Anti mouse igm hrp

Blood collected from mice tail vein was centrifuged at 300× g for 5 min at 4 °C and the serum was used for quantifying the production of IgM and IgG antibodies by ELISA. ELISA plates were coated with EVs (5.0 µg/mL) or P. brasiliensis yeast cells (1.0 × 10⁶ cells/well) and, after incubation and washing, were blocked with 1% BSA in PBS. After washing, plates were incubated with mice sera diluted 1:100 in PBS. Plates were washed and then incubated with anti-mouse IgM-HRP or anti-mouse IgG-HRP (Southern Biotech, Birmingham, AL, USA). After washing, the reaction was developed by adding o-phenylenediamine dihydrochloride (OPD) (400 µg/mL—Sigma-Aldrich, Darmstadt, HE, Germany) and hydrogen peroxide in citrate buffer (pH = 5). The 96 well-flat bottom plates were read on a spectrophotometer at 490 nm wavelength.

Pertussis and tetanus IgG titers were measured using Bordetella pertussis and Tetanus toxoid IgG ELISA kits according to the manufacturer’s instructions (Immuno-Biological Laboratories, Inc., Minneapolis, USA). Per manufacturer’s instruction, immunity to pertussis was defined as a pertussis IgG titer higher than 20 U/mL. For citrullination ELISAs, the precoated wells of pertussis and tetanus toxoid IgG ELISA kits were incubated with citrullination buffer (100mM Tris-HCl pH7.5, 1mM DTT, and 5mM CaCl₂) alone or with buffer and 0.01μg/mL peptidylarginine deiminase (PAD) 4 and 0.01μg/mL PAD2 overnight at 37°C similar to previously [30 (link)]. Wells were washed three times before proceeding with the ELISA per manufacturer’s instruction. As a negative control, non-precoated 96 well plates (EIA/RIA Plate High Binding, Costar, Corning, USA) were exposed to buffer alone or buffer with PAD enzymes as above and used in ELISA to detect IgG in sera that binds to PAD enzymes. To assess citrullination efficiency, the pertussis and tetanus precoated wells exposed to buffer alone or buffer with PAD enzymes were washed three times before proceeding with the ELISA with these modifications: mouse anti-citrulline IgM (clone F95, EMD Millipore, Darmstadt, Germany) diluted 1:200 as primary antibody and anti-mouse IgM-HRP (SouthernBiotech, Birmingham, USA) diluted 1:5000 as secondary antibody.

Serum samples were collected on the indicated days and stored in BD Vacutainers; 96-well plates were coated with 2 μg/mL of either recombinant Spike_RBD or Spike_RBD (K417T, E484K, N501Y) proteins in phosphate-buffered saline (PBS) overnight at 4 °C and incubated in blocking buffer (0.05% Tween20 + 2% bovine serum albumin in PBS). Plates were incubated with diluted serum samples for 2 h at room temperature. Plates were then washed four times with PBS and incubated with goat anti-mouse IgG-HRP, anti-mouse IgM-HRP, anti-mouse IgG1-HRP, or IgG2b-HRP (SouthernBiotech) at 1:10,000 or with IgA-HRP at 1:2,000 in blocking buffer for 1 h. Plates were developed with 3,3',5,5'-tetramethylbenzidine liquid substrate reagent (Sigma). The reaction was stopped with 1 N HCl, and absorbance was read at 450 nm.

The antibody titers for various isotypes in the sera of NP-KLH immunized mice were quantified relative to a sample pooled from sera of NP-KLH immunized mice. Nunc MaxiSorp™ plates (ThermoFisher) were coated with 1µg/ml of NP-(14)-BSA or NP-(2)-BSA (Biosearch). An initial dilution of 1:500 of the serum was prepared followed by a series of 1:5 dilutions. For the quantification of anti-ds-DNA-antibodies, high-binding half-area plates (Corning) were coated with Poly-L-lysin (Sigma-Aldrich), followed by 25 ng/ml calf thymus dsDNA (Sigma-Aldrich). An initial dilution of 1:10 of the sera was prepared, following a series of 1:2 dilutions. The following isotype-specific detection antibodies were used: donkey anti-mouse IgG-HRP (JacksonImmunoResearch), goat anti-mouse IgG1-HRP, anti-mouse IgG2b-HRP, anti-mouse IgG2c-HRP, anti-mouse IgG3-HRP, anti-mouse IgM-HRP (all from SouthernBiotech). Total quantification of IgG, was performed using a mouse-IgG-Kit (Roche) according to the manufacturer’s instructions.

In parallel to dendritic cell challenge, a same-day plating of the same lot of bacterial cells was used to analyze Lewis Y produced by bacterial cells. Cells were resuspended in H. pylori lysis buffer with Protease inhibitor [20 mM Tris-HCl, pH 7.4, 1% Igepal, 150 mM NaCl, 1 mM EDTA, Protease inhibitor (MilliporeSigma)] and were frozen for 30 minutes to lyse cells. The protein concentration of lysates was determined using a Lowry assay, and lysate concentrations were standardized to 2.5 mg/ml. Samples were electrophoresed via SDS-PAGE on a Mini-PROTEAN TGX Stain-Free Precast 12% acrylamide gel with 4% stacking layer (Bio-Rad). Coomassie stain was used to evaluate protein loading. For Lewis Y detection, electrophoresed samples were transferred to nitrocellulose for western blot analysis. The nitrocellulose membrane with transferred samples was probed with anti-Lewis Y antibody (Abcam, Waltham, MA) followed by anti-mouse IgM HRP (Southern Biotech, Birmingham, AL), then treated with luminol/peroxidase reagent and visualized using a Syngene G box (Cambridge, UK).