INTENDED USE AND TEST PRINCIPLE
This TNF-α ELISA kit is intended Laboratory for Research use only ａnd is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow ａnd the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of TNF-α in the sample, this TNF-α ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples ａnd allow the operator to produce a standard curve of Optical Density versus TNF-α concentration. The concentration of TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.

SAMPLE COLLECTION AND STORAGES
Serum - Use a serum separator tube ａnd allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum ａnd assay immediately or aliquot ａnd store samples at -20℃. Avoid repeated freeze-thaw cycles
Plasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.
Tissue homogenates - For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed ａnd then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.
Cell culture supernates, tissue homogenate ａnd other biological fluids - Remove particulates by centrifugation ａnd assay immediately or aliquot ａnd store samples at -20℃. Avoid repeated freeze-thaw cycles.
Note: The samples shoule be centrifugated dequately ａnd no hemolysis or granule was allowed.

CALCULATION OF RESULTS
1.This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (X) axis versus the corresponding concentration on the horizontal (Y) axis.
2.First, calculate the mean O.D. value for each standard ａnd sample. All O.D. Values are subtracted by the mean value of the balnk well before result interpretation. Construct the standard curve using graph paper or statistical software.
3.To determine the amount in each sample, first locate the O.D. value on the Y-axis ａnd extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis ａnd read the corresponding concentration.
4.Any variation in operator, pipetting ａnd washing technique, incubation time or temperature, ａnd kit age can cause variation in result. Each user should obtain their own standard curve.
5.Intra-assay CV(%) is less than 10% ａnd Inter-assay CV(%) is less than 15%.
6.Assay range: 2.5 pg/mL – 80 pg/mL.
7. Sensitivity: The minimum detectable dose of Chicken TNF-α is typically less than 0.1 pg/mL.
8. Cross-reactivity: This assay recognizes recombinant ａnd natural Chicken TNF-α. No significant cross-reactivity or interference was observed.
9. Storage: 2-8℃ (Use frequently); six months (-20℃)。
10. Standard curve