Affinity chromatography antibody—enzyme

Some typical biological interactions, frequently used in affinity chromatography are enzyme to substrate analogue, inhibitor, or cofactor antibody to antigen, virus, or cell lectin to polysaccharide, glycoprotein, cell surface receptor, or cell nucleic acid to complementary base sequence, histones, or nucleic acid polymerase hormone or vitamin to receptor, or carrier protein glutathione to glutathione-S-transferase (GST) or GST fusion proteins and metal ions to poly (His) fusion proteins, or native proteins with histidine, cysteine and/or tryptophan residues on their surfaces. 

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

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. 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

Compared with enzymes fewer reports are available on immobilization of antibody (Ab) in sol-gels and their applications in immunosensing. Immobilization of Abs on a solid support was first reported in 1967 [128] and the technology has widespread application in affinity chromatography and other areas. However, the major problem associated with covalent immobilization of antibody on solid surface is partial loss of biological activity due to the random orientation of the asymmetric macromolecules,... 

Instead of dialysis or gel filtration, an affinity chromatography on Concanavalin A Sepharose (cf. Protocol 3.6.2.4) is recommended, because on the one hand, no conjugated antibody is removed, and on the other hand, the sugar used for elution stabilizes the enzyme-antibody conjugate in solution. 

The ligands used for enzyme purification can be specific to the desired enzyme (substrate, substrate analogue, enzyme inhibitor, antibody), specific for different classes of enzyme (AMP, NAD, PLP) or of limited predefined specificity (dye affinity chromatography withProcion, Cibacron dyes). 

Affinity chromatography can be applied to the isolation and purification of virtually all biological macromolecules. It has been used to purify nucleic acids, enzymes, transport proteins, antibodies, hormone receptor proteins, drug-binding proteins, neurotransmitter proteins, and many others. 

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