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Handbook of Affinity Chromatography - CRC Press Book

The Handbook of Affinity Chromatography reflects upon important factors to consider in the development of affinity methods, such as the choice of support material, immobilization methods, and application or elution conditions. It reviews common affinity methods and explores the latest preparative, analytical, and biophysical applications, including the use of affinity chromatography with other separation techniques and analytical systems. This basis seamlessly supports the discussion of recent developments in techniques including the use of affinity ligands in capillary electrophoresis, mass spectrometry, microanalytical systems, and optical biosensors.


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New chapters feature expanded discussions on molecularly imprinted polymers and biomimetic ligands, chromatographic immunoassays, affinity-based immunoassays, affinity-based chiral stationary phases, and affinity ligands in multidimensional systems. Engaging the collaboration of 48 scientists and students from 23 laboratories and organizations to present the latest information on affinity methods, the Handbook of Affinity Chromatography illustrates a wide range of applications and theory for scientists, students, and laboratory workers throughout the fields of chemistry and biology.

An alternative approach that can be used with systems that have weak-to-moderate strength interactions is to use small affinity columns and fast flow rates, which can allow this type of study to be conducted without the need for a displacing agent 59 , In addition, a step change in a factor such as the mobile phase pH can sometimes be used to promote rapid release of the solute, as might be used to compare elution conditions for an affinity column 17 , The peak decay method has been used in HPAC with small affinity columns to determine the dissociation rates of AGP for amitriptyline, lidocaine, and nortriptyline This method has also been used with affinity monolith columns to determine the dissociation rate constants for HSA with a number of drugs 67 , A pH step change has been employed with the peak decay method to characterize the dissociation rate of thyroxine from antibodies and aptamers that can bind to this hormone The split-peak method is a technique that can be used with affinity columns to look at systems with strong binding and relatively slow rates of dissociation 7 , 8 , In this technique conditions are used in which only part of an injected solute has sufficient time to bind to the affinity column.

The result is a situation in which the injection of even a pure sample of the solute results in 2 peaks, one of which is highly retained and the second of which elutes as a nonretained fraction. The change in the relative size of these peaks is then examined as the injection flow rate is varied and used to determine the association rate constant or mass transfer rate for the solute in the column 7 , 8 , The split-peak method has been used in several studies to examine the binding of immunoglobulins to small affinity columns that contain protein A or protein G 59 , This method has been used to optimize a clinical assay based on HPAC for the rapid measurement of IgG-class antibodies in serum by using protein A columns Forms of this method have also been employed to examine the binding of HSA to immobilized anti-HSA antibodies and to characterize the rate of binding by thyroxine with antibodies or aptamers 59 , Ultrafast affinity extraction is a newer method that can examine both the rate and degree of binding for a biological interaction 59 , 71 — This method looks at an interaction in solution through the use of a small affinity column that can rapidly capture the nonbound form of a solute in an injected mixture of this solute and a soluble binding agent.

The relative size of the captured fraction is used to determine the rate or extent of the biological interaction. The immobilized agent used to capture the free form of the solute may be an antibody 72 , 74 , 75 or a more general binding agent such as HSA i. Conditions are used so that the time the sample spends in the column is small or comparable to the time needed for dissociation of the solute from its soluble binding agent.

This often involves the use of affinity microcolumns and flow rates that produce sample residence times in the column that span from the low millisecond range up to a few seconds Ultrafast affinity extraction has been used for systems that range from weak-to-strong binding 59 , 72 , For instance, this method has been used with columns that contained immobilized antibodies for determining the free fraction of warfarin in mixtures of this drug with HSA 74 and, in combination with flow-based displacement immunoassays, the free fractions of thyroxine and phenytoin in clinical samples 72 , This method has been used with columns containing HSA to measure the equilibrium constants for a soluble form of this protein with R- or S- warfarin, S- ibuprofen, and imipramine Ultrafast affinity extraction has also been used in a 2-column system and with a chiral stationary phase to measure the free fractions and binding constants of R - and S - warfarin in drug-protein mixtures and serum Fig.

This particular example uses an affinity microcolumn containing immobilized HSA for ultrafast affinity extraction. Reproduced from Zheng et al. This review discussed a variety of ways in which affinity chromatography has been used to examine biological interactions of interest in clinical or pharmaceutical analysis. Particular attention was given to more recent work and techniques that have involved the use of HPAC. The general principles behind techniques such as zonal elution and frontal analysis were described, showing how these approaches can be used to examine the strength of a biological interaction, the number and types of sites that are involved in these processes, and the effects of one solute on another during these interactions.

Several means for examining the kinetics of a biological interaction by HPAC and affinity chromatography were also considered. Systems that have been examined by these methods have ranged from the binding of drugs and hormones to proteins or receptors to the analysis of antibody-antigen, enzyme-inhibitor, and sugar-lectin interactions 6 — 10 , 12 , There are many features of these methods that have made them attractive for such work.

For instance, as shown in this review, these techniques can be used with many binding agents or formats and can be coupled with numerous detection methods, spanning from absorbance measurements to MS 8 — 10 , This is often possible using approaches with label-free detection, although these methods can also be combined with suitable tags for the study of trace analytes 8 , 72 , The speed and precision of these methods, especially when used in HPAC, are other valuable features. The ability to often reuse the same immobilized binding agent i.

Many of the recent developments in this field will probably continue and further expand the capabilities of these methods. For instance, it is expected that columns based on monoliths and supports for ultraperformance liquid chromatography will continue to be adapted for use in HPAC and affinity chromatography 6 , 10 , 20 — This should allow even more rapid assays to be created with these methods and will provide greater ease in coupling affinity columns with MS or other analytical techniques 5 , 12 , 19 , 38 — Further work in the miniaturization of affinity columns and systems is also anticipated 5 , 10 , 23 , This work has already led to the possibility of carrying out binding studies on relatively exotic binding agents e.

These efforts, in turn, have resulted in the proposed use of HPAC as a tool in personalized medicine Another trend that is expected to continue is the creation of new formats for affinity-based binding assays. A specific example that was described is ultrafast affinity extraction, which can provide a direct measure of the strength or rate of biological interactions in solution and which can even examine these processes directly in clinical samples 59 , 72 — The combination of these recent tools and formats with those that are already available should result in even more future applications for affinity chromatography and HPAC in the characterization of biological interactions for clinical studies or pharmaceutical analysis.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: a significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; b drafting or revising the article for intellectual content; and c final approval of the published article.

Chromatographic Science: Handbook of Affinity Chromatography Vol. 63 (1993, Hardcover)

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Research Funding: D. Skip to main content. Review Article Review. David S. DOI: View this table: View inline View popup. Table 1. Comparison of data obtained by frontal analysis for examining the binding of R -propranolol and S -propranolol with an HPAC column containing immobilized LDL left and a general model showing the types of interactions each of these enantiomers had with LDL right.

Site-Specific Binding and Competition Studies Another use of zonal elution in HPAC and affinity chromatography has been in characterizing the competition that one compound may have with another for a given binding agent. Screening Drug Candidates A related application of HPAC and affinity chromatography has been as a tool for screening drug candidates and potential binding partners for biological targets.


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Characterization of Binding Sites The use of affinity chromatography and HPAC in competition studies is one way in which the interaction sites of a particular drug or solute can be identified and studied on a protein or other type of binding agent 8 , Kinetic Methods Yet another way affinity chromatography and HPAC can be used is to provide data on the kinetics of a biological interaction. Scheme for the simultaneous isolation of a free drug fraction and separation of the various chiral forms of a drug in this fraction by using ultrafast affinity extraction and an HPLC chiral stationary phase.

Conclusion and Future Directions This review discussed a variety of ways in which affinity chromatography has been used to examine biological interactions of interest in clinical or pharmaceutical analysis. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: D.

Affinity Chromatography: A Historical Perspective

Hage, Eastern Analytical Symposium and Abbvie. Expert Testimony: None declared. Patents: None declared. Other Remuneration: D. Hage, Elsevier. References 1. Tietz textbook of clinical chemistry and molecular diagnostics. St Louis MO : Saunders ; Clarke W , editor. Contemporary practice in clinical chemistry. Williams MA , Daviter T , editors. Protein-ligand interactions, methods and applications. New York : Springer ; Biophysical chemistry, part 2: techniques for the study of biological structure and function, part 2.

San Francisco : Freeman ; Emerging technologies for fragment screening.

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Fragment-based drug discovery: lessons and outlook. Hage DS , editor. Handbook of affinity chromatography , 2nd Ed. Chaiken IM. Analytical affinity chromatography. Quantitative affinity chromatography: practical aspects. In: Hage DS , editor.

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Quantitative affinity chromatography: recent theoretical developments. Analysis of biomolecular interactions using affinity microcolumns: a review.

Affinity chromatography

Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: binding of glimepiride to normal or glycated human serum albumin. J Chromatogr A ; : — Schriemer DS.

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Biosensor alternative: frontal affinity chromatography. Anal Chem ; 76 : A — 8A. Entrapment of alpha 1 -acid glycoprotein in high-performance affinity columns for drug-protein binding studies. J Chromatogr B ; : — Identification and analysis of stereoselective drug interactions with low density lipoprotein by high-performance affinity chromatography. Anal Biochem Chem ; : — Analysis of drug interactions with high-density lipoprotein by high-performance affinity chromatography.

Anal Biochem ; : — Analysis of drug interactions with very low density lipoprotein by high-performance affinity chromatography. On-column entrapment of alpha 1 -acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography. Anal Bioanal Chem ; : — Chromatographic analysis of drug interactions in the serum proteome.