操(cao)作(zuo)步驟(zou):

1. 標(biao)(biao)準(zhun)品的稀(xi)釋與加(jia)(jia)(jia)(jia)樣：在(zai)(zai)酶標(biao)(biao)包被板上設標(biao)(biao)準(zhun)品孔(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)，在(zai)(zai)di一(yi)、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品100μl，然后(hou)在(zai)(zai)di一(yi)、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品稀(xi)釋液50μl，混(hun)勻；然后(hou)從di一(yi)孔(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取100μl分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)三、第(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品稀(xi)釋液50μl，混(hun)勻；然后(hou)在(zai)(zai)第(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)先各(ge)(ge)取50μl棄(qi)掉(diao)，再(zai)(zai)(zai)各(ge)(ge)取50μl分(fen)(fen)別(bie)(bie)取50μl分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)七、第(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)七、第(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品稀(xi)釋液50μl，混(hun)勻后(hou)從第(di)(di)(di)(di)七、第(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)五(wu)(wu)(wu)、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)五(wu)(wu)(wu)、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品稀(xi)釋液50ul，混(hun)勻；混(hun)勻后(hou)從第(di)(di)(di)(di)五(wu)(wu)(wu)、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)分(fen)(fen)別(bie)(bie)取50μl加(jia)(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)九、第(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品稀(xi)釋液50μl，混(hun)勻后(hou)從第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取50μl棄(qi)掉(diao)。（稀(xi)釋后(hou)各(ge)(ge)孔(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)(jia)樣量都為50μl，濃度分(fen)(fen)別(bie)(bie)為180 ng/L，120 ng/L ，60 ng/L，30 ng/，15 ng/L）。

2. 加樣(yang)：分別(bie)設(she)空(kong)白(bai)孔(kong)(kong)（空(kong)白(bai)對(dui)照孔(kong)(kong)不(bu)(bu)加樣(yang)品(pin)(pin)及(ji)酶標(biao)試劑，其(qi)余各步操作相同）、待測(ce)樣(yang)品(pin)(pin)孔(kong)(kong)。在酶標(biao)包被(bei)板上待測(ce)樣(yang)品(pin)(pin)孔(kong)(kong)中先加樣(yang)品(pin)(pin)稀釋液40μl，然后再(zai)加待測(ce)樣(yang)品(pin)(pin)10μl（樣(yang)品(pin)(pin)最終(zhong)稀釋度為5倍）。加樣(yang)將樣(yang)品(pin)(pin)加于酶標(biao)板孔(kong)(kong)底(di)部，盡(jin)量不(bu)(bu)觸及(ji)孔(kong)(kong)壁(bi)，輕輕晃動混勻。

3. 溫育：用封板膜封板后置(zhi)37℃溫育30分鐘。

4. 配(pei)液(ye)：將30（48T的20倍(bei)）倍(bei)濃縮洗(xi)滌液(ye)用蒸餾水(shui)30（48T的20倍(bei)）倍(bei)稀釋(shi)后備(bei)用。

5. 洗滌：小心揭掉(diao)封(feng)板膜(mo)，棄去(qu)液體，甩(shuai)干(gan)(gan)，每孔加滿洗滌液，靜置30秒后棄去(qu)，如此重(zhong)復5次，拍干(gan)(gan)。

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

7. 溫(wen)育：操作同(tong)3。

8. 洗(xi)滌：操作(zuo)同(tong)5。

9. 顯色(se)：每孔先加入(ru)顯色(se)劑A50μl，再加入(ru)顯色(se)劑B50μl，輕輕震蕩混勻，37℃避光顯色(se)15分(fen)鐘.

10. 終止(zhi)：每孔加終止(zhi)液50μl，終止(zhi)反(fan)應（此時藍色立轉(zhuan)黃(huang)色）。

11. 測定：以空白空調零，450nm波長依序測量各(ge)孔的吸光(guang)度（OD值）。 測定應在加終(zhong)止液后15分鐘(zhong)。