Development of a Multiplexed Flow Cytometer Receptor Assay

It is well known that many chemicals may disturb the endocrine system. These endocrine-disrupting chemicals (EDCs) can cause adverse effects on human health and wildlife. In 1996, Congress directed the U.S. Environmental Protection Agency (EPA) to develop the Endocrine Disruptor Screening Program (EDSP) to determine if chemicals used in commerce may have hormonal effects in humans. There are more than 87,000 chemicals used in commerce that need to be evaluated for potential risks. In addition, there are probably an equal number of metabolites and breakdown products that need to be evaluated, many of which potentially could pollute the environment and cause disruption of the endocrine system. The wide variety of potentially estrogenic compounds makes it difficult to predict estrogenicity on a structural basis. A good analytical system should be able to detect EDCs that currently are unknown. At present, animal models are available to study the estrogenic activity of a compound; their disadvantages include high cost and time consumption, as well as the use of animals. There also are in vitro systems, many of them on cellular systems, such as the E-screen, YES assay, and vitellogenin production. Although the cellular assays are effective tools, they require extended periods to obtain a result, and difficulties can arise when real samples are analyzed due to ubiquitous cytotoxic compounds, such as humic acid, which can interfere with cell proliferation. The radioreceptor assay is an effective technique, but it requires the use of radioactivity. Therefore, there is an opportunity to develop an assay system¿in support of the EDSP¿for screening chemicals that determines whether a chemical interacts with the endocrine system simultaneously in a single and reliable high-throughput assay. There also is a market opportunity to develop assays to screen for numerous EDCs in the same sample during the routine testing of water, food, and environmental samples. This research project focuses on the development of a new multiplexed analytical technique using antibodies and receptors in combination with flow cytometry to determine whether a chemical in a sample may interact with the endocrine system. The results of this research will lead to a high-throughput, sensitive, and cost-effective assay in which the estrogenic (bio-effect) activity of compounds can be determined simultaneously in a single sample.