操作步驟:

1. 標(biao)準(zhun)(zhun)品(pin)的(de)稀(xi)釋(shi)(shi)與加(jia)樣：在(zai)酶標(biao)包被(bei)板上設標(biao)準(zhun)(zhun)品(pin)孔(kong)10孔(kong)，在(zai)di一、第(di)(di)(di)(di)二孔(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別加(jia)標(biao)準(zhun)(zhun)品(pin)100μl，然(ran)后(hou)(hou)(hou)在(zai)di一、第(di)(di)(di)(di)二孔(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)；然(ran)后(hou)(hou)(hou)從di一孔(kong)、第(di)(di)(di)(di)二孔(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取(qu)100μl分(fen)(fen)別加(jia)到第(di)(di)(di)(di)三(san)孔(kong)和(he)第(di)(di)(di)(di)四(si)孔(kong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)四(si)孔(kong)分(fen)(fen)別加(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)；然(ran)后(hou)(hou)(hou)在(zai)第(di)(di)(di)(di)三(san)孔(kong)和(he)第(di)(di)(di)(di)四(si)孔(kong)中(zhong)(zhong)(zhong)(zhong)先各(ge)(ge)取(qu)50μl棄掉(diao)，再(zai)(zai)各(ge)(ge)取(qu)50μl分(fen)(fen)別取(qu)50μl分(fen)(fen)別加(jia)到第(di)(di)(di)(di)七(qi)(qi)、第(di)(di)(di)(di)八(ba)孔(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)七(qi)(qi)、第(di)(di)(di)(di)八(ba)孔(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別加(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)后(hou)(hou)(hou)從第(di)(di)(di)(di)七(qi)(qi)、第(di)(di)(di)(di)八(ba)孔(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)到第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)孔(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)孔(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別加(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)50ul，混(hun)(hun)勻(yun)；混(hun)(hun)勻(yun)后(hou)(hou)(hou)從第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)孔(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)分(fen)(fen)別取(qu)50μl加(jia)到第(di)(di)(di)(di)九、第(di)(di)(di)(di)十孔(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)分(fen)(fen)別加(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)后(hou)(hou)(hou)從第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取(qu)50μl棄掉(diao)。（稀(xi)釋(shi)(shi)后(hou)(hou)(hou)各(ge)(ge)孔(kong)加(jia)樣量(liang)都為50μl，濃度(du)分(fen)(fen)別為180 ng/L，120 ng/L ，60 ng/L，30 ng/，15 ng/L）。

2. 加樣(yang)(yang)：分別設空白孔(kong)（空白對(dui)照孔(kong)不加樣(yang)(yang)品(pin)及酶標試劑(ji)，其余各步操作相同）、待(dai)測(ce)樣(yang)(yang)品(pin)孔(kong)。在酶標包被板(ban)上待(dai)測(ce)樣(yang)(yang)品(pin)孔(kong)中先加樣(yang)(yang)品(pin)稀釋(shi)液(ye)40μl，然后再加待(dai)測(ce)樣(yang)(yang)品(pin)10μl（樣(yang)(yang)品(pin)最(zui)終稀釋(shi)度為5倍）。加樣(yang)(yang)將樣(yang)(yang)品(pin)加于(yu)酶標板(ban)孔(kong)底部，盡量不觸及孔(kong)壁，輕(qing)輕(qing)晃動(dong)混勻。

3. 溫育：用封(feng)板膜封(feng)板后置37℃溫育30分鐘(zhong)。

4. 配液(ye)：將30（48T的20倍）倍濃縮(suo)洗滌液(ye)用(yong)蒸餾水30（48T的20倍）倍稀(xi)釋后備(bei)用(yong)。

5. 洗滌：小心揭掉封板膜(mo)，棄(qi)去(qu)液體(ti)，甩干(gan)，每孔加滿洗滌液，靜置30秒后棄(qi)去(qu)，如此重復5次，拍干(gan)。

6. 加酶：每孔(kong)加入(ru)酶標試劑50μl，空白孔(kong)除外。

7. 溫育：操(cao)作同3。

8. 洗滌：操作同(tong)5。

9. 顯色：每孔先加入(ru)顯色劑A50μl，再加入(ru)顯色劑B50μl，輕輕震(zhen)蕩混勻，37℃避光(guang)顯色15分鐘.

10. 終(zhong)止：每孔(kong)加終(zhong)止液50μl，終(zhong)止反應（此時藍(lan)色立轉黃(huang)色）。

11. 測(ce)定：以空白空調零，450nm波(bo)長依序(xu)測(ce)量各(ge)孔的(de)吸光度（OD值）。 測(ce)定應在加終止(zhi)液后15分(fen)鐘。

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