Separation and characterization of anti-benzylpenicilloyl (BPO) antibodies. I. Biochemical and biophysical properties of anti-BPO-IgG obtained by affinity and subsequent ion-exchange chromatography.
Anti-BPO antibodies were purified by means of affinity chromatography using AH-Sepharose 4B coated with covalently bound BPO groups. Specific elution was achieved by the hapten analogue BPO-epsilon-aminocaproic acid (BPO-EACA); desorption of the remaining antibody was performed thereafter by 0.1 M acetic acid. The resulting antibody fractions--hapten-eluted antibody (H-Ab) and acid eluted antibody (A-Ab), respectively--were further separated by ion-exchange chromatography which led to the appearance of 3 subfractions in the case of H-Ab (H1, H2, H3) and 2 subfractions in the case of A-Ab (A1 and A2). In liquid isoelectrofocusing an inhomogeneous pattern resulted. The bulk of antibodies focused between pH 6.5 and 7.0. The average avidity of H-Ab was found to be higher than that of A-Ab suggesting that avidity may influence the elution pattern in affinity chromatography. The hydrophobic influence of the "spacer" and/or interactions of antibodies directed against the hydrophobic regions of the BPO group may explain why a considerable part of the antibodies could be recovered from the immunosorbent only by acid elution.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000941:Antigens; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003956:Dialysis; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D010400:Penicillin G / Q000276:immunology*; D011817:Rabbits
D000906:Antibodies; D000941:Antigens; D007106:Immune Sera; D007074:Immunoglobulin G; D010400:Penicillin G