sepharose and Ascites

The paper deals with convenient and fast methods for purification of monoclonal antibodies (MAbs) to carbohydrate antigens Le(y) and Le(d) from the cell culture and ascite fluid by ion-exchange chromatography on S-Sepharose and salt-promoted chromatography on a "thiophilic" adsorbent. One-step procedure on S-Sepharose of MAbs to Le(y) (IgG and IgM) provides significant purification (over 90% of contaminants were removed), while a purification factor for IgM to Le(d) is much lower. Highly purified IgM to Le(d) could be obtained by a two-step purification procedure including "thiophilic-adsorption" chromatography and gel-filtration (90-98% of contaminants from the cell culture and ascite fluid were removed). The preparations of IgG and IgM retain their initial antibody activity.

Topics: Adsorption; Animals; Antibodies, Monoclonal; Ascites; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin G; Immunoglobulin M; Lewis Blood Group Antigens; Mice; Mice, Inbred BALB C; Molecular Weight; Sepharose

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.

Topics: Animals; Antibodies, Monoclonal; Ascites; Carrier Proteins; Cattle; Chromatography, Affinity; Chromatography, High Pressure Liquid; Collectins; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Gels; Humans; Immunoglobulin G; Immunoglobulin M; Sepharose

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.

Topics: Ascites; Chromatography, Affinity; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Haptoglobins; Hemoglobins; Humans; Immunodiffusion; Molecular Weight; Peroxidases; Receptors, Concanavalin A; Sepharose; Trypsin