大鼠前列(lie)腺(xian)素Ｅ２（PGE2）ELISA試(shi)劑盒說明書

本試劑盒僅供研究使用。

藥品名稱(cheng)：

通用名：大鼠前列腺素Ｅ２（PGE2）酶聯免疫分析試劑盒

使(shi)用目的：

本試劑盒用于測定大鼠血清，血漿及相關組織樣本中前(qian)列腺素Ｅ２（PGE2）含量。

實(shi)驗原理

本試劑盒應用雙抗體夾心法測定標本中大鼠前列腺素Ｅ２（PGE2）水平。用純化的大鼠前列腺素Ｅ２（PGE2）抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入前列腺素Ｅ２（PGE2），再與HRP標記的羊抗鼠抗體結合，形成抗體-抗原-酶標抗體復合物，經過*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸的作用下轉化成zui終的黃色。顏色的深淺和樣品中的前列腺素Ｅ２呈正相關。用酶標儀在450nm波長下測定吸光度（OD值），通過標準曲線計算樣品中大鼠前列腺素(su)Ｅ２（PGE2）濃度。

試劑盒(he)組(zu)成

標本要求(qiu)

1．不能檢測含NaN3的(de)樣(yang)品，因NaN3抑(yi)制辣根過氧(yang)化物酶的(de)（HRP）活性。

2．標本(ben)采集后(hou)盡(jin)早進(jin)(jin)行(xing)提(ti)(ti)取，提(ti)(ti)取按相關文獻進(jin)(jin)行(xing)，提(ti)(ti)取后(hou)應盡(jin)快進(jin)(jin)行(xing)實驗。若(ruo)不能馬(ma)上進(jin)(jin)行(xing)試驗，可將標本(ben)放于-20℃保存，但應避免反(fan)復凍融

操作步驟

1. 標(biao)(biao)(biao)準(zhun)品(pin)的稀釋與加(jia)(jia)(jia)(jia)樣(yang)：在酶標(biao)(biao)(biao)包被板上設標(biao)(biao)(biao)準(zhun)品(pin)孔(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)，在*、第(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)100μl，然后(hou)在*、第(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)稀釋液(ye)(ye)50μl，混勻；然后(hou)從(cong)*孔(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)(ge)取100μl分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)，再(zai)在第(di)(di)(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)稀釋液(ye)(ye)50μl，混勻；然后(hou)在第(di)(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)先各(ge)(ge)取50μl棄掉(diao)，再(zai)各(ge)(ge)取50μl分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在第(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)稀釋液(ye)(ye)50ul，混勻；混勻后(hou)從(cong)第(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)(ge)取50μl分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在第(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)稀釋液(ye)(ye)50μl，混勻后(hou)從(cong)第(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)(fen)(fen)別(bie)取50μl加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)九、第(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在第(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)分(fen)(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)稀釋液(ye)(ye)50μl，混勻后(hou)從(cong)第(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)(ge)取50μl棄掉(diao)。（稀釋后(hou)各(ge)(ge)孔(kong)(kong)(kong)(kong)加(jia)(jia)(jia)(jia)樣(yang)量都(dou)為50μl，濃(nong)度分(fen)(fen)(fen)(fen)(fen)別(bie)為360ng/L，240ng/L ，120ng/L，60ng/L，30ng/L）。
2. 加(jia)(jia)樣(yang)(yang)：分別設空(kong)白孔(kong)（空(kong)白對照(zhao)孔(kong)不(bu)加(jia)(jia)樣(yang)(yang)品(pin)及酶標(biao)試(shi)劑，其余各步操作相同）、待測(ce)樣(yang)(yang)品(pin)孔(kong)。在(zai)酶標(biao)包被板(ban)上(shang)待測(ce)樣(yang)(yang)品(pin)孔(kong)中(zhong)先加(jia)(jia)樣(yang)(yang)品(pin)稀釋液40μl，然后再加(jia)(jia)待測(ce)樣(yang)(yang)品(pin)10μl（樣(yang)(yang)品(pin)zui終(zhong)稀釋度為5倍）。加(jia)(jia)樣(yang)(yang)將樣(yang)(yang)品(pin)加(jia)(jia)于酶標(biao)板(ban)孔(kong)底(di)部，盡量不(bu)觸及孔(kong)壁，輕輕晃動(dong)混勻。
3. 溫(wen)育：用封板(ban)膜封板(ban)后置37℃溫(wen)育30分鐘。  
4. 配液：將20倍(bei)濃縮洗滌液用(yong)蒸餾水20倍(bei)稀釋后(hou)備用(yong)
5. 洗(xi)滌(di)(di)：小心(xin)揭掉(diao)封板膜，棄(qi)去液體，甩干(gan)，每孔加(jia)滿洗(xi)滌(di)(di)液，靜置30秒(miao)后棄(qi)去，如(ru)此(ci)重復(fu)5次，拍(pai)干(gan)。
6. 加酶(mei)：每孔加入(ru)酶(mei)標試(shi)劑50μl，空白孔除外。
7. 溫育：操作同3。
8. 洗(xi)滌(di)：操(cao)作同(tong)5。
9. 顯(xian)色：每孔(kong)先加入(ru)顯(xian)色劑(ji)A50μl，再加入(ru)顯(xian)色劑(ji)B50μl，輕輕震蕩混勻，37℃避光顯(xian)色15分鐘(zhong).
10. 終(zhong)(zhong)止(zhi)：每孔(kong)加終(zhong)(zhong)止(zhi)液50μl，終(zhong)(zhong)止(zhi)反(fan)應（此時藍色立轉(zhuan)黃色）。
11. 測(ce)定(ding)：以空(kong)白空(kong)調零，450nm波(bo)長依序測(ce)量各孔(kong)的吸(xi)光度（OD值）。 測(ce)定(ding)應在加終止液后15分(fen)鐘以內進行。

計(ji)算

  以(yi)標(biao)(biao)(biao)(biao)準(zhun)(zhun)物(wu)的(de)濃(nong)度為橫坐標(biao)(biao)(biao)(biao)，OD值為縱坐標(biao)(biao)(biao)(biao)，在坐標(biao)(biao)(biao)(biao)紙(zhi)上繪出標(biao)(biao)(biao)(biao)準(zhun)(zhun)曲線，根據(ju)樣品的(de)OD值由標(biao)(biao)(biao)(biao)準(zhun)(zhun)曲線查出相應的(de)濃(nong)度；再(zai)乘以(yi)稀釋倍數；或用標(biao)(biao)(biao)(biao)準(zhun)(zhun)物(wu)的(de)濃(nong)度與OD值計算出標(biao)(biao)(biao)(biao)準(zhun)(zhun)曲線的(de)直線回歸方程(cheng)式，將樣品的(de)OD值代入方程(cheng)式，計算出樣品濃(nong)度，再(zai)乘以(yi)稀釋倍數，即為樣品的(de)實際濃(nong)度。

注(zhu)意(yi)事項

1．試(shi)劑盒從冷藏環境中(zhong)取出應在室溫平(ping)衡15-30分鐘后方可使用，酶標包被板開封(feng)(feng)后如未(wei)用完(wan)，板條應裝入密封(feng)(feng)袋中(zhong)保(bao)存。

2．濃洗滌液(ye)可能會有結晶(jing)析出，稀釋時可在(zai)水(shui)浴中加(jia)溫助溶，洗滌時不影響結果。

3．各步加(jia)(jia)樣(yang)(yang)(yang)均應使用加(jia)(jia)樣(yang)(yang)(yang)器，并經(jing)常校(xiao)對其準確性，以避(bi)免試驗誤差。一次加(jia)(jia)樣(yang)(yang)(yang)時間控制在5分(fen)鐘內，如標本數量多，推薦使用排槍加(jia)(jia)樣(yang)(yang)(yang)。

1. 請每次測(ce)定(ding)的同時(shi)做(zuo)標準曲線，做(zuo)復孔。如標本中待測(ce)物(wu)質含量過高（樣本OD值大于標準品(pin)孔*孔的OD值），請先用樣品(pin)稀釋液(ye)稀釋一定(ding)倍數(shu)（n倍）后再測(ce)定(ding)，計算(suan)時(shi)請zui后乘以(yi)總稀釋倍數(shu)（×n×5）。
2. 封板膜只限一次性使用，以避(bi)免交(jiao)叉污染(ran)。

6．底物請避光保存。

7．嚴(yan)格按照說明書的操作進(jin)行，試驗(yan)結果判定(ding)必須以酶標儀讀數為準(zhun).

8．所有樣品，洗滌液和各種廢棄物(wu)都應(ying)按(an)傳染物(wu)處理。

9．本試(shi)劑不(bu)同批號組(zu)分不(bu)得(de)混(hun)用。

10. 如與英文(wen)說明書有異，以英文(wen)說明書為準。

檢(jian)測范(fan)圍：

0.5 pg/ml-100 pg/ml

靈(ling)敏度：

0.25 pg/ml

發表外文文獻二篇

規格：

96人(ren)份/盒

保存(cun)條(tiao)件及有(you)效期

1．試劑(ji)盒保存(cun)：；2-8℃。

2．有效(xiao)期：6個月