操作步驟:

1. 標(biao)(biao)準(zhun)品(pin)(pin)的稀(xi)釋(shi)與(yu)加(jia)(jia)樣：在(zai)酶標(biao)(biao)包被板上設標(biao)(biao)準(zhun)品(pin)(pin)孔(kong)10孔(kong)，在(zai)di一、第(di)(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)中(zhong)分(fen)(fen)別(bie)加(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)100μl，然后(hou)在(zai)di一、第(di)(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)中(zhong)加(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)液(ye)(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)；然后(hou)從di一孔(kong)、第(di)(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)中(zhong)各(ge)取100μl分(fen)(fen)別(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)(di)四(si)(si)孔(kong)，再在(zai)第(di)(di)(di)(di)(di)(di)(di)(di)三、第(di)(di)(di)(di)(di)(di)(di)(di)四(si)(si)孔(kong)分(fen)(fen)別(bie)加(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)液(ye)(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)；然后(hou)在(zai)第(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)(di)四(si)(si)孔(kong)中(zhong)先各(ge)取50μl棄掉，再各(ge)取50μl分(fen)(fen)別(bie)取50μl分(fen)(fen)別(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)中(zhong)，再在(zai)第(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)中(zhong)分(fen)(fen)別(bie)加(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)液(ye)(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)后(hou)從第(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)中(zhong)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)(di)六(liu)孔(kong)中(zhong)，再在(zai)第(di)(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)(di)六(liu)孔(kong)中(zhong)分(fen)(fen)別(bie)加(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)液(ye)(ye)50ul，混(hun)(hun)(hun)勻(yun)(yun)；混(hun)(hun)(hun)勻(yun)(yun)后(hou)從第(di)(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)(di)六(liu)孔(kong)中(zhong)各(ge)分(fen)(fen)別(bie)取50μl加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)九、第(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)中(zhong)，再在(zai)第(di)(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)分(fen)(fen)別(bie)加(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)液(ye)(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)后(hou)從第(di)(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)中(zhong)各(ge)取50μl棄掉。（稀(xi)釋(shi)后(hou)各(ge)孔(kong)加(jia)(jia)樣量(liang)都為50μl，濃度(du)分(fen)(fen)別(bie)為180 ng/L，120 ng/L ，60 ng/L，30 ng/，15 ng/L）。

2. 加(jia)樣(yang)：分(fen)別(bie)設空(kong)白孔(kong)(kong)（空(kong)白對(dui)照孔(kong)(kong)不(bu)加(jia)樣(yang)品(pin)(pin)(pin)(pin)及(ji)酶標(biao)(biao)試(shi)劑，其余各步操作相同(tong)）、待(dai)測樣(yang)品(pin)(pin)(pin)(pin)孔(kong)(kong)。在酶標(biao)(biao)包被(bei)板上待(dai)測樣(yang)品(pin)(pin)(pin)(pin)孔(kong)(kong)中先(xian)加(jia)樣(yang)品(pin)(pin)(pin)(pin)稀釋液40μl，然后再加(jia)待(dai)測樣(yang)品(pin)(pin)(pin)(pin)10μl（樣(yang)品(pin)(pin)(pin)(pin)最終稀釋度為5倍）。加(jia)樣(yang)將(jiang)樣(yang)品(pin)(pin)(pin)(pin)加(jia)于酶標(biao)(biao)板孔(kong)(kong)底部，盡量不(bu)觸及(ji)孔(kong)(kong)壁，輕輕晃動混勻。

3. 溫(wen)育：用封板膜封板后置37℃溫(wen)育30分(fen)鐘(zhong)。

4. 配液：將30（48T的(de)20倍）倍濃縮洗滌液用(yong)蒸餾水30（48T的(de)20倍）倍稀釋后備用(yong)。

5. 洗滌(di)：小心揭(jie)掉封板(ban)膜(mo)，棄去(qu)液體(ti)，甩干，每孔加滿洗滌(di)液，靜置30秒(miao)后棄去(qu)，如此重復5次，拍干。

6. 加酶(mei)：每孔加入酶(mei)標試劑50μl，空白孔除外。

7. 溫育：操作同3。

8. 洗滌：操作同5。

9. 顯(xian)色(se)：每孔先(xian)加入(ru)顯(xian)色(se)劑A50μl，再加入(ru)顯(xian)色(se)劑B50μl，輕輕震(zhen)蕩(dang)混勻，37℃避(bi)光顯(xian)色(se)15分鐘(zhong).

10. 終(zhong)止(zhi)：每(mei)孔(kong)加終(zhong)止(zhi)液50μl，終(zhong)止(zhi)反應（此時藍色立(li)轉(zhuan)黃色）。

11. 測定：以空(kong)(kong)白空(kong)(kong)調零，450nm波長(chang)依序測量各孔的吸光度（OD值）。 測定應在加終止液后15分鐘。

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