Biotin Conjugated Human CSPG4 Recombinant Rabbit Monoclonal Antibody [PSH10-79] - Detector

Sandwich ELISA analysis of Human CSPG4 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA723244) diluted in carbonate/bicarbonate buffer, at a concentration of 5 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 CSPG4 protein (HA211047) starting from 20,000 pg/ml to 0 pg/ml and detect antibody (HA723247B, 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.

Sandwich ELISA analysis of Human CSPG4 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA723245) diluted in carbonate/bicarbonate buffer, at a concentration of 5 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 CSPG4 protein (HA211047) starting from 20,000 pg/ml to 0 pg/ml and detect antibody (HA723247B, 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.

Interpolated concentrations of native CSPG4 in A-375, SK-MEL-28, SK-BR-3 and MCF7 cell culture supernatant.

The concentrations of CSPG4 were measured in duplicates, interpolated from the CSPG4 standard curve and corrected for sample dilution. Undiluted samples are A-375 cell culture supernatant 25%, SK-MEL-28 cell culture supernatant 100%, SK-BR-3 cell culture supernatant 100% and MCF7 cell culture supernatant 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean CSPG4 concentration was determined to be 164.9 ng/ml in A-375 cell culture supernatant, 17.2 ng/ml in SK-MEL-28 cell culture supernatant and undetectable in SK-BR-3 and MCF7 cell culture supernatant.

Interpolated concentrations of spiked CSPG4 in human cell culture media samples.

The concentrations of CSPG4 were measured in duplicates, interpolated from the CSPG4 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).