人皮質(zhì)醇（Cortisol）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：本試劑盒用于測定人血清、血漿、組織勻漿及相關(guān)液體樣本中皮質(zhì)醇（Cortisol）的含量。

實(shí)驗(yàn)原理：

本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中人皮質(zhì)醇（Cortisol）水平。用純化的人皮質(zhì)醇（Cortisol）捕獲抗體包被微孔板，制成固相抗體，往包被的微孔中依次加入人皮質(zhì)醇（Cortisol），再與HRP標(biāo)記的檢測抗體結(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)化成最終的黃色。顏色的深淺和樣品中的人皮質(zhì)醇（Cortisol）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(jì)算樣品中人皮質(zhì)醇（Cortisol）含量。

試劑盒組成：

注：標(biāo)準(zhǔn)品濃度依次為：400、200、100、50、25、0 nmol/L.

樣本處理及要求：

1. 血清：室溫血液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如出現(xiàn)沉淀，應(yīng)再次離心。

2. 血漿：應(yīng)根據(jù)標(biāo)本的要求選擇EDTA或檸檬酸鈉作為抗凝劑，混合10-20分鐘后，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)該再次離心。

3. 尿液：用無菌管收集，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。

4. 細(xì)胞培養(yǎng)上清：檢測分泌性的成份時(shí)，用無菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。檢測細(xì)胞內(nèi)的成份時(shí)，用PBS（PH7.2-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100萬/ml左右。通過反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。保存過程中如有沉淀形成，應(yīng)再次離心。

5. 組織標(biāo)本：切割標(biāo)本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存?zhèn)溆谩?biāo)本融化后仍然保持2-8℃的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標(biāo)本勻漿充分。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。分裝后一份待檢測，其余冷凍備用。

6. 標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20℃保存，但應(yīng)避免反復(fù)凍融.

7. 不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。

操作步驟

1. 標(biāo)準(zhǔn)品的加樣：設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μL；。

2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、待測樣品孔。在酶標(biāo)包被板上待測樣品孔中先加樣品稀釋液40μl，然后再加待測樣品10μl（樣品最終稀釋度為5倍）。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動(dòng)混勻。

3. 加酶：每孔加入酶標(biāo)試劑100μl，空白孔除外。

4. 溫育：用封板膜封板后置37℃溫育60分鐘。

5. 配液：將20倍濃縮洗滌液用蒸餾水20倍稀釋后備用。

6. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復(fù)5次，拍干。

7. 顯色：每孔先加入顯色劑A50μl，再加入顯色劑B50μl，輕輕震蕩混勻，37℃避光顯色15分鐘.

8. 終止：每孔加終止液50μl，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。

9. 測定：以空白孔調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。 測定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行。

注意事項(xiàng)：

1． 試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30分鐘后方可使用，酶標(biāo)包被板開封后如未用完，板條應(yīng)裝入密封袋中保存。樣本在使用前也要在室溫平衡60分鐘。

2． 濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。

3． 各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)間最好控制在5分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。

4． 請(qǐng)每次測定的同時(shí)做標(biāo)準(zhǔn)曲線，最好做復(fù)孔。如標(biāo)本中待測物質(zhì)含量過高（樣本OD值大于標(biāo)準(zhǔn)品孔第一孔的OD值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測定，計(jì)算時(shí)請(qǐng)最后乘以總稀釋倍數(shù)（×n×5）。

5． 封板膜只限一次性使用，以避免交叉污染。

6． 底物請(qǐng)避光保存。

7． 嚴(yán)格按照說明書的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).

8． 所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。

9． 本試劑不同批號(hào)組分不得混用。

10. 如與英文說明書有異，以英文說明書為準(zhǔn)。

計(jì)算：

以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)，    

在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的OD      

值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；再乘以稀釋      

倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與OD值計(jì)算出標(biāo)      

準(zhǔn)曲線的直線回歸方程式，將樣品的OD值      

代入方程式，計(jì)算出樣品濃度，再乘以稀釋      

倍數(shù)，即為樣品的實(shí)際濃度。                  

（此圖僅供參考）

試劑盒性能：

1.樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值為0.95以上。

2.批內(nèi)變異系數(shù)與批間變異系數(shù)應(yīng)分別小于10%和15% 。

檢測范圍：                                              

12.5 nmol/L - 400 nmol/L

靈敏度：                                              

檢測濃度小于1.0 nmol/L

保存條件及有效期：

1.試劑盒保存： 2-8℃。

2．有效期： 6個(gè)月

Human Cortisol

Drug Names

Generic Name：Human Cortisol (Cortisol) ELISA Kit.

Purpose

This kit allows for the determination of Cortisol concentrations in Human serum, plasma, tissue homogenates and other biological fluids.

Principle of the assay

The kit assay Human Cortisol level in the sample, use Purified Human Cortisol antibody to coat microtiter plate wells, make solid-phase antibody, then add Cortisol to the wells, Combined antibody which With HRP labeled, 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 Cortisol in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

Note: Standard concentration was followed by:

400、200、100、50、25、0 nmol/L.

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.

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. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

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.add enzyme：Add HRP-Conjugate reagent 100μl to each well, except blank well.

4.Incubate: After closing plate with Closure plate membrane ,incubate for 60 min at 37℃.

5.Configurate liquid: 20-fold wash solution diluted 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 15 min at 37℃

8.Stop the reaction：Add Stop Solution 50μl 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

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 .

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

=

Assay range

12.5 nmol/L - 400 nmol/L

Sensitivity

The minimum detectable dose is typically less than 1.0 nmol/L

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.