Tetramethylbenzidine tmb

96-well microtiter plates were coated with 100 µL 10 µg/mL recombinant FRα in 0.05 M sodium bicarbonate (pH 9.6) and the plate was kept overnight at 4 °C. After washing the plate three times with TBST, the plate was blocked with 3% BSA at 37 °C for 2 h. For polyclonal phage ELISA, after each round of screening, the phage eluate was amplified and 100 μL 10¹⁰ phages diluents were added to each well and incubated for 2 h. To analysis individual phage clones, each phage clone was amplified and 10¹⁰ phages were added to each well (100 µL /well) and incubated for 2 hours at 37 °C. After washing the plate for 3 times with TBST and 3 times with TBS, 100 µL of HRP-conjugated anti-M13 antibody (1:10000, Sino Biological, Inc., Beijing) was added and the plate was incubated for 1 hour at 37 °C. After washing, the tetramethylbenzidine (TMB) (Sangon Biotech Co., Ltd., Shanghai) substrate (100 μL/well) was added to the wells and the reaction lasted for 15 minutes. The reactions were stopped with 2 M sulfuric acid (50 μL/well). The absorbance of each well at 450 nm was detected with an automated ELISA reader.

Biotin-labelled pHLAs were generated through refolding as per the previous report [26 (link)]. A 96-well EIA/RIA plate (Corning Incorporated, Corning, NY, USA) was coated with streptavidin (2 μg/mL, 0.1 mL) in coating buffer (0.5 M carbonate/bicarbonate buffer, pH = 9.6) at 4 °C overnight. After incubation, the plate was washed with PBST (PBS containing 0.05% Tween 20) 3 times and was then blocked with blocking buffer (5% skim powder in PBST) at 37 °C for 1 h. The wells were then incubated with different biotin-labelled pHLAs at 37 °C for 1 h. The plate was then washed 4 times with PBST before a 1 h incubation with different concentrations of NY-TCR/aCD3 at 37 °C followed by a 0.5 h incubation with HRP-conjugated goat anti-human IgG (H + L) (Beyotime Biotechnology, Shanghai, China) at 37 °C after 4 washes with PBST. Finally, the samples were detected using tetra-methyl-benzidine (TMB, Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China)) after 6 washes with PBST, and the reaction was stopped using 2 M H₂SO₄ followed by the measuring the absorbance at 450 nm. The results were presented as the mean ± S.D (n = 3)

96-well polystyrene microtiter plates were coated with a solution of target protein (5 μg/mL) in 0.05 M sodium bicarbonate (pH 9.6) and the plate was kept overnight at 4 °C. After washing the plate three times, the plate was blocked with 0.1 M PBS (pH 7.4) containing 3% NFM at 37 °C for 2 hours (200 μL/well). Individual phage clones were amplified and 10¹⁰ phages were added to each well (100 μL/well) and incubated for 2 hours at 37 °C. After washing the plate for 3 times with TBST and 3 times with TBS, 100 μL of HRP-conjugated anti-M13 antibody (1:5000, Sino Biological, Inc., Beijing) was added and the plates were incubated for 2 hours at 37 °C. After washing three times, the tetramethylbenzidine (TMB) (Sangon Biotech Co., Ltd., Shanghai) substrate (100 μL/well) was added and the reaction lasted for 15 minutes. The reactions were stopped with 2 M sulfuric acid (50 μL/well). The absorbance of each well at 450 nm was detected with an automated ELISA reader. Phage clones with high absorbance were selected for sequencing.