Immunochromatography on insolubilized antibodies of very low affinity: application to immunoadsorbence of bovine alpha-fetoprotein.
Cross-reactive antibodies were utilized to prepare immunoadsorbents possessing a very low affinity to bovine alpha-fetoprotein (AFP). A goat anti-human AFP serum cross-reactive with bovine AFP was first depleted of antibodies reactive with bovine AFP in immunodiffusion. The remaining antibodies from this serum and gamma-globulin from a sheep antiserum against rabbit AFP, without prior absorption, were coupled to Sepharose. Chromatography of fetal calf serum on these adsorbents resulted in retardation of bovine AFP relative to other proteins. A major part of the AFP eluted from the columns with phosphate-buffered saline. The rest eluted as a sharp peak with a small quantity of 4 or 6 M urea. The elution of AFP with the initial column buffer has made it possible to prepare pure AFP that has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. Elimination of the washing step and the ease of elution has allowed purification of gram amounts of AFP. The fact that immunoadsorbents prepared from antibodies with no detectable reactivity in immunodiffusion still caused delayed elution in chromatography suggests that this procedure may be useful in search of proteins cross-reactive with a known protein.
D000818:Animals; D000906:Antibodies; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex; D002417:Cattle; D003429:Cross Reactions; D006041:Goats; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D005779:Immunodiffusion; D007164:Immunosorbents; D011817:Rabbits; D012756:Sheep; D012995:Solubility; D000509:alpha-Fetoproteins / Q000276:immunology* / Q000302:isolation & purification
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D007164:Immunosorbents; D000509:alpha-Fetoproteins