Human/Monkey CD14 Recombinant Rabbit Monoclonal Antibody [PSH28-59] - BSA and Azide free (Detector)

Sandwich ELISA analysis of Human CD14 protein matched pair antibodies

Capture: HA724796, Human/Monkey CD14 Rabbit mAb [PSH28-58]
Detector: HA724797, Human/Monkey CD14 Rabbit mAb [PSH28-59]

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA724796) 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 CD14 protein (HA211512) starting from 8,000 pg/mL to 0 pg/mL and detect antibody (HA724797, Biotin, 0.05 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 CD14 in human samples.

Capture: HA724796, Human/Monkey CD14 Rabbit mAb [PSH28-58]
Detector: HA724797, Human/Monkey CD14 Rabbit mAb [PSH28-59]

Interpolated concentration of native CD14 was measured in duplicate at different sample concentrations. Undiluted samples were 0.5% cell supernatant. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean CD14 concentration was determined to be 4,464 ng/mL in human serum and 10.6 ng/mL in HepG2 cell culture supernatant. There was no detectable signal in Raji cell culture supernatant.

Interpolated concentrations of native CD14 in cynomolgus monkey serum.

Capture: HA724796, Human/Monkey CD14 Rabbit mAb [PSH28-58]
Detector: HA724797, Human/Monkey CD14 Rabbit mAb [PSH28-59]

Interpolated concentration of native CD14 was measured in duplicate at different sample concentrations and interpolated from the CD14 standard curves. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean CD14 concentration was determined to be 535 ng/mL in cynomolgus monkey serum.

Interpolated concentrations of spiked CD14 in cell culture media samples.

Capture: HA724796, Human/Monkey CD14 Rabbit mAb [PSH28-58]
Detector: HA724797, Human/Monkey CD14 Rabbit mAb [PSH28-59]

The concentrations of CD14 were measured in duplicates, interpolated from the CD14 standard curves and corrected for sample dilution. Diluted samples are as follows: 50% cell culture media with FBS. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).