A general affinity chromatographic method for preparing monospecific antibody to mammalian serum haptoglobin

A general affinity chromatographic method for preparation of monospecific antibody to serum haptoglobin of any species is described. Hemoglobin prepared from the species to be immunized is coupled to an organomercurical substituted agarose gel support (Affi-Gel 501). The immobilized hemoglobin binds haptoglobin with great affinity and allows removal of other serum proteins by extensive washing. The haptoglobin-hemoglobin complexes are then specifically eluted by buffers containing dithiothreitol or other thiols and are further purified by chromatography on concanavalin A-agarose and Sephacryl S-200 columns. The pure complexes are very effective immunogens. Potent monospecific antisera to rabbit and to human haptoglobin have been prepared. The potential usefulness of affinity chromatography support media with specifically cleavable ligand sites in studies of haptoglobin and in other biological studies is discussed.