Identification and quantitation of capsular antigen in capsulated and noncapsulated strains of Haemophilus influenzae type b by crossed-immunoelectrophoresis

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Abstract
Sonicated preparations of capsulated Haemophilus influenzae type b, two of its spontaneous mutants, one containing patches of capsules (class I variant) and the other noncapsulated (class II variant), and a noncapsulated strain of H. influenzae type d were analyzed by crossed-immunoelectrophoresis using unadsorbed antiserum to capsulated H. influenzae type b. Twenty common antigens were present in all four cultures. Two type b-specific antigens were also identified in the three H. influenzae type b cultures when antiserum adsorbed with H. influenzae type d sonicates (AdR) was used. One of these, the type b capsular antigen, cross-reacted with an antigen of Bacillus pumilus. Further adsorption of AdR with the B. pumilus sonicate reduced, but did not eliminate, the antibodies to the type b capsular antigen, although all antibodies to B. pumilus were removed. Sonication sheared the type b capsular antigen, resulting in an increase in its electrophoretic mobility in agarose gel. The capsular antigen from all three H. influenzae type b cultures had the same electrophoretic characteristics. Reproducible quantitation of sheared and unsheared capsular antigen was demonstrated by rocket immunoelectrophoresis. As little as 2.5 ng of polyribophosphate pentose was identified and measured. Capsulated H. influenzae type b contained 78 ng of polyribophosphate pentose per mug of cell protein; class I contained 20 ng and class II contained 4 ng. The small amount of type b capsular antigen present in the class II variant may account for its lack of detection in this organism before now.