High-performance liquid chromatography of amino acids, peptides and proteins: XCIII. Comparison of methods for the purification of mouse monoclonal immunoglobulin M autoantibodies

Abstract

A comparison of methods for the purification of naturally occurring mouse monoclonal autoantibodies, of the immunoglobulin M (IgM) isotype, has been performed to determine the optimal strategies for the isolation of IgM from ascites fluid and in vitro tissue culture hybridoma supernatants. In order to quantify each purification procedure, the concentration of IgM in eluted fractions was determined by using a double-sandwich μ-chain-specific anti-IgM enzyme-linked immunosorbent assay, and the purity of the IgM was determined by a bicinchoninic acid-based protein assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The most efficient single-step purification was based on size-exclusion chromatography on high-resolution Superose 6 HR 10 30 fast protein liquid chromatography (FPLC) columns. This procedure resulted in recoveries of monoclonal IgMs of ca. 71–86% with purities between 68 and 86%. Single-step chromatography of monoclonal IgM, on Superose 6 FPLC columns resulted in a 21-fold purification of IgM, prepared by the in vitro culture of hybridoma cells in dialysis membrane. Size-exclusion chromatography, performed with Sephacryl S-300 columns, resulted in reduced resolution of monoclonal IgM, with yields of ca. 57–80% and purity of ca. 42–58% compared with the high-resolution Superose 6 FPLC columns. “Non-ideal” size-exclusion chromatography on Superose 6 FPLC columns resulted in selective retention of monoclonal IgMs and elution of IgM with high-ionic-strength buffers in the trailing peak. Recovery of IgM with this strategy was high ( ca. 82–92%) but the purity was not comparable to the single-step fractionation of IgM on Superose 6 FPLC columns. Single-step anion- and cation-exchange and mixed-mode hydroxyapatite chromatography resulted in only partial purification of monoclonal IgM with the applied procedures. With these latter separation techniques, monoclonal IgM was eluted with a variety of other ascites fluid or supernatant proteins, including those with apparent molecular weights identical to those of mouse IgG and albumin. Sequential purification of monoclonal IgMs by Mono Q anion exchange, followed by Superose 6 FPLC columns, resulted in a 2- to 3-fold purification of IgM but did not separate IgM from high-molecular-weight contaminants with apparent molecular weights similar to those of α 2-macroglobulin and IgG. Enrichment of monoclonal IgM from ascites fluid by ammonium sulphate precipitation revealed increasing IgM recovery with increasing ammonium sulphate final concentrations up to 60%. Isolation of IgM, based on euglobulin properties, following dialysis in either 2% (w/v) boric acid (pH 6.0), 5 m M Tris-HCl (pH 7.8), or distilled water, resulted in low IgM yields (⩽ 10%) and purity ( ca. 10–20%). Precipitation of monoclonal IgM from ascites fluid with polyethylene glycol 6000 (PEG 6000) resulted in ca. 63% recovery and purification of 4- to 5-fold, indicating that this procedure may be advantageous for enrichment or concentration of IgM. Antibody reactivity with intracellular and cell surface murine autoantigens, as determined by indirect immunofluorescence, was maintained following all purification procedures. The results indicate that optimal purification of monoclonal IgMs, on the laboratory scale, was performed in a single step by size-exclusion chromatography on Superose 6 HR 10 30 FPLC columns. This procedure will allow the isolation of naturally occurring mouse monoclonal IgM autoantibodies for the characterization of their autoreactive specificities.