Mouse iga elisa kit

The fresh feces (0.1 g) was collected in the colon of mice after the sacrifice, suspended in phosphate-buffered saline, vortexed at 4 °C for 20 min, and centrifuged (10 min, 16,000 g, and 4 °C). The resulting supernatants were used for the assay of IgA. The IgA level was determined using mouse IgA ELISA kit (Invitrogen, Carlsbad, USA).

To determine RBD-, S2-, and M2e-specific antibodies in mice sera, the sera of immunized mice were 3× serially diluted in primary antibody diluent (complete RPMI 1640 media with 0.2% (v/v) Tween 20). Enzyme-linked immunosorbent assay (ELISA) 96-well plates (NUNC Maxisorp, Thermo Scientific, Waltham, MA, USA) were coated with recombinant proteins RBD, S2, or M2e (0.75 µg/mL) in coupling buffer (pH 9.6, 50 mM sodium carbonate-bicarbonate) at 4 °C overnight. After blocking at 37 °C for 2 h, the ELISA plates were washed and incubated with the diluted sera at 37 °C for 1 h. Anti-mouse IgG-HRP antibodies (GE Healthcare, Cat#NA931; 1:5000) were used to detect the antibodies binding to RBD, S2, or M2e. After incubation with the substrate tetramethylbenzidine (TMB) solution (Mandel Scientific, Guelph, ON, Canada) and termination with stop solution, the absorbance at 450 nm (OD450) of each well was measured. IgA antibody levels were determined by using the mouse IgA ELISA kit (Thermo fisher, Cat#88-50450-88, Waltham, MA, USA). The endpoint titers of mouse sera were calculated using the interpolation in GraphPad Prism 9.0 with a cutoff of 2.5 times the mean-negative.

Fecal samples were collected by placing each mouse in a separate clean box for 3–5 min 1 day before euthanization, and freshly defecated fecal pellets, uncontaminated with urine, were sampled, snap-frozen in liquid nitrogen, and stored at −80°C till further analysis. The wet weight of feces samples ranged from 10 to 32 mg (median: 17.5 mg). Extraction buffer [PBS (pH = 7.4), protease inhibitor cocktail (Sigma Aldrich, Germany), and 0.01% sodium azide] was added to each sample at a ratio of 1 mL buffer to 1 g feces. Samples were thoroughly homogenized by a homogenizer (Bertin Technologies, France) at 6,000 g for 20 s. Fecal suspensions were centrifuged at 14,000 g for 10 min at 4°C and stored at −20°C until further use. The optimum sample dilution was tested, and 200 times dilution was selected for the IgA measurement using an ELISA kit (Mouse IgA ELISA kit, Thermo Fisher Scientific, The Netherlands) according to the manufacturer’s instructions.