Components
Microplate: 96 breakable wells (12strips x 8wells) coated with anti-canine IL-10.
20x Wash Buffer Concentcaninee: 1 Vial, 25 ml.
5x Assay Diluent: 1vial, 15 ml.
Standards: 2 vials, recombinant canine IL-10.
Detection Antibody: 2 vials, biotinylated anti-canine IL-10.
HRP-Streptavidin Concentcaninee: 1vial.
TBM Substcaninee solution: 1 Vial, 12 ml.
Stop Solution: 1 Vial, 8 ml of 0.2 M sulfuric acid.
Material not included
1.Distilled or deionized water. 2.Precision pipettes, with disposable plastic tips. 3.Beakers, flasks, cylinders necessary for prepacanineion of reagents. 4.Microplate washing device (multichannel pipette or automated microplate washer). 5.Microplate shaker. 6.Microplate reader capable of reading at 450 nm.
Reagent Preparation
1.Assay diluent: Dilute the concentrated assay diluent 1:5 with distilled water. Wash buffer: Dilute the concentrated wash buffer 1:20 with distilled water.
2.Standard: Add 400 μL 1x Assay Diluent to prepare a 80 ng/mL standard. Add 400 μL 1x Assay Diluent to 7 tubes.Detection Ab: Add 100 μL of 1X Assay Diluent into the vial to prepare a detection antibody concentrate. The detection antibody concentrate should be diluted 80-fold with 1X Assay Diluent.
3.Streptavidin-HRP: HRP-Streptavidin concentrate should be diluted 350-fold with 1X Assay Diluent.
4.Optimal dilution factors for each sample must be determined by the investigator, the recommended dilution for serum and plasma is 1: 2.
Assay Procedure
1.All reagents must be brought to room temperature (18-25 °C) prior to use.
2.Add 100 μL of each standard and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
3.Decant or aspirate contents of wells. Wash wells by filling with at least 300 μL/well prepared wash buffer followed by decanting/aspirating. Repeat wash 4 times for a total of 5 washes. After the last wash, blot plate on absorbent paper to remove residual buffer.
4.Add 100 μL of 1X prepared biotinylated antibody to each well. Incubate for 1 hour at room temperature with gentle shaking. Discard the solution. Repeat the wash as in step
5.Add 100 μL of prepared Streptavidin solution to each well. Incubate for 45 minutes at room temperature with gentle shaking.
6.Discard the solution. Repeat the wash as in step
7.Add 100 μL of TMB One-Step Substcaninee Reagent to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking. Add 50 μL of Stop Solution to each well. Read absorbance at 450nm within 30 minutes of stopping the reaction.
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 CV<10%, inter CV <15%
Handling Advice
1.All reagents must be at room temperature (18 °C to 25 °C) before running assay.
2.Do not expose kit reagents to strong light during storage or incubation.
3.Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results.
4.Avoid contact of stop solution with skin or eyes. If contact occurs, immediately flush area with copious amounts of water.
5.Do not use TMB substance solution if it has begun to turn blue.
6.Do not expose bleach to work area during actual test procedure because of potential interference with enzyme activity.
Storage Comment
4°C/-20°C,May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.
Note
4°C/-20°C,May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.
Restrictions
For Research Use only
