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當(dāng)前位置:齊一生物科技(上海)有限公司>>ELISA試劑盒>>雞ELISA試劑盒>> 雞抗肌內(nèi)膜抗體IgA(EMAIgA)ELISA試劑盒
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更新時(shí)間:2014-11-18 15:52:52瀏覽次數(shù):250次
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雞抗肌內(nèi)膜抗體IgA(EMAIgA)ELISA試劑盒本試劑盒用于測(cè)定血清,血漿及相關(guān)液體樣本中含量。
實(shí)驗(yàn)原理:
本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中該產(chǎn)品水平。用純化的本產(chǎn)品抗體包被微孔板,制成固相抗體,往包被單抗的微孔中依次加入本產(chǎn)品抗原,再與HRP標(biāo)記的本產(chǎn)品抗體結(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zui終的黃色。顏色的深淺和樣品中的本產(chǎn)品呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度(OD值),通過標(biāo)準(zhǔn)曲線計(jì)算樣品中該產(chǎn)品濃度。
試劑盒組成:
試劑盒組成 48孔配置 96孔配置 保存
說明書 1份 1份
封板膜 2片(48) 2片(96)
密封袋 1個(gè) 1個(gè)
酶標(biāo)包被板 1×48 1×96 2-8℃保存
標(biāo)準(zhǔn)品:1800ng/L 0.5ml×1瓶 0.5ml×1瓶 2-8℃保存
標(biāo)準(zhǔn)品稀釋液 1.5ml×1瓶 1.5ml×1瓶 2-8℃保存
酶標(biāo)試劑 3 ml×1瓶 6 ml×1瓶 2-8℃保存
樣品稀釋液 3 ml×1瓶 6 ml×1瓶 2-8℃保存
顯色劑A液 3 ml×1瓶 6 ml×1瓶 2-8℃保存
顯色劑B液 3 ml×1瓶 6 ml×1瓶 2-8℃保存
終止液 3ml×1瓶 6ml×1瓶 2-8℃保存
濃縮洗滌液 (20ml×20倍)×1瓶 (20ml×30倍)×1瓶 2-8℃保存
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抑制辣根過氧化物酶的(HRP)活性。
雞抗肌內(nèi)膜抗體IgA(EMAIgA)ELISA試劑盒操作步驟:
1.標(biāo)準(zhǔn)品的稀釋與加樣:在酶標(biāo)包被板上設(shè)標(biāo)準(zhǔn)品孔10孔,在*、第二孔中分別加標(biāo)準(zhǔn)品100μl,然后在*、第二孔中加標(biāo)準(zhǔn)品稀釋液50μl,混勻;然后從*孔、第二孔中各取100μl分別加到第三孔和第四孔,再在第三、第四孔分別加標(biāo)準(zhǔn)品稀釋液50μl,混勻;然后在第三孔和第四孔中先各取50μl棄掉,再各取50μl分別加到第五、第六孔中,再在第五、第六孔中分別加標(biāo)準(zhǔn)品稀釋液50ul,混勻;混勻后從第五、第六孔中各取50μl分別加到第七、第八孔中,再在第七、第八孔中分別加標(biāo)準(zhǔn)品稀釋液50μl,混勻后從第七、第八孔中分別取50μl加到第九、第十孔中,再在第九第十孔分別加標(biāo)準(zhǔn)品稀釋液50μl,混勻后從第九第十孔中各取50μl棄掉。(稀釋后各孔加樣量都為50μl,濃度分別為1200 ng/L,800 ng/L ,400 ng/L,200ng/L, 100 ng/L)。
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(cè)樣品孔。在酶標(biāo)包被板上待測(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zui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30(48T的20倍)倍濃縮洗滌液用蒸餾水30(48T的20倍)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,使用排搶加樣。
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)zui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。
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)與批見應(yīng)分別小于9%和11%
雞抗肌內(nèi)膜抗體IgA(EMAIgA)ELISA試劑盒保存條件及有效期:
1.試劑盒保存;2-8℃。
2.有效期:6個(gè)月
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