操作步驟:
1. 標(biao)準(zhun)品(pin)的稀(xi)(xi)釋(shi)與加(jia)樣(yang):在酶標(biao)包被板(ban)上設(she)標(biao)準(zhun)品(pin)孔(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong),在di一、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別加(jia)標(biao)準(zhun)品(pin)100μl,然(ran)后(hou)在di一、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)加(jia)標(biao)準(zhun)品(pin)稀(xi)(xi)釋(shi)液(ye)50μl,混(hun)勻(yun);然(ran)后(hou)從di一孔(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)(qu)(qu)100μl分(fen)別加(jia)到第(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong),再(zai)在第(di)(di)(di)(di)三、第(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)分(fen)別加(jia)標(biao)準(zhun)品(pin)稀(xi)(xi)釋(shi)液(ye)50μl,混(hun)勻(yun);然(ran)后(hou)在第(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)中(zhong)(zhong)先各(ge)取(qu)(qu)(qu)(qu)50μl棄掉,再(zai)各(ge)取(qu)(qu)(qu)(qu)50μl分(fen)別取(qu)(qu)(qu)(qu)50μl分(fen)別加(jia)到第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)中(zhong)(zhong),再(zai)在第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別加(jia)標(biao)準(zhun)品(pin)稀(xi)(xi)釋(shi)液(ye)50μl,混(hun)勻(yun)后(hou)從第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)加(jia)到第(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)中(zhong)(zhong),再(zai)在第(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別加(jia)標(biao)準(zhun)品(pin)稀(xi)(xi)釋(shi)液(ye)50ul,混(hun)勻(yun);混(hun)勻(yun)后(hou)從第(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)分(fen)別取(qu)(qu)(qu)(qu)50μl加(jia)到第(di)(di)(di)(di)九、第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)中(zhong)(zhong),再(zai)在第(di)(di)(di)(di)九第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)分(fen)別加(jia)標(biao)準(zhun)品(pin)稀(xi)(xi)釋(shi)液(ye)50μl,混(hun)勻(yun)后(hou)從第(di)(di)(di)(di)九第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)(qu)(qu)50μl棄掉。(稀(xi)(xi)釋(shi)后(hou)各(ge)孔(kong)(kong)(kong)(kong)加(jia)樣(yang)量都(dou)為50μl,濃度分(fen)別為180 ng/L,120 ng/L ,60 ng/L,30 ng/,15 ng/L)。
2. 加樣:分(fen)別設空白(bai)孔(空白(bai)對照(zhao)孔不加樣品(pin)(pin)(pin)及酶(mei)標試劑,其余各步操作相同)、待(dai)測樣品(pin)(pin)(pin)孔。在酶(mei)標包被板上待(dai)測樣品(pin)(pin)(pin)孔中先加樣品(pin)(pin)(pin)稀(xi)釋(shi)液40μl,然后再(zai)加待(dai)測樣品(pin)(pin)(pin)10μl(樣品(pin)(pin)(pin)最終稀(xi)釋(shi)度為5倍)。加樣將樣品(pin)(pin)(pin)加于酶(mei)標板孔底部(bu),盡量不觸及孔壁,輕輕晃動混勻(yun)。
3. 溫育(yu):用封(feng)板(ban)(ban)膜封(feng)板(ban)(ban)后置(zhi)37℃溫育(yu)30分鐘(zhong)。
4. 配液(ye):將30(48T的(de)20倍(bei))倍(bei)濃(nong)縮(suo)洗滌液(ye)用蒸餾水30(48T的(de)20倍(bei))倍(bei)稀釋(shi)后備用。
5. 洗滌(di)(di):小心揭(jie)掉封(feng)板膜(mo),棄(qi)去液體,甩干(gan)(gan),每孔加滿洗滌(di)(di)液,靜(jing)置30秒后棄(qi)去,如此重復5次,拍干(gan)(gan)。
6. 加酶:每孔(kong)加入酶標試劑50μl,空白孔(kong)除(chu)外。
7. 溫育(yu):操(cao)作(zuo)同(tong)3。
8. 洗滌(di):操作同5。
9. 顯(xian)色(se)(se):每孔先加入顯(xian)色(se)(se)劑A50μl,再加入顯(xian)色(se)(se)劑B50μl,輕輕震蕩混(hun)勻(yun),37℃避光顯(xian)色(se)(se)15分(fen)鐘.
10. 終(zhong)止(zhi)(zhi):每孔加終(zhong)止(zhi)(zhi)液(ye)50μl,終(zhong)止(zhi)(zhi)反應(此時藍(lan)色立轉(zhuan)黃(huang)色)。
11. 測(ce)定(ding):以空白(bai)空調零,450nm波長依序測(ce)量各孔的(de)吸光度(OD值)。 測(ce)定(ding)應在加終止液后15分鐘(zhong)。雌二醇單克隆抗體
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