A number of pneumococcal polysaccharide (PS)- protein conjugate vaccines are in clinical studies. We have therefore continued studies on various aspects of antibody assay standardization. Different methods of coating the pneumococcal polysacccahrides to different polystyrene ELISA plates were compared and the optimum binding for each type PS was determined. Co-mixing with mHSA was found to be better for most types tested except for type 3 PS. Pre-coating wells with mHSA is best for type 3, because the specificity of antibodies binding were much more type 3 specific. Lot 89SF, the reference standard serum was therefore reassayed for this type. The IgG value was within 20% CV of that assigned by Lederle-Praxis research laboratories. Better inhibition also is achieved using this method of attachment. Column chromatography in Sephacryl S-500 for molecular sizing was done using several pneumococcal PS types including types 3, 4, 14, and the common C PS. Fractions were assayed by inhibition of binding to immune serum to C Ps and type PS. The phosphorous assay was performed as a marker for C PS. The C PS appears to be associated with the high molecular weight type PS's, and we are investigating whether the C PS is covalently bound to the type PS. Monoclonal antibodies to Types 9V, 14 and 3 are currently being used to study the multiplicity of epitopes present on a given PS. Phagocytosis assays will also be done to study correlation of pneumococcal antibodies to protection.
