Hrp conjugated goat anti mouse igg1 antibody

Enzyme-linked immunosorbent assay (ELISA) plates were purchased from AGC (Tokyo, Japan). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgE antibody was purchased from Southern Biotech (Birmingham, AL, USA). HRP-conjugated goat anti-mouse IgG1 antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA). 3,3,5,5-Tetramethylbenzidine (TMB) peroxidase substrate was purchased from Kirkegaard & Perry Laboratories (KPL; Gaithersburg, MD, USA). Coomassie brilliant blue (CBB) R-250, Bradford protein assay reagent, and pentobarbital sodium salt were purchased from Nacalai Tesque (Kyoto, Japan). Enhanced chemiluminescence (ECL) western blotting substrates and X-ray films (Amersham Hyperfilm MP) were purchased from GE Healthcare (Chalfont St. Giles, UK). Luminata Crescendo Western HRP Substrate was purchased from Merck Millipore (Burlington, MA, USA). Immunoreactive enhancers, Can Get Signal solutions 1 and 2, were purchased from TOYOBO (Osaka, Japan). All other chemicals used in this study were of the highest purity available.

A 96-well flat-bottom half area plate (Costar) was coated with 10 μg/ml mBSA in coating buffer (15 mM Na2CO3 and 35 mM NaHCO3) for 1 h and blocked with 2% FCS (PAA Laboratories GmbH) in PBS for 2 h. Serum from AIA mice (day 10) was diluted 1:150 with blocking buffer and incubated for 2 h. HRP conjugated goat anti-Mouse IgG1-antibody (Bethyl) was diluted 1:10.000 in blocking buffer and loaded for further 1 h. The horseradish peroxidase activity was induced by TMB-substrate (Merck), stopped with 6% orthophosphoric acid and the signal was assessed using a Wallac 1420 Victor2 Microplate Reader (Perkin Elmer) at 450 nm. All incubation steps were performed at RT.

Enzyme-linked immunosorbent assay (ELISA) plates were purchased from AGC (Tokyo, Japan). Horseradish-peroxidase (HRP)-conjugated goat antimouse IgE antibody was purchased from Southern Biotech (Birmingham, AL, USA). HRP-conjugated goat antimouse IgG1-antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA). 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD, USA). Coomassie Brilliant Blue R-250, Bradford protein assay reagent, and pentobarbital sodium salt were purchased from Nacalai Tesque (Kyoto, Japan). Enhanced chemiluminescence (ECL) Western blotting substrate and X-ray films (Amersham Hyperfilm MP) were purchased from GE Healthcare (Chalfont St. Giles, UK). Immunoreactive enhancers, CanGet Signal solutions 1 and 2, were purchased from TOYOBO (Osaka, Japan). All other chemicals used in this study were of the highest purity available.