Human Interferon gamma Recombinant Rabbit Monoclonal Antibody [PSH01-47] - BSA and Azide free (Detector)

Standard curve of human IFNG matched pair antibodies:

Capture: HA721683, Interferon gamma Rabbit mAb [PSH01-45]
Detector: HA721685, Interferon gamma Rabbit mAb [PSH01-47]

Sandwich ELISA analysis of human IFNG matched pair antibodies
Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA721683) diluted in carbonate/bicarbonate buffer, at a concentration of 4 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1% BSA blocking buffer, and incubated with serial diluted IFNG protein starting from 2500 pg/ml to 0 pg/ml and detect antibody (HA721685, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Standard curve of human IFNG matched pair antibodies:

Sandwich ELISA analysis of human IFNG matched pair antibodies
Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody [PSH01-46] diluted in carbonate/bicarbonate buffer, at a concentration of 4 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1%BSA blocking buffer, and incubated with serial diluted IFNG protein starting from 2500 pg/ml to 0 pg/ml and detect antibody [PSH01-47]-Biotin (0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Interpolated concentrations of native IFN-gamma in human PBMC cell culture supernatant.

Capture: HA721683, Interferon gamma Rabbit mAb [PSH01-45]
Detector: HA721685, Interferon gamma Rabbit mAb [PSH01-47]

PBMC cells were stimulated with 10 µg/ml PHA-M or vehicle control and incubated for 48 hours. The concentrations of IFN-gamma were interpolated from the IFN-gamma standard curves and corrected for sample dilution. Undiluted samples are PHA-M stimulated PBMC supernatant 10% and unstimulated PBMC supernatant 10%. The mean IFN-gamma concentration was determined to be 12,543 pg/ml in PHA-M stimulated PBMC supernatant and undetectable in the unstimulated PBMC supernatant.

Sandwich ELISA analysis of Cynomolgus IFNG matched pair antibodies

Capture: HA721683, Human Interferon gamma Rabbit mAb [PSH01-45]
Detector: HA721685, Human Interferon gamma Rabbit mAb [PSH01-47]

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA721683) diluted in carbonate/bicarbonate buffer, at a concentration of 4 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1% BSA blocking buffer, and incubated with serial diluted Recombinant Human Interferon gamma protein (HA210762) starting from 4,000 pg/ml to 0 pg/ml and detect antibody (HA721685, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Interpolated concentrations of native IFN-gamma in cynomolgus monkey PBMC cell culture supernatant samples treated with or without PMA(50 ng/ml) and Ionomycin(1µg/ml) for 24 hours.

Capture: HA721683, Human Interferon gamma Rabbit mAb [PSH01-45]
Detector: HA721685, Human Interferon gamma Rabbit mAb [PSH01-47]

The concentrations of IFN-gamma measured in duplicate and interpolated from the IFN-gamma standard curve and corrected for sample dilution. Undiluted samples are as follows: stimulated PBMC surpernatant 50% and unstimulated PBMC supernatant 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean IFN-gamma concentration was determined to be 1,046 pg/ml in stimulated PBMC culture supernatant and undetectable in the unstimulated PBMC culture surpernatant.