IOCyto Detect™ TNF alpha Rat Uncoated ELISA Kit (CAT#: ITS-0622-CR192)

10 x 96 Tests

Capture Antibody
Detection Antibody
Standard
ELISA/ELISPOT Coating Buffer
Assay Diluent
Detection enzyme
Substrate Solution
Certificate of Analysis
96 Well Plate

Buffers
Pipettes and pipettors
Refrigerator
96-well ELISA plate reader (microplate spectrophotometer)
Microplate shaker
ELISA plate washer

16 pg/mL

100 µL

Less than 36 hours

96 well plate

1. Coated buffer (1X)
Dilute PBS (10X) at 1:10 in deionized water.
2. Capture antibody
Antibodies (250X) were captured in the coated buffer (1X) with 1:250 dilution.
3. 5X ELISA/ELISPOT dilution
Dilute a concentrate (5X) of 1:5 in deionized water.
4. Standard
The protein standard was reconstructed by adding distillate water. The redissolved volume is indicated on the label of the standard bottle. Let the standard redissolve for 10-30 minutes. Swirl or mix gently to ensure complete uniform dissolution.
5. Detect antibody
Detection of antibodies in 1:250 diluted ELISA/ELISPOT solution (250X).
6. Enzyme
Dilute HRP concentrate with 1:100 in ELISA/ELISPOT diluted solution (100X).

1. ELISA plate with captured antibody in coated buffer. Sealed plate and incubated overnight at 4°C.
2. Drain the hole and wash it 3-5 times with washing buffer. Leaving time for soaking during each washing step can improve washing results. Imprinted plate on blotting paper to remove any residual buffer.
3. The well was sealed with ELISA/ELISPOT Diluent (1X). Incubate at room temperature for 1-2 hours.
4. Prepare standards (see Test Protocols).
5. Drain with washing buffer and wash at least once.
6. Double the series dilution of the top standard products to make standard curves with 8 points in total. To do this, ELISA/ELISPOT Diluent (1X) was added to the Wells, leaving the remaining holes empty. The highest standard concentration is added to the remaining empty Wells. Transfer the top standard from hole A to hole B. Mix the contents of both B Wells by repeated suction and injection, then transfer 1to two wells C. Be careful not to scratch the surface of the micropores. Continue this process 4-6 times.
7. Add the sample to the appropriate well.
8. Add ELISA/ELISPOT Diluent (1X) into the blank well.
9. Seal the plate and incubate at room temperature for 2-3 hours.
10. Prepare for antibody test (see Test Protocol).
11. Drain and rinse as in Step 2. Repeat the cleaning for a total of 3-5 times. Allow time for soaking in each wash step to improve washing effect. Imprinted plate on blotting paper to remove any residual buffer.
12. Add diluted test antibody to all Wells.
13. Seal the plate and incubate at room temperature for 1-2 hours.
14. Prepare streptavidin-HRP (see Test Protocol).
15. Drain and rinse as in Step 2. Repeat the cleaning for a total of 3-5 times. Setting aside a soaking time (about 1-2 minute) in each washing step can improve the washing effect. Imprinted plate on blotting paper to remove any residual buffer.
16. Add diluted Streptavidin-HRP.
17. Seal the plate and incubate at room temperature for 30-45 minutes.
18. Drain and wash as described in step 2, making sure to allow 1-2 minutes of soaking time before draining. Repeat a total of 6-8 washes.
19. Add 1X TMB solution.
20. Incubate at room temperature for 15-30 minutes.
21. Add the stop solution.

Plates were read at 450 nm. If there is wavelength subtraction, subtract 570nm from the 450nm value and analyze the data.

16-2,000 pg/mL

Uncoated ELISA Kit

All products are supplied for research and laboratory use only.

All blood components and biological materials should be treated with precautions as potentially hazardous. Strictly follow the management principles of the Centers for Disease Control and Prevention, Occupational Safety and Health Administration for handling and disposing of infectious agents.

The ELISA Kits are shipped at 2 to 8°C. Upon receipt, store the kits at 2 to 8°C.

Stability If properly stored, all components are stable for up to 12 months. For expiry dates for the entire kit, see the kit label. The expiration date of each ingredient is shown on the bottle label. The expiration date of a kit ingredient is guaranteed only if the ingredient is properly stored and, in the event of repeated use of an component, the reagent will not be contaminated by the first treatment.

Each production lot of this ELISA kit is quality tested to meet criteria such as sensitivity, specificity, precision and lot-to-lot consistency. See the manual for more information on validation.

Wet or Dry Ice

TNF; DIF; TNF-alpha; TNFA; TNFSF2; Tumour necrosis factor; TNF-α; tumor necrosis factor; TNLG1F; Tumor necrosis factor alpha

Tumor necrosis factor (TNF, cachexin, or cachectin; often called tumor necrosis factor alpha or TNF-α) is an adipokine and a cytokine. TNF is a member of the TNF superfamily, which consists of various transmembrane proteins with a homologous TNF domain. As an adipokine, TNF promotes insulin resistance, and is associated with obesity-induced type 2 diabetes. As a cytokine, TNF is used by the immune system for cell signaling. If macrophages (certain white blood cells) detect an infection, they release TNF to alert other immune system cells as part of an inflammatory response.