Immunoglobulin affinity column

Purification of immunoglobulins has usually involved complex fractionation procedures that have yielded results that have not always been entirely satisfactory, particularly when the immunoglobulins such as IgE are present in low concentrations. In addition to the use of antiimmunoglobulin affinity columns [143-145], other procedures have also been developed. 

Suitable affinity columns of this type have been designed for use with HPLC and this offers an extremely rapid method of analyzing or purifying antibodies. However, the method will detect any mammalian immunoglobulin which means that the immunoglobulin content of the serum used in the growth medium may interfere with analysis. 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

Figure 26-16 shows the isolation of the protein immunoglobulin G (IgG) by affinity chromatography on a column containing covalently bound protein A. Protein A binds to... 

The complement protein, Cl q, has an 18-fold higher affinity for pentamenc IgM than IgG. This property may be used to purify IgM selectively from MAb ascites. Immobilized Cl q binds IgM at 4°C, and the immunoglobulin is eluted simply and isocratically (using the same buffer) by bringing the column to room temperature for 2 h. 

Protein A or Protein G affinity chromatography Immunoglobulins show specific affinity for these proteins which can be obtained complexed to Sepharose (Pharmacia). It is a simple matter so apply antiserum diluted in 20 mM phosphate buffer to a small column and subsequently elute the pure IgG using a glycine buffer pH 2.7. 

The ideal column of immunoaffinity chromatography would be made using monoclonal anti-immunoglobulin antibodies. These might be selected for their appropriate epitope affinity, specificity, and association constant to release the monoclonal antibody under defined gentle, nondenaturing conditions. 

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