University of North Florida
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Contact Info

Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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David S. Hage

Abbrev:
Hage, D.S.
Other Names:
Address:
Department of Chemistry, University of Nebraska, 738 Hamilton Hall, Lincoln, NE 68588 USA
Phone:
402-472-2744
Fax:
402-472-9402

Citations 6

"Automated Protein Assay Using Flow Injection Analysis"
J. Chem. Educ. 1998 Volume 75, Issue 8 Pages 1025-1028
Carrie A. C. Wolfe, Matthew R. Oates and David S. Hage

Abstract: A simple flow injection analysis system is described that can be used in undergraduate instrumental anal. labs for performing bicinchoninic acid protein assays. This system can either be used to illustrate the principles of flow injection anal., or it can serve as a means for introducing students to a common method used in analyzing biological related samples.
Bicinchoninic acid Protein Spectrophotometry Education

"Affinity Chromatography: A Review Of Clinical Applications"
Clin. Chem. 1999 Volume 45, Issue 5 Pages 593-615
David S. Hage

Abstract: Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific: analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Same traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on-or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes.

"Chromatographic Competitive Binding Immunoassays: A Comparison Of The Sequential And Simultaneous Injection Methods"
Biomed. Chromatogr. 2003 Volume 17, Issue 2-3 Pages 188-200
Mary Anne Nelson, Wanda S. Reiter, David S. Hage*

Abstract: Two approaches for performing competitive binding immunoassays by HPLC and other flow-based systems are the simultaneous and sequential injection methods. Both these techniques make use of a column with a limited amount of antibody, onto which is injected a sample and a fixed amount of a labeled analyte analog. An indirect measure of the unlabeled analyte in the sample is then obtained by looking at the amount of analog in either the nonretained or retained peaks. In the simultaneous injection mode, the sample and labeled analog are applied at the same time to the column, while in the sequential mode the sample is injected first, followed by the analog. This results in a difference in the analytical characteristics of these two approaches. This study used chromatographic theory and previous data obtained for injections of human serum albumin (HSA) onto an anti-HSA antibody column to compare the response, detection limits, range, and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method always provided the best lower limit of detection and sensitivity. However, the simultaneous mode had a broader dynamic range and higher upper limit of detection. From these observations, several guidelines were developed regarding the use and selection of such assays for new applications.

"Chromatographic Immunoassays"
Anal. Chem. 2001 Volume 73, Issue 7 Pages 198A-205A
David S. Hage and Mary Anne Nelson

Abstract: When a simple and selective method is required, there are few analytical techniques that compare with immunoassays. This group of methods uses antibodies or antibody-related molecules as reagents for chemical analysis (1, 2). Immunoassays are among the most specific of the analytical techniques, provide low detection limits, and can be used for a wide range of substances. These features, plus the fact that many samples need little or no pretreatment, have made immunoassays common in clinical testing, pharmaceutical analysis, environmental monitoring, and food safety testing. There are many ways in which immunoassays can be performed, but a recent approach based on LC, known as a chromatographic or flow immunoassay, has received particular attention (3 10). We examine this method s basic principles, discuss various formats in which it can be performed, and describe the potential advantages, limitations, and applications.
Preconcentration

"Development Of A Theoretical Model For Chromatographic-based Competitive Binding Immunoassays With Simultaneous Injection Of Sample And Label"
Anal. Chem. 1999 Volume 71, Issue 15 Pages 2965-2975
David S. Hage, David H. Thomas, A. Roy Chowdhuri, and William Clarke

Abstract: This study examined the theory and behavior of an HPLC-based chromatography competitive binding immunoassay with the simultaneous injection of sample and a labeled analyte analog. Equations based on nonlinear chromatography theory were derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. These equations related the assay response (B/Bo) to the columns binding capacity, the moles of injected analyte or labeled analog, and the flow rate/adsorption kinetics of the system. There was good agreement between the predicted theoretical response and experimental data obtained for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. This theory was also successful in describing the changes that occurred in the calibration curve when the flow rate or amt. of labeled analog applied to the column was varied. A comparison was made between the results of this study and previous theoretical work that examined the behavior of a related, sequential injection competitive binding method. On the basis of the results reported in this work, several general guidelines were developed for the design and optimization of simultaneous injection methods for use in such areas as clinical testing, pharmaceutical anal., and environmental monitoring.
Adsorption

"Determination Of Urinary Albumin Using High Performance Immunoaffinity Chromatography And Flow Injection Analysis"
Anal. Chem. 1994 Volume 66, Issue 23 Pages 4265-4271
Peggy F. Ruhn, Jay D. Taylor, and David S. Hage

Abstract: The detection of low levels of albumin in urine can be an important aid in the early diagnosis of kidney disease. In this study, an automated system for urine analysis was developed that could simultaneously measure both albumin and creatinine, an indicator of urine output and volume. Albumin was determined by use of a column that contained immobilized anti-albumin antibodies. An online flow injection analysis system was used to measure creatinine as this solute eluted nonretained from the antibody column. The total analysis time of the system was 5 min per injection. Limits of detection for albumin and creatinine in a 20 µL sample (at S/N = 2) were 3 and 5 mg/L, respectively. The response for these compounds covered the range of clinical interest. Within-run precision for both analytes was±1-2% at normal urine concentrations. The results of this method showed good agreement with those obtained by reference techniques. This approach can be adapted for the detection of other urine components by changing the type of antibody column used in the system.
Albumin Creatinine Urine LC Immunoassay Method comparison Immobilized antibody