绵羊基质金属蛋白酶-9（MMP-9）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

45μg/L -1200μg/L

使用目的：

本试剂盒用于测定绵羊血清、血浆及相关液体样本中基质金属蛋白酶-9（MMP-9）含量。

实验原理

本试剂盒应用双抗体夹心法测定标本中绵羊基质金属蛋白酶-9（MMP-9）水平。用纯化的绵羊基质金属蛋白酶-9（MMP-9）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基质金属蛋白酶-9（MMP-9），再与HRP标记的基质金属蛋白酶-9（MMP-9）抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物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最终的黄色。颜色的深浅和样品中的基质金属蛋白酶-9（MMP-9）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绵羊基质金属蛋白酶-9（MMP-9）浓度。

试剂盒组成

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。

2.        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（样品最终稀释度为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。

3.        温育：用封板膜封板后置37℃温育30分钟。  

4.        配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用

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.        加酶：每孔加入酶标试剂50μl，空白孔除外。

7.        温育：操作同3。

8.        洗涤：操作同5。

9.        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.

10.    终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。

11.    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。

操作程序总结：

计算

  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。一次加样时间最好控制在5分钟内，如标本数量多，推荐使用排枪加样。

4． 请每次测定的同时做标准曲线，最好做复孔。如标本中待测物质含量过高（样本OD值大于标准品孔第一孔的OD值），请先用样品稀释液稀释一定倍数（n倍）后再测定，计算时请最后乘以总稀释倍数（×n×5）。

5． 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

Human     MMP-9

FOR RESEARCH USE ONLY

Assay range：45μg/L -1200μg/L                96determinations

Purpose

This kit allows for the determination of MMP-9 concentrations in Human serum, cellculture supernates and other biological fluids

Principle of the assay

The kit assay Human MMP-9 level in the sample，use Purified Human MMP-9 antibody to coat microtiter plate wells, make solid-phase antibody, then add MMP-9 to wells, Combined MMP-9 antibody which With HRP labeled goat anti-Human become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction 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 of Human MMP-9 inthe samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

Specimen requirements

1.  extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in-20 ℃to preserve, Avoid repeated freeze-thaw cycles.

2.  Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1.  Dilute and add sample:Dilute Original density Standard as follow table:

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 dilution is 5-fold), add sample to wells , don’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 at37℃.

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, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except  blank well.

7.incubate：Operation with 3.

8.washing：Operation with 5.

9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at37℃

10.Stop the reaction：Add Stop Solution50μl to 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 description

Calculate

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find 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 temperature, ELISA 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 diluente and multiplied by the dilution factor.（×n×5）.

5.  Closure plate membrane only 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 the microtiter plate reader as 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