大鼠极低密度脂蛋白VLDL酶联免疫分析试剂盒使用说明书

大鼠极低密度脂蛋白（VLDL）酶联免疫分析试剂盒使用说明书

本试剂盒仅供研究使用。

检测范围：                                                          96T

3μg/ml-120μg/ml

 

使用目的：

本试剂盒用于测定大鼠血清、血浆及相关液体样本中极低密度脂蛋白（VLDL）含量。

实验原理

本试剂盒应用双抗体夹心法测定标本中大鼠极低密度脂蛋白（VLDL）水平。用纯化的大鼠极低密度脂蛋白（VLDL）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入极低密度脂蛋白（VLDL），再与HRP标记的极低密度脂蛋白（VLDL）抗体结合，形成抗体-抗原-酶标抗体复合物，经过洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成终的黄色。颜色的深浅和样品中的极低密度脂蛋白（VLDL）呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中大鼠极低密度脂蛋白（VLDL）浓度。

大鼠极低密度脂蛋白（VLDL）酶联免疫分析试剂盒使用说明书

试剂盒组成

 

1	30倍浓缩洗涤液	20ml×1瓶	7	终止液	6ml×1瓶
2	酶标试剂	6ml×1瓶	8	标准品（240μg/ml）	0.5ml×1瓶
3	酶标包被板	12孔×8条	9	标准品稀释液	1.5ml×1瓶
4	样品稀释液	6ml×1瓶	10	说明书	1份
5	显色剂A液	6ml×1瓶	11	封板膜	2张
6	显色剂B液	6ml×1/瓶	12	密封袋	1个

标本要求

1．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

2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。

操作步骤

1. 标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。

 

120μg/ml	5号标准品	150μl的原倍标准品加入150μl标准品稀释液
60μg/ml	4号标准品	150μl的5号标准品加入150μl标准品稀释液
30μg/ml	3号标准品	150μl的4号标准品加入150μl标准品稀释液
15μg/ml	2号标准品	150μl的3号标准品加入150μl标准品稀释液
7.5μg/ml	1号标准品	150μl的2号标准品加入150μl标准品稀释液

 

 

大鼠极低密度脂蛋白（VLDL）酶联免疫分析试剂盒使用说明书

1. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。
2. 温育：用封板膜封板后置37℃温育30分钟。  
3. 配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用
4. 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。
5. 加酶：每孔加入酶标试剂50μl，空白孔除外。
6. 温育：操作同3。
7. 洗涤：操作同5。
8. 显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色15分钟.
9. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。
10. 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15分钟以内进行。

操作程序结：

 

计算

大鼠极低密度脂蛋白（VLDL）酶联免疫分析试剂盒使用说明书

  以标准物的浓度为横坐标，OD值为纵坐标，在坐标纸上绘出标准曲线，根据样品的OD值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。

注意事项

1．试剂盒从冷藏环境中取出应在室温平衡15-30分钟后方可使用，酶标包被板开封后如未用完，板条应装入密封袋中保存。

2．浓洗涤液可能会有结晶析出，稀释时可在水浴中加温助溶，洗涤时不影响结果。

3．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。一次加样时间好控制在5分钟内，如标本数量多，使用排枪加样。

1. 请每次测定的同时做标准曲线，好做复孔。如标本中待测物质含量过高（样本OD值大于标准品孔孔的OD值），请先用样品稀释液稀释一定倍数（n倍）后再测定，计算时请后乘以稀释倍数（×n×5）。
2. 封板膜只限一次性使用，以避免交叉污染。

6．底物请避光保存。

7．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.

8．所有样品，洗涤液和各种废弃物都应按传染物处理。

9．本试剂不同批号组分不得混用。

10. 如与英文说明书有异，以英文说明书为准。

保存条件及有效期

1．试剂盒保存：；2-8℃。

2．有效期：6个月

 

 

 

 

大鼠极低密度脂蛋白（VLDL）酶联免疫分析试剂盒使用说明书

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RD

Rat VLDL

 

FOR RESEARCH USE ONLY

 

Assay range：3μg/mL– 120μg/mL                96determinations

Purpose

This kit allows for the determination ofVLDL concentrationsin Ratserum cellculture supernatesand otherbiological fluids

 

Principle of the assay

The kitassay RatVLDLlevel in the sample，use Purified RatVLDLantibody to coat microtiter plate wells make solid-phase antibody then addVLDLto wellsCombinedVLDLantibody which With HRP labeledbecome antibody - antigen - enzyme-antibody complex after washing CompletelyAdd TMB substrate solutionTMB substrate becomes blue color At HRP enzyme-catalyzedreaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm.The concentration ofRatVLDLin the samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

 

1	wash  solution	20ml×1bottle	7	Stopp Solution	6ml×1 bottle
2	HRP-Conjugate reagent	6ml×1 bottle	8	Standard（240μg/ml）	0.5ml×1 bottle
3	Microelisa stripplate	12well×8strips	9	Standard diluent	1.5ml×1bottle
4	Sample diluent	6ml×1 bottle	10	Instruction	1
5	Chromogen Solution A	6ml×1 bottle	11	Closure plate membrane	2
6	Chromogen Solution B	6ml×1 bottle	12	Sealed bags	1

Specimen requirements

1. extractas soon as possible after Specimen collectionand according to the relevant literature and should be experiment as soon as possible after the extraction. If it can’tspecimen can be kept in -20 ℃ to preserve Avoid repeated freeze-thaw cycles.
2. Can’t detect the sample which contain NaN3 becauseNaN3inhibits HRP active.

Assay procedure

1. Dilute and add sample:DiluteOriginal densityStandard as follow table:

 

120μg/ml	5Standard	150μlOriginal densityStandard+150μlStandard diluent
80μg/mL	4Standard	150μl5 Standard+150μlStandard diluent
40μg/mL	3Standard	150μl 4Standard+150μlStandard diluent
20μg/mL	2Standard	150μl 3 Standard+150μlStandard diluent
10μg/mL	1Standard	150μl 2 Standard+150μlStandard diluent

2.add sample：Set blank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well then add testing sample 10μl (sample final dilutionis 5-fold)add sample to wellsdon’t touch the well wall as far as possible and Gently mix.

3.Incubate: After closing plate with Closure plate membrane incubate for 30 min at 37℃.

4.Configurate liquid: 30-fold(or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.

5.washing：Uncover Closure plate membrane discardLiquid dry by swing add washing buffer to every well still for 30s then drainrepeat 5 times dry by pat.

6.add enzyme：AddHRP-Conjugate reagent 50μl to each well except  blank well.

7.incubate：Operation with 3.

8.washing：Operation with 5.

9.color：AddChromogen Solution A50ul andChromogen Solution B to each wellevade the light preservationfor 15 min at 37℃

10.Stop the reaction：Add Stop Solution50μlto each well Stop the reaction(the blue color change to yellow color).

11.assay：take blank well as zero Read absorbance at 450nm after Adding Stop Solution and within 15min.

Steps deion

 

StandardSample diluent

 

 

 

AddStandardSample diluentincubate for 30 min at 37℃.

 

 

 

Wash 5 timeAddHRP-Conjugate reagent incubate for 30 min at 37℃.

 

 

 

Wash 5 timesAdd Chromogen Solution Aand Bincubate for 30 min at 37℃.

 

 

 

AddStopp Solution

 

 

 

Read absorbance at 450nm within 15 min

 

 

 

calculate

Calculate

Take the standard density as the horizontal the OD value for the verticaldraw the standard curve on graph erFind out the corresponding density according to the sample OD value by the Sample curve multiplied by the dilution multiple or calculate the straight line regression equation of the standard curve with the standard density and the OD value with the sample OD value in the equation calculate the sample density multiplied by the dilution factor the result is the sample actual density.

Important notes

1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperatureELISA plates coated if has not use up after opened the plate should be stored in Sealed bag.
2. washing buffer will Crystallization separation it can be heated the water helps dissolve when dilute . Washing does not affect the result.
3. add Sample with sampler Each step And proofread its accuracy frequently avoids the experimental error. add sample within 5 min if the number of sample is much recommend to use Volley .
4. if the testing material content is excessively higher(The sample OD is bigger than the first standard well )please dilute Sample (n-fold) Please diluenteandmultiplied by the dilution factor.（×n×5）.
5. Closure plate membraneonly limits the disposable use to avoid cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly The test result determination must take themicrotiter plate readeras a standard.
8. All samples washing buffer and each kind of reject should according to infective material process.
9. Do not mix reagents with those from other lots.

 

Storage and validity

1．Storage：  2-8℃.

2．validity：six months