Purification and characterisation of anti-pneumococcal capsular polysaccharide IgG immunoglobulins

- A 3-step column chromatography protocol for anti-PCP IgG purification is proposed.
- The anti-PCP IgG preparation compares well to international serum standards.
- The procedure may provide an alternative method for the preparation of reference materials.

ObjectivesThe production of reference materials for quantifying pneumococcal antibody concentrations relies upon large scale vaccination. An alternative simple, reproducible protocol has been developed for the affinity purification of 23 serotype anti-pneumococcal capsular polysaccharide (PCP) IgG immunoglobulins.Design & MethodsThe purification protocol utilised IgG fractionation, capsular polysaccharide (CPS) adsorption, and affinity chromatography using Pneumovax®-Sepharose. Purification efficiency and method reproducibility were assessed by comparison of 4 batches of anti-PCP IgG. Immunoglobulin composition was determined using nephelometry and functionality was evaluated using VaccZyme™ ELISAs.ResultsAnti-PCP IgG preparations were ≥ 95% pure by SDS-PAGE analysis with no contaminating IgA or IgM immunoglobulins or IgG antigen specific antibodies towards haemophilus influenzae b, diphtheria toxoid or tetanus toxoid. The predominant IgG subclass in the preparation was IgG2.ConclusionsThis novel purification procedure produced highly specific anti-PCP IgG preparations that compared well to both Lot 89SF and 007sp international serum standards and could be used as an alternative method for the production of reference materials.