Components
Assay plate x1
Standard x2
Sample Diluent 1 x 20ml
Assay Diluent A 1 x 10ml
Assay Diluent B 1 x 10ml
Detection Reagent A 1 x 120μl
Detection Reagent B 1 x 120μl
Wash Buffer(25 x concentrate) 1 x 30ml
Substrate 1 x 10ml
Stop Solution 1 x 10ml
Plate sealer for 96 wells x5
Instruction 1x
Material not included
Microplate reader.
Pipettes and pipette tips.
EP tube Deionized or distilled water.
Reagent Preparation
Wash Buffer -Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.
Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. The undiluted standard serves as the high standard (10.0 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL).
Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 10.0 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (10.0 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL).
Assay Procedure
1.Add 100 μL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 °C.
2.Remove the liquid of each well, don't wash. Add 100 μL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37 °C.
3.Aspirate each well and wash, repeating the process three times for a total of three washes. let it sit for 1~2 minutes. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4.Add 100 μL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hour at 37 °C.
5.Repeat the aspiration/wash process for 5 times as conducted in step three.
6.Add 90 μL of Substrate Solution to each well. Incubate within 15-30 minutes at 37 °C. Protect from light.
7.Add 50 μL of Stop Solution to each well.
8.Determine the optical density of each well at once, 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.
Precaution of Use
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Storage Comment
The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.Other reagents are kept according to the labels on vials. But for long term storage, please keep the whole kit at -20°C. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit.
Note
The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.Other reagents are kept according to the labels on vials. But for long term storage, please keep the whole kit at -20°C. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit.
Restrictions
For Research Use only
Datasheet