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http://www.rndsystems.com/pdf/314-bp.pdf

ORDERING INFORMATION
Catalog Number: MAB726
Clone: 22210
Lot Number: AYF04
Size: 500 µg
Formulation: 0.2 µm filtered solution in PBS
with 5% trehalose
Storage: -20o
 C
Reconstitution: sterile PBS
Specificity: human TNF RII
extracellular domain
Immunogen: E. coli-derived rhTNF RII
extracellular domain
Ig class: mouse IgG1
Applications: ELISA capture
Neutralization of bioactivity
Western blot1

Monoclonal
Anti-human TNF RII/TNFRSF1B Antibody
Preparation
This antibody was produced from a hybridoma resulting from the fusion of a mouse
myeloma with B cells obtained from a mouse immunized with purified, E. coli-derived,
recombinant human tumor necrosis factor receptor type II （rhTNF RII） extracellular domain.
The IgG fraction of the tissue culture supernatant was purified by Protein G affinity
chromatography. TNF RII, also known as p75 or p80 TNF R, is a prototypic member of the
TNF receptor superfamily and has been designated TNFRSF1B. It binds TNF with high
affinity and is expressed primarily in cells of the immune system.
Formulation
Lyophilized from a 0.2 µm filtered solution in phosphate-buffered saline （PBS） with 5%
trehalose.
Reconstitution
Reconstitute with sterile PBS. If 1 mL of PBS is used, the antibody concentration will be
500 µg/mL.
Storage
Lyophilized samples are stable for twelve months from date of receipt when stored
at -20° C to -70° C. Upon reconstitution, the antibody can be stored at 2° - 8° C for 1 month
without detectable loss of activity. Reconstituted antibody can also be aliquotted and stored
frozen at -20° C to -70° C in a manual defrost freezer for six months without detectable
loss of activity. Avoid repeated freeze-thaw cycles.Specificity
This antibody was selected for use as a capture antibody in human TNF RII sandwich
ELISAs.
Applications
Neutralization of Human TNF RII Bioactivity - The exact concentration of antibody
required to neutralize human TNF RII activity is dependent on the cytokine concentration,
cell type, growth conditions and the type of activity studied. To provide a guideline, R&D
Systems has determined the neutralization dose for this antibody under a specific set of
conditions. The Neutralization Dose50 （ND50） for this antibody is defined as that
concentration of antibody required to yield one-half maximal inhibition of the cytokine
activity on a responsive cell line, when that cytokine is present at a concentration just high
enough to elicit a maximum response.
The ND50 for this lot of anti-human TNF RII antibody was determined to be approximay
0.5 - 1.5 µg/mL in the presence of 0.3 µg/mL of rhTNF RII and 0.25 ng/mL of rhTNF-α,
using the mouse L929 cell line as an assay. The specific conditions are described in the
figure legends.
Western Blot - This antibody can be used at 1 - 2 µg/mL with the appropriate secondary
reagents to detect human TNF RII. The detection limit for rhTNF RII is approximay
0.5 ng/lane under non-reducing conditions. In western blots, this antibody does not
cross-react with rhTNF RI, rmTNF RI or rmTNF RII.
ELISA Capture - This antibody can be used as a capture antibody in a human TNF RII
ELISA in combination with biotinylated, human TNF RII affinity purified polyclonal detection
antibody （Catalog # BAF726）. A general protocol is provided on the next page. Using plates
coated with 100 µL/well of the capture antibody at 2 µg/mL, in combination with 100 µL/well
of the detection antibody, an ELISA for sample volumes of 100 µL can be obtained. To
arrive at the optimal dose range for this ELISA, set up a two-fold dilution series of the
protein standard starting with 0.5 ng/mL. In this format, no cross-reactivity was observed
Figure 1: Human TNF RII inhibits the TNF-induced mouse L-929 cell lysis in a dose-
dependent manner. The ED50 for this effect is typically 0.05 - 0.1 µg/mL, in the presence of
0.25 ng/mL rhTNF-α and 1 µg/mL of actinomycin D.
Figure 2: Approximay 0.5 - 1.5 µg/mL of the antibody will neutralize 50% of the bioactivity
due to 0.3 µg/mL of rhTNF RII and 0.25 ng/mL of rhTNF-α.