操作步驟:

1. 標(biao)準(zhun)品的稀(xi)(xi)(xi)釋(shi)與(yu)加(jia)(jia)(jia)樣(yang)(yang)：在酶標(biao)包被板上設標(biao)準(zhun)品孔(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)，在di一(yi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)二(er)(er)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)準(zhun)品100μl，然(ran)后(hou)在di一(yi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)二(er)(er)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)(jia)標(biao)準(zhun)品稀(xi)(xi)(xi)釋(shi)液(ye)50μl，混(hun)勻；然(ran)后(hou)從di一(yi)孔(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)二(er)(er)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各取100μl分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)，再(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)三、第(di)(di)(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)準(zhun)品稀(xi)(xi)(xi)釋(shi)液(ye)50μl，混(hun)勻；然(ran)后(hou)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)先各取50μl棄(qi)掉，再(zai)各取50μl分(fen)(fen)(fen)(fen)別(bie)取50μl分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)準(zhun)品稀(xi)(xi)(xi)釋(shi)液(ye)50μl，混(hun)勻后(hou)從第(di)(di)(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)準(zhun)品稀(xi)(xi)(xi)釋(shi)液(ye)50ul，混(hun)勻；混(hun)勻后(hou)從第(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各分(fen)(fen)(fen)(fen)別(bie)取50μl加(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)九、第(di)(di)(di)(di)(di)(di)(di)(di)(di)十(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)(di)(di)十(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)標(biao)準(zhun)品稀(xi)(xi)(xi)釋(shi)液(ye)50μl，混(hun)勻后(hou)從第(di)(di)(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)(di)(di)十(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各取50μl棄(qi)掉。（稀(xi)(xi)(xi)釋(shi)后(hou)各孔(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)樣(yang)(yang)量(liang)都為(wei)50μl，濃度分(fen)(fen)(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)：分別(bie)設空白孔(kong)(kong)（空白對照(zhao)孔(kong)(kong)不加(jia)樣(yang)品及酶(mei)標(biao)試劑，其余各步操作相同）、待測(ce)(ce)樣(yang)品孔(kong)(kong)。在酶(mei)標(biao)包被板上(shang)待測(ce)(ce)樣(yang)品孔(kong)(kong)中先加(jia)樣(yang)品稀(xi)釋液40μl，然后(hou)再加(jia)待測(ce)(ce)樣(yang)品10μl（樣(yang)品最終稀(xi)釋度(du)為(wei)5倍）。加(jia)樣(yang)將樣(yang)品加(jia)于酶(mei)標(biao)板孔(kong)(kong)底部，盡量不觸及孔(kong)(kong)壁(bi)，輕輕晃動混(hun)勻。

3. 溫(wen)育(yu)：用封(feng)板(ban)(ban)膜封(feng)板(ban)(ban)后置37℃溫(wen)育(yu)30分鐘。

4. 配液：將30（48T的20倍）倍濃(nong)縮洗滌(di)液用蒸餾水30（48T的20倍）倍稀(xi)釋后備用。

5. 洗(xi)滌：小心揭(jie)掉封(feng)板膜，棄去液體，甩(shuai)干(gan)，每孔加滿(man)洗(xi)滌液，靜置30秒(miao)后棄去，如此重復5次(ci)，拍干(gan)。

6. 加酶：每孔加入酶標試劑50μl，空白(bai)孔除(chu)外。

7. 溫育：操作同3。

8. 洗滌：操作同5。

9. 顯色：每孔先(xian)加(jia)入顯色劑A50μl，再加(jia)入顯色劑B50μl，輕(qing)輕(qing)震蕩混勻，37℃避光顯色15分鐘.

10. 終(zhong)止：每孔(kong)加終(zhong)止液(ye)50μl，終(zhong)止反應（此時藍色立轉黃色）。

11. 測定(ding)：以空(kong)白空(kong)調零(ling)，450nm波長依序測量各孔(kong)的吸(xi)光度(du)（OD值）。 測定(ding)應在加(jia)終止液(ye)后15分鐘。

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