Rat TNF-Alpha ELISA Assay

Product Overview:

Rat TNF alpha (TNF-α) ELISA assay kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of rat TNF alpha (TNF-α) concentrations in cell culture supernates, serum, and plasma.

Assay Principle:

The rat TNF alpha (TNF-α) ELISA assay kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF alpha has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF alpha present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for TNF alpha is added to the wells and binds to the combination of capture antibody TNF-alpha in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of TNF alpha present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven TNF alpha (TNF-α) standard dilutions and TNF alpha sample concentration determined.

Sensitivity:

15 pg/ml

Dynamic Range:

31.2 pg/ml-2000 pg/ml

Incubation Time:

3.5 hours

Sample Type:

Serum, plasma, cell culture

Sample Size:

100 µl

Specimen Collection and Handling:

Cell culture supernates – remove particulates by centrifugation.
Serum – use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum, avoid hemolysis and high blood lipid samples.
Plasma – recommended EDTA as an anticoagulant in plasma. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.
Dilute samples at the appropriate multiple (recommended to do pre-test to determine the dilution factor).