Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

Abstract

Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins.

Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits

“…1). The heavy component had a buoyant density of 1.22 g/cm3, which is similar to the buoyant densities of the outer membranes of other gram-negative bacteria (7,20), including P. haemolytica (26), and the light component had a buoyant density of 1.12 g/cm3. Analysis of both heavy and light components by SDS-PAGE showed that the protein profiles of the heavy and light components were different (Fig.…”

“…A 5-ml sample of this culture harvested by centrifugation twice at 20,000 x g for 10 min each time at 4°C and washed in phosphate-buffered saline (PBS) (0.01 M PBS, 0.85% NaCl, pH 7.2) was suspended in 1 ml of PBS. P. multocida cells were labeled with the lactoperoxidase-catalyzed radioiodination method (7). Labeled cells were sus-pended in PBS to a specific activity of 2 x 108 cpm/ml and used immediately in RIP experiments.…”

“…In this study, we adapted the staphylococcal protein A-dependent radioimmunoprecipitation (RIP) method (7) to identify those proteins present in the outer membrane of P. multocida that are immunogenic in rabbits and to identify the subset of outer membrane proteins of P. multocida that are exposed on the cell surface and accessible to protective antibodies in intact whole cells (6). The latter subset of outer membrane proteins has the greater potential to be protective.…”

Host discrimination of Mycoplasma pneumoniae proteinaceous immunogens.

“…Although the literature suggests that M. pneumoniae stimulates cellular, humoral, and secretory immune responses (13,18,(38)(39)(40)(41), we chose to examine the host humoral response to specific mycoplasma proteins. The RIP technique, previously shown to be a powerful tool for analyzing the immune response to proteinaceous immunogens of pathogens (29,30,32,(42)(43)(44), was used to examine acute and convalescent human and hamster sera. Preinfection hamster and acute human sera usually precipitate a small number of mycoplasma proteins in the soluble-antigen RIP assay.…”

Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence

“…The Hib strains used in this study were obtained from pediatric research centers located throughout the United States and have been described previously (9). Bacterial media (brain heart infusion [BHI; Difco Laboratories, Detroit, Mich.] supplemented with Levinthal base [BHIs] [1]) and growth conditions have been described previously (13). Hib strains were stored at -70°C in a mixture of equal parts of glycerol and BHI.…”