Biotin Conjugated Human IGFBP3 Recombinant Rabbit Monoclonal Antibody [PSH12-17] - Detector

Sandwich ELISA analysis of human IGFBP3 matched pair antibodies

Capture: HA723432, Human Leptin Rabbit mAb [PSH12-16]
Detector: HA723433, Human Leptin Rabbit mAb [PSH12-17]

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA723432) 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 IGFBP3 protein (HA210585) starting from 4,000 pg/ml to 0 pg/ml and detect antibody (HA723433, Biotin, 0.05 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.

Interpolated concentrations of native IGFBP-3 in A431, A549 and HT-29 cell culture supernatant.

Capture: HA723432, Human Leptin Rabbit mAb [PSH12-16]
Detector: HA723433, Human Leptin Rabbit mAb [PSH12-17]

The concentrations of IGFBP-3 were measured in duplicates, interpolated from the IGFBP-3 standard curve and corrected for sample dilution. Undiluted samples are A431 cell culture supernatant 2%, A549 cell culture supernatant 25% and HT-29 cell culture supernatant 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean IGFBP-3 concentration was determined to be 131.3 ng/ml in A431 cell culture supernatant, 8.04 ng/ml in A549 cell culture supernatant and undetectable in HT-29 cell culture supernatant.

Interpolated concentrations of spiked IGFBP-3 in human cell culture media samples.

Capture: HA723432, Human Leptin Rabbit mAb [PSH12-16]
Detector: HA723433, Human Leptin Rabbit mAb [PSH12-17]

The concentrations of IGFBP-3 were measured in duplicates, interpolated from the IGFBP-3 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).