Isolation of specific IgM monoclonal antibodies by affinity chromatography using alkaline buffers.
Comparative studies on the efficacies of various buffers in eluting absorbent-bound antibodies revealed the denaturative effect of 3 M KSCN on all three IgM antibodies but not on an IgG protein, and the generally weak eluting power of glycine-HCl buffer, pH 2.5, on these monoclonal antibodies. A recently described medium consisting of 50% (v/v) ethylene glycol in an alkaline buffer, pH 10.5, was found to be relatively efficient for elution in all cases. However, subsequent studies on one of the IgM antibodies showed that alkali alone could effect elution, with recovery of active protein improving on increasing the pH, till the maximum (38%) at pH 11.0, after which denaturation occurred. Addition of ethylene glycol to the medium facilitated the elution; however, at pH greater than 10.0, the solvent potentiated the denaturative effect of the medium. Since pH 11.0 was found to be the highest pH in which all three IgM antibodies examined were stable, 0.1 M glycine-NaOH buffer, pH 11.0, may be a useful eluent for IgM (and other) antibodies in general.
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002021:Buffers; D002846:Chromatography, Affinity; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D005998:Glycine; D006863:Hydrogen-Ion Concentration; D007075:Immunoglobulin M / Q000302:isolation & purification*; D011489:Protein Denaturation; D012972:Sodium Hydroxide
D000911:Antibodies, Monoclonal; D002021:Buffers; D005026:Ethylene Glycols; D007075:Immunoglobulin M; D012972:Sodium Hydroxide; D019855:Ethylene Glycol; D005998:Glycine