人細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）酶聯(lián)免疫(ELISA)分析試劑盒使用說(shuō)明書

樊克生物專業(yè)供應(yīng)  

本試劑盒僅供研究使用。

檢測(cè)范圍：    48T       25 ng/L -800 ng/L

使用目的：

本試劑盒用于測(cè)定人血清、血漿及相關(guān)液體樣本細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）含量。

實(shí)驗(yàn)原理

本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）水平。用純化的人細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）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細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152），再與HRP標(biāo)記的細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過(guò)徹底洗滌后加底物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細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中人細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4（CTLA-4/CD152）濃度。

試劑盒組成

標(biāo)本要求

1．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

2．不能檢測(cè)含NaN3的樣品，因NaN3抑制辣根過(guò)氧化物酶的（HRP）活性。

操作步驟

1. 標(biāo)準(zhǔn)品的稀釋：本試劑盒提供原倍標(biāo)準(zhǔn)品一支，用戶可按照下列圖表在小試管中進(jìn)行稀釋。

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）、標(biāo)準(zhǔn)孔、待測(cè)樣品孔。在酶標(biāo)包被板上標(biāo)準(zhǔn)品準(zhǔn)確加樣50μl，待測(cè)樣品孔中先加樣 品稀釋液40μl，然后再加待測(cè)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. 溫育：用封板膜封板后置37℃溫育30分鐘。   

4. 配液：將20倍濃縮洗滌液用蒸餾水20倍稀釋后備用

5. 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。

6. 加酶：每孔加入酶標(biāo)試劑50μl，空白孔除外。

7. 溫育：操作同3。

8.洗滌：操作同5。

9. 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.

10.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）。

11.測(cè)定：以空白空調(diào)零，450nm波長(zhǎng)依序測(cè)量各孔的吸光度（OD值）。 測(cè)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(kāi)封后如未用完，板條應(yīng)裝入密封袋中保存。

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(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線，做復(fù)孔。如標(biāo)本中待測(cè)物質(zhì)含量過(guò)高（樣本OD值大于標(biāo)準(zhǔn)品孔孔的OD值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測(cè)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(shuō)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(shuō)明書有異，以英文說(shuō)明書為準(zhǔn)。

保存條件及有效期

1．試劑盒保存：；2-8℃。

2．有效期：6個(gè)月

The performance of kit:

1 sensitivity: minimum detection concentration is less than 1 standard. Linearity of dilution. Sample linear regression and the expected concentration correlation coefficient R value is 0.990.

2: no specific reaction with other cytokines.

3 repeatability: plate, plate between the coefficients of variation were less than 10%.

Human type III procollagen amino terminal peptide ELISA kit steps:

1 before use, all reagents and mixing. Do not allow liquid to produce a large number of bubbles, so as to avoid adding a large number of bubbles, resulting in the addition of the error.

2 according to determine the number of sample number plus standard strip number. Each standard and blank hole is recommended to do the hole. Each sample can be made according to its own quantity, and can be used as a hole in the hole.

3 diluted after standard 50ul in reaction hole, added to the sample 50 UL in reaction hole to be measured. Immediately joined the 50 UL antibody biotin. Cover the membrane plate, gently oscillating mixing, 37 degrees Celsius for 45 minutes.

4 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. Repeat this operation 4 times. If the washing machine with washing, washing times increased once.

5 per hole adding chain affinity enzyme -HRP 100ul, gently oscillating mixing, 37 degrees 30 minutes incubation.

6 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. Repeat this operation 4 times. If the washing machine with washing, washing times increased once.

7 per hole adding substrate A, B 50ul, gently oscillating mixing, 37 degrees 5 minutes incubation. Avoid light.

8 remove ELISA plate, quickly add 50ul terminated liquid, adding the stop solution immediately after the determination results.

9 od determination of each hole at the wavelength of 450nm.

The result of judgment and analysis:

1, instrument value: Yu Bo 450nm ELISA od read the hole on the instrument

2, to the OD value as a vertical coordinate (y), corresponding ot standard concentrations as a horizontal coordinate (x), do have corresponding curve, sample ot content can be according to the OD value by standard curve conversion out corresponding concentration, multiplied by the dilution multiple; or with the standard concentration and the OD value calculated the regression equation of the standard curve, the sample OD value in the equation to calculate the sample concentration, multiplied by the dilution factor is the actual concentration of the sample.

3, detection range: 0-100ng/ml

4, sensitivity: 0.39ng/ml