Components
Reagent (Quantity): Assay plate (1), 2 Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A 1×120µl Detection Reagent B 1×120µl Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)
Reagent Preparation
Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 50 ng/mL. The undiluted standard serves as the high standard (50 ng/mL). The Sample Diluent serves as the zero standard (0 ng /mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.
Assay Procedure
1.Prepare all reagents, working standards and samples as directed in the previous sections.
2.Add 100 uL of Standard, Control, or sample per well. Incubate for 2 hours at 37 °C.
3.Remove the liquid of each well, don't wash.
4.Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C.
5.Aspirate each well and wash, repeating the process three times for a total of three washes.
6.Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7.Repeat the aspiration/wash as in step.
8.Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9.Add 50 uL of Stop Solution to each well.
10.Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Calculation of Results
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit.
Handling Advice
The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with those from other lots or sources.
If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.
Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/temperature and kit age can cause variation in binding.
This assay is designed to eliminate interference by soluble receptors, binding proteins and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Storage Comment
The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
Note
The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
Restrictions
For Research Use only
