Tween 20

ELISA was used to evaluate the sensitization profiles of cases and controls against Bt allergen extract. All procedures were done at room temperature unless otherwise stated. Bt extract was diluted in a 50-mM carbonate-bicarbonate buffer (pH 9.6) to have a final concentration of 10 µg/mL. A 50-µL diluted coating Bt extract was then added per well onto an ELISA plate (Corning Costar Inc., NY, USA) and was incubated overnight at 4°C with gentle constant shaking. Plates were blocked with 100 µL of 1% BSA (Fisher BioReagents, MA, USA) diluted in 1× PBS for 1 hour. A 5× diluted human plasma (50 µL) in 1% BSA was added to each well in duplicates and the plates were incubated for 2 hours. Afterwards, 100 µL of diluted Horseradish peroxidase conjugated with antihuman IgE was added per well and plates were incubated for an hour. Lastly, colorimetric reaction was performed by adding 100 µL of TMB (Sigma-Aldrich, Saint Louis, MO, USA) per well. In between steps, ELISA plates were washed 3 times with 1× PBS with 0.05% Tween 20 (Loba Chemie Pvt Ltd, IN) using an ELISA Washer (Lisawash 3000, IN). Absorbance at 605 nm was read 30 minutes after the addition of TMB on the ELISA plate reader (SPECTROstar Nano, USA).

Inhibition of IgE reactivity using ELISA was performed to determine the inhibition capacity of rBlo t 5 against Bt allergen extract. Seven Bt extract and rBlo t 5-positive samples were used. All procedures were done at room temperature unless otherwise stated. A 10-µg/mL concentration of Bt extract diluted in a 50-mM carbonate-bicarbonate buffer (pH 9.6) was coated onto ELISA plates (Corning Costar Inc., NY, USA) at 50 µL per well and was incubated overnight at 4°C. Simultaneously, each plasma was preabsorbed with 5 µg/mL of rBlo t 5 and was also incubated overnight at 4°C. The incubated plate was blocked with 1% BSA (Fisher BioReagents, MA, USA) diluted in 1× PBS for 1 hour. The preabsorbed plasma, along with the corresponding unabsorbed plasma in duplicates, was added to the plate and incubated for 2 hours. Afterwards, 100 µL of diluted horseradish peroxidase conjugated with antihuman IgE was added per well and was incubated for an hour. Lastly, colorimetric reaction was performed by adding 100 µL of TMB (Sigma-Aldrich, Saint Louis, MO, USA) per well. In between steps, ELISA plates were washed 3 times with 1× PBS with 0.05% Tween 20 (Loba Chemie Pvt Ltd, IN) using an ELISA Washer (Lisawash 3000, IN). Absorbance at 605 nm was read 30 minutes after the addition of TMB on the ELISA plate reader (SPECTROstar Nano, USA).

EA was purchased from Yucca Enterprises Pvt. Ltd, Mumbai, India. Capmul MCM C8, and captex 500 were gifted by Abitec Corp., USA. Capryol 90, labrafil M 2125 CS, labrafil M 2130 CS, labrafac CC, labrasol, lauroglycol FCC, transcutol P were kindly gifted by Gattefosse, France. Oleic acid, tween 80, tween 20, span 20, propylene glycol, PEG 400 and PEG 200 were purchased from Loba Chemie Pvt. Ltd., Mumbai. Cremophor RH 40 was a gift from BASF, Germany.