Masters Theses

Date of Award

5-1990

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Thomas C. Montie

Committee Members

Robert Moore, Jeffrey Becker

Abstract

Pseudomonas aeruginosa is an opportunistic gram negative rod having a single polar flagellum. It can be a source of nosocomial infections, and immunocompromised patients are especially vulnerable to life-threatening infections due to bacteremia or sepsis. A high incidence of antibiotic resistance has made consideration of immunotherapy an alternative or supplemental treatment. The presence of only two flagellar antigen (FLAg) types in Pseudomonas aeruginosa offers the possibility of developing a simple monoclonal antibody (MAb) cocktail. Polyclonal antibodies have been used in animal protection studies and to classify FLAgs as either type-a or type-b. Type-b FLAgs are homologous, whereas type-a FLAgs share a common ao epitope with various combinations of a1, a2, a3 or a4 subantigens. Knowing the degree of cross-reactivity among a-types would be helpful for immunization studies and for typing unknown strains. This project was undertaken in order to characterize monoclonal antibodies produced against type-a FLAg in their reaction with isolated flagella preparations and native flagella on whole cells of various type-a strains. Purified flagella from strain 170018 (a0 a3 a4) were injected into Balb/c mice for antibody production. Mouse spleen cells were fused with SP2/0-Ag14 myeloma cells to produce hybridomas. After limiting dilutions and screening, two antibodies, 53B and 29, were selected for further study. They were reacted with purified flagella from six standard type-a strains, representing various subtypes, and one type-b strain by ELISA and/or immunoblot. Also whole cells from the same strains were tested by colony blot, agglutination and motility inhibition assays. Type-a MAb 53B, selected for detailed study, showed no cross-reactivity with the type-b strain and reacted with all type-a strains in ELISA. However, among a-types the intensity of cross-reactivity of the common epitope (a0) was varied. Immunoblot and colony blot assays did not detect type a0 a3 that was least reactive in ELISA. There appeared to be a correlation between relative molecular weight of FLAGs and cross-reactivity of the common epitope, with lower molecular weight FLAgs (45,000 and 47,000) being more reactive. When whole cells were tested in agglutination and motility assays, three strains (170018, 5940, 1210) were agglutinated and two (170018, 5940) demonstrated motility inhibition. Thus, detection of antibody binding to FLAg was not always sufficient to cause agglutination or motility inhibition of flagella in the native state. A second type-a antibody, MAb 29, did not react with one type-a strain in ELISA or colony blots. However, four strains (170018, 5940, 1210, 7191) were positive for agglutination and motility inhibition. Slight cross-reactivity with the b-type was observed by ELISA and colony blots. Immunodiffusion showed that MAb 53B underwent an isotype class switch from IgG1 to IgG2 a. This was confirmed by ELISA using isotype specific secondary antibodies. Specificity for the antigen binding site appeared to be unaltered when compared to the pre-switched IgG1 in ELISA and agglutination. MAb 29 was typed as IgG2 a. Two MAbs were produced which reacted at different sites and with different specificities to type-a FLAg. MAb 53B would appear useful for diagnostics and typing of unknown strains because all a-types were detected, though not to the same degree, and no cross-reactivity with type-b was observed. MAb 23 might be more useful in animal protection studies since two-thirds of the strains demonstrated motility inhibition.

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