Components
Pre-coated, ready to use 96-well strip plate
Plate sealer for 96 wells
Standard
Standard Diluent
Detection Reagent A
Detection Reagent B
Assay Diluent A
Assay Diluent B
TMB Substrate
Stop Solution
Wash Buffer (30 x concentrate)
Instruction manual
Material not included
1.Microplate reader with 450 ± 10nm filter.
2.Precision single or multi-channel pipettes and disposable tips.
3.Tubes for diluting samples.
4.Deionized or distilled water.
5.Absorbent paper for blotting the microtiter plate.
6.Container for Wash Solution
7.0.01Mol/L (or 1x) Phosphate Buffered Saline(PBS), pH 7.0-7.2.
Reagent Preparation
1.Bring all kit components and samples to room temperature (18-25 °C) before use.
2.Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently.
3.Dilute Detection Reagent A and Detection Reagent B to the working concentration 100-fold with Assay Diluent A and B, respectively.
4.Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5.TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
Assay Procedure
1.Add 100μL each of dilutions of standard, blank and samples into the appropriate wells, respectively. Cover with the Plate sealer. Incubate for 1 hour at 37 °C.
2.Remove the liquid of each well, don't wash.
3.Add 100μL of Detection Reagent A working solution to each well, cover the wells with the plate sealer and incubate for 1 hour at 37 °C.
4.Aspirate the solution and wash with 350μL of 1x Wash Solution to each well and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. Invert the plate and blot it against absorbent paper.
5.Add 100μL of Detection Reagent B working solution to each well, cover the wells with the plate sealer and incubate for 30 minutes at 37 °C.
6.Repeat the aspiration/wash process for total 5 times as conducted in step 4.
7.Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at 37 °C. Protect from light.
8.Add 50μL of Stop Solution to each well. Mix the liquid by tapping the side of the plate.
9.Remove any drop of water and fingerprint on the bottom of the plate. Then, run the microplate reader and conduct measurement at 450nm immediately.
Calculation of Results
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with target concentration on the y-axis and absorbance on the x-axis.
Assay Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level of target were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level of target were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV < 10%
Inter-Assay: CV < 12%
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.
Handling Advice
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.
Storage Comment
For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon receipt while the others should be at 4 °C.
For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.
Note
For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon receipt while the others should be at 4 °C.
For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.
Restrictions
For Research Use only
