本试剂盒只能用于科学研究，不得用于医学诊断。

大鼠低氧诱导因子（HIF）定量检测试剂盒（ELISA）

使用说明书

【试剂盒名称】

大鼠低氧诱导因子（HIF）定量检测试剂盒（ELISA）

【试剂盒用途】

定量检测大鼠血清、血浆及相关液体样本中低氧诱导因子（HIF）的含量。

【检测原理】

本试剂盒采用双抗体一步夹心酶联免疫吸附法（ELISA）。将标准品、待测样本和酶标工作液加入到预先包被大鼠低氧诱导因子（HIF）抗体的透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分。依次加入显色剂A、B液，显色剂（TMB）在辣根过氧化物酶（HRP）催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样品中大鼠低氧诱导因子（HIF）浓度呈正相关，450nm波长下测定OD值，根据标准品和样品的OD值，计算样本中大鼠低氧诱导因子（HIF）含量。

【试剂盒组成】

备注：标准品用标准品稀释液依次稀释为：128、64、32、16、8、4 pg/mL

【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

【操作步骤】

1、准备：从冰箱取出试剂盒，室温复温平衡30分钟。

2、配液：用蒸馏水将20倍浓缩洗涤液稀释成原倍的洗涤液。

3、加标准品和待测样本：取足够数量的酶标包被板，固定于框架上，分别设置标准品孔、待测样本孔和空白对照孔，记录各孔位置，在标准品孔中加入标准品50μL；待测样本孔中先加入待测样本10μL，再加*40μL（即样本稀释5倍）；每孔再加入酶标工作液100μL；空白对照孔不加。

4、温育：37℃水浴锅或恒温箱温育60min。

5、洗板：弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次（也可用洗板机按说明书操作洗板）。

6、显色：每孔先加入显色剂A 液50μL，再加入显色剂B液 50μL，37℃避光显色15min。

7、终止：取出酶标板，每孔加终止液50μL，终止反应（颜色由蓝色立转黄色）。

8、测定：以空白孔调零，在终止后15分钟内，用450nm波长测量各孔的吸光值（OD值）。

9、计算：根据标准品的浓度及对应的OD值，计算出标准曲线的直线回归方程，再根据样本的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。zui终浓度为实际测定浓度乘以稀释倍数。

【样本要求】

1、样本不能含叠氮钠（NaN₃），因为叠氮钠（NaN₃）是辣根过氧化物酶（HRP）的抑制剂。

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。若不能立即试验，可将标本放于-20℃保存，但应避免反复冻融。

3、样本应充分离心，不得有溶血及颗粒。

【注意事项】

1、实验严格按照说明书的操作进行，实验结果判定必须以酶标仪读数为准。

2、酶标包被板开封后如未用完，应立即装入密封袋中加干燥剂保存。

3、建议所有的标准品、样本和空白对照都做双份检测，取平均值，以减小实验误差。

4、若显色过浅，可适当延长底物温育时间。

5、为避免交叉污染，标准品、样本和空白对照每加一个就要更换一次吸头；酶标工作液、*和底物等公共组分，要悬臂加样，不得碰到微孔；不得重复使用封板膜。

6、试剂盒保质期内使用，不同批号的试剂不得混用。

7、底物B对光敏感，避免长时间暴露于光下。

【操作程序总结】

准备试剂，样品和标准品

加入准备好的样品、标准品和酶标工作液，37℃反应60分钟

洗板5次，加入显色液A、B，37℃显色15分钟

加入终止液

15分钟之内读OD值

计算

【检测范围】

4-128pg/mL

【规格】

96人份/盒

【贮藏】

2-8℃，避光防潮保存。

【有效期】

6个月

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Rat hypoxia-inducible factor （HIF） ELISA Kit instruction

Kit name

Rat hypoxia-inducible factor （HIF） ELISA Kit

Intended use

The kit is used to assay the content of Rat hypoxia-inducible factor （HIF）in Rat serum，blood plasma and other related tissue liquid.

Test principle

The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay （ELISA） to assay the level of Rat hypoxia-inducible factor （HIF） in samples. Add Rat hypoxia-inducible factor  （HIF） to pre-coated Rat hypoxia-inducible factor （HIF） monoclonal antibody microelisa well, incubation; washing. Add HRP tagged Rat hypoxia-inducible factor （HIF） antibodies. After another incubation and washing, remove the unbound enzyme, add Chromogen Solution A and B, the color of the liquid change into blue, and the color finally become yellow at the effect of acid. The depth of the color is positively correlated with concentration of the Rat hypoxia-inducible factor （HIF） in samples.

Materials supplied

Note: Standard was diluent with Standard diluent followed by: 128、64、32、16、8、4pg/mL

Materials required but not supplied

1.         37 ℃ incubator

2.         Standard microplate reader

3.         Precision pipettes and Disposable pipette tips

4.         Distilled water

5.         Disposable tubes for sample dilution

6.         Absorbent paper

Assay procedure

1.        Prepare: The kit takeing out from the environment of 2-8℃ should be balanced 30 minutes at less in the room temperature before using.

2.        Diluent: Diluent the 20×wash solution.

3.        Add standard and Sample: Set Standard wells, testing sample wells and blank wells. Add Diluted standard 50μl to standard well; Add Sample dilution 40μl to testing sample well which on Assay plate, then add testing sample 10μl （sample final dilute degree is 5 times）, Add HRP-conjugate reagent 100μl to each well, except the blank well.

4.        Incubation: Incubate 60 minutes at 37 ℃ in incubator.

5.        Wash: Discard Liquid, drying, filling in diluted washing liquid to each well, oscillation for 1 min, discard the washing liquid with absorbent paper Pat dry. Repeat four times, Pat dry.

6.        Add chromogen solution A and B: Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix, incubate for 15 min at 37℃.

7.        Add Stop Solution: Add Stop Solution 50μl to each well, Stop the reaction（the blue color change to yellow immediay）.

8.        Take blank well as zero, measure the optical densit （OD） at 450 nm after adding Stop Solution and within 15 min.

9.        According to standard concentration and the corresponding OD values calculated standard curve linear regression equation, then the OD values according to the sample on the regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be multiplied by the total dilution.

Specimen requirements

1.      Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

2.      Extract as soon as possible after Specimen collection, Extracted according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can not be tested immediay, specimen can be kept in -20℃ to preserve, but repeated freezing and thawing should be avoided.

3.      The samples shoule be centrifugated dequay and no hemolysis or granule was allowed.

Important notes

1.         The operation should be carried out in strict accordance with instructions and test results must be based on microplate reader to determine readings shall prevail.

2.         If the microelisa stripplate has not used up after open, it should be stored in the sealed bag.

3.         Recommended that all standard materials, test samples are doing double to minish the Experimental error.

4.         Please multiply total dilution times when calculation. 5 times is the best dilute time according to this ELISA Kit design.

5.         If the testing material content in the sample is excessively high, please use Special dilution to dilute certain multiple, then assay.

6.         If the color too shallow, It may be appropriate to extend the substrate incubation time.

7.         Add Sample with sampler each step and proofread its accuracy frequently to avoid the experimental error. In order to avoid cross-contamination, avoid to reusing the suction head and closure plate membrane.

8.         Use the kit in validity, not mix the reagents of different batches.

9.         Chromogen Solution B is light-sensitive, avoid prolonged exposure to light,

Summary procedures

Preparing reagents, samples and standard

Add prepared sample and standard, adding HRP-Conjugate Reagent incubated 60 minutes at 37 ℃

Plate washed five times, adding Chromogen Solution A and B incubated 15 minutes at 37 ℃

Add stop solution

Measure within 15min

Calculation

Assay range：4-128pg/mL

Package size: 96 determinations

Storage：  2-8℃.

validity： six months.