英文：Human Decorin ELISA Kit
中文： 人核心蛋白聚糖（Decorin) Elisa试剂盒



备注：
经过大量正常标本检验，标本的正常浓度值均在试剂盒提供的检测范围内，实验过程中直接取50μL样本上样即可。当有部分样本值超过最大标准品浓度时，可用样本稀释液将标本进行适当稀释后再进行实验。

注意事项

1. 严格按照规定的时间和温度进行温育以保证准确结果。所有试剂都必须在使用前达到室温20-25℃。使用后立即冷藏保存试剂。
2. 洗板不正确可以导致不准确的结果。在加入底物前确保尽量吸干孔内液体。温育过程中不要让微孔干燥掉。
3. 消除板底残留的液体和手指印，否则影响OD值。
4. 底物显色液应呈无色或很浅的颜色，已经变蓝的底物液不能使用。
5. 避免试剂和标本的交叉污染以免造成错误结果。
6. 在储存和温育时避免强光直接照射。
7. 平衡至室温后再打开密封袋以防水滴凝聚在冷板条上。
8. 任何反应试剂不能接触漂白溶剂或漂白溶剂所散发的强烈气体。任何漂白成分都会破坏试剂盒中反应试剂的生物活性。
9. 不能使用过期产品。
10. 如果可能传播疾病，所有的样品都应管理好，按照规定的程序处理样品和检测装置。

试剂准备
试剂盒从冷藏环境中取出应在室温平衡后方可使用。
20×洗涤缓冲液的稀释：蒸馏水按1：20稀释，即1份20×洗涤缓冲液加19份蒸馏水。

操作步骤

1. 从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。
2. 设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；
3. 样本孔中加入待测样本50μL；空白孔不加。
4. 除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶（HRP）标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。
5. 弃去液体，吸水纸上拍干，每孔加满洗涤液（350μL），静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次（也可用洗板机洗板）。
6. 每孔加入底物A、B各50μL，37℃避光孵育15min。
7. 每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。
   
   Samplecollection and storages
   Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles
   Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
   Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
   Note: The samples should be centrifugated adequately and no hemolysis or granule was allowed.
   Materials required but not supplied
   1. Standard microplate reader(450nm)
   2. Precision pipettes and Disposable pipette tips.
   3. 37 ℃ incubator
   Precautions
   1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer.
   2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided.
   3.  Mix all reagents before using.
   Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature( 20-25°C)
   Materials supplied

   Name	96determinations	48determinations
   Microelisa stripplate	12*8strips	12*4strips
   Standard	0.3ml	0.3ml
   Sample diluent	6.0ml	3.0ml
   HRP-Conjugate reagent	10.0ml	5.0ml
   20X Wash solution	25ml	15ml
   Chromogen Solution A	6.0ml	3.0ml
   Chromogen Solution 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: Standard concentration was followed by:
   20、10、5、2.5、1.25、0 ng/mL.
   Reagent preparation
   20×wash solution:Dilute with Distilled or deionized water 1:20.
   Assay procedure
   1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.
   2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
   3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.
   4. Add100μlofHRP-conjugate reagent to each well,cover with an adhesive stripandincubatefor60 minutes at37°C.
   5. Aspirate each well and wash, repeating the process four times for a total of five washes.Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating ordecanting. Invert the plate and blot it against clean paper towels.
   6. 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.
   7. Add 50μl Stop Solution to each well. The color in the wells should change from blue toyellow. If the color in the wells is green or the color change does not
   
   
   appear uniform,gently tap the plate to ensure thorough mixing.
   8. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes.
   Calculation of results
8. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
9. First, 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 before result interpretation. Construct the standard curve using graph paper or statistical software.
10. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
11. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
12. The sensitivity by this assay is 0.1 ng/mL.
13. Standard curve
14. 
    
    Storage： 2-8℃.
    validity： six months.
    
    FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!