Purification and characterization of an anti-pneumococcal capsular polysaccharide IgG antibody preparation
Abstract number: P1924
Lock E., Worthington P., Foyle L., Garner L., McGough J., Barber R., Stubbs P.D., Alcock F., Hughes R., Wallis G.L., Parker A.R.
Objectives:Streptococcus pneumoniae is a major human pathogen causing pneumonia, sepsis, meningitis and otitis media. The major virulence factor of S. pneumoniae is the capsular polysaccharide (PS), of which there are 90 different types each inducing their own serotype specific antibodies. Since anti-PS antibodies are highly protective, efforts have focused on using the PS as immunogens for the development of pneumococcal vaccines. Standardisation of quantitative PS antibody measurement is of utmost importance between different laboratories. The objective of the study was to purify specific anti-PS IgG antibodies against the 23-valent PNEUMOVAX vaccine (Merck and Co. Inc).
Methods: Pooled plasma from multiple donors (6 to 10) were clotted. Three independent pools were prepared. Total IgG was purified using ammonium sulphate precipitation and anion-exchange chromatography. Following adsorption of contaminating non-specific PS, pneumococcal capsular polysaccharide(PCP)-specific antibodies were obtained by affinity purification using PNEUMOVAX covalently linked to Sepharose. The purified PCP specific IgG was characterised for purity, serotype composition, function, and then quantified.
Results: SDS-PAGE analysis suggested that the purified PCP specific IgG was at least 95% pure. Functionally, the preparation contained no contaminating Haemophilus influenzae type b, diphtheria toxoid or tetanus toxoid or contaminating non-specific PS antibodies. The major IgG subclasses in the preparation were IgG2 > IgG1 with negligible levels of IgG3 and IgG4. Immunoglobulin classes IgA and IgM were absent. Serotype analysis indicated that this preparation reacted with all 23 serotypes present in the PNEUMOVAX vaccine.
Conclusion: Determination of accurate antibody titre is essential for serodiagnosis, epidemiological investigation and evaluation of vaccine response. We show that it is possible to purify a sample containing 23 serotype specific antibodies produced in response to PNEUMOVAX vaccination which could be used to aid standardisation of different technologies to measure antibodies to PCP.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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