Horseradish peroxidase hrp

Anti-CTB-specific IgA levels in fecal extracts were determined using a previously described method [22 (link)]. Briefly, MaxiSorp ELISA plates (Nalgene Nunc International, Thermo Fisher, Waltham, MA, USA) were coated overnight with 1 µg/mL of CTB-KDEL. After blocking, 100 µL of fecal samples in appropriate dilutions were added to the ELISA plates. The IgAs bound to the plates were detected using goat anti-mouse IgA antibodies conjugated with horseradish peroxidase (HRP; SouthernBiotech, Birmingham, AL, USA) and a TMB Super Sensitive One Component HRP Microwell Substrate (SurModics BioFx). Absorbance was measured at 450 nm with a microplate reader (BIotek Synergy H1, Winooski, VT, USA) after the reaction was stopped. Dilutions of purified mouse IgA standards were used to optimize the ELISA. The titer was defined as the greatest dilution factor of the sample with positive OD450 reading after subtracting an average background value.

96-well ELISA plates were coated with recombinant gE protein at 0.5 µg/mL and incubated overnight at 2–8 °C. Next day, plates were washed and blocked for 2 h at room temperature using 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Serial 5-fold dilutions of the serum samples were made, starting at a 1:100 or 1:500 or 1:2500 dilution depending upon the timepoint being measured. After discarding the blocking solution, diluted serum samples were then transferred to blocked plates and incubated for 2 h at room temperature. After incubation, plates were washed with PBS containing 0.05% Tween-20 (PBS-Tween). Goat anti-Mouse IgG (H + L) secondary antibody conjugated with Horseradish peroxidase (HRP) (SouthernBiotech, 1:5000 dilution) was added to the plates for detection and incubated for 1 h at room temperature. Plates were developed with Tetramethylbenzidine (TMB) substrate, and reaction was stopped with 1 M Phosphoric acid (H₃PO₄). Absorbance was read at a wavelength of 450 nm using a Biotek Synergy HTX plate reader. Antibody titers were interpolated based on a cut off OD value of 0.2 derived from OD values of blank wells containing no sera.