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Sandwich ELISA analysis of human CTGF matched pair antibodies
Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA723296) 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 CTGF protein (HA211051) starting from 6,000 pg/ml to 0 pg/ml and detect antibody (HA723297, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 50 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 CTGF in HeLa and MCF7 cell culture supernatant.
The concentrations of CTGF were measured in duplicates, interpolated from the CTGF standard curve and corrected for sample dilution. Undiluted samples are HeLa cell culture supernatant 100% and MCF7 cell culture supernatant 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean CTGF concentration was determined to be 9.9 ng/ml in HeLa cell culture supernatant and undetectable in MCF7 cell culture supernatant.
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Interpolated concentrations of spiked CTGF in human cell culture media samples.
The concentrations of CTGF were measured in duplicates, interpolated from the CTGF 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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