BD Pharmingen™ Biotin Mouse Anti-Human CD120a

Immunofluorescent Staining and Flow Cytometric Analysis: Biotin Mouse Anti-Human CD120a (Cat. No. 550900) can be used for immunofluorescent staining (≤ 1 µg antibody/10×6 cells) and flow cytometric analysis of nucleated human cells to measure their expressed levels of surface TNFRI. A two-layer staining protocol is recommended for maximizing the detection of TNFRI expressed by cells as detailed in the figure legend. As a demonstration of specificity, the binding of MABTNFR1-B1 to TNFRI is inhibited when human TNFRI+ target cells are preincubated with saturating levels of recombinant human TNF at 4°C, i.e., when the TNFRI are bound with ligand. Based on our data, a recombinant human TNF level above 50 ng/10×6 cells is sufficient to completely inhibit the binding of MABTNFR1-B1 (0.06 µg/10×6 cells). Please note also that as a consequence of in vivo or in vitro activation, cell surface TNFRI can either be shed by cells or transiently expressed at higher levels. As a result, cellular activation can affect the cell's overall expressed level of surface TNFRI.

ELISA Detection: Biotin Mouse Anti-Human CD120a serves as the detection antibody in a sandwich ELISA for measuring human soluble TNFRI protein levels. Biotin Mouse Anti-Human CD120a antibody can be paired with the Purified Mouse Anti-Human CD120a (Cat No. 552535) using recombinant human soluble TNFRI as the standard. This detection antibody should be titrated between 0.5 - 1 µg/ml to determine its optimal concentration for ELISA detection. To obtain linear standard curves, doubling dilutions of recombinant soluble human TNFRI ranging from 200 to 15 pg/ml are recommended for inclusion in each ELISA plate.  For specific methodology, please visit the protocols section under "ELISA and ELISPOT" at our website, http://www.bdbiosciences.com/us/s/resources.