（本試劑(ji)盒僅供體(ti)外研究(jiu)使用、不用于臨床診斷(duan)!）

實驗原理：本試劑盒應用(yong)雙(shuang)抗(kang)體夾心法測(ce)定標(biao)本中(zhong)小鼠(shu)CD蛋白(損壞性關(guan)(guan)節炎)（YKL-40）水(shui)平(ping)。用(yong)純化(hua)(hua)的(de)(de)(de)小鼠(shu)CD蛋白(損壞性關(guan)(guan)節炎)（YKL-40）抗(kang)體包被(bei)微孔板，制成(cheng)固相(xiang)抗(kang)體，往包被(bei)單抗(kang)的(de)(de)(de)微孔中(zhong)依次加入(ru)YKL-40，再與HRP標(biao)記的(de)(de)(de)羊抗(kang)鼠(shu)抗(kang)體結合，形成(cheng)抗(kang)體-抗(kang)原-酶(mei)標(biao)抗(kang)體復合物，經過*洗滌后(hou)加底(di)物TMB顯色。TMB在HRP酶(mei)的(de)(de)(de)催化(hua)(hua)下轉(zhuan)化(hua)(hua)成(cheng)藍色，并在酸的(de)(de)(de)作用(yong)下轉(zhuan)化(hua)(hua)成(cheng)zui終的(de)(de)(de)黃色。顏色的(de)(de)(de)深淺和樣(yang)品中(zhong)的(de)(de)(de)YKL-40呈正相(xiang)關(guan)(guan)。用(yong)酶(mei)標(biao)儀(yi)在450nm波長下測(ce)定吸光度(du)（OD值），通過標(biao)準曲線計算樣(yang)品中(zhong)小鼠(shu)CD蛋白(損壞性關(guan)(guan)節炎)（YKL-40）濃度(du)。

試劑盒(he)組成(cheng)：

操作步(bu)驟

1. 標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)的稀(xi)釋(shi)(shi)與加樣：在(zai)(zai)(zai)(zai)酶標(biao)(biao)(biao)包被(bei)板上設標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)孔(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)，在(zai)(zai)(zai)(zai)*、第(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分別(bie)(bie)加標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)100μl，然(ran)后(hou)在(zai)(zai)(zai)(zai)*、第(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)加標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)(ye)50μl，混勻(yun)；然(ran)后(hou)從*孔(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)(ge)取100μl分別(bie)(bie)加到(dao)第(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)，再(zai)在(zai)(zai)(zai)(zai)第(di)(di)(di)(di)三、第(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)分別(bie)(bie)加標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)(ye)50μl，混勻(yun)；然(ran)后(hou)在(zai)(zai)(zai)(zai)第(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)先各(ge)(ge)取50μl棄(qi)(qi)掉，再(zai)各(ge)(ge)取50μl分別(bie)(bie)加到(dao)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)(zai)(zai)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分別(bie)(bie)加標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)(ye)50ul，混勻(yun)；混勻(yun)后(hou)從第(di)(di)(di)(di)五、第(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)(ge)取50μl分別(bie)(bie)加到(dao)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)(zai)(zai)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分別(bie)(bie)加標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)(ye)50μl，混勻(yun)后(hou)從第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分別(bie)(bie)取50μl加到(dao)第(di)(di)(di)(di)九、第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)(zai)(zai)第(di)(di)(di)(di)九第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)(kong)分別(bie)(bie)加標(biao)(biao)(biao)準(zhun)(zhun)(zhun)品(pin)稀(xi)釋(shi)(shi)液(ye)(ye)50μl，混勻(yun)后(hou)從第(di)(di)(di)(di)九第(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)(ge)取50μl棄(qi)(qi)掉。（稀(xi)釋(shi)(shi)后(hou)各(ge)(ge)孔(kong)(kong)(kong)(kong)(kong)加樣量都為(wei)50μl，濃(nong)度分別(bie)(bie)為(wei)180pg/ml，120pg/ml ，60pg/ml，30pg/ml ，15pg/ml）。
2. 加(jia)(jia)樣(yang)(yang)(yang)：分別設空白(bai)孔(kong)（空白(bai)對照孔(kong)不加(jia)(jia)樣(yang)(yang)(yang)品(pin)(pin)及(ji)酶(mei)(mei)標試劑(ji)，其余(yu)各步操作相同）、待(dai)測(ce)樣(yang)(yang)(yang)品(pin)(pin)孔(kong)。在酶(mei)(mei)標包被板上待(dai)測(ce)樣(yang)(yang)(yang)品(pin)(pin)孔(kong)中先加(jia)(jia)樣(yang)(yang)(yang)品(pin)(pin)稀釋(shi)液40μl，然后再加(jia)(jia)待(dai)測(ce)樣(yang)(yang)(yang)品(pin)(pin)10μl（樣(yang)(yang)(yang)品(pin)(pin)zui終稀釋(shi)度為5倍）。加(jia)(jia)樣(yang)(yang)(yang)將樣(yang)(yang)(yang)品(pin)(pin)加(jia)(jia)于酶(mei)(mei)標板孔(kong)底部，盡量不觸及(ji)孔(kong)壁(bi)，輕(qing)輕(qing)晃動混勻。
3. 溫育：用(yong)封板膜(mo)封板后置37℃溫育30分鐘。
4. 配液(ye)：將20倍(bei)濃縮洗滌液(ye)用蒸(zheng)餾水20倍(bei)稀釋后(hou)備用。
5. 洗滌：小(xiao)心(xin)揭掉封(feng)板膜，棄去液體，甩干，每孔加滿洗滌液，靜置(zhi)30秒后棄去，如此重(zhong)復5次，拍干。
6. 加酶(mei)：每孔加入酶(mei)標試劑50μl，空(kong)白孔除外。
7. 溫育：操作同3。
8. 洗滌：操作同(tong)5。
9. 顯(xian)色(se)(se)：每(mei)孔先加入(ru)顯(xian)色(se)(se)劑(ji)A50μl，再加入(ru)顯(xian)色(se)(se)劑(ji)B50μl，輕輕震蕩混勻(yun)，37℃避光顯色15分鐘(zhong).
10. 終(zhong)止：每孔加終(zhong)止液(ye)50μl，終(zhong)止反應（此(ci)時藍色立轉黃色）。
11. 測定(ding)(ding)：以空白空調零，450nm波長依序測量(liang)各孔(kong)的(de)吸光度(du)（OD值）。 測定(ding)(ding)應在加終止液(ye)后15分鐘以內進行。