CHARACTERIZATION OF A MONOCLONAL-ANTIBODY TO THE CAPSULE OF HAEMOPHILUS-INFLUENZAE TYPE-B, GENERATED BY IN-VITRO IMMUNIZATION

Monoclonal antibodies to polyribosylribitolphosphate (PRP), the capsul ar polysaccharide of Haemophilus influenzae type b (Hib), are useful t ools in the investigation of the molecular and cellular mechanisms cau sing Hib meningitis. A better understanding of these mechanisms may le ad to improved therapeutic strategies. A number of different in vivo i mmunization techniques in BALB/c mice were used, which did not however reveal detectable serum levels of antibodies to PRP. Therefore a modi fied in vitro immunization technique, originally established for in vi tro immunization of human B lymphocytes, was used for this weak immuno gen in mice. After 5 days of in vitro stimulation with purified PRP th e splenic lymphocytes of BALB/c mice were fused with the mouse myeloma line P3-X63-Ag8.653. One hybridoma produced an IgM antibody (12E7) wh ich recognized the capsular polysaccharide in ELISA and specifically l abelled all tested Hib strains in immune fluorescent microscopy. The b lotted polysaccharide PRP was immunostained with monoclonal antibody 1 2E7. Preincubation of Hib with this antibody enhanced the oxygen radic al metabolism of polymorphnuclear leucocytes in a chemiluminescence as say. There was no cross-reactivity with the supernatants of other Haem ophilus influenzae serotypes and other bacterial species, as shown by counterimmunoelectrophoresis.