Chromatographic Immunoassays & Affinity Microcolumns

There a many possible applications for 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. We have published many papers on this topic and this work was the subject of a review and a cover article that appeared in an April 2001 issue of Analytical Chemistry.

One recent application of our work in this area 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.

Another example is to measure free drug/hormone fractions by using of an ultrafast immunoextraction/displacement assay. This method is based on using of sandwich microcolumns that contain a small layer of antibodies which bind the drug/hormone. Free fraction of drug/hormone can be extracted by this affinity column in very short periods of time (e.g., 80-120 ms).  The free drug portion in the solution can be isolated when a drug-protein mixture passing through this type of column. The result is found to give good correlation with traditional methods like ultrafiltration but requires much less analysis time, makes this approach attractive for use in pharmacological studies and a tool in clinical chemistry for personalized medicine.

Examples of Research with Chromatographic Immunoassays and Affinity Microcolumns

  1. David S. Hage and Mary Anne Nelson, “Chromatographic Immunoassays”, Anal. Chem., 73 (2001) 198A-205A.
  2. William Clarke and David S. Hage, “Development of Sandwich HPLC Microcolumns for Millisecond Extractions of Analytes”, Anal. Chem., 73 (2001) 1366-1373.
  3. William Clarke, A. Roy Choudhuri and David S. Hage, “Analysis of Free Drug Fractions by Ultra-fast Immunoaffinity Chromatography”, Anal. Chem., 73 (2001) 2157-2164.
  4. David S. Hage, “High-Performance Affinity Chromatography: A Powerful Tool for Studying Serum Protein Binding”, J. Chromatogr. B, 768 (2002) 3-30
  5. William Clarke, John E. Schiel, Annette Moser and David S. Hage, “Analysis of Free Hormone Fractions by an Ultrafast Immunoextraction/Displacement Immunoassay: Studies Using Free Thyroxine as a Model System”, Anal.  Chem., 77 (2005) 1859-1866.
  6. Tao Jiang, Rangan Mallik and David S. Hage, “Affinity Monoliths for Ultrafast Immunoextraction”, Anal. Chem., 77 (2005) 2362-2372.
  7. Mary Anne Nelson, Arther Gates, Maud Dodlinger and David S. Hage, “Development of a Portable Immunoextraction/RPLC System for Field Studies of Herbicide Residues”, Anal. Chem., 76 (2004) 805-813.
  8. Annette C. Moser and David S. Hage, “Chromatographic Immunoassays”, In: Handbook of Affinity Chromatography, (D. S. Hage, Editor), CRC Press/Taylor & Francis, 2006, Chapter 29.
  9. Corey M. Ohnmacht, John E. Schiel and David S. Hage, “Analysis of Free Drug Fractions using Near Infrared Fluorescent Labels and An Ultrafast Immunoextraction/Displacement Assay”, Anal. Chem., 78 (2006) 7547-7556.

Fg. 1
General design of a sandwich microcolumn.

Fg. 2
Typical chromatogram obtained for the use of affinity microcolumns in an ultrafast immunoextraction/displacement assay for determining the free fraction of phenytoin in samples.