Specificity for the mu-chain of mouse IgM is determined by Ouchterlony Double Diffusion (ODD), prior to agarose bead coupling. The antibody preparation is specific for mouse IgM when tested against purified mouse IgA, IgG (all subclasses) and IgM myeloma proteins. Identity and purity of the antibody is established by immunoelectrophoresis (IEP), prior to agarose bead coupling. Electrophoresis of the product followed by diffusion against anti-goat IgG and anti-goat whole serum results in single arcs of precipitation in the gamma region.
The IgG fraction of goat anti-mouse IgM (mu-chain specific) antiserum is covalently attached to cyanogen bromide activated cross-linked beaded agarose. Five to ten milligrams of IgG fraction is bound per milliliter of resin. After equilibration, a minimum of 0.4 mg of mouse IgM can be bound and eluted per milliliter of packed resin.
A two milliliter column of antibody-agarose is prepared using four milliliters of the antibody-agarose suspension. The column is equilibrated in 0.01 M sodium phosphate buffer, pH 7.2, containing 0.5 M NaCl (PB). The antigen solution to be bound is applied slowly and followed by a PB wash. Unbound effluent fractions are collected and assayed for protein content (Lowry). The column is then stripped by washing with 0.1 M glycine, 0.15 M NaCl, pH 2.4, or 0.5 M acetic acid, 0.15 M NaCl, pH 2.4. Fractions containing protein are collected, brought to neutral pH and assayed for protein content (Lowry). Regeneration
Goat Anti-Mouse IgM-Agarose may be regenerated and used for future adsorptions. Strip the agarose with ten column volumes of 0.1 M glycine, 0.15 M sodium chloride, pH 2.4, or 0.5 M acetic acid, 0.15 M sodium chloride, pH 2.4, then wash with 0.01 M sodium phosphate buffer, pH 7.2, containing 0.5 M sodium chloride (PB).