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Home List of Titles Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae
Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/227353
- Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae
- Kyd, J. M.; Taylor, D.; Cripps, A. W.
- Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell- mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.
- Publication type
- Journal article
- Infection and Immunity, Vol. 62, no. 12 (Dec 1994), pp. 5652-5658
- Publication year
- FOR Code(s)
- 06 Biological Sciences; 07 Agricultural and Veterinary Sciences; 11 Medical and Health Sciences
- Antibodies; Antigen specificity; Bacterial typing techniques; Bronchitis; Dodecyl sulfate sodium; Haemophilus influenzae; Immunisation; Lymphocyte proliferation; Outer membrane proteins; Peyer's patches; Polyacrylamide gel electrophoresis; Proteins; Sarcosine; Vaccines
- American Society for Microbiology
- Publisher URL
- Copyright © 1994, American Society for Microbiology.
- Research Projects
Haemophilus influenzae: dissection of the antibody response, National Health and Medical Research Council grant number 910104
- Peer reviewed