Application:Enzyme-linked immunosorbent assay for Antigen Detection
Test method:Double-antibody Sandwich
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 Erythropoietin (EPO). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Erythropoietin (EPO). 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 Erythropoietin (EPO), 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 Erythropoietin (EPO) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Sensitivity:The minimum detectable dose of this kit is typically less than 35.22pg/mL
tissue homogenates and other biological fluids
This assay has high sensitivity and excellent specificity for detection of Erythropoietin (EPO).
No significant cross-reactivity or interference between Erythropoietin (EPO) and analogues was observed.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Erythropoietin (EPO) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Erythropoietin (EPO) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
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.
1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.