ELISA Detection: Biotin Rat Anti-Human CD120b (Cat. No. 552477) serves as the detection antibody in a sandwich ELISA for measuring human TNFRII protein levels. Biotin Rat Anti-Human CD120b (Cat. No. 552477) can be paired with purified MR2-2 anti-human TNFRII using the recombinant human soluble TNFRII 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 TNFRII ranging from 1000 to 10 pg/ml are recommended for inclusion in each ELISA plate. For specific methodology, please visit our website, http://www.bdbiosciences.com/us/s/resources, and go to the protocols section under "ELISA and ELISPOT".
Note: This ELISA antibody pair shows no cross-reactivity with the following recombinant human cytokines: CD14,CD40, CD40L, IL-1Ra, IL-1RII, IL-1α, IL-1β, IL-2, IL-3, IL-4, sIL-4R, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, TNF, LT-α, sTNFRI, IFNγ, TRAIL, GM-CSF, TGFβ, Trx. This ELISA antibody pair also shows no cross-reactivity with recombinant mouse sTNFRI, recombinant mouse sTNFRII, recombinant mouse TNF.
Immunofluorescent Staining and Flow Cytometric Analysis: The Purified Rat Anti-Human CD120b (Cat. No. 551311) antibody can be used for the immunofluorescent staining (≤ 1 µg antibody/10e6 cells) and flow cytometric analysis of human nucleated cells to measure their expressed levels of surface TNFRII. An appropriate immunoglobulin is Purified Rat IgG2b, κ Isotype Control (Cat. No. 555846). Other formats available for immunofluorescent staining include, PE Rat Anti-Human CD120b (Cat. No. 552418). Please note also that as a consequence of in vivo or in vitro activation, cell surface TNFRII 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 TNFRII.