Purification and Characterization of Salmonella Antigens

Purified COPS and FliC monomers for use as vaccine antigens for immunization and antibody measurements by enzyme-linked immunosorbent assays (ELISAs) were generated from S. Enteritidis reagent strain CVD 1943, as described [20 (link), 23 (link)]. Endotoxin removal was confirmed by endpoint limulus amebocyte lysate assay with an Endosafe PTS system (Charles River, MA). Nucleic acid removal was assessed by A260 nm for COPS and Quant-IT Sybr Green assay (Life Technologies, CA) for FliC. Removal of host cell protein in the COPS preparation was confirmed with the bicinchoninic acid (BCA) assay (Thermo, MA) and by SDS-PAGE with Coomassie staining for FliC. S. Enteritidis COPS identity and molecular size were confirmed by Dionex HPAEC-PAD and HPLC-SEC respectively as described [16 (link)]. Flagellin identification was accomplished by SDS-PAGE/Western blot analysis with monoclonal antibody CA6IE2, and confirmation of monomeric form by HPLC-SEC as described [23 (link)].

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Baliban S.M., Curtis B., Toema D., Tennant S.M., Levine M.M., Pasetti M.F, & Simon R. (2018). Immunogenicity and efficacy following sequential parenterally-administered doses of Salmonella Enteritidis COPS:FliC glycoconjugates in infant and adult mice. PLoS Neglected Tropical Diseases, 12(5), e0006522.

Publication 2018

Endosafe PTS system BCA) assay HPAEC-PAD

Amebocyte lysate Antibody Assay Bicinchoninic acid Cell Cops Endotoxin Enzyme linked immunosorbent assays Flagellin Hplc Immunization Limulus Monoclonal antibody Nucleic acid Protein River Sds page Strain Sybr green Vaccine antigens Western blot analysis

Corresponding Organization : University of Maryland, Baltimore

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Variable analysis

independent variables

Purified COPS and FliC monomers

dependent variables

Immunization
Antibody measurements by enzyme-linked immunosorbent assays (ELISAs)

control variables

Endotoxin removal confirmed by endpoint limulus amebocyte lysate assay
Nucleic acid removal assessed by A260 nm for COPS and Quant-IT Sybr Green assay for FliC
Removal of host cell protein in the COPS preparation confirmed with the bicinchoninic acid (BCA) assay and by SDS-PAGE with Coomassie staining for FliC
S. Enteritidis COPS identity and molecular size confirmed by Dionex HPAEC-PAD and HPLC-SEC
Flagellin identification accomplished by SDS-PAGE/Western blot analysis with monoclonal antibody CA6IE2, and confirmation of monomeric form by HPLC-SEC

positive controls

None specified

negative controls

None specified

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