Hrp conjugated goat anti mouse iga

The levels of IgG, IgG1 and IgG2a in serum were measured by an ELISA assay as described previously [15 (link), 27 (link)]. Plates were coated with 5 μg/ml purified rCsCP (0.05 M carbonate-bicarbonate buffer, pH 9.6) at 100 μl per well and incubated at 4 °C overnight. After washing with PBS-T three times, the plates were blocked with 5% skimmed milk for 2 h at 37 °C. After washing routine program, the plates were incubated with serum samples (1:100) for 2 h at 37 °C and subsequently with HRP-conjugated goat anti-mouse IgG (1:2000, Santa Cruz, USA), IgG1 (1:2000, Santa Cruz, USA), or IgG2a (1:2000, Invitrogen, Carlsbad, USA), respectively, for 1 h at RT. Next, 100 μl tetramethylbenzidine (TMB, BD, Franklin Lakes, USA) solution was added to the plates for 10–15 min and stopped by 50 μl of 2 M H₂SO₄, and the absorbance was detected at 450 nm. Before changing the incubation solution, the plates were washed with PBS-T 3 times.
Similarly, IgA levels in sera (1:20), intestinal lavage mucus (1:20), and bile (1:100) were also detected. HRP-conjugated goat anti-mouse IgA (1:2000, Southern Biotech, Birmingham, USA) was employed as a secondary antibody.

Serum levels of IgA levels were measured through sandwich ELISA method as described previously (7 (link)). In brief, plates were coated overnight with 2.5 μg/ml goat F(ab’)₂ anti-mouse Ig (SouthernBiotech, 1012-01, 1:400) in sodium carbonate buffer (pH 9.6). After washing, the plates were blocked with 1% BSA for 2 hours. Then diluted serum samples and mouse IgA (SouthernBiotech, 0106-01, the first standard concentration is 100ng/ml), used as the standard, were added and incubated for 1 hour. Following another round of washing, the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA (SouthernBiotech, 1040-05, 1:5000, 2ug/ml) antibodies for 1 hour. To determine serum levels of IgA-IgG complexes, a cross-capture ELISA was performed. The 96-well plates were coated with 2.5 μg/ml of goat anti-mouse IgA (SouthernBiotech, 1040-01, 1:400) overnight. After washing and blocking, the diluted serum samples were added and allowed to bind for 1 hour. Then the HRP-conjugated goat anti-mouse IgG antibody (SouthernBiotech, 1030-05, 1:5000, 0.2ug/ml) was applied. All reactions were carried out at room temperature. Finally, the plates were developed using 3,3’,5,5’-tetramethylbenzidine (TMB) and the reactions were stopped with 1 M sulfuric acid. The results were analyzed using an ELISA reader (Bio-Rad 550) at 450 and 570 nm wavelengths.

The number of RBD-specific IgA secreting cells was enumerated by ELISPOT assay as previously described [24 (link)]. Briefly, MultiScreen IP filter plates (96-well) (Millipore, Billerica, MA, USA) were coated with RBD protein (2 μg/well) in PBS at 4 °C overnight. After washing, the plates were blocked with 200 μL/well of RPMI 1640 medium supplemented with 10% FBS for 2 h at room temperature. Afterward, threefold dilutions of splenocytes isolated from immunized mice were added into each well and cultivated at 37 °C with 5% CO₂ for 5 h. After incubation, the cells were removed and the plates were washed with PBST 3 times. To detect IgA-secreting cells, HRP-conjugated goat anti-mouse IgA (Southern Biotech, Birmingham, AL, USA) was added and incubated for 1 h at room temperature. After 3 washes, the signals were developed by staining with DAB (SigmaFast DAB tablet, Sigma, St. Louis, MO, USA). The spots were scanned and counted on an ImmunoSpot S6 Ultimate Reader.