The bound BLAs will infect the helper bacteria, due to the ability of the bacteriophage to bind to the helper bacteria.  The bacteriophage will replicate inside the helper bacteria, and force the helper bacteria to make the reporter gene (beta-galactosidase).  Once the bacteriophage replication cycle is complete (1 hour), the helper bacteria are lysed, releasing the beta-galactosidase.  The substrate is added at the same time as the helper bacteria.  The substrate can be colorimetric, fluorescent, or luminescent in nature.  (8)  Depending on the substrate, the required instrument (visual, fluorometer, luminometer) is used to measure the signal. 

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.

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.  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 target.  In addition, the method described here, in which assays capable of detection many different targets 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.  The luminescent substrates allow for extremely sensitive detection of the target.  We are currently developing the BALISA 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.