To obtain murine cold agglutinin (CA) and cryoglobulin antibodies, BALB/c mice were hyperimmunized with heat-killed type XIV Streptococcus pneumoniae. The spleen cells of these mice were fused with either the P3 NS1/Ag4. 1 or P3 X63/Ag.653 cell line. Several stable hybridomas were obtained that produced monoclonal antibodies (Mab) that reacted with rabbit and human erythrocytes only at temperatures below 37 degrees C. Three of these Mab were also cryoglobulins, as evidenced by their insolubility at reduced temperature. All of the antibodies studied were IgM(k) and reacted with purified type XIV S. pneumoniae polysaccharide at room temperature. With one exception, all antibodies were specific for N-acetyl-lactosamine, the immunodominant sugar residue expressed on type XIV polysaccharide. Inhibition experiments demonstrated that both CA activity and cryoprecipitation were inhibited by the same sugar compounds in the same order of efficiency. The data presented strongly suggest these CA antibodies are cross-reactive members of a S. pneumoniae-specific population. Cryoprecipitation persisted in antibodies purified under conditions that would exclude the presence of trapped serum antigens. It is therefore proposed that the cryoprecipitation observed is a result of the interactions of the antibody combining sites with carbohydrate residues of adjacent antibody molecules.

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