For the quantitative determination of activated objective concentrations in serum, plasma, tissue homogenate, cell culture supernates and other biological fluids.
For Research use only and is not for use in diagnostic or therapeutic procedures. If you have any questions, please contact the Shanghai Elisa Biotech Co.,Ltd.
PRINCIPLE OF THE ASSAY
The kit uses a double‐antibody sandwich enzyme‐linked immunosorbent assay (ELISA) to analyze the level of objective in samples. Add standard and sample to wells pre‐coated with one objective antibody at the same time add second HRP‐conjugated objective antibody to bind the analyte, followed by incubation and washing procedures to remove unbound substance. Finally, HRP substrates are added, incubated for detection, and a blue color is developed. Reaction is stopped and color turns to yellow when Stopping Solution (acidic) is added. The yellow color intensity proportionally correlates to the concentration of the concentration of the objective in samples.
SAMPLE COLLECTION AND STORAGES
Serum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates, tissue homogenate and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles.
Sample preparation - Typically, serum, plasma and other biological fluids samples do not require dilution. If samples generate values higher than the highest standard, please dilute the samples with Sample Diluent and repeat the assay.
Notes: Serum and plasma to be used within 7 days may be stored at 2-8℃, otherwise samples must be stored at -20 or -80℃ to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.
All reagents provided are stored at 2-8°C. Refer to the expiration date on the label.
MATERIALS REQUIRED BUT NOT SUPPLIED
1. Standard microplate reader capable of measuring absorbance at 450 nm
2. Automated Washing
3. 37℃ incubator
4. Clean tube and Eppendorf tube
5. Distilled or deionized water
6. Precision pipettes, disposable pipette tips, multi-channel pipettes and Absorbent paper
1. The operation should be carried out in strict accordance with the provided instructions.
2. To preserve unused strip-wells, it should be stored in the sealed bag.
3. Always avoid foaming when mixing or reconstituting protein solutions.
4. Pipette reagents and samples into the center of each well.
5. The samples should be transferred into the assay wells within 15 minutes of dilution.
6. We recommended that all standard, testing samples are tested in duplicate to minimize the test errors.
7. If the blue color too shallow after 15 minutes incubation with the substrates, it may be appropriate to extend the incubation time.
8. Avoid cross‐contamination by changing tips, using separate reservoirs for each reagent, avoid using the suction head without extensive wash.
9. Do not mix the reagents from different batches
10. Stop Solution should be added in the same order of the Substrate solution.
11. Chromogenic Substrate B is light-sensitive, please avoid prolonged exposure to light.
12. The kit should be kept at 2-8℃ and cannot be used after expiration date. The standards should be kept at -20℃after receiving.
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 completely 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 Solution (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.
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℃.
1. First, secure the desired number of coated wells in the holder. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
2. Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.
3. Add 100μl of HRP-conjugate reagent 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.(See WASHING METHOD)
5. Add chromogen solution A 50μl and chromogen solution 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
The standard curve is generated by plotting the average O.D. (450nm) obtained for each of the standard O.D.values on the vertical (X) axis versus the corresponding concentration on the horizontal (Y) axis. Calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard (Blank well) before result interpretation. Construct the standard curve using graph paper or statistical software. Remember to multiply total dilution times when calculation.
Typical standard curve data
( reference in general, not for this kit in particular )
PROCEDURES IN SUMMARY
Prewarm all reagents to room temperature before assay.
Prepare reagents, samples in clean tubes or 96-well plate.
Transfer standard and samples to assay plate/strip, and add HRP-Conjugate reagent and incubate 60 minutes at 37℃.
Plate-wash four times, add Substrate A and B, incubate 15 minutes at 37℃ Add stop solution.
Measure within 15 minutes
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