Components
Microplate: 96 breakable wells (12strips x 8wells) coated with anti-human E-selectin.
20x Wash Buffer Concentrate: 1 Vial, 25 ml.
5x Assay Diluent: 1vial, 15 ml.
Standards: 2 vials, recombinant human E-selectin.
Detection Antibody: 2 vials, biotinylated anti-human E-selectin.
HRP-Streptavidin Concentrate: 1vial.
TBM Substrate 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 preparation 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: Briefly spin standard vial before use. Add 300 μL 1x Assay Diluent to prepare a 100 ng/mL standard. Gently vortex to mix. Take 100 μL standard into a tube, then add 400 μL 1x Assay Diluent to prepare a 20,000pg/mL stock standard solution. Add 400 μL 1x Assay Diluent to 7 tubes.
3.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.
4.Streptavidin-HRP: HRP-Streptavidin concentrate should be diluted 200-fold with 1X Assay Diluent.
5.Sample: 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 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.
Assay Precision
Intra CV<10%, inter CV <15%
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
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
