Human LIF Recombinant Rabbit Monoclonal Antibody [PSH05-96] - BSA and Azide free (Capture)

Sandwich ELISA analysis of human LIF matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722495) 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 standard Human LIF protein (HA210736) starting from 2000 pg/ml to 0 pg/ml and detect antibody (HA722496, 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 LIF in cell culture supernatant and serum samples.

LIF was measured in 100% cell supernatant. The concentrations of LIF were interpolated from the LIF standard curves. The mean LIF concentration was determined to be 148 pg/mL in MDA-MB-231 cell supernanant. There was no detectable signal in T-47D cell supernatant and human serum.

Interpolated concentrations of native LIF in human A375 cell culture supernatant samples.

Interpolated concentration of native LIF was measured in duplicate at different sample concentrations. Undiluted samples were 12.5% cell supernatant. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2).

Interpolated concentrations of spiked LIF in human cell culture media samples.

The concentrations of LIF were measured in duplicates, interpolated from the LIF 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).