价格详细资料：试剂盒价格电议，齐一生物销售：021－6034 8496；181214 53965；173021 04490,齐一生物科技（上海）有限公司提供ELISA试剂盒受到了广大研究所.中科院科研单位一致肯定和认同。齐一生物大品牌保证，倾力为国内外科研院校实验室提供*优质产品。

【人内脂素/内脏脂肪素(visfatin)ELISA试剂盒】注意事项

1. 当混合蛋白溶液时应尽量轻缓，避免起泡。

2. 洗涤过程非常重要，不充分的洗涤易造成假阳性。

3. 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。

5. 如标本中待测物质含量过高，请先稀释后再测定，计算时请*后乘以稀释倍数。

6. 在配制标准品、检测溶液工作液时，请以相应的稀释液配制，不能混淆。

7. 底物请避光保存。

8. 不要用其它生产厂家的试剂替换试剂盒中的试剂。

FOR RESEARCH USE ONLY

Drug Names

Generic Name：

This kit can be used for determination of serum, plasma ａnd liquid samples Organization Content.

The experimental principle:

The product levels were measured in samples of the kit by double antibody sandwichmethod. The product with the purified antibody coated microtiter plate, made of solid phase antibody, to package is the product antigen monoclonal antibodies are then added to the micropores, the product and then with HRP labeled antibody binding, the formation of antibody - antigen - antibody complex enzyme label, after thorough washing with TMB chromogenic substrate. TMB in the HRP enzyme catalytic conversion into the blue, and in the action of acid into the final yellow. This product is positively related to the depth of color and in the samples. Instrument measured absorbance in the 450nm wavelength with ELISA (OD), the product concentration in the samples was calculated by standard curve.

Materials provided with the kit

Materials provided with the kit 96 determinations Storage

User manual   1  

Closure plate membrane   2  

Sealed bags   1  

Microelisa stripplate    1   2-8℃

Standard：360ng/L 0.5ml×1 bottle   2-8℃

Standard diluent  1.5ml×1 bottle   2-8℃

HRP-Conjugate reagent    6ml×1 bottle 2-8℃

Sample diluent    6ml×1 bottle 2-8℃

Chromogen Solution A 6ml×1 bottle 2-8℃

Chromogen Solution B 6ml×1 bottle 2-8℃

Stop Solution 6ml×1 bottle 2-8℃

wash  solution    （20ml×30 fold）

×1bottle  2-8℃

Specimen requirements

1.    serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. 【人内脂素/内脏脂肪素(visfatin)ELISA试剂盒】

2.    plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.

3.    Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax ａnd cerebrospinal fluid Reference to it.

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

5.    Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain samples at 2-8℃ after melting,add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.

6.    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.

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

Assay procedure

1.Dilute ａnd add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first ａnd the second well, then add Standard dilution 50μl to the first ａnd the second well, mix; take out 100μl form the first ａnd the second well then add it to the third ａnd the forth well separately. then add Standard dilution 50μl to the third ａnd the forth well ,mix ; then take out 50μl from the third ａnd the forth well discard, add 50μl to the fifth ａnd the sixth well ,then add Standard dilution 50μl to the fifth ａnd the sixth well, mix ; take out 50μl from the fifth ａnd the sixth well ａnd add to the seventh ａnd the eighth well, then add Standard dilution 50μl to the seventh ａnd the eighth well ,mix ; take out 50μl from the seventh ａnd the eighth well ａnd add to the ninth ａnd the tenth well, add Standard dilution 50μl to the ninth ａnd the tenth well, mix , take out 50μl from the ninth ａnd the tenth well discard(add Sample 50μl to each well after Diluting ,(density: 240ng/L,160ng/L ,80ng/L,40ng/L, 20ng/L)

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. 【人内脂素/内脏脂肪素(visfatin)ELISA试剂盒】

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.

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 mins, if the number of sample is much , recommend to use Volley . 【人内脂素/内脏脂肪素(visfatin)ELISA试剂盒】

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.

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.

Storage ａnd validity

1．Storage：  2-8℃.

2．validity： six months.

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