操作步驟:

1. 標(biao)準(zhun)品(pin)(pin)的稀(xi)釋(shi)(shi)與加(jia)樣：在(zai)酶標(biao)包被板上設標(biao)準(zhun)品(pin)(pin)孔(kong)(kong)(kong)10孔(kong)(kong)(kong)，在(zai)di一(yi)、第(di)(di)(di)(di)二孔(kong)(kong)(kong)中分(fen)(fen)(fen)(fen)別加(jia)標(biao)準(zhun)品(pin)(pin)100μl，然后(hou)(hou)(hou)(hou)(hou)在(zai)di一(yi)、第(di)(di)(di)(di)二孔(kong)(kong)(kong)中加(jia)標(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)(shi)液(ye)(ye)(ye)50μl，混(hun)勻(yun)；然后(hou)(hou)(hou)(hou)(hou)從(cong)(cong)di一(yi)孔(kong)(kong)(kong)、第(di)(di)(di)(di)二孔(kong)(kong)(kong)中各(ge)(ge)取(qu)100μl分(fen)(fen)(fen)(fen)別加(jia)到第(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)，再(zai)在(zai)第(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)四孔(kong)(kong)(kong)分(fen)(fen)(fen)(fen)別加(jia)標(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)(shi)液(ye)(ye)(ye)50μl，混(hun)勻(yun)；然后(hou)(hou)(hou)(hou)(hou)在(zai)第(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)中先各(ge)(ge)取(qu)50μl棄(qi)掉(diao)，再(zai)各(ge)(ge)取(qu)50μl分(fen)(fen)(fen)(fen)別取(qu)50μl分(fen)(fen)(fen)(fen)別加(jia)到第(di)(di)(di)(di)七、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)中，再(zai)在(zai)第(di)(di)(di)(di)七、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)中分(fen)(fen)(fen)(fen)別加(jia)標(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)(shi)液(ye)(ye)(ye)50μl，混(hun)勻(yun)后(hou)(hou)(hou)(hou)(hou)從(cong)(cong)第(di)(di)(di)(di)七、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)中加(jia)到第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)中，再(zai)在(zai)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)中分(fen)(fen)(fen)(fen)別加(jia)標(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)(shi)液(ye)(ye)(ye)50ul，混(hun)勻(yun)；混(hun)勻(yun)后(hou)(hou)(hou)(hou)(hou)從(cong)(cong)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)中各(ge)(ge)分(fen)(fen)(fen)(fen)別取(qu)50μl加(jia)到第(di)(di)(di)(di)九(jiu)、第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)中，再(zai)在(zai)第(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)分(fen)(fen)(fen)(fen)別加(jia)標(biao)準(zhun)品(pin)(pin)稀(xi)釋(shi)(shi)液(ye)(ye)(ye)50μl，混(hun)勻(yun)后(hou)(hou)(hou)(hou)(hou)從(cong)(cong)第(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)中各(ge)(ge)取(qu)50μl棄(qi)掉(diao)。（稀(xi)釋(shi)(shi)后(hou)(hou)(hou)(hou)(hou)各(ge)(ge)孔(kong)(kong)(kong)加(jia)樣量都為(wei)50μl，濃度(du)分(fen)(fen)(fen)(fen)別為(wei)180 ng/L，120 ng/L ，60 ng/L，30 ng/，15 ng/L）。

2. 加樣(yang)(yang)(yang)(yang)：分別設(she)空(kong)白(bai)孔(kong)（空(kong)白(bai)對照孔(kong)不加樣(yang)(yang)(yang)(yang)品及酶(mei)標試劑，其(qi)余(yu)各步操(cao)作相同）、待測樣(yang)(yang)(yang)(yang)品孔(kong)。在(zai)酶(mei)標包(bao)被板(ban)上(shang)待測樣(yang)(yang)(yang)(yang)品孔(kong)中先加樣(yang)(yang)(yang)(yang)品稀釋(shi)液40μl，然后再加待測樣(yang)(yang)(yang)(yang)品10μl（樣(yang)(yang)(yang)(yang)品最(zui)終(zhong)稀釋(shi)度為5倍）。加樣(yang)(yang)(yang)(yang)將樣(yang)(yang)(yang)(yang)品加于酶(mei)標板(ban)孔(kong)底(di)部，盡量不觸及孔(kong)壁，輕輕晃動混勻。

3. 溫育：用封板膜封板后置37℃溫育30分鐘。

4. 配液：將30（48T的20倍(bei)）倍(bei)濃(nong)縮洗滌液用蒸(zheng)餾水(shui)30（48T的20倍(bei)）倍(bei)稀釋(shi)后備(bei)用。

5. 洗(xi)滌(di)：小心揭掉封板膜(mo)，棄(qi)(qi)去液(ye)體，甩干(gan)，每孔加滿(man)洗(xi)滌(di)液(ye)，靜置30秒后(hou)棄(qi)(qi)去，如此重復(fu)5次，拍(pai)干(gan)。

6. 加(jia)(jia)酶(mei)：每孔加(jia)(jia)入酶(mei)標(biao)試劑50μl，空白孔除外。

7. 溫(wen)育：操作同3。

8. 洗滌：操作同(tong)5。

9. 顯(xian)色(se)：每孔先加(jia)入(ru)顯(xian)色(se)劑A50μl，再(zai)加(jia)入(ru)顯(xian)色(se)劑B50μl，輕輕震蕩混勻，37℃避光顯(xian)色(se)15分鐘.

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

11. 測定：以(yi)空白空調零(ling)，450nm波長依(yi)序測量各孔(kong)的吸光度（OD值）。 測定應在(zai)加終止液后15分鐘。