Annette C. Moser

Immunoaffinity chromatography: an introduction to applications and recent developments

Immunoaffinity chromatography (IAC) combines the use of LC with the specific binding of antibodies or related agents. The resulting method can be used in assays for a particular target or for purification and concentration of analytes prior to further examination by another technique. This review discusses the history and principles of IAC and the various formats that can be used with this method. An overview is given of the general properties of antibodies and of antibody-production methods. The supports and immobilization methods used with antibodies in IAC and the selection of application and elution conditions for IAC are also discussed. Several applications of IAC are considered, including its use in purification, immunodepletion, direct sample analysis, chromatographic immunoassays and combined analysis methods. Recent developments include the use of IAC with CE or MS, ultrafast immunoextraction methods and the use of immunoaffinity columns in microanalytical systems.

Capillary electrophoresis-based immunoassays: Principles and quantitative applications

The use of CE as a tool to conduct immunoassays has been an area of increasing interest over the last decade. This approach combines the efficiency, small sample requirements, and relatively high speed of CE with the selectivity of antibodies as binding agents. This review examines the various assay formats and detection modes that have been reported for these assays, along with some representative applications. Most CE immunoassays in the past have employed homogeneous methods in which the sample and reagents are allowed to react in solution. These homogeneous methods have been conducted as both competitive binding immunoassays and as noncompetitive binding immunoassays. Fluorescent labels are most commonly used for detection in these assays, but enzyme labels have also been utilized for such work. Some additional work has been performed in CE immunoassays with heterogeneous methods in which either antibodies or an analog of the analyte is immobilized to a solid support. These heterogeneous methods can be used for the selective isolation of analytes prior to their separation by CE or to remove a given species from a sample/reagent mixture prior to analysis by CE. These CE immunoassays can be used with a variety of detection modes, such as fluorescence, UV/Vis absorbance, chemiluminescence, electrochemical measurements, MS, and surface plasmon resonance.

Evaluation of alternatives to warfarin as probes for Sudlow site I of human serum albumin: Characterization by high-performance affinity chromatography

Warfarin is often used as a site-specific probe for examining the binding of drugs and other solutes to Sudlow site I of human serum albumin (HSA). However, warfarin has strong binding to HSA and the two chiral forms of warfarin have slightly different binding affinities for this protein. Warfarin also undergoes a slow change in structure when present in common buffers used for binding studies. This report examined the use of four related, achiral compounds (i.e., coumarin, 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, and 4-hydroxycoumarin) as possible alternative probes for Sudlow site I in drug binding studies. High-performance affinity chromatography and immobilized HSA columns were used to compare and evaluate the binding properties of these probe candidates. Binding for each of the tested probe candidates to HSA was found to give a good fit to a two-site model. The first group of sites had moderate-to-high affinities for the probe candidates with association equilibrium constants that ranged from 6.4 x 10(3)M(-1) (coumarin) to 5.5 x 10(4)M(-1) (4-hydroxycoumarin) at pH 7.4 and 37 degrees C. The second group of weaker, and probably non-specific, binding regions, had association equilibrium constants that ranged from 3.8 x 10(1)M(-1) (7-hydroxy-4-methylcoumarin) to 7.3 x 10(2)M(-1) (coumarin). Competition experiments based on zonal elution indicated that all of these probe candidates competed with warfarin at their high affinity regions. Warfarin also showed competition with coumarin, 7-hydroxycoumarin and 7-hydroxy-4-methycoumarin for their weak affinity sites but appeared to not bind and/or compete for all of the weak sites of 4-hydroxycoumarin. It was found from this group that 4-hydroxycoumarin was the best alternative to warfarin for examining the interactions of drugs at Sudlow site I on HSA. These results also provided information on how the major structural components of warfarin contribute to the binding of this drug at Sudlow site I.

Evaluation of indole‐based probes for high‐throughput screening of drug binding to human serum albumin: Analysis by high‐performance affinity chromatography

There has been growing interest in the use of rapid and selective separation methods such as high-performance affinity chromatography (HPAC) or affinity capillary electrophoresis (ACE) for the characterization of drug-protein interactions. L-Tryptophan is commonly used in these and other methods as a site-selective probe for examining the binding of small solutes and drugs at Sudlow site II on the protein HSA. However, solutions of L-tryptophan can be unstable and are generally prepared fresh daily for these studies. In this report, HPAC was used to examine other indole compounds as possible replacements for L-tryptophan as a site-selective probe for use in the high-throughput screening of drug binding to HSA; the implications of these results in the use of such compounds in ACE were also considered. The probe candidates that were tested included indole-3-acetic acid, indole-3-carboxylic acid, indole-3-butyric acid, indole-3-propionic acid, indole-3-methanol, 3-acetylindole, and 3-methylindole. All of these compounds were found by (1)H NMR and UV-Vis spectroscopy to be stable for up to 3 wk at room temperature when kept in a pH 7.4, 0.067 M phosphate buffer. The binding of these compounds was examined by using columns that contained immobilized HSA. 3-Acetylindole was found to be the best candidate in this group for use as an alternative probe to L-tryptophan for Sudlow site II. This probe had the same binding site and a similar affinity to L-tryptophan but was more stable in aqueous solution, making it suitable for high-throughput screening of drug-HSA binding in both HPAC and ACE.

BIOINTERACTION ANALYSIS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: KINETIC STUDIES OF IMMOBILIZED ANTIBODIES

A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7-12x10(6)M(-1) at pH 7.0 and 25 degrees C. Split-peak analysis gave association rate constants of 1.4-12x10(5)M(-1)s(-1) and calculated dissociation rate constants of 0.01-0.4s(-1) under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056-0.17s(-1). A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4x10(-4)s(-1). This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports.

Biointeraction analysis of immobilized antibodies and related agents by high-performance immunoaffinity chromatography

A method is described based on high-performance immunoaffinity chromatography for examining the interactions of immobilized antibodies or related binding agents with their targets. It is shown how this method can be used to obtain information on the binding, elution and regeneration kinetics of immobilized binding agents, such as those used with immunoaffinity supports. The theory behind this approach is briefly described and it is demonstrated how both the kinetic and thermodynamic properties of a biointeraction can be determined experimentally through this method. Several applications are used to illustrate this technique, including antibody-antigen interactions and the binding of aptamers with their targets in the presence of silica-based supports. The same approach can be adapted for use with other types of targets, binding agents and support materials.

Clinical applications of capillary electrophoresis based immunoassays

Immunoassays have long been an important set of tools in clinical laboratories for the detection, diagnosis, and treatment of disease. Over the last two decades, there has been growing interest in utilizing CE as a means for conducting immunoassays with clinical samples. The resulting method is known as a CE immunoassay. This approach makes use of the selective and strong binding of antibodies for their targets, as is employed in a traditional immunoassay, and combines this with the speed, efficiency, and small sample requirements of CE. This review discusses the variety of ways in which CE immunoassays have been employed with clinical samples. An overview of the formats and detection modes that have been employed in these applications is first presented. A more detailed discussion is then given on the type of clinical targets and samples that have been measured or studied by using CE immunoassays. Particular attention is given to the use of this method in the fields of endocrinology, pharmaceutical measurements, protein and peptide analysis, immunology, infectious disease detection, and oncology. Representative applications in each of these areas are described, with these examples involving work with both traditional and microanalytical CE systems.

Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA-Immobilized Enzymes

Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.

Studies of Antibody-Antigen Interactions by Capillary Electrophoresis: A Review

Antibody-antigen interactions are vital in immunoassay development and can determine detection limits and analysis times. Capillary electrophoresis (CE) is a powerful technique that can be used to quantify antibody-antigen interactions. These CE methods range from simple separations of a premixed antibody and antigen sample applied as a short plug to allow for separation of complex, free antibody, and free antigen to more complex systems which inject complexed samples in the presence of antibody or antigen; or even injections of antibody and antigen sequentially. The objective of this review is to identify and describe various CE techniques which have been used to study antibody-antigen interactions. A brief discussion of linear and nonlinear curve fitting is also included.