There a many possible applications for these affinity-based techniques in chemical analysis.  One area that our group has been studying for many years is the use of antibodies in HPLC systems.  This has resulted in the creation of methods we refer to as “chromatographic immunoassays”.  These methods can be used for rapid and selective measurements of drugs, hormones, proteins, and peptides in biological samples.  This makes this approach appealing for use in clinical testing, pharmaceutical analysis, and biotechnology.  Chromatographic immunoassays can also be combined with other analytical methods, like reversed-phase HPLC or capillary electrophoresis, for the simultaneous analysis of several compounds within a given class of chemicals.

One recent application of our work has been the development of an automated, portable system for the determination of triazine herbicides in water.  This method combines the use of a small antibody column with a traditional reversed-phase HPLC column and UV/vis absorbance detection.  We have recently evaluated the use of this system in the field and have shown that it can be provide results at a site of a river or stream within 10 min of sample injection.  This same method can be altered for measurement of other herbicides or pollutants by changing the type of antibody column that is used in the system.

Other examples of chromatographic immunoassays that have been developed by our group include techniques for L-thyroxine, parathyroid hormone, fluorescein-labeled proteins, and human serum albumin.  We have explored and developed many assay formats in this work, including methods that make use of competitive binding assays, sandwich immunoassays, displacement immunoassays, and one-site immunometric assays.  As part of these studies, our group has been one of the first to consider the theory of these methods.   The result has been a series of equations and computer models that we have successfully developed to describe the response and behavior of these assays under various operating conditions.

Studies of Biological Interactions

Another way in which we use affinity ligands is to study the reactions that take place between proteins and other biological agents.  We are especially interested in using immobilized proteins as models for the same proteins in humans or in animals.  These immobilized proteins are used to create a synthetic system that mimics the interactions of these proteins with drugs or other agents.  This makes this approach of great interest for screening possible drug candidates and for predicting the behavior of drug in the body.

One protein we have studied extensively is human serum albumin (HSA).  HSA is the most abundant protein in serum and plays an important role in delivering many types of drugs throughout the body.  We attach HSA within HPLC columns and routinely use these columns to examine how this protein binds to various drugs, hormones, and other small solutes.  Chemicals that we have studied with these columns have included warfarin, thyroxine, tryptophan, ibuprofen, digitoxin, clomiphene, tamoxifen, phenytoin, and carbamazepine.  We have also created a similar column based on the serum protein alpha-1 acid glycoprotein (AGP) that can be used to examine the interactions of this protein with various drugs.   

   As part of this work, we have developed several new approaches for examining the protein binding of drugs and small solutes by HPAC and ACE.  Examples include approaches we have reported for the study of low solubility compounds, agents with multiple binding sites on a protein, and drugs that have allosteric interactions with a binding protein.  In recent work we have also described an ultrafast technique that looks directly at the non-bound fraction of a drug or hormone in a protein sample.  This method, which makes use of millisecond-scale separations on an antibody-based column, has great potential as a new tool for the analysis of clinical and pharmaceutical samples.

Biochemical Immobilization and Characterization

A part of our research has involved the optimization or creation of new schemes for immobilizing or modifying biological agents.  For instance, our group has developed a silica-based material that can be used for the site-selective immobilization of antibodies.  We have also optimized and studied techniques for the immobilization of HSA, AGP, and proteolytic enzymes.  We use a variety of tools to study immobilized proteins as part of this work.  These tools include protein assays, infrared spectroscopy, solid-state nuclear magnetic resonance spectroscopy, and mass spectrometry.  Both HPAC and capillary electrophoresis are also used as part of this work.