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人弓形蟲(Tox)Elisa試劑盒

時(shí)間:2011/12/14閱讀:556
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人弓形蟲(Tox)Elisa試劑盒說(shuō)明書
人弓形蟲(Tox)Elisa試劑盒僅供研究使用,。
檢測(cè)范圍:96T
使用目的:本試劑盒用于測(cè)定人血清,、血漿及相關(guān)液體樣本中弓形蟲(Tox)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)本中人弓形蟲(Tox)表達(dá),。用純化的人弓形蟲(Tox)抗體包被微孔板,,制成固相抗體,,可與樣品中弓形蟲(Tox)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)記的弓形蟲(Tox)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)本中人弓形蟲(Tox)的存在與否。
人弓形蟲(Tox)Elisa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 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.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,。
2.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
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。
計(jì)算和結(jié)果判定:
臨界值(CUT OFF)計(jì)算:臨界值=陰性對(duì)照孔平均值+0.15
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
陽(yáng)性判定:樣品OD值≥ 臨界值(CUT OFF)者為弓形蟲(Tox)陽(yáng)性
陰性判定:樣品OD值< 臨界值(CUT OFF)者為弓形蟲(Tox)陰性。
注意事項(xiàng):
1.操作嚴(yán)格按照說(shuō)明書進(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

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