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Mouse Angiotensin converting enzyme,ACE ELISA Kit
点击次数：1711发布时间：2012/11/14 22:14:41
更新日期：2013/11/5 9:37:43
所 在 地：中国大陆
产品型号：小鼠血管紧张素转化酶(ACE)ELISA Kit
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小鼠血管紧张素转化酶（ACE）ELISA Kit
Mouse Angiotensin converting enzyme,ACE ELISA Kit
用途： For research use only. Not for diagnostic use.
原理 ： This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for ACE has been pre-coated onto a microplate. Standards ａnd samples are pipetted into the wells ａnd any ACE present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for ACE is added to the wells. After washing, avidin conjugated Horseradish Peroxidase （HRP） is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells ａnd color develops in proportion to the amount of ACE bound in the initial step. The color development is stopped ａnd the intensity of the color is measured.
特异性： This assay has high sensitivity ａnd excellent specificity for detection of Mouse ACE. No significant cross-reactivity or interference between Mouse ACE ａnd analogues was observed.
精密度： Intra-assay Precision （Precision within an assay）: CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision （Precision between assays）: CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
样本搜集及储存： Serum: Use a serum separator tube （SST） ａnd allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g. Remove serum ａnd assay immediately or aliquot ａnd store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of collection. Assay immediately or aliquot ａnd store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
检测步骤： Bring all reagents ａnd samples to room temperature before use. Centrifuge the sample again after thawing before the assay. It is recommended that all samples ａnd standards be assayed in duplicate.
1. Prepare all reagents, working standards, ａnd samples as directed in the previous sections.
2. Refer to the Assay Layout Sheet to determine the number of wells to be used ａnd put any remaining wells ａnd the desiccant back into the pouch ａnd seal the ziploc, store unused wells at 4°C.
3. Add 100μl of standard ａnd sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C. A plate layout is provided to record standards ａnd samples assayed.
4. Remove the liquid of each well, don＇t wash.
5. Add 100μl of Biotin-antibody （1x） to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C. （Biotin-antibody （1x） may appear cloudy. Warm up to room temperature ａnd mix gently until solution appears uniform.）
6. Aspirate each well ａnd wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer （200μl） using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, ａnd let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash Buffer by aspirating ordecanting. Invert the plate ａnd blot it against clean paper towels.
7. Add 100μl of HRP-avidin （1x） to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
8. Repeat the aspiration/wash process for five times as in step 6.
9. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at 37°C. Protect from light.
10. Add 50μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher ａnd less accurate.
结果计算： Using the professional soft "Curve Expert 1.3" to make a standard curve is recommended, which can be downloaded from our web.
Average the duplicate readings for each standard 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 ACE concentrations versus the log of the O.D. ａnd the best fit line can be determined by regression analysis. 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.
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