Preparation of immunosorbents by cross-linking antigens

5% solution of GA is added in a ratio of 0.5 ml per 100 mg protein. The proteins should be at a concentration of about 20 mg/ml, in 200 mM sodium acetate buffer, pH 5.0, or in 100 mM phosphate buffer, pH 7.4. The gel, which forms almost immediately, is kept for 3 h at room temperature, homogenized with a Potter homogenizer in 200 mM phosphate buffer, pH 7.4 (20-40 

For every particular antigen-antibody system the maximum binding of antibody per ml of gel and the optimum conditions (ionic strength, pH, temperature, solvent conditions) for the elution of the antibodies should be determined in a systematic way in pilot studies. 

The column is washed with 500 mM phosphate buffer, pH 7.4, containing 100 mM NaCl, and antibodies are adsorbed in the same buffer. Best results are obtained (Eveleigh and Levy, 1977) by using a relative low ligand concentration (5 mg/ml) and 0.5% Tween 80 to eliminate non-specific interactions. 

Whole antiserum or physicochemically purified Ig is mixed with the immunosorbent in 100 mM phosphate buffer, pH 7.4 at 4°C overnight. Adsorbed antibodies are eluted batchwise, after washing of the adsorbent until the optical density of the supernatant is less than 0.05 at 280 nm. Alternatively, the immunosorbent may be packed in a column after removal of the fine particles (Avrameas and Ternynck, 1969), 

Best dissociation conditions vary with the antibodies (Section 8.2). Most techniques depend on the deformation of the interacting surfaces of the antibody and antigen, obtained with chaotropic ions (3.5 M potassium thiocyanate in 100 mM phosphate buffer, pH 6.6), organic acids with low surface tension (propionic acid, acetic acid. Section 8.2), denaturants (8 M urea, 7 M guanidine-HCl), and pH extremes (100 mM glycine-HCl, pH 2.5). 

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