Immunoaffinity chromatography can be considered as a subset of affinity chromatography, in which an immunoglobulin is used to bind analyte molecules. Because immunoglobulins show exquisite specificities towards antigens used in their production, immunoaffinity columns are ideal for the purification of the targeted analytes. Either monoclonal or polyclonal antibodies, produced using a variety of techniques (1), may be used as a source of immunoglobulin for immunoaffinity columns.

Immunoaffinity columns are extremely versatile and have been used for the isolation and concentration of a diverse number of analytes from a wide array of matrices (2). Analytes may include macromolecules such as proteins and receptors or small molecules such as environmental toxins, antibiotics, or pesticides. Matrices may include animal tissues or excreta, plant extracts, cell culture medium, or virtually any milieu encountered in biological work. Because of its value as a research tool, immunoaffinity chromatog-raphy has found extensive use by the pharmaceutical industry to purify therapeutic proteins, the food safety community to purify small amounts of toxins from food and as a general tool for analytical chemists to purify analytes for subsequent instrumental analysis.

The general principle of immunoaffinity chromatography is illustrated in Fig. 1. The analyte in the sample matrix is loaded onto the column, the column is washed to remove interfering substances, and the analyte is eluted from the column for subsequent use. The column is the heart of the purification system and must bind the analyte specifically enough to allow other substances to be rinsed off the column, allow the elution of the analyte under conditions that do not elute interferences, and permit the column to be regenerated multiple times for subsequent use.

Important considerations when generating immunoaffinity columns include the conjugation of antibody to the supporting (column) material, packing the column, developing suitable washing and elution protocols, and constructing appropriate tests to evaluate column performance. Of primary importance to the utility of an

Fig. 1. Diagram of immunoaffinity column.

immunoaffinity column is the suitability of the antibody selected for immobilization onto the column support. The antibody should have adequate affinity toward the analyte so that analyte binding occurs with sufficient selectivity as to not bind interfering matrix components. Solvent conditions under which the analyte is eluted from the affinity column must be of sufficient strength to remove all the analyte, but mild enough so that the antibody is not denatured during elution. The generation of a suitable antibody is not the subject of this chapter, but there is a vast amount of literature on this subject (3,4).

Conjugation of the antibody to a supporting material should allow for a column with flow properties compatible with low-pressure chromatography and the antibody should retain its essential analyte binding characteristics. The selected chromatographic support must have low levels of nonspecific binding, a high surface area, and good liquid flow properties. In addition, the support must be capable of being modified with a bifunctional molecule; one end of the molecule will react to the support and the other end will react with the antibody. This discussion will focus on the use of cyanogen bro mide-activated Sepharose which has been designed to react with free amino groups (i.e., lysine) present in antibodies. A number of alternate supports, listed in Table 1, are available (see Note 1) that bind with different functional groups found on the antibody, most commonly either amino groups or carbohydrates located on the Fc portion of the antibody. The procedure to use these supports may be found in the company literature or on their website.

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