Goat anti mouse igg hrp

Indirect ELISA was used to determine antigen‐specific antibody levels in murine and human serum samples as previously described elsewhere.³⁰ For recRBD‐specific ELISAs, recRBD (Genscript) was coated onto microtitre MaxiSorp plates (Thermo Fisher, Waltham, MA, USA) at a concentration of 1 μg mL⁻¹. For peptide‐specific ELISA, streptavidin at 5 μg mL⁻¹ (Sigma‐Aldrich) was first coated onto microtitre MaxiSorp plates followed by coating with biotinylated peptides at 1 μg mL⁻¹. CP samples and mouse sera were both tested at various serial dilutions. Goat anti‐mouse IgG‐HRP at 1:3000 dilution (Bio‐Rad Laboratories, Hercules, CA, USA) was used to determine mouse IgG levels whereas goat anti‐human IgG‐HRP at 1:3000 dilution (Bio‐Rad Laboratories) or goat anti‐human IgM‐HRP at 1:10 000 dilution (Thermo Fisher, Hercules, CA, USA) was used to determine human antibody levels. SIGMAFAST OPD substrate (Sigma‐Aldrich) was added according to manufacturer's instructions and incubated at room temperature for 20 min.

Cells were harvested in RIPA-lite lysis and resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described.^{15 (link)} Immunoblotting was performed with the following antibodies: PR (#sc-166169; Santa Cruz Biotechnology), pS294 PR (made in-house), IGF1Rβ (#3027 S; Cell Signaling Technology, Danvers, MA, USA), IR (#sc-57342; Santa Cruz Biotechnology), IRS-1 (Millipore #06-248), FOXO1 (#2880 S; Cell Signaling Technology), GAPDH (Santa Cruz Biotechnology #0411), goat anti-rabbit IgG-HRP (BioRad), and goat anti-mouse IgG-HRP (BioRad). Blots were developed using an ECL reagent (Super Signal West Pico PLUS; Pierce, Waltham, MA, USA) and imaged by film. Densitometric quantification of protein levels was measured in ImageJ.

BALB/c mice (female, 4–6 weeks) were immunised intramuscularly on day 0, 21 and 28 with 30 μg of peptide-DT or peptide-CRM conjugates formulated in alhydrogel (Alum, Brenntag Biosector, Denmark) in 1:1 ratio (v/v), as previously described^{25 (link)}. For the combination vaccine, 30 μg of each conjugate was admixed in a ratio of 1:1 by weight and formulated in Alum as previously described^{9 (link)}. Control mice received saline with adjuvant alone. For carrier priming experiments, mice were immunised three times with DT and then rested for 4 weeks before they received three immunisations with MJ8CombiVax. A parallel cohort of naïve mice was also vaccinated with MJ8CombiVax. In all experiments, one-week post last immunisation mice were bled via submandibular vein and serum isolated. The serum samples were stored at −20 °C until used. ELISA was used to determine murine Ag-specific IgG antibody titers as previously described^{34 (link)}. Goat anti-mouse IgG-HRP (Bio-Rad laboratories) was used to determine Ab titers. Titre was defined as the highest dilution that gave an optical density (OD) reading of more than three standard deviations (SD) above the mean OD of control wells containing normal mouse sera at the same dilution^{34 (link)}.