Igg2a

Plates were coated overnight at 4°C with 4 μg/mL of goat polyclonal anti-rat IgM or IgG antibodies (supplied by Jackson ImmunoResearch Laboratories). After several washing steps, saturation with PBS/5% BSA was performed for 2 hours at room temperature. Diluted samples and isotype control (rat IgM, IgG1, IgG2a, and IgG2b supplied by BD Biosciences), used to determine the standard curves, were incubated for 90 minutes at 37°C. HRP-conjugated anti-rat IgM, IgG1, IgG2a, or IgG2b (supplied by Bethyl Laboratories) was incubated for 90 minutes at 37°C to allow specific detection of rat immunoglobulin isotypes. Tetramethylbenzidine (TMB; BD Biosciences) was used as HRP chromogenic substrate before the reaction was stopped by addition of H₂SO₄. Optical density at 450 nm was determined using a Tecan Spark plate reader.

Whole blood from each mouse was collected and centrifuged at 2500 rpm for 20 minutes. Serum was collected and stored at −80°C before analysis. Enzyme-linked immunosorbent assay (ELISA) plate wells were coated with Der p (10 µg) in coating buffer (15 mM Na₂CO₃ and 35 mM NaHCO₃, pH 9.6) at 4°C overnight, washed with 0.05% Tween 20 in PBS (PBST), and blocked with 0.5% bovine serum albumin (BSA; Sigma-Aldrich). Sera or BALF samples were diluted to appropriate concentrations and added to wells at 4°C overnight. After washing with PBST, goat anti-mouse IgA conjugated horseradish peroxidase (HRP; 1∶10000, Novus Biologicals), goat anti-mouse IgG1 conjugated HRP, or goat anti-mouse IgG2a or conjugated HRP (IgG1, 1∶10000, IgG2a, 1∶5000, Bethyl) was added. Finally, color was developed with TMB (tetramethylbenzidine) substrate (Clinical Science Products).

IgG1, IgG2a, and IgE ELISA kits were purchased from Bethyl Laboratories (Montgomery, TX, USA) and were used according to the manufacturer's recommended protocol. Antigen (OVA)-specific IgA, IgM, IgG, and IgG subclass antibody titers were measured by using an indirect competitive enzyme-linked immunosorbent assay (ELISA) protocol based on previously described methods (27 (link)).

Mice (n = 10-14 per group) were anesthetized with isoflurane and bled from the orbital plexus just before sacrificed. Serum samples were stored at −80°C. Serum titers of four IgG subclasses against mTg were detected by ELISA as described in our previous studies [1 (link), 15 (link), 47 ]. Serially diluted (five-fold) serum samples were added into the 96-well ELISA plates (Nunc) which had been coated with mTg (1 μg/well) and blocked with PBS-1% BSA (Sigma), and then incubated at room temperature for 2h. Anti-Tg IgG subclasses were detected using horseradish peroxidase-conjugated goat anti-mouse IgG1 (1:30,000), IgG2a (1:30,000), IgG2b (1:15,000), or IgG3 (1:250) (all from Bethyl, TX, U.S.). Plates were developed with tetramethylbenzidine (TMB, Amresco, OH, U.S.) and absorbance at 450 nm was measured using a 96-well plate reader (Bio-Rad, CA, U.S.). These determinations were performed in duplicate and their mean values were used for statistical analysis.

Antibody concentrations in collected blood and supernatant were determined using IgE (BioLegend 432401), IgM (Thermo Fisher Scientific 88-50470-22), IgG1 (Bethyl Laboratories E90-105), IgG2a (Bethyl Laboratories E99-107), and IgG2b (Bethyl Laboratories E99-109) ELISA kits. Freshly collected blood samples were prepped for ELISA by first allowing samples to clot and then centrifuging at 1500g for 15 minutes. The clear plasma, not including the buffy coat, was collected and frozen (–20°C) for subsequent ELISA. The serum samples were diluted at 1:50 for IgE ELISAs or 1:1000 for IgG1, IgG2a, IgG2b, and IgM ELISAs in the respective kit sample diluent. Supernatant collected from in vitro cultures was used undiluted for the IgE ELISA and diluted 1:5 for all other ELISAs. Differences in ELISA dilutions were equalized prior to analysis. Kit protocols were followed for standards and addition of capture antibody, detection antibody, TMB, and stop solution (2N sulfuric acid). For OVA-specific ELISAs, 96-well ELISA plates were instead coated with 1 μg/mL OVA (MilliporeSigma) and incubated overnight at 4°C, and then kit protocols were followed (61 (link)). Absorbance was measured at 450 nm wavelength.

ELISA plates were pre-coatedprecoated overnight with pseudotyped HIVluc-MERS virus at 4 °C. The coated plates were washed three times with PBS-T (PBS containing 0.1% Tween 20) and then blocked with blocking buffer (5% skim milk in PBS-T) for 2 h at 25 °C. Plates were incubated with the twofold serial dilution of mouse serum starting from 1:100 and incubated at 25 °C for 2 h. Then, the plates were washed four times with PBS-T and incubated with HRP-conjugated anti-mouse IgG (Bethyl Laboratories, 1:1000), IgG1 (Bethyl Laboratories, 1:1000), or IgG2a (Bethyl Laboratories, 1:1000) at 25 °C for 1 h. After four washes with PBS-T, the plates were incubated with a 0.03% 3,3′,5,5′-tetramethylbenzidine solution (Koma Biotech) for 15 min at 25 °C. The reaction was stopped with 1 N H₂SO₄. The optical density (OD) at a wavelength of 450 nm was measured using an ELISA plate reader (Bio-Rad). The endpoint titer was defined as the last serum dilution at which the OD value was above the average of negative-control wells (secondary antibody only).

ELISA plates were coated with 50 μl per well of purified rCsPmy protein (5 μg/ml) in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) and blocked with PBST containing 5% skimmed milk at 37 °C for 2 h. After washing three times with PBST, the plates were incubated with serum (1:200 dilutions) for 1 h at 37 °C. Subsequently, HRP-conjugated rabbit anti-mice IgG (1:8000 dilutions, Proteintech Group, Chicago, USA), IgG1 or IgG2a (1:2000 dilutions, Bethyl, Montgomery, USA) were added as secondary antibodies. Plates were incubated for 1 more hour at 37 °C and then reacted with the substrate TMB (BD, Franklin Lakes, USA). After 10 min incubation in dark, reactions were stopped by adding 50 μl of 2 M H₂SO₄ and the absorbance was measured at 450 nm by a microplate reader.
ELISA detection of sIgA in intestinal mucus, bile or feces was done with the similar method as above. After blocking with PBST containing 5% skim milk, intestinal mucus (1:50 dilutions), bile (1:100 dilutions), or extracted fecal supernatant (1:10 dilutions) were subjected to the wells. Finally, sIgA levels were detected using HRP-goat anti-mice IgA antibody (1:2000 dilution).