HIV Detection using the Peptide Beacon Architecture
Kevin Joseph Cash, University of California, Santa Barbara
Molecular Biology
2007
Current, rapid HIV tests that are administered to patients take between 5 minutes to nearly an hour. These tests are colorimetric tests and detect the presence of HIV antibodies for a cost on the order of $20 per test. Preliminary positive test results are followed by a secondary test (commonly Western Blot) to confirm the results. The secondary test requires more time (days to weeks) as well as expensive equipment, centralized testing and trained technicians. The development of a rapid (<5 minute) and inexpensive test for HIV would be of significant benefit to hospitals and clinics.
Peptide beacons (PBs) are a class of biosensors recently developed that utilize peptides modified with fluorophores and fluorescence modulators to detect a target. Upon binding to a target (such as an HIV antibody) the peptide undergoes binding induced folding. This changes the fluorescence signature of the PB, consequently allowing the detection of the target. There are two fluorescent PB systems, one based on pyrene excimer fluorescence and another based on fluorescence from a ruthenium tris(bipyridine) system. In the pyrene excimer system, target binding interrupts excimer formation, decreasing fluorescence. In the ruthenium system, binding changes the peptide dynamics, decreasing fluorescence quenching and increasing the fluorescent signal. Both systems take less than a minute for sample fluorescence measurement and can be performed with widely-available, bench-top fluorimeters.
There are three specific aims for this research project. The first is the application of the PB architecture to multiple HIV antibody/epitope pairs. This will allow improved HIV detection than simply relying on a single antibody. The second aim is to employ time-resolved fluorescence to minimize the fluorescent background of realistic samples, such as blood. This will allow the detection of HIV antibodies in whole blood. The third aim is direct detection of component proteins of the HIV virus. This will allow HIV detection before the body has produced antibodies against HIV while the viral load is still high.
The execution of this research project begins with specific aim 1. Four antibody/epitope pairs are selected for use (antibodies targeting p17, protease and gp41) for the construction of PBs. Pyrene PBs representing each of the four pairs will be synthesized first in order to screen each system for successful detection. The ruthenium PBs will then be constructed for successful systems, and used for the second specific aim. The third aim, direct detection of HIV gp120, will be carried on concurrently with the second step.
This research project is significant for HIV detection in California as well as for society at large. The PB architecture is potentially a detection system that has very widespread applications in hospitals and clinics for minimal capital expenditure as well as a low cost for each test. This will allow more patients to be screened, as well as almost instantaneous feedback to the patient, eliminating the need for a subsequent visit to ascertain the results. This will help to maximize treatment options for the patient while minimizing transmission of the disease, both of which will help the health system of California. Additionally, this research could open new avenues of research into other diseases, increasing research funding in the state and potential advances in the treatment of many other diseases.
