Efficient separation of IgG from IgM antibodies via conjugated surfactant micelles

Abstract

Immunoglobulin-G (IgG) (∼150 kDa) antibodies confer longer term immunity against bacterial or viral infections than the heavier IgM’s (∼900 kDa), which are generally detectable in blood circulation in response to more recently acquired infections. There may be, however, a time overlap, which is problematic for diagnostic purposes, in the interests of which it is essential to separate IgM's from IgG’s. We describe a purification platform, functioning at pH 6.5, containing Tween-20, or Brij-O20, non-ionic detergent micelles, mixed with the sugar-rich detergent dodecyl maltoside (DDM), amino acid monomer tyrosine (Tyr), and conjugated by the amphiphilic complex [(bathophenanthroline)₃: Fe²⁺]. Using conjugated Brij-O20 micelles, with input molar ratio IgG: IgM 9:1, IgG is recovered at 10 °C with 85–90% yield, (by SDS-PAGE densitometry) and ≥95% purity (also by SDS-PAGE), while IgM's are recovered at lower yields (28–34%) and contain small amounts of co-extracted IgG's. Addition of E. coli lysate as an artificial contamination background does not reduce the yield or purity of the recovered IgG. Tween-20/DDM/Tyr micelles lead to IgG purity ≥95% similar to that of Brij-O20, but with lower process yields (64–70%, by densitometry). Chromatographic separation with Protein A or Protein G resins leads to yields comparable to those obtained with Brij-O20 micelles, but with lower purity.