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Appendices:
Appendix A: Juices These juices were obtained: R.W. KNUDSEN – Just Cranberries Ingredients: filtered water, concentrated cranberry juice Black River – Montmorency Cherry Juice Ingredients: Unclarified Montmorency Cherry Juice
(Enzyme-linked Immunosorbent Assay) ELISA Set Assay Protocol 1. Microwells were coated with 100 μl per well of Capture Antibody (anti-human IL-8 capture antibody) diluted in Coating Buffer (carbonate coating buffer) according to lot-specific Instruction/Analysis Certificate. Plate was sealed and incubated overnight at 4°C. 2. Wells were aspirated and washed 3 times with ≥ 200 μl/ well Wash Buffer. After the last wash, the plate was inverted and blotted on an absorbent paper to remove any residual buffer. 3. Plates were blocked with ≥ 200 μl/well Assay Diluent. They were incubated at room temperature (RT) for 1 hour. 4. They were aspirated and washed well. 5. Standard and sample dilutions were prepared in Assay Diluent. 6. 100 μl of each standard, sample and control were pipetted into appropriate wells. Plate was sealed and incubated for 2 hours at room temperature. 7. Aspirate/wash as in step 2, but with 5 total washes. 8. Add 100 μl of prepared Working Detector (Detection Antibody + Avidin-HRP reagent) to each well. Seal plate and incubate for 1 hour at room temperature. 9. Aspirate/wash as in step 2, but with 7 total washes. Note: In this final wash step, soak wells in wash buffer for 30 seconds to 1 minute for each wash. 10. Add 100 μl of Substrate Solution to each well. Incubate plate (without plate sealer) for 30 minutes at room temperature in the dark. 11. Add 50 μl of Stop Solution to each well. 12. Read Absorbance at 450 nm within 30 minutes of stopping reaction. If wavelength correction is available, substract A 570 nm from A 450 nm.
Goldsby, Richard A., Thomas J. Kindt, and Barbara A. Osborne. Kuby Immunology. 4th ed. New York: Freeman, 2000. |