人丙肝（HCV）酶聯(lián)免疫分析
試劑盒使用說(shuō)明書(shū)
本試劑盒僅供研究使用。
96T
使用目的：
本試劑盒用于測(cè)定人血清、血漿及相關(guān)液體樣本中丙肝（HCV）表達(dá)。
實(shí)驗(yàn)原理
本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人丙肝（HCV）表達(dá)。用純化的人乙肝（HBV）抗
體包被微孔板，制成固相抗體，可與樣品中丙肝（HCV）相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗
原和其他成分后再與HRP 標(biāo)記的丙肝（HCV）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，
經(jīng)過(guò)*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。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度 （OD值） ，與 CUTOFF值相比較，
從而判定標(biāo)本中人丙肝（HCV）的存在與否。
試劑盒組成
1 30 倍濃縮洗滌液 20ml×1瓶 7 終止液 6ml×1 瓶
2 酶標(biāo)試劑 6ml×1瓶 8 陽(yáng)性對(duì)照 0.5ml×1 瓶
3 酶標(biāo)包被板 12 孔×8條 9 陰性對(duì)照 0.5ml×1 瓶
4 樣品稀釋液 6ml×1瓶 10 說(shuō)明書(shū) 1 份
5 顯色劑A 液 6ml×1瓶 11 封板膜 2 張
6 顯色劑B液 6ml×1/瓶 12 密封袋 1 個(gè)
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. 編號(hào)：將樣品對(duì)應(yīng)微孔按序編號(hào)，每板應(yīng)設(shè)陰性對(duì)照2 孔、陽(yáng)性對(duì)照2 孔、空白對(duì)照1
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）
2. 加樣：分別在陰、陽(yáng)性對(duì)照孔中加入陰性對(duì)照、陽(yáng)性對(duì)照 50µl。然后在待測(cè)樣品孔先
加樣品稀釋液 40µl，然后再加待測(cè)樣品 10µl。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30 倍濃縮洗滌液用蒸餾水30 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(mǎn)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?xún)?nèi)進(jìn)行。
操作程序總結(jié)：
計(jì)算和結(jié)果判定：
試驗(yàn)有效性：陽(yáng)性對(duì)照孔平均值≥1.00; 陰性對(duì)照平均值≤0.10
臨界值（CUT OFF）計(jì)算：臨界值=陰性對(duì)照孔平均值+0.15
陰性判定：樣品OD 值< 臨界值（CUT OFF）者為丙肝（HCV）陰性
陽(yáng)性判定：樣品OD 值≥ 臨界值（CUT OFF）者為丙肝（HCV）陽(yáng)性
。
注意事項(xiàng)
1．操作嚴(yán)格按照說(shuō)明書(shū)進(jìn)行，本試劑不同批號(hào)組分不得混用。 2．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。
3．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。
4． 封板膜只限一次性使用，以避免交叉污染。
5．底物請(qǐng)避光保存。
6．試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)，使用雙波長(zhǎng)檢測(cè)時(shí)，參考波長(zhǎng)為630nm
7．所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。終止液為 2M 的硫酸，使用時(shí)必須
注意安全。
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 HCV
FOR RESEARCH USE ONLY
96 determinations
Purpose
This kit allows for the determination of HCV expression in Human serum, and other
biological fluids.
Principle of the assay
The kit assay HCV level in the sample, use Purified HCV antibody to coat microtiter plate
wells, make solid-phase antibody, then add HCV to wells, Combined With HCV, after washing
and removing non-combinative antibody and other components ,then Combined HCV antibody
which with HRP labeled become antibody – antigen - enzyme- antibody complex, after washing
Compley, 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. Compared with
the CUTOFF value, according to this to judge HCV exist in the sample or not.
Materials provided with the kit
1 wash solution 20ml×1bottle 7 Stopp Solution 6ml×1 bottle
2 HRP-Conjugate reagent 6ml×1 bottle 8 Positive control 0.5ml×1 bottle
3 Microelisa stripplate 12well×8strips 9 Negative control 0.5ml×1bottle
4 Sample diluent 6ml×1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml×1 bottle 11
Closure plate
membrane
2
6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1
Specimen requirements
1. 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. 2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1.Number: to sample correspond microtitration well and Number Sequence, each plate should
be set feminine comparison 2 wells, masculine comparison 2 wells, blank comparison 1
well（don’t add sample and HRP-Conjugate reagent to blank comparison well, other each step
the operation are same）.
2.add sample：separay add Positive control and Negative control 50µl to the Positive and
Negative well . add Sample dilution 40µl to testing sample well, then add testing sample 10µl.
add sample to the bottom of ELISA plates coated well , 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.Configurate liquid: 30-fold （or 20-fold）wash solution diluted 30-fold （or 20-fold） with distilled
water until 600ml,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 50µlto each well, except the 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.
Determine the result Test validity: the average of Positive control well≥1.00; the average of Negative control well
≤0.10.
Calculate Critical（CUT OFF） : Critical= the average of Negative control well + 0.15.
Negative control: sample OD< Calculate Critical（CUT OFF） is HCV Negative control.
Positive control: ample OD≥ Calculate Critical（CUT OFF） is HCV Positive control.
Important notes
1.Please according to use instruction strictly, Do not mix reagents with those from other lots.
2.The kit takes out from the refrigeration environment should be balanced 15-30 minutes in
the room temperature then use, ELISA plates coated if has not use up after opened, the
plate should be stored in Sealed bag.
3.washing buffer will Crystallization separation, it can be heated the water helps dissolve
when dilute . Washing does not affect the result.
4.Closure plate membrane only limits the disposable use, in order to avoid the overlapping
pollution
5.The substrate please evade the light preservation.
6.The test result determination must take the microtiter plate reader as a standard, when use
dual-wavelength to assay, Reference wavelength is 630nm.
7.All samples, washing buffer and each kind of reject should according to infective material
process. Stopp Solution is 2M sulphuric acid. You must pay attention to safe when use .
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.
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