Anti mouse igg or iga

IgG and IgA antibody levels for MV140, MV140-containing E. coli strain (V121), or UTI89 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) from serum and urine samples obtained at indicated timepoints. Briefly, 96-well non-tissue culture-treated plates (Greiner Bio-One, Monroe, NC, USA) were pretreated with poly-L-lysine (Sigma-Aldrich) for 1 h under UV light. Then, plates were coated with the heat-inactivated MV140, V121 E. coli or UTI89 (all at 450 FTU) overnight at 4 °C, and, subsequently, incubated with 50 µL of mouse serum for 2 h at room temperature (RT). In the case of the urine samples, 50 µL of pooled concentrated urine were used per well. After a washing step, Igs were detected using a horseradish peroxidase-labeled antibody (anti-Mouse IgG or IgA from Sigma). After a final washing step, the peroxidase substrate (OPD, 3 mg/mL from Sigma-Aldrich) was added in 0.1 M citrate buffer with 0.03% H₂0₂—pH 5.5. The enzymatic reaction was allowed to develop for 30 min and stopped by adding a 10% HCl solution. Plates were read at 492 nm (Synergy Mx, Biotek, Agilent Technologies, Durham, NC, USA), and antibody concentration was expressed as arbitrary units (AUs) per mL, calculated from the optical density (OD) at 492 nm using the following formula: AU/mL = [(OD × dilution factor] × 10.

Changes in humoral immune responses upon oral administration of recombinant yeast cells were analyzed by estimating the antibodies induced through indirect ELISAs using NUNC Maxisorp 96-well ELISA plates coated with 100 ng/well of E. coli-expressed recombinant scEDIII protein. The plates were washed three times with PBS + 0.05% Tween 20 and blocked with 1% BSA in PBS for 2 h at 37 °C. Following the washes, twofold serial dilutions were performed after adding 100 μL/well of sera from immunized mice (the starting dilution points were 1:25 for serum IgG and 1:2 for fecal sIgA), and the plates were incubated overnight at 4 °C. Alkaline phosphatase (AP)-conjugated secondary antibodies (anti-mouse IgG or IgA; Sigma-Aldrich) diluted 1:5000 in PBS containing 0.5% BSA were added and incubated for 2 h at 37 °C followed by a washing step. To detect the response, 100 μL/well of phosphatase substrate (S0942, Sigma-Aldrich) was added and incubated for 15 min at room temperature. The reaction was stopped with 2 M H₂SO₄ and the optical density was measured at 405 nm using a microplate reader (Multiskan™ GO Microplate Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA).