Affinity chromatography elution conditions

Affinity chromatography A form of chromatography in which separation is achieved by utilizing highly specific biochemical interactions, such as steric-or charge-related conditions, between me analyte and a molecule immobilized on a column. It is different from most forms of chromatography in mat analytes do not continuously elute from me column - only mose mat interact wim me stationary phase are retained and mus separated from omer components of me mixture under investigation. These immobilized materials are eluted from me column after all omer materials have been removed. 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

Initially, the antibodies should be purified prior to prepare the immunoaffinity column. Precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration chraoma-tography or affinity chromatography may be employed with the aim of antibody purification. Activated beads which are coated with bacterial proteins A or G may be used as the support material. Some parameters may be changed for the elution of the sample solution for example the ionic conditions of mobile phase may be changed or chaotropic buffers may be used [11]. 

Molecular exclusion chromatography is based on the inability of large molecules to enter small pores in the stationary phase. Small molecules enter these pores and therefore exhibit longer elution times than large molecules. Molecular exclusion is used for separations based on size and for molecular mass determinations of macromolecules. In affinity chromatography, the stationary phase retains one particular solute in a complex mixture. After all other components have been eluted, the desired species is liberated by a change in conditions. 

Successful application of affinity chromatography requires careful design of experimental conditions. The essential components, which are outlined below, are (1) creation and preparation of a stationary matrix with immobilized ligand and (2) design of column development and eluting conditions. 

Isolation of IgG by affinity chromatography involves application of serum to a column of matrix-bound ligand, washing to remove non-IgG components and elution of IgG by changing the conditions such that the ligand-IgG interaction is disrupted. 

Adsorption techniques such as ion exchange, HIC, chromatofocusing, reversed-phase chromatography, and affinity chromatography can have high capacity factors because experimental conditions can be manipulated so that the elution volume for a peak can exceed the total bed volume (VM as is the case with peaks 2 and 3 in Fig. B4.2.4.) However, in gel filtration, which is a nonad-sorptive technique, all peaks must elute within the volume Vy- V(h as is the case with peak 1 in Fig. B4.2.4. 

An example of virus clearance factors in chromatographic processes frequently used for purification of antibodies is given in Table 17. Lower clearance factors for protein A affinity chromatography have been found by Mariani and Tarditi128 when compared to results found with protein G by Walter and Allgaier.237 An explanation of this fact can be found in the fact that protein G requires harsher elution conditions than protein A. 

With a ready source of immunogen in the form of peptide, it is straightforward to purify antipeptide antibodies by affinity chromatography. The peptide is covalently coupled to agarose and the crude antibody passed down the column. Unbound material is washed away and the bound antibody eluted under denaturing conditions, for example, low pH (pH 2.5), high pH (pH... 

Following affinity chromatography of the MIANS-ricin A-chain digest over the anti-MIANS column, the eluted fraction was run over the C,g RP-HPLC column utilizing gradient elution conditions. Individual peptides were collected for amino acid analysis (data not shown) and for sequencing. 

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