Anti igg isotypes

ELISA plate wells were coated with 100 ng of either TcdA rRBD or OVA overnight and then blocked with 5% nonfat dry milk (w/v) in PBS. Mouse antisera serially diluted 2-fold with PBS containing 1% BSA (Calbiochem, Darmstadt, Germany) were added to the wells, followed by incubation at room temperature (RT) for 2 h. Each group of pre-immunized serum was diluted fifty-fold with PBS. After washing three times with PBST, 50 ng/mL of either anti-IgG isotypes (Invitrogen, Carlsbad, CA) or anti-IgA (Invitrogen, Carlsbad, CA) HRP-conjugated IgG (KPL, Gaithersburg, MD)-specific antibodies diluted in PBS containing 1% BSA was added to the wells and incubated at RT for 1 h. After washing three times with PBST, the plates were treated with TMB peroxidase substrate (KPL) at RT in the dark for 20 min. To determine the anti-RBD or anti-OVA titers, the OD_{450 nm} absorbance was measured using a spectrophotometer.

ELISA plate wells were coated either with 100 ng of A-rRBD or OVA at 4 °C overnight, then blocked with 5 % nonfat dry milk (w/v) in PBS. Mouse antisera 2-fold serially diluted with PBS containing 1 % BSA (Calbiochem, Darmstadt, Germany) were added to the wells followed by incubation at room temperature (RT) for 2 h. After washing with 3 × PBST, either anti-IgG isotypes (Invitrogen, Carlsbad, CA.) or anti-IgA (Invitrogen, Carlsbad, CA) HRP-conjugated IgG (KPL, Gaithersburg, MD) specific antibodies diluted in PBS containing 1 % BSA were added to the wells and incubated at RT for 1 h. After washing with 3 × PBST, the plates were treated with TMB peroxidase substrate (KPL) at room temperature in the dark for 20 min. To determine anti-A-rRBD or anti-OVA titer, OD_450nm absorbance was measured using a spectrophotometer (Spectra max M2, Molecular Devices, Sunnyvale, CA).