操作步驟:

1. 標(biao)準(zhun)(zhun)品(pin)(pin)(pin)的稀(xi)(xi)釋與加(jia)(jia)樣(yang)(yang)：在(zai)酶標(biao)包被板上(shang)設標(biao)準(zhun)(zhun)品(pin)(pin)(pin)孔(kong)(kong)(kong)10孔(kong)(kong)(kong)，在(zai)di一(yi)、第(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)中(zhong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)100μl，然(ran)后(hou)(hou)在(zai)di一(yi)、第(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)中(zhong)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混勻(yun)；然(ran)后(hou)(hou)從(cong)di一(yi)孔(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)中(zhong)各(ge)(ge)(ge)(ge)取100μl分(fen)別(bie)(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)三(san)(san)孔(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)(di)(di)三(san)(san)、第(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混勻(yun)；然(ran)后(hou)(hou)在(zai)第(di)(di)(di)(di)(di)(di)三(san)(san)孔(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)中(zhong)先各(ge)(ge)(ge)(ge)取50μl棄掉(diao)，再(zai)(zai)各(ge)(ge)(ge)(ge)取50μl分(fen)別(bie)(bie)取50μl分(fen)別(bie)(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)中(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)中(zhong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混勻(yun)后(hou)(hou)從(cong)第(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)中(zhong)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)中(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)中(zhong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50ul，混勻(yun)；混勻(yun)后(hou)(hou)從(cong)第(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)中(zhong)各(ge)(ge)(ge)(ge)分(fen)別(bie)(bie)取50μl加(jia)(jia)到第(di)(di)(di)(di)(di)(di)九(jiu)(jiu)、第(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)中(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)(di)(di)(di)九(jiu)(jiu)第(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混勻(yun)后(hou)(hou)從(cong)第(di)(di)(di)(di)(di)(di)九(jiu)(jiu)第(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)中(zhong)各(ge)(ge)(ge)(ge)取50μl棄掉(diao)。（稀(xi)(xi)釋后(hou)(hou)各(ge)(ge)(ge)(ge)孔(kong)(kong)(kong)加(jia)(jia)樣(yang)(yang)量都(dou)為(wei)(wei)50μl，濃度分(fen)別(bie)(bie)為(wei)(wei)180 ng/L，120 ng/L ，60 ng/L，30 ng/，15 ng/L）。

2. 加(jia)(jia)(jia)(jia)樣(yang)(yang)(yang)：分別(bie)設空白(bai)孔(kong)(kong)（空白(bai)對照孔(kong)(kong)不(bu)(bu)加(jia)(jia)(jia)(jia)樣(yang)(yang)(yang)品及酶標試劑，其余各步操作相同）、待(dai)測樣(yang)(yang)(yang)品孔(kong)(kong)。在(zai)酶標包(bao)被板上(shang)待(dai)測樣(yang)(yang)(yang)品孔(kong)(kong)中先加(jia)(jia)(jia)(jia)樣(yang)(yang)(yang)品稀釋液40μl，然(ran)后再加(jia)(jia)(jia)(jia)待(dai)測樣(yang)(yang)(yang)品10μl（樣(yang)(yang)(yang)品最終稀釋度(du)為5倍）。加(jia)(jia)(jia)(jia)樣(yang)(yang)(yang)將樣(yang)(yang)(yang)品加(jia)(jia)(jia)(jia)于(yu)酶標板孔(kong)(kong)底部，盡量不(bu)(bu)觸及孔(kong)(kong)壁，輕輕晃動混(hun)勻(yun)。

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

4. 配液(ye)：將30（48T的(de)20倍(bei)）倍(bei)濃縮洗(xi)滌液(ye)用(yong)蒸餾水30（48T的(de)20倍(bei)）倍(bei)稀釋后備用(yong)。

5. 洗(xi)(xi)滌(di)(di)：小心揭掉封板膜，棄去液體，甩(shuai)干(gan)，每孔加滿洗(xi)(xi)滌(di)(di)液，靜置(zhi)30秒后棄去，如此重復(fu)5次，拍干(gan)。

6. 加酶：每孔加入酶標試劑50μl，空白孔除外。

7. 溫育：操作(zuo)同(tong)3。

8. 洗(xi)滌：操作(zuo)同5。

9. 顯(xian)(xian)色(se)：每(mei)孔先加入顯(xian)(xian)色(se)劑A50μl，再(zai)加入顯(xian)(xian)色(se)劑B50μl，輕(qing)(qing)輕(qing)(qing)震蕩混勻，37℃避光顯(xian)(xian)色(se)15分鐘.

10. 終止(zhi)：每孔(kong)加終止(zhi)液50μl，終止(zhi)反應(ying)（此時藍(lan)色立轉黃色）。

11. 測(ce)定(ding)：以空白空調(diao)零，450nm波長(chang)依序(xu)測(ce)量各孔的吸光(guang)度(du)（OD值）。 測(ce)定(ding)應在(zai)加終止液(ye)后15分鐘。