Each mAb was diluted in 50 mM of carbonate buffer (pH 9.6) to a concentration of 10 μg/mL, and then added to an ELISA plate (AGC TECHNO GLASS, Shizuoka, Japan). To immobilize the antibodies, the plate was incubated overnight at 4°C. Wells were blocked with PBS containing 2% (w/v) skim milk for 1 h at room temperature (RT). After three washes with PBS containing 0.05% (v/v) Tween-20 (PBS-T), 100 μL of antigen protein (1 ng/mL) diluted with PBS-T or blank (PBS-T alone) was added and incubated for 60 min at RT. After three washes with PBS-T, 100 μL of each mAb conjugated with horseradish peroxidase (HRP) was added into each well and incubated for 60 min at RT. Antibody labeling was performed using the Peroxidase Labeling Kit -NH2 (Dojindo Laboratories, Kumamoto, Japan). After three washes with PBS-T, 100 μL of ABTS substrate solution (Kirkegaard & Perry Laboratories, Washington, DC, USA) was added and incubated for 30 min at RT. Absorbance at 415/492 nm was measured on a plate reader, and the signal-to-noise ratio (S/N) was calculated.
Elisa plate
Manufactured by AGC Techno Glass
Sourced in Japan
The ELISA plate is a laboratory equipment used for enzyme-linked immunosorbent assay (ELISA) testing. It is a flat plate with multiple wells, typically made of polystyrene, that can hold small volumes of liquid samples for analysis. The wells are designed to capture specific antigens or antibodies, which can then be detected and quantified using specialized reagents and equipment.
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2 protocols using elisa plate
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ELISA Assay for Antibody Screening
2
ELISA Protocol for Antibody Characterization
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Each mAb was diluted in 50 mM of carbonate buffer (pH 9.6) to a concentration of 10 μg/mL, and then added to an ELISA plate (AGC TECHNO GLASS, Shizuoka, Japan). To immobilize the antibodies, the plate was incubated overnight at 4 °C. Wells were blocked with PBS containing 2% (w/v) skim milk for 1 h at room temperature (RT). After three washes with PBS containing 0.05% (v/v) Tween-20 (PBS-T), 100 μL of antigen protein (8 ng/mL) diluted with PBS-T or blank (PBS-T alone) was added, and the mixture was incubated for 60 min at RT. After three washes with PBS-T, 100 μL of each mAb, conjugated with horseradish peroxidase (HRP), was added into each well and incubated for 60 min at RT. Antibody labeling was performed using the Peroxidase Labeling Kit—NH2 (Dojindo Laboratories, Kumamoto, Japan). After three washes with PBS-T, 100 μL of ABTS substrate solution (Kirkegaard and Perry Laboratories, Washington, DC, USA) was added and the mixture was incubated for 30 min at RT. Absorbance at 405/490 nm was measured on GloMax Discover System (Promega), and the signal-to-noise ratio (S/N) was calculated.
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