Specificity
This assay has high sensitivity and excellent specificity for detection of Tumor Necrosis Factor Alpha (TNFa). No significant cross-reactivity or interference between Tumor Necrosis Factor Alpha (TNFa) and analogues was observed.
Recovery
Matrices listed below were spiked with certain level of recombinant Tumor Necrosis Factor Alpha (TNFa) and the recovery rates were calculated by comparing the measured value to the expected amount of Tumor Necrosis Factor Alpha (TNFa) in samples.
| Matrix | Recovery Range(%) | Average Recovery(%) |
| serum(n=5) | 88-97 | 94 |
| EDTA plasma(n=5) | 80-102 | 83 |
| heparin plasma(n=5) | 79-104 | 91 |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Tumor Necrosis Factor Alpha (TNFa) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Tumor Necrosis Factor Alpha (TNFa) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%
Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Tumor Necrosis Factor Alpha (TNFa) 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) | 90-99% | 98-105% | 80-105% | 94-101% |
| EDTA plasma(n=5) | 92-101% | 86-96% | 86-93% | 88-101% |
| heparin plasma(n=5) | 78-97% | 90-99% | 91-99% | 85-103% |
Stability
The stability of 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. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.
Test Principle
The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Tumor Necrosis Factor Alpha (TNFa). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Tumor Necrosis Factor Alpha (TNFa). 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 Tumor Necrosis Factor Alpha (TNFa), 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 Tumor Necrosis Factor Alpha (TNFa) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
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