Production and characterization of monoclonal antibodies to exotoxin A from Pseudomonas aeruginosa

Abstract

Hybridomas secreting monoclonal antibodies specific for exotoxin A from Pseudomonas aeruginosa strain PA103 were derived from the fusion of spleen cells from mice immunized with: (i) purified exotoxin A, (ii) Formalin-treated exotoxin A, (iii) exotoxin A covalently coupled to Sepharose 4B, or (iv) P. aeruginosa-infected mice. All hybridomas were screened and selected by using an enzyme-linked immuno-adsorbent assay. All antibody isotypes were represented (immunoglobulins G, A, and M) as determined by enzyme-linked immunoadsorbent assay. The most productive fusions resulted from immunization with antigens coupled to an insoluble matrix, such as Sepharose 4B, or by infection of mice. Several hybridomas were selected and cloned by limiting dilution. The specificity of the monoclonal antibodies for exotoxin A was demonstrated by indirect immunoprecipitation of 125I-labeled exotoxin A followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and by the immunoblotting technique. The protective ability of certain monoclonal antibodies was demonstrated in vitro by toxin neutralization in tissue culture and in vivo by prolonged survival time in the burned mouse infection model, after passive immunization. One monoclonal antitoxin displayed specificity for PA103-derived exotoxin yet failed to react with exotoxin purified from PAO-PR1 or PAO1, suggesting that structural differences exist between these exotoxins.