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Sandwich ELISA analysis of human IL-1RA matched pair antibodies
Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722788) diluted in carbonate/bicarbonate buffer, at a concentration of 2 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 IL-1RA protein (HA210837) starting from 2000 pg/ml to 0 pg/ml and detect antibody (HA722790B, 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.
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Interpolated concentrations of native IL-1RA in human samples.
Interpolated concentration of native IL-1RA was measured in duplicate at different sample concentrations and interpolated from the IL-1RA standard curves. Undiluted samples were 50% samples. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean IL-1RA concentration was determined to be 4983 pg/mL in human urine.
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Interpolated concentrations of native IL-1RA in human cell culture supernatant samples.
Interpolated concentration of native IL-1RA was measured in duplicate at different sample concentrations and interpolated from the IL-1RA standard curves. Undiluted samples were 100% cell supernatant. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean IL-1RA concentration was determined to be 3068 pg/mL in THP-1 and 374 pg/ml in HepG2 in cell culture supernatant.
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Interpolated concentrations of spiked IL-1RA in human cell culture media samples.
The concentrations of IL-1RA were measured in duplicates, interpolated from the IL-1RA standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).
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