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。                 

（此图仅供参考）

试剂盒性能：

1.样品线性回归与预期浓度相关系数R值为0.95以上。

2.批内与批见应分别小于9%和11%

检测范围：                                             

6pg/mL -200 pg/mL

保存条件及有效期：

1.试剂盒保存：；2-8℃。

2．有效期：6个月

Ratoccludin

Drug Names

Generic Name：RatoccludinELISA Kit.

Purpose

This kitallows for the determinationof occludin concentrations in Rat serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay Rat occludin level in the sample，use Purified Rat occludin antibody to coat microtiterplate wells, make solid-phase antibody, then add occludin to wells, Combined occludinantibody which With HRP labeled ,become antibody - antigen - enzyme-antibodycomplex, after washing Completely, Add TMB substrate solution,TMBsubstrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by theaddition of a sulphuric acid solution and the color change is measuredspectrophotometrically at a wavelength of 450 nm. The concentration of occludinin the samples is thendetermined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

Specimen requirements

1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain.

2. plasma-use suitedEDTA, citrate or heparinized plasmaas an anticoagulant,mix 10-20 mins ,centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain.

3. Urine-collectsue a sterile container, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugalagain.The Operation of Hydrothorax and cerebrospinal fluidReference to it.

4. cell culture supernatant-detect secretory components,collect sue a sterile container, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant,detect the composition of cells, Dilut cellsuspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeatedfreeze-thaw cycles, damage cells and release of intracellularcomponents, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugalagain.

5. Tissuesamples- After cutting samples, check theweight,add PBS（PH7.2-7.4）, Rapidly frozen with liquidnitrogen, maintainsamples at 2-8℃aftermelting,add PBS（PH7.4）,Homogenized by hand or Grinders, centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant.

Assay procedure

1.Dilute and add sample toStandard: set 10 Standard wells on the ELISA plates coated, add Standard100μl to the first and the second well, then add Standard dilution 50μl to thefirst and the second well, mix; take out 100μl form the first and the secondwell then add it to the third and the forth well separately. then add Standarddilution 50μlto the third and the forth well ,mix ; then take out 50μl from the third andthe forth well discard, add 50μl to the fifth and the sixth well ,then addStandard dilution50μl to the fifth and the sixth well, mix ; takeout 50μl from the fifth and the sixth well and add to the seventh and theeighth well, then add Standard dilution 50μl to theseventh and the eighth well ,mix ; take out 50μl from the seventh and theeighth well and add to the ninth and the tenth well, add Standard dilution 50μl to theninth and the tenth well, mix , take out 50μl from the ninth and the tenth welldiscard(add Sample 50μl to each well after Diluting ,(density:150pg/mL，100pg/mL，50pg/mL，25pg/mL，12.5pg/mL)

2.add sample：Setblank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent,other each step operation is same). testing sample well. add Sample dilution40μl 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.Configurateliquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) withdistilled water and reserve.

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

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

7.incubate：Operationwith 3.

8.washing：Operationwith 5.

9.color：Add Chromogen Solution A50ul andChromogen Solution B to each well, evade the light preservationfor 15 min at 37℃

10.Stop thereaction：Add Stop Solution50μl to each well, Stop the reaction(theblue color change to yellow color).

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

Important notes

1.  The kit takes out from the refrigerationenvironment should be balanced 15-30 minutes in the room temperature, ELISA platescoated if has not use up after opened, the plate should be stored in Sealedbag.

2.  washing buffer will Crystallization separation,it can be heated the water helps dissolve when dilute . Washing does not affectthe result.

3.  add Sample with sampler Each step, And proofreadits accuracy frequently, avoids the experimental error. add sample within 5min, if the number of sample is much , recommend to use Volley .

4.  if the testing material content is excessivelyhigher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.（×n×5）.

5.  Closure platemembrane onlylimits 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 microtiterplate reader as astandard.

8.  All samples, washing buffer and each kind ofreject should according to infective material process.

9.  Donot mix reagents with those from other lots.

Calculate

Assay range

6pg/mL -200 pg/mL

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.