Introduction

The binding between an antibody and its target ligand or antigen is among the strongest interactions known in biology. One of the consequences of this is that the specific antibody-ligand interaction will predominate and in many instances the presence of other molecules in the antibody preparation is of little consequence and prior purification is therefore not necessary. In some instances, however, purification is necessary or desirable as the other proteins in serum may interfere.

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

Successful protein purification results from the exploitation of one or more of the many characteristics responsible for the wide diversity within the protein population, such as charge, size, hydro-phobicity, or biological affinity. Fortunately, in some properties immunoglobulins are distinctive and as a consequence an acceptable degree of purity can often be achieved using relatively straightforward procedures.

One property readily exploited is the high isoelectric point (IEP) associated with the immunoglobulin family. They have an IEP in the region of 8.6 in contrast to most proteins, which have values typically in the region of pH 6.0-7.0, while that of the predominant serum protein and major "contaminant," serum albumin has an IEP less than 5. Therefore, at most pH values, the charge carried by immunoglobulins will differ from the majority of the other serum proteins, and this will allow the use of high capacity techniques, such as ion-exchange chromatography.

The exploitation of biological activity through affinity chroma-tography can be an extremely powerful purification method. A number of bacteria produce immunoglobulin-binding proteins as part of their defence against antibodies. Such proteins, the best-known of which is protein A from Staphylococcus aureus, bind to the constant region of the antibody molecule and thus their usefulness is independent of antibody specificity (1). In some circumstances the antigen, or a close structural analog, may be used as the basis for purification, but here the strong binding affinity may be counter productive because it is often difficult to displace the antibody without the use of denaturing conditions.

Other conventional methods of protein purification also have their place. Precipitation techniques, such as ammonium sulfate or polyethylene glycol fractionation, do not give a high degree of purity on their own but do concentrate the protein and in combination with other techniques, such as ion-exchange chromatography, can be very useful. Separation on the basis of size is not a high capacity technique but can be applicable in the purification of IgM, which, with a molecular weight approaching a million, is much larger than the majority of other serum proteins.

There is no one procedure or combination of procedures that is applicable to all cases. The class of antibody to be purified has to be considered. Conventional procedures for the production of polyclonal antisera will generally result in immunoglobulin G, and procedures for the generation of monoclonal antisera may result in any of the immunoglobulin classes. Immunoglobulins from different species have broadly similar properties, but protein A, for example, will not bind all of the subclasses of human IgG, nor is it particularly effective in the purification of IgG from rat, sheep, or goat (2). Thus a certain amount of trial and error may be required to reach the best protocol for a particularly demanding application, but the methods described here should provide an adequate purity for most purposes.

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