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
Microplate reader with 450 nm filter.
Precision single or multi-channel pipettes and disposable tips.
Tubes for diluting samples.
Deionized or distilled water.
Absorbent paper for blotting the microtiter plate.
Container for Wash Solution
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.Prepare all reagents, samples and standards,
2.Add 100μL standard or sample to each well. Incubate 1 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.
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
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 unopened 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 opened kit: When the kit is opened, the remaining reagents still 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 reseal along entire edge of zip-seal.
For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.
Note
For unopened 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 opened kit: When the kit is opened, the remaining reagents still 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 reseal along entire edge of zip-seal.
For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.
Restrictions
For Research Use only
