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禽腦脊髓炎抗體(AE-Ab)Elisa試劑盒說明書

時間:2011/10/25閱讀:568
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本試劑盒僅供研究使用。檢測范圍:96T.使用目的:本試劑盒用于測定禽血清、血漿及相關(guān)液體樣本中腦脊髓炎抗體(AE-Ab)表達(dá)。實驗原理:本試劑盒應(yīng)用雙抗原夾心法測定標(biāo)本中禽腦脊髓炎抗體(AE-Ab)表達(dá)。用純化的抗原包被微孔板,制成固相抗原,可與樣品中腦脊髓炎抗體(AE-Ab)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)記的抗原結(jié)合,形成抗原-抗體-抗原復(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。用酶標(biāo)儀在450nm 波長下測定吸光度(OD 值),與CUTOFF 值相比較,從而判定標(biāo)本中禽腦脊髓炎抗體(AE-Ab)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 陽性對照 0.5ml×1 瓶,3 酶標(biāo)包被板 12 孔×8 條 9 陰性對照 0.5ml×1 瓶,4 樣品稀釋液 6ml×1 瓶 10 說明書 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,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)行實驗。若不能馬上進(jìn)行試驗,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. 編號:將樣品對應(yīng)微孔按序編號,每板應(yīng)設(shè)陰性對照2 孔、陽性對照2 孔、空白對照1孔(空白對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、陽性對照孔中加入陰性對照、陽性對照50μl。然后在待測樣品孔先加樣品稀釋液40μl,然后再加待測樣品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)(此時藍(lán)色立轉(zhuǎn)黃色)。11. 測定:以空白空調(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。操作程序總結(jié):計算和結(jié)果判定,試驗有效性:陽性對照孔平均值≥1.00; 陰性對照平均值≤0.10臨界值(CUT OFF)計算:臨界值=陰性對照孔平均值+0.15,陰性判定:樣品OD 值< 臨界值(CUT OFF)者為禽腦脊髓炎抗體(AE-Ab)陰性,陽性判定:樣品OD 值≥ 臨界值(CUT OFF)者為禽腦脊髓炎抗體(AE-Ab)陽性。注意事項:1.操作嚴(yán)格按照說明書進(jìn)行,本試劑不同批號組分不得混用。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.濃洗滌液可能會有結(jié)晶析出,稀釋時可在水浴中加溫助溶,洗滌時不影響結(jié)果。4. 封板膜只限一次性使用,以避免交叉污染。5.底物請避光保存。6.試驗結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn),使用雙波長檢測時,參考波長為630nm7.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 個月。

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