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| Product name: | Mouse Tumor Necrosis Factor Alpha ELISA Kit |
| Detection range: | 0.156-10ng/mL |
| Sensitivity: | 0.064ng/mL |
| Type: | Ready-to-use TNFα ELISA kit |
| Synonyms: | DIF; TNF-A; TNFSF2; Cachectin; Tumor Necrosis Factor Ligand Superfamily Member 2 |
| Species: | Mouse |
| Sample type: | serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. |
| Experimental method: | Sandwich |
| Shelf life: | 16 months |
| UniProt entry: | 21926 |
| NCBI gene entry: | P06804 |
| Component: | 1. Pre-coated, ready to use 96-well strip plate 1 2. Plate sealer for 96 wells 2 3. Standard 2 4. Standard Diluent 1×10 mL 5. Detection Solution A 1×6 mL 6. Detection Solution B 1×6 mL 7. TMB Substrate 1×4.5 mL 8. Stop Solution: 1×3 mL 9. Wash Buffer (30× concentrate): 1×10 mL |
| Documents: | Please contact us to request the manual |
Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to the antigen. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the antigen. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain the antigen, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of the antigen in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Recovery
Matrices listed below were spiked with certain level of recombinant antigen and the recovery rates were calculated by comparing the measured value to the expected amount of the antigen in samples.
| Matrix | Recovery range (%) | Average(%) |
| serum (n=5) | 80-102 | 91 |
| EDTA plasma(n=5) | 81-100 | 90 |
| heparin plasma(n=5) | 80-89 | 84 |
Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of the antigen and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
| Sample | 1：2 | 1：4 | 1：8 | 1：16 |
| serum(n=5) | 87-91% | 87-107% | 74-101% | 92-97% |
| EDTA plasma(n=5) | 90-105% | 84-101% | 90-101% | 79-108% |
| heparin plasma(n=5) | 84-95% | 92-105% | 82-105% | 89-91% |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level antigen were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level antigen were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%Inter-Assay: CV<12%
Stability
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
Note: To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.
Assay procedure summary
1. Prepare all reagents, samples and standards;
2. Add 50µL standard or sample to each well. And then add 50µL prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37℃;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
7. Add 50µL Stop Solution. Read at 450 nm immediately.
