Goat anti mouse iga hrp

The antibodies of serum and irrigating solution specific to OBGs, intimin, Stx1 and Stx2 were measured by enzyme linked immunosorbent assay. 100 μl OBGs (0.1 mg/ml), purified intimin protein, Stx1 or Stx2 (0.1 mg/ml) were coated in 96-well plates overnight at 4 °C, respectively. Goat anti-mouse IgA-HRP (Sigma, 1:5000) or goat anti-mouse IgG-HRP (Sigma, 1:5000) used as the detection antibodies. The reactions were developed with TMB and stopped with 2 M H₂SO₄. The absorbance at 450 nm was detected.

Luminal IgA levels from the small intestine were measured by an enzyme-linked immunosorbent assay (ELISA). The small intestine was removed from mice after necropsy and incubated overnight at 4 °C in PBS with 0.01% sodium azide and 1% protease inhibitor cocktail (Sigma-Aldrich). The IgA containing supernatants were transferred to a new tube after centrifugation at 16,000×g for 10 min and stored at − 80 °C. For the sandwich ELISA, maxisorb 96-well plates were coated with 2 µg/ml sheep anti-mouse IgA (Sigma-aldrich) for 16 h at 4 °C. After washing with PBS-Tween 20 (0.05%) (PBST), plates were subsequently blocked with 2% bovine serum albumin (BSA) in PBST. Luminal extracts were added to the plates in a 1/10 dilution and incubated for 1 h at room temperature followed by a washing step with PBST. Detection of IgA was performed by adding goat anti-mouse IgA-HRP (Sigma-Aldrich) in a 1/1000 dilution in PBST followed by a visualisation by ABTS in ABTS buffer (Roche). Finally, optical density was measured at 405 nm with a Tecan plate reader moderated by conjugate control levels measured at 492 nm.

The IgA/IgG of serum and irrigating solution specific to OBGs and proteins (intimin, EVP1, and protein CVP1) were measured by enzyme-linked immunosorbent assay. The presence of serum IgG and of the subtypes IgG1, IgG2a, IgG2b, and IgG3 specific to vaccine candidates was determined by indirect ELISA. The BGs (EBGs and CBGs, 0.1 mg/mL) and purified proteins (intimin, EVP1, and CVP1, 0.1 mg/mL) were coated in 96-well plates overnight at 4 °C, respectively. Serum samples were serially diluted in 2-fold dilutions from 1:10 to 1:20480. The endpoint dilution titer was calculated as the serum dilution resulting in an absorbance reading of 0.2 units above background. Goat anti-mouse IgA-HRP (Sigma, 1:5000) or goat anti-mouse IgG-HRP (Sigma, 1:5000) were used as the detection antibodies. The reactions were developed with TMB (3,3’,5,5’-Tetramethylbenzidine) and stopped with 2 M H₂SO₄. The absorbance at 450 nm was detected.