大鼠环孢素A（CsA）Elisa试剂盒说明书

大鼠环孢素A（CsA）Elisa试剂盒说明书
本试剂盒仅供研究使用。
检测范围： 96T
20ng/mL -480ng/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环孢素A（CsA）含量。
实验原理
大鼠环孢素A（CsA）Elisa试剂盒说明书
本试剂盒应用双抗体夹心法测定标本中大鼠环孢素A（CsA）水平。用纯化的大鼠环孢素
A（CsA）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环孢素A（CsA），再
与HRP 标记的环孢素A（CsA）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zui终的黄
色。颜色的深浅和样品中的环孢素A（CsA）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环孢素A（CsA）浓度。
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（960 ng/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。
480ng/mL 5 号标准品150μl 的原倍标准品加入150μl 标准品稀释液
240ng/mL 4 号标准品150μl 的5 号标准品加入150μl 标准品稀释液
120ng/mL 3 号标准品150μl 的4 号标准品加入150μl 标准品稀释液
60ng/mL 2 号标准品150μl 的3 号标准品加入150μl 标准品稀释液
30ng/mL 1 号标准品150μl 的2 号标准品加入150μl 标准品稀释液
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zui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
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，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zui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
Rat Cyclosporine A（CsA）
FOR RESEARCH USE ONLY
Assay range：20ng/mL -480ng/mL 96 determinations
Purpose
This kit allows for the determination of CsA concentrations in Rat serum, cell culture
supernates and other biological fluids
Principle of the assay
The kit assay Rat CsA level in the sample，use Purified Rat CsA antibody to coat
microtiter plate wells, make solid-phase antibody, then add CsA to wells, Combined CsA
antibody which With HRP labeled,become antibody - antigen - enzyme-antibody complex, after
washing Compley, 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 Rat CsA in 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
（960ng/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
RD
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:
480ng/mL 5 Standard 150μl Original density Standard+150μl Standard diluent
240ng/mL 4 Standard 150μl 5 Standard+150μl Standard diluent
120ng/mL 3 Standard 150μl 4 Standard+150μl Standard diluent
60ng/mL 2 Standard 150μl 3 Standard +150μl Standard diluent
30ng/mL 1 Standard 150μl 2 Standard +150μl Standard diluent
2.add sample：Set blank wells separay （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 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, 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 at 37℃
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
Standard, Sample diluent
Add Standard, Sample diluent, incubate for 30 min at 37℃.
Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.
Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37℃.
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
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大鼠环孢素A（CsA）Elisa试剂盒说明书

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