48T 组织 小鼠细胞色素C氧化酶(COX)酶联免疫检测试剂盒使用说明书

小鼠细胞色素C氧化酶(COX)酶联免疫检测试剂盒使用说明书种属小鼠适用样本组织保存条件 2-8℃仅用于科研，不得用于医学诊断使用前仔细阅读本说明书。

本酶联免疫试剂盒是基于生物素双抗体夹心技术原理，来检测小鼠细胞色素C氧化酶(COX)，只能用于研究用途，不得用于医学诊断。

一、用途：用于小鼠组织及相关液体样本中细胞色素C氧化酶(COX)的测定。

二、工作原理本试剂盒采用的是生物素双抗体夹心酶联免疫吸附法 (ELISA) 测定样品中细胞色素C氧化酶(COX)的水平。

向预先包被了小鼠细胞色素C氧化酶(COX)单克隆抗体的酶标孔中加入细胞色素C氧化酶(COX)，温育；温育后加入生物素标记的抗COX抗体，再与链霉亲和素-HRP 结合，形成免疫复合物，再经过温育和洗涤，去除未结合的酶，然后加入底物A、B，产生蓝色，并在酸的作用下转化成最终的黄色。

颜色深浅与样品中细胞色素C氧化酶(COX)的浓度呈正相关。

三、试剂盒组成编号产品名称48T 96T1 标准品（80ng/ml）0.5ml 0.5ml2 标准品稀释液3ml3ml3 酶标包被板12孔×4条12孔×8条4 链霉亲和素-HRP3ml6ml5 浓缩洗涤液20×20ml30×20ml6 生物素标记的抗-COX抗体1ml1ml7 显色剂A液3ml6ml8 显色剂B液3ml6ml9 终止液3ml6ml10 说明书1份1份11 封板膜2张(48T)2张(96T)12 密封袋1个1个四、需要而未提供的试剂和器材1、37℃恒温箱2、标准规格酶标仪3、精密移液器及一次性吸头4、蒸馏水5、一次性试管6、吸水纸五、注意事项1、从2-8℃取出的试剂盒，在开启试剂盒之前要室温平衡至少30分钟。

酶标包被板开封后如未用完，板条应装入密封袋中保存。

2、各步加样均应使用加样器，并经常校对其准确性，以避免试验误差。

3、严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准。

4、为避免交叉污染，要避免重复使用手中的吸头和封板膜。

5、不用的其它试剂应包装好或盖好。

不同批号的试剂不要混用。

保质期之前使用。

6、底物B对光敏感，避免长时间暴露于光下。

六、洗板方法手工洗板方法：甩掉酶标板内的液体；在实验台上铺垫几层吸水纸，酶标板朝下用力拍几次；将稀释后的洗涤液至少0.35ml注入孔内，浸泡1-2分钟。

根据需要，重复此过程数次。

自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中。

七、标本要求1、不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。

2、标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。

若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融。

八、操作程序1、标准品的稀释：（本试剂盒提供原倍标准品一支，用户请按照说明自行在小试管中倍比稀释）：40ng/ml （5号标准品） 120μl的原倍标准品加入120μl的标准品稀释液20ng/ml （4号标准品） 120μl的5号标准品加入120μl的标准品稀释液10ng/ml （3号标准品） 120μl的4号标准品加入120μl的标准品稀释液5ng/ml （2号标准品） 120μl的3号标准品加入120μl的标准品稀释液2.5ng/ml （1号标准品） 120μl的2号标准品加入120μl的标准品稀释液2、根据待测样品数量加上标准品的数量决定所需的板条数。

每个标准品和空白孔建议做复孔。

每个样品根据自己的数量来定，能使用复孔的尽量做复孔。

3、加样：①、空白孔：空白对照孔不加样品，生物素标记的抗COX抗体，链霉亲和素-HRP，只加显色剂A&B和终止液，其余各步操作相同；②、标准品孔：加入标准品50μl，链霉素-HRP50μl（标准品中已事先整合好生物素抗体，故不加）；③、待测样品孔：加入样本40μl，然后各加入抗-COX抗体10μl、链酶亲和素-HRP50μl，盖上封板膜，轻轻振荡混匀，37℃温育60分钟。

④、配液：将浓缩洗涤液用蒸馏水稀释成1×应用液，备用。

⑤、洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。

⑥、显色：每孔先加入显色剂A 50μl，再加入显色剂B 50μl，轻轻震荡混匀，37℃避光显色10分钟。

⑦、终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。

⑧、测定：以空白空调零，450nm波长依序测量各孔的吸光度（OD值）。

测定应在加终止液后10分钟以内进行。

⑨、根据标准品的浓度及对应的OD值计算出标准曲线的回归方程，再根据样品的OD值在回归方程上计算出对应的样品浓度。

也可以使用各种应用软件来计算。

九、操作程序总结：准备试剂，样品和标准品检测范围：0.2ng/ml→60ng/ml 规格：48T/盒保存：2-8℃有效期：6个月（2-8℃）Mouse Cytochrome C oxidase(COX)Elisa Assay Kit InstructionSpecies MouseSamples TissueStorage 2-8℃For research use onlyNot for use in diagnostic proceduresPlease carefully read this Instruction before using. This ELISA kit is based on the principle of double-antibody sandwich technique to detect Mouse Cytochrome C oxidase (COX).Be used only for research purposes, not be used for medical diagnosis.1、INTENDED USEThis kit is used to assay the Cytochrome C oxidase (COX) in the sample of Mouse’s tissue and other related tissue Liquid.2、TEST PRINCIPLEThe kit uses a double-antibody sandwich enzyme-linked immuno-sorbent assay (ELISA) to assay the level of Cytochrome C oxidase (COX) in samples. Add Cytochrome C oxidase (COX) to monoclonal antibody Enzyme well which is pre-coated with Mouse Cytochrome C oxidase(COX) monoclonal antibody, incubation; then, add Cytochrome C oxidase (COX) antibodies labeled with biotin, and combined with Streptavidin-HRP to form immune complex; then carry out incubation and washing again to remove the uncombined enzyme. Then add Chromogen Solution A, B, the color of the liquid changes into the blue, And at the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the Substance Cytochrome C oxidase(COX) of sample were positively correlated.3、MATERIALS SUPPLIED IN THE TEST KITNumber Item 48T 96T1 Standard（80ng/ml）0.5ml 0.5mldiluent 3ml 3ml2 Standard12well×4 strips 12well×8 strips3 MicroelisaStripplate4 Str- HRP-Conjugate Reagent 3ml 6mlsolution 20×20ml30×20ml5 Wash6 Biotin-COX Ab 1ml 1mlA 3ml 6ml7 ChromogenSolution8 Chromogen Solution B 3ml 6mlSolution 3ml 6ml9 Stop10 Instruction 1 111 Closure plate membrane 2(48T) 2(96T)bags 1 112 Sealed4、MATERIALS REQUIRED BUT NOT SUPPLIED① 37℃ incubator② Standard Enzyme reader③ Precision pipettes and Disposable pipette tips④ Distilled water⑤ Disposable tubes for sample dilution⑥ Absorbent paper5、IMPORTANT NOTES1、Beening taken out from the 2-8℃ environment, the kit should be balanced 30 minutes in theambient temperature then use. If the Coated plates of Enzyme haven’t been used up after opened, the remaining plates should be stored in Sealed bag.2、For each step, add Sample with sample injector which should be calibrated frequently, in order toavoid unnecessary experimental tolerance.3、The operation shall be carried out accordance to the Instructions strictly. And test results must bebased on the readings of the Enzyme reader.4、In order to avoid cross-contamination, it is forbidden to re-use the suction head and seal platemembrane in your hands.5、The idle agents shall be put up or covered. Do not use reagent with different batches. And use thembefore expired date6、The substrate B is light-sensitive. Prolonged exposure to light is forbidden.6、WASHING METHODManually washing method: shake away the remain liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside down strongly. Inject at least 0.35ml after-dilution washing solution into the well, and marinate 1~2 minutes. Repeat this process according to your requirements.Automatic washing method: if there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance.7、SPECIMEN REQUIREMENTS1、Don’t detect the samples with NaN3 which could inhibit HRP activity of the horseradish peroxidase.2、Extract as soon as possible after Specimen collection. The extraction should conform with therelevant literature, and then carry out the experiment as soon as possible. If it is impossible to test immediately, preserve the specimen in -20℃ enviroment. And repeated freezing and thawing is forbidden.8、ASSAY PROCEDURE1、Standard dilution: （This test kit will supply one original Standard reagent, please dilute it byyourself according to the Instruction.）40ng/ml StandardNo.5 120μl Original Standard + 120μl Standard diluentsNo.4 120μl Standard No.5 + 120μl Standard diluents 20ng/ml StandardNo.3 120μl Standard No.4 + 120μl Standard diluents 10ng/ml Standard5ng/ml StandardNo.2 120μl Standard No.3 + 120μl Standard diluentsNo.1 120μl Standard No.2 + 120μl Standard diluents2.5ng/ml Standard2、The quantity of the plates depends on the quantities of to-be-tested samples and the standards. It issuggested to duplicate each standard and blank well. Every sample shall be made according to your required quantity, and try to use the duplicated well as possible.3、Inject samples:① Blank well: don’t add samples and COX-antibody labeled with biotin, Streptavidin-HRP, onlyChromogen solution A and B, and stop solution are allowed; other operations are the same.② Standard wells: add standard 50μl, Streptavidin-HRP 50μl (since the standard already hascombined biotin antibody, it is not necessary to add the antibody);③ To be test wells: add sample 40μl, and then add both COX-antibody10μl and Streptavidin-HRP50μl. Then seal the sealing memberance, and gently shaking, incubated 60 minutes at 37℃.④ Confection: dilute the wash solution with distilled water to 1×working solution for use.⑤ Washing: remove the memberance carefully, and drain the liquid, shake away the remaining water.⑥ Add chromogen solution A 50μl,then chromogen solution B 50μl to each well. Gently mixed,incubate for 10 min at 37℃ away from light.⑦ Stop: Add Stop Solution 50μl into each well to stop the reaction(the blue changes into yellowimmediately).⑧ Final measurement: Take blank well as zero,measure the optical densit (OD) under 450nmwavelength which should be carried out within 10min after adding the stop solution.⑨ According to standards’ concentration and the corresponding OD values, calculate out the standardcurve linear regression equation, and then apply the OD values of the sample on the regression equation to calculate the corresponding sample’s concentration. It is acceptable to use kinds of software to make calculations.9、SUMMARY PROCEDURESPreparing reagents, samples and standards℃℃measure the OD value within 10minAssay range：0.2ng/ml→60ng/ml Package size：48T perboxStorage：2-8.℃Validity：Six months (2-8)℃。