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 mu-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
alpha 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%.(ABSTRACT TRUNCATED AT 400 WORDS)