Serum HDM-specific IgE ELISA was performed as described previously(15 (link)). 96-well
Nunc MaxiSorp flat-bottom plates (Thermo-Fisher Scientific, Waltham, MA) were coated with 10 ug/mL HDM in sodium bicarbonate
buffer (pH 9.5) for 48 hours at 4°C. Following coating, plates came to room temperature for 15 minutes and were washed six
times with 0.05% PBS/Tween 20 (PBST) and blocked for one hour at 37°C with BD OptEIA Assay Diluent (BD
Biosciences, San Diego, CA; Cat #555213). After washes, serum samples were added in two-fold serial dilutions (range 1:20
to 1:2560) and incubated at room temperature for 1.5 hours.
Plates were washed eight times with PBST and labeled with Biotin-SP-conjugated goat anti- mouse IgE (Southern Biotech,
Birmingham, AL; Cat #1110-08) diluted 1:5000 in blocking buffer for one hour at room temperature. Plates were then washed
six times and incubated with streptavidin HRP (BD Biosciences, San Jose, CA; Cat #554066) diluted 1:1000 in blocking
buffer at room temperature for thirty minutes. Plates were washed seven times with PBST and incubated with
3,3',5,5'-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD; Cat #50-76-01) for 20 minutes in the dark at room temperature.
The reaction was stopped using 1 M phosphoric acid at equal volume to TMB and the plates were read at dual 450nm-570nm wavelengths
using an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA).
Nunc MaxiSorp flat-bottom plates (Thermo-Fisher Scientific, Waltham, MA) were coated with 10 ug/mL HDM in sodium bicarbonate
buffer (pH 9.5) for 48 hours at 4°C. Following coating, plates came to room temperature for 15 minutes and were washed six
times with 0.05% PBS/Tween 20 (PBST) and blocked for one hour at 37°C with BD OptEIA Assay Diluent (BD
Biosciences, San Diego, CA; Cat #555213). After washes, serum samples were added in two-fold serial dilutions (range 1:20
to 1:2560) and incubated at room temperature for 1.5 hours.
Plates were washed eight times with PBST and labeled with Biotin-SP-conjugated goat anti- mouse IgE (Southern Biotech,
Birmingham, AL; Cat #1110-08) diluted 1:5000 in blocking buffer for one hour at room temperature. Plates were then washed
six times and incubated with streptavidin HRP (BD Biosciences, San Jose, CA; Cat #554066) diluted 1:1000 in blocking
buffer at room temperature for thirty minutes. Plates were washed seven times with PBST and incubated with
3,3',5,5'-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD; Cat #50-76-01) for 20 minutes in the dark at room temperature.
The reaction was stopped using 1 M phosphoric acid at equal volume to TMB and the plates were read at dual 450nm-570nm wavelengths
using an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA).