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上海仁捷生物科技有限公司
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ELISA
ENZYME LINKED IMMUNOSORBENT ASSAY
INTENDED USE AND TEST PRINCIPLE
This IL-6 ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of IL-6 in the sample, this IL-6 ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus IL-6 concentration. The concentration of IL-6 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
SAMPLE COLLECTION AND STORAGES
Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximay 1000×g. Assay freshly prepared serum immediay or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8℃ within 30 minutes of collection. Remove plasma and assay immediay or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
Tissue homogenates - For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.
Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediay or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
Note: The samples shoule be centrifugated dequay and no hemolysis or granule was allowed.
MATERIALS REQUIRED BUT NOT SUPPLIED
1. 37 ℃ incubator
2. Standard microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable pipette tips and Absorbent paper
4. Distilled or deionized water
REAGENTS PROVIDED
All reagents provided are stored at 2-8°C. Refer to the expiration date on the label.
Name | 96 determinations | 48 determinations |
MICROTITER PLATE | 8*12strips | 8*6strips |
STANDARD(6 vial) | 0.3ml/vial | 0.3ml/vial |
SAMPLE DILUENT | 6.0ml | 3.0ml |
ENZYME CONJUGATE | 10.0ml | 5.0ml |
WASH SOLUTION | 25ml | 15ml |
SUBSTRATE A | 6.0ml | 3.0ml |
SUBSTRATE B | 6.0ml | 3.0ml |
STOP SOLUTION | 6.0ml | 3.0ml |
Closure plate membrane | 2 | 2 |
User manual | 1 | 1 |
Sealed bags | 1 | 1 |
Note:
1. Standard concentration was followed by: 160, 80, 40, 20, 10, 0 pg/mL.
2. If samples generate values higher than the highest standard, please dilute the samples with Sample Diluent and repeat the assay.
PRECAUTIONS
REAGENT PREPARATION AND STORAGE
Wash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8°C.
ASSAY PROCEDURE
1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microtiter plate.
2. Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.
3. Add 100μl of Enzymeconjugate to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the Microtiter Plate 4 times.
Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well compley with Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame.
Automated Washing - Aspirate all wells, then wash plates four times using Wash Buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.
5. Add Substrate A 50μl and Substrate B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
6. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
7. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
CALCULATION OF RESULTS
7. Sensitivity: The minimum detectable dose of Rat IL-6 is typically less than 1.0 pg/mL.
8. Cross-reactivity: This assay recognizes recombinant and natural Rat IL-6. No significant cross-reactivity or interference was observed.
9. Storage: 2-8℃ (Use frequently); six months (-20℃)。
10. Standard curve
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
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