Ab97245

Pierce Streptavidin High Binding Capacity (Thermo Fisher Scientific, Waltham, MA) coated plates were washed 3 times with PBST (137 mM NaCl, 2.7 mM KCl, 10.15 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4, 0.05% (v/v) Tween 20). Biotinylated M2e peptide (Peptide 2.0 Inc., Chantilly, VA) at 10 mg mL^-1 in PBS was adsorbed to the plates, 100 μL per well for 2 h at room temperature. Plates were then washed 3 times with PBST. Plates were incubated with mouse sera initially at 100-fold dilution followed by four-fold serial dilutions with PBST containing 0.5% (w/v) skim milk (90 min, 37°C). After washing 4 times with PBST, HRP-conjugated goat anti-mouse IgG1 or IgG2a (Ab97240 or Ab97245, respectively, Abcam, Cambridge, UK) was added at 20 000- or 10 000-fold dilution, respectively, followed by incubation (90 min, 37°C). Plates were washed 4 times with PBST and developed (0.4 mg mL^-1 o-phenylenediamine dihydrochloride (Sigma-Aldrich, St Louis, MA), 50 mM phosphate citrate buffer containing 0.03% (w/v) sodium perborate) prior to absorbance measurement at 450 nm. End point titers were determined as the highest dilution of serum for which the OD was 3 standard deviations above the mean optical density of blank wells.

To investigate the IgG subtypes of the induced anti-HER2 antibodies in mouse serum, an ELISA was performed as described previously (see Section 2.9). In brief, recombinant human HER2 (2 µg/mL in PBS) was coated on 96-well MaxiSorp plates (Thermo Fisher) overnight at 4 °C. Afterwards, the plates were blocked using PBS-T supplemented with 0.3% milk powder (AmericanBio) for 1 h at room temperature. Serum samples were two-fold serially diluted and incubated for 2 h at room temperature. After washing three times with PBS-T, anti-mIgG1-HRP (abcam, ab97240), anti-mIgG2a-HRP (abcam, ab97245), and anti-mIgG2b-HRP (abcam, ab97250) antibodies (1 mg/mL) were diluted 1:3000 in blocking buffer and were added for 1 h at room temperature. Plates were washed four times and were developed using OPD substrate (Sigma). The enzymatic reaction was stopped by adding 100 µL of 3-M hydrochloric acid (Thermo Fischer), and optical density was measured at 490 nm using a plate reader (BioTek). Raw data were analyzed in Microsoft Excel (Version 2003) and GraphPad Prism 7. Results were normalized to blank and are plotted as IgG1:IgG2a:IgG2b ratios of total induced anti-HER2 Abs.

VZV‐specific total immunoglobulin G (IgG), IgG1, and IgG2a in mouse serum and total IgG, IgG1, and IgG2 in guinea pig serum were measured by eELISA. The 96‐well plates (Nunc Maxisorp^TM; Thermo Fisher Scientific) were coated with 50 ng well⁻¹ VZV gE for mice and 1000 PFU well⁻¹ VZV for guinea pigs and incubated overnight at 4°C. The wells were then blocked with 200 μL of 5% (v/v) skim milk for 1 hour at room temperature (RT). Diluted serum samples and VZV gE Ab (No. 127‐10031; RayBiotech, Inc, Peachtree Corners, GA) were added to the plates and incubated for 2 hours at RT. The wells were then washed three times with 200 μL phosphate‐buffered saline (PBS) mixed with 0.05% (v/v) Tween 20 (PBST). The following antibodies were then added: anti‐mouse IgG (ab97265; Abcam, Cambridge, UK), IgG1 (ab97240; Abcam), and IgG2a Ab (ab97245; Abcam) or anti‐guinea pig IgG (ab9608; Abcam), IgG1 (ABIN457757; Antibodies, Cambridge, UK), and IgG2 Ab (GAGp/IgG2/PO; Nordic MUbio, Susteren, The Netherlands). The mixtures were then incubated for 1 hour at 37°C. After washing, 3,3′,5′5′‐tetramethylbenzidine (TMB) substrate was added to the wells and the mixtures were incubated for 15 minutes. A stop solution was then added to halt the reaction. Optical densities were measured at 450 nm in a microplate reader.

Enzyme‐linked immunosorbent assay (ELISA) plates were coated with individual peptides or rEg.P29 at 10 μg/ml and incubated overnight at 4°C. The plates were washed five times with 0.05% Tween 20 PBS (PBST) and blocked with 5% skim milk powder in PBST at 37°C for 1 h. After washing five times with PBST, the plates were incubated with primary serum (1:100) in PBS (with 10% FBS) for 2 h and washed five times with PBST for 2–3 min. One hundred microliters each of horseradish peroxidase (HRP)‐conjugated anti‐mouse IgM, IgG, IgA, IgG1, IgG2a, IgG2b, IgG2c, and IgG3 (Cat nos.: ab97230, ab97023, ab97235, ab97240, ab97245, ab97250, ab97255, and ab97260, respectively; Abcam), and IgE (Cat no.: PA1‐84764; Invitrogen) were added enzyme plates and incubated at 37°C for 1 h. After washing, 3,3′,5,5′‐Tetramethylbenzidine (TMB) was added for 8–10 min, and the reaction was stopped by 2 M H₂SO₄. The absorbance was measured at 450 nm within 15 min using an ELISA reader (Thermo Fisher Scientific).

Edmonston strain-derived MV antigens (Jena Bioscience) or recombinant S protein (ABIN6952426, Antibodies Online) were coated on NUNC MAXISORP 96-well immuno-plates (Thermo Fisher) at 1 µg/ml. Coated plates were incubated overnight at 4 °C. After washing and blocking, sera from immunized mice were serially diluted in the binding buffer and incubated on plates for 1 h at 37 °C. After washing steps, an HRP-conjugated goat anti-mouse IgG (H + L) antibody (Jackson ImmunoResearch, 115-035-146, 1:5000 dilution) was added for 1 h at 37 °C. Antibody binding was detected by the addition of the TMB substrate (Eurobio) and the reaction was stopped with 100 µl of 30% H₂SO₄. The optical densities were recorded at 450 and 620 nm wavelengths using the EnSpire 2300 Multilabel Plate Reader (Perkin Elmer). Endpoint titers for each individual serum sample were calculated as the reciprocal of the last dilution giving twice the absorbance of the negative control sera. Isotype determination of the antibody responses was performed using HRP-conjugated isotype-specific (IgG1 or IgG2a) goat anti-mouse antibodies (AB97240 and AB97245, Abcam, 1:5000).

Binding properties of murine sera to monomer RBD or S protein were determined by ELISA. 96-well plates (3590; Corning, USA) were coated overnight with 3 μg/mL of monomer RBD or S protein (Sino Biological, China) in 0.05 M carbonate–bicarbonate buffer, pH 9.6, and blocked in 5% skim milk in PBS. Serum or bronchoalveolar lavage fluid (BALF) samples were serially diluted and added to each well. The plates were incubated for 2 h and then washed. The plates were incubated with goat anti-mouse IgG-HRP antibody (Abcam, ab6789, for IgG titration), goat anti-mouse IgA-HRP antibody (Abcam, ab97235, for IgA titration), goat anti-mouse IgG1-HRP antibody (Abcam, ab97240, for IgG1 titration), or goat anti-mouse IgG2a-HRP antibody (Abcam, ab97245, for IgG2a titration), incubated for 1.5 h and then washed. The plates subsequently developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. Reactions were stopped with 2 M hydrochloric acid, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer, USA). The endpoint titres were defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.5-fold of the background values. Antibody titre below the limit of detection was determined as half the limit of detection.