Practical Protein Chemistry - A. Darbre 1989

Affinity Chromatography of Proteins
General Techniques of Affinity Chromatography
Immobilized Antibodies

The so-called “immunoadsorption” principle was employed in one of the earliest studies on Affinity Chromatography. First, the purified antigen used for immunization is coupled to an insoluble matrix, after which the specific Antibodies present in the serum are captured on the resulting affinity sorbent. Desorption is carried out using a concentrated solution of the antigen or its fragments (haptens). If the latter are not available in sufficient quantities for elution, low-pH Buffer solutions or solutions containing guanidine-HCl or urea are used instead. This approach to immunoaffinity chromatography yields a heterogeneous population of antibodies or antibody-secreting lymphocytes. At the same time, preparing the immunosorbent and performing the elution require a substantial amount of purified antigen.

When the available amount of the target antigen is only sufficient for the initial immunization, an alternative and relatively recently developed variation of immunoaffinity chromatography is used. The resulting antibodies are immobilized on an insoluble matrix, and the antigen-containing extract is applied to the Column. Desorption is performed in the standard manner. However, when preparing the sorbent, as well as during Adsorption and Elution, one must take into account the high lability of antibodies in the presence of Denaturing Agents.

Monospecificity of antibodies is a crucial factor when employing them as ligands. Such antibodies can traditionally be obtained only by immunizing animals with highly purified antigen preparations. Recently, however, a more efficient Modification of the method has been developed to bypass these difficulties. Mice are immunized with a mixture of Antigens, antibody-producing Spleen Cells are harvested, and then fused with transformed myeloma cells. This yields stable, antibody-secreting Cell hybrids known as hybridomas. Each individual clone of such cells produces antibodies directed against only a single antigenic determinant of a specific antigen, thereby exhibiting strict monospecificity. The Monoclonal Antibodies produced by the hybridoma can be obtained in large quantities either from transformed cells in vitro or in vivo within the mouse peritoneal cavity. Antibodies obtained using this method have been successfully utilized for the Isolation and Purification of various lymphocyte surface antigens via affinity chromatography.

The efficacy of monoclonal antibodies is well demonstrated by the Isolation of the rat thymocyte common leukocyte antigen (L-C antigen) [7]. In The First stage, affinity chromatography on immobilized lentil lectin combined with Gel filtration in the presence of deoxycholate yields a partially purified preparation of thymocyte membrane glycoprotein with a Molecular Weight of 100,000 (representing a 400- to 900-fold purification degree). Following mouse immunization with this preparation, spleen cells producing antibodies against the L-C antigen are isolated and fused with myeloma cells to generate hybridomas. Cell clones producing monoclonal antibodies are screened using a radioactive-label binding assay combined with inhibition by the partially purified antigen preparation. Affinity chromatography on immobilized monoclonal antibodies yields a highly purified L-C antigen preparation, as confirmed by Electrophoresis. The relatively high antigen yield following column elution reported in [7] is attributed to the fact that monoclonal antibodies interact with only a single antigenic determinant on the antigen molecule, unlike conventional polyclonal antibodies directed against multiple distinct determinants. It should also be noted that mouse monoclonal antibodies typically exhibit a lower affinity for the antigen compared to hyperimmune rabbit antibodies.