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Sample Volume
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100 μL
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Assay Time
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1 - 4.5 h
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Plate
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Pre-coated
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Protocol
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1. Prepare all reagents, samples and standards
2. Add 100 μL standard or sample to each well. Incubate 2 hours at 37 °C
3. Aspirate and add 100 μL prepared Detection Reagent A. Incubate 1 hour at 37 °C
4. Aspirate and wash 3 times
5. Add 100 μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C
6. Aspirate and wash 5 times
7. Add 90 μL Substrate Solution. Incubate 15-25 minutes at 37 °C
8. Add 50 μL Stop Solution. Read at 450nm immediately.
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Reagent Preparation
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Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.
Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 20ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (20ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL).
Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.
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Sample Preparation
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Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2°C - 8°C within 30 minutes of collection. Store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20°C After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20°C.
Cell culture supernates and Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Note:
1. Samples to be used within 5 days may be stored at 2-8°C , otherwise samples must stored at -20°C (1 month) or -80°C (2 months) to avoid loss of bioactivity and contamination.
2. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.
3. Influenced by the factors including cell viability, cell number and also sampling time, samples from cell culture supernatant may not be detected by the kit
4. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
5. When performing the assay slowly bring samples to room temperature.
6. Do not use heat-treated specimens.
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Assay Procedure
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The microtiter plate provided in this kit has been pre-coated with an antibody specific to the index. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the index. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain the index, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of the index in the samples is then determined by comparing the O.D. of the samples to the standard curve.
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Calculation of Results
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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. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the MUC-5AC concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 1.3. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Important note:
1. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit
2. The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
3. Kits from different batches may be a little different in detection range, sensitivity and color developing time.Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for information.
4. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results.
5. Do not remove microtiter plate from the storage bag until needed.
6. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
7. Use fresh disposable pipette tips for each transfer to avoid contamination.
8. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
9. Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
10. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
11. Kits from different manufacturers for the same item might produce different results, since we haven’t compared our products with other manufacturers.
12. The instruction manual also suit for the kit of 48T, but all reagents of 48T kit is reduced by half.
13. Valid period: six months.
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Assay Precision
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- Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
- Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
- CV(%) = SD/meanX100
- Intra-assay: CV<10%
- Inter-assay: CV<12%
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Restrictions
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For Research Use only
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