Grouping of haemolytic streptococci by monoclonal antibodies: determinant specificity, cross-reactivity and affinity

Streptococcal group polysaccharide (CHO), A-, A-variant-, B-, C-, D- and G-specific monoclonal antibodies were prepared by the hybridoma technique employing spleen cells of several inbred mouse strains which are either high or low responders to the group A-CHO. The isotypes of these reagents were restricted to the class mu and IgG subclasses gamma 3 and--in small numbers--gamma 1. Two distinct categories of antibodies were identified for all but group D specificity: those which agglutinate suspended bacteria but do not precipitate purified soluble antigen, and those which show both agglutinating and precipitating properties. The group D antibodies described here were only of the latter category. The reactions were inhibitable by haptens in as far as these were known. Cross-reactions were observed in group-A-specific antibodies with E and L polysaccharides. Most G-CHO-specific antibodies cross-reacted with B-CHO. Association constants determined by fluorescence quenching measurements were for binding of complete A and C polysaccharides in the range of 10(6) to greater than 10(8) M-1, and for hapten binding by A-, Av- and C-CHO-specific antibodies in the range of 10(3) to 10(4) M-1. These results support a model of steric arrangements of antigenic determinants on A-variant bacteria and solubilized antigen [42] and allow its extension to streptococcal groups A, B, C and G. This model explains the observed functional differences by postulating single, terminal determinants which interact with the prevailing non-precipitating antibodies and internal repeating determinants which react with precipitins, respectively. No significant differences were found in the reactivity patterns to these streptococcal group antigens between strains of mice in terms of their ability to respond with high or low serum antibody titres to group A-CHO. On the other hand, within high and low responder strains, different kinetics of the optimal timing of fusion after initiation of the secondary immune reaction by boosting was observed. Low responders were most efficiently used for fusion 1.5 days later than high-responder spleen cells. This feature is interpreted to indicate an earlier proliferation of B lymphocytes in high responders, due to either an improved responsiveness to T-lymphocyte help or a reduced reactivity with T suppressor cells in comparison to low-responder B lymphocytes.