For Research Use Only

僅供研究用

貨號：QY-M30038

小鼠胰島素(INS)酶聯(lián)免疫分析(ELISA)

試劑盒使用說明書

本試劑僅供研究使用 目的：本試劑盒用于測定小鼠血清，細(xì)胞上清及相關(guān)液體樣本中胰島素(INS)的含量。

實驗原理：

本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中小鼠胰島素(INS)水平。用純化的小鼠抗-胰島素(INS)抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入胰島素(INS),再與 HRP 標(biāo)記的胰島素（INS）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過*洗滌后加底物 TMB 顯色。TMB 在 HRP 酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺和樣品中的胰島素(INS)呈正相關(guān)。用酶標(biāo)儀在 450nm 波長下測定吸光度（OD 值），通過標(biāo)準(zhǔn)曲線計算樣品中小鼠胰島素(INS)濃度。

Specimen requirements

N

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 separay. 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: 12 mU/L,8mU/L ,4 mU/L,2 mU/L, 1 mU/L)

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

4.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃. 5.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.

6.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.

7.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 10 min at 37℃

8.Stop the reaction ： Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).

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

Important notes

• 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.
• washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.
• 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 .
• 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）.
• Closure plate membrane only limits the disposable use, to avoid cross-contamination.
• The substrate evade the light preservation.
• Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.
• All samples, washing buffer and each kind of reject should according to infective material process.
• Do not mix reagents with those from other lots.

Assay range

0.3 mU/L -15 mU/L

Storage and validity

1．Storage： 2-8℃.

2．validity： six months.

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