Flagellin Hyperimmunization Serum IgG Purification

Flagellin immunization was carried out as previously described (Kobayashi et al., 2009 (link)). Briefly, C57BL/6 mice were hyperimmunized by two intraperitoneal (i.p.) injections with 10 μg of flagellin from Salmonella typhimurium (InvivoGen) in Incomplete Freund’s Adjuvant (IFA) (Sigma Aldrich) two weeks apart. Mice were culled at four weeks and total serum IgG, as well as control serum IgG from unimmunized mice, was purified using Pierce Protein G purification columns (Thermo Fisher Scientific), as per the manufacturer’s instructions.

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Castro-Dopico T., Dennison T.W., Ferdinand J.R., Mathews R.J., Fleming A., Clift D., Stewart B.J., Jing C., Strongili K., Labzin L.I., Monk E.J., Saeb-Parsy K., Bryant C.E., Clare S., Parkes M, & Clatworthy M.R. (2019). Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis. Immunity, 50(4), 1099-1114.e10.

Publication 2019

Salmonella typhimurium Incomplete Freund’s Adjuvant (IFA)

C57bl mice Flagellin Freund adjuvant Immunization Mice Protein g Salmonella typhimurium Serum

Corresponding Organization : Wellcome Sanger Institute

Other organizations : Medical Research Council, MRC Laboratory of Molecular Biology, Cambridge University Hospitals NHS Foundation Trust

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Variable analysis

independent variables

Flagellin from Salmonella typhimurium (10 μg)

dependent variables

Total serum IgG

control variables

Mouse strain (C57BL/6)
Injection route (intraperitoneal)
Incomplete Freund's Adjuvant (IFA)
Immunization schedule (two injections, two weeks apart)
Time point of sample collection (four weeks)

controls

Positive control: Serum IgG from mice immunized with flagellin
Negative control: Serum IgG from unimmunized mice

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