Application:Enzyme-linked immunosorbent assay for Antigen Detection
Test method:Competitive Inhibition
Storage conditions:Reproductive science
Delivery conditions:Hormone metabolism
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to 17-Hydroxyprogesterone (17-OHP) has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled 17-Hydroxyprogesterone (17-OHP) and unlabeled 17-Hydroxyprogesterone (17-OHP) (Standards or samples) with the pre-coated antibody specific to 17-Hydroxyprogesterone (17-OHP). After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of 17-Hydroxyprogesterone (17-OHP) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of 17-Hydroxyprogesterone (17-OHP) in the sample.
Sensitivity:The minimum detectable dose of this kit is typically less than 87.3pg/mL
cell culture supernates and other biological fluids
This assay has high sensitivity and excellent specificity for detection of 17-Hydroxyprogesterone (17-OHP).
No significant cross-reactivity or interference between 17-Hydroxyprogesterone (17-OHP) and analogues was observed.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level 17-Hydroxyprogesterone (17-OHP) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level 17-Hydroxyprogesterone (17-OHP) 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 50µL standard or sample to each well.
And then add 50µL prepared Detection Reagent A immediately.
Shake and mix. Incubate 1 hour at 37°C;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
7. Add 50µL Stop Solution. Read at 450 nm immediately.