操作(zuo)步驟(zou)：

1. 標(biao)(biao)準(zhun)(zhun)品的稀(xi)(xi)釋(shi)與(yu)加(jia)(jia)(jia)樣：在(zai)酶(mei)標(biao)(biao)包被板上設標(biao)(biao)準(zhun)(zhun)品孔(kong)(kong)(kong)10孔(kong)(kong)(kong)，在(zai)di一、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品100μl，然后在(zai)di一、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品稀(xi)(xi)釋(shi)液(ye)(ye)50μl，混勻(yun)；然后從di一孔(kong)(kong)(kong)、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)100μl分(fen)(fen)別(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)，再在(zai)第(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)四孔(kong)(kong)(kong)分(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品稀(xi)(xi)釋(shi)液(ye)(ye)50μl，混勻(yun)；然后在(zai)第(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)先各(ge)取(qu)50μl棄掉，再各(ge)取(qu)50μl分(fen)(fen)別(bie)取(qu)50μl分(fen)(fen)別(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再在(zai)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品稀(xi)(xi)釋(shi)液(ye)(ye)50μl，混勻(yun)后從第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再在(zai)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品稀(xi)(xi)釋(shi)液(ye)(ye)50ul，混勻(yun)；混勻(yun)后從第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)分(fen)(fen)別(bie)取(qu)50μl加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)九、第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再在(zai)第(di)(di)(di)(di)九第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)分(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品稀(xi)(xi)釋(shi)液(ye)(ye)50μl，混勻(yun)后從第(di)(di)(di)(di)九第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)50μl棄掉。（稀(xi)(xi)釋(shi)后各(ge)孔(kong)(kong)(kong)加(jia)(jia)(jia)樣量都為(wei)50μl，濃度分(fen)(fen)別(bie)為(wei)180 ng/L，120 ng/L ，60 ng/L，30 ng/，15 ng/L）。

2. 加(jia)樣(yang)(yang)(yang)：分別設(she)空白(bai)孔(kong)（空白(bai)對照孔(kong)不加(jia)樣(yang)(yang)(yang)品(pin)(pin)及酶標(biao)試(shi)劑，其余各(ge)步操作相(xiang)同）、待(dai)測(ce)樣(yang)(yang)(yang)品(pin)(pin)孔(kong)。在酶標(biao)包被板(ban)上待(dai)測(ce)樣(yang)(yang)(yang)品(pin)(pin)孔(kong)中先(xian)加(jia)樣(yang)(yang)(yang)品(pin)(pin)稀釋液40μl，然后再加(jia)待(dai)測(ce)樣(yang)(yang)(yang)品(pin)(pin)10μl（樣(yang)(yang)(yang)品(pin)(pin)最終稀釋度為(wei)5倍）。加(jia)樣(yang)(yang)(yang)將樣(yang)(yang)(yang)品(pin)(pin)加(jia)于酶標(biao)板(ban)孔(kong)底部，盡量不觸及孔(kong)壁，輕輕晃動混(hun)勻。

3. 溫育：用封板膜(mo)封板后置37℃溫育30分鐘。

4. 配液(ye)：將30（48T的20倍）倍濃縮洗滌(di)液(ye)用(yong)蒸餾水30（48T的20倍）倍稀釋后備用(yong)。

5. 洗滌：小心(xin)揭掉封板(ban)膜，棄(qi)去液體，甩干(gan)，每孔加滿洗滌液，靜置30秒后(hou)棄(qi)去，如此(ci)重復5次，拍干(gan)。

6. 加酶(mei)：每孔加入(ru)酶(mei)標(biao)試劑50μl，空白孔除外。

7. 溫育(yu)：操作同3。

8. 洗(xi)滌：操作同5。

9. 顯色(se)：每孔先加入(ru)顯色(se)劑(ji)A50μl，再加入(ru)顯色(se)劑(ji)B50μl，輕(qing)輕(qing)震(zhen)蕩混勻，37℃避光顯色(se)15分鐘.

10. 終止(zhi)：每孔加終止(zhi)液50μl，終止(zhi)反(fan)應(ying)（此時藍色(se)立轉黃色(se)）。

11. 測(ce)定：以空(kong)白空(kong)調(diao)零，450nm波長依(yi)序測(ce)量各孔的吸光度（OD值）。 測(ce)定應在加終止液后15分鐘。