Three monoclonal antibodies (MO8-X3C, MO8-X3E and MO8-X4B) directed against filamentous hemagglutinin (FHA) have been produced. We have characterized these monoclonal antibodies and investigated their role in inhibiting attachment of mammalian cells to FHA. The monoclonal antibodies have been purified by HPLC and used in ELISA and Western Blots to screen for FHA. MO8-X3C and MO8-X3E, both IgG1 subtype, can inhibit the attachment of Chinese hamster ovary (CHO) cells to purified FHA when this protein is coated on plastic wells. MO8-X3C has also been shown to inhibit the attachment of the whole bacteria, B. pertussis, to CHO cell monolayers. Furthermore, this monoclonal antibody, MO8-X3C, can also inhibit the invasion of HeLa cells.by B. pertussis. These antibodies should be useful reagents for screening B. pertussis mutants for expression of FHA and in regulation and characteriazation of pertussis acellular vaccines. FHA is one of the primary candidates being considered for future pertussis acellular vaccines. Determining the binding epitope for these MAbs on FHA should help elucidate the cell attachment site of FHA. We are using the epitope scanning pin technology, where 15 amino acid peptides are synthesized on activated pins and with increments of 4 amino acids along the FHA protein sequence, the entire sequence is covered. We will also study the role of these mAb directed against FHA in passive immunization of mice against B. pertussis infection to further understand the pathogenesis of B. pertussis and the different mechanisms of protection. We have also recently found cross reactivity with these anti-FHA MAbs and two high molecular weight outer membrane proteins from nontypable Hemophilus influenza. We are further characterizing these proteins which contain some homology with FHA at the protein level.
