AFFINITY PURIFICATION OF IMMUNOGLOBULIN-M USING A NOVEL SYNTHETIC LIGAND

While monoclonal antibodies of the G class can be conveniently purifie d by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several pro blems, since specific and cost-effective ligands for IgM are not avail able. A synthetic peptide (TG19318), deduced from the screening of a c ombinatorial peptide library, was characterized previously by our grou p for its binding properties for immunoglobulins of the G class and it s applicability as a synthetic ligand for polyclonal and monoclonal Ig G purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobiliz ing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very conve nient one-step purification of monoclonal IgMs directly from crude sou rces, loading the samples on the columns equilibrated with saline buff ers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a bu ffer change to 0.1 M acetic acid or 0.05-0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85-95%, as determined by densitometric scanning of sodium dodecyl s ulfate-polyacrylamide gels of purified fractions, and by gel permeatio n analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support. (C) 1998 Elsevier Science B.V. All rights reserv ed.