Preparation and Use of Immunoaffinity Columns with Monoclonal Antibodies Without Purification from Ascites and Tissue-Culture Medium

Until recently, the primary methods available for preparing immunoaffinity columns with both polyclonal and monoclonal antibodies (MAbs) have required purification of the antibodies from serum, ascites, or tissue-culture medium prior to coupling to the affinity matrix beads or gel. Advances in the chemistry of selective coupling of molecules present in complex solutions to matrices, however, have introduced methods that are especially useful for preparing immunoaffinity columns with MAbs. Also, with pure MAbs, this activated gel may be an attractive alternative to the often-used CNBr-activated Sepharose, because the chemistry is different. First, the resulting covalent linkage is stable at high and low pH in contrast to the unstable linkage formed by CNBr-activated Sepharose. Second, the vinyl group—the active partner—is less reactive, resulting in a lower degree of crosslinking, a phenomenon that might destroy the binding activity of the antibody in the process of coupling and immobilization.