小鼠組織因子(TF) ELISA試劑盒使用說明書

本(ben)試劑僅供(gong)研究使用       目(mu)的：本試劑盒用(yong)于測定小鼠血(xue)清，血(xue)漿及(ji)相關(guan)液體樣本中(zhong)組織因子(zi)(TF)的含量。

實驗原理：

    本試劑盒(he)應用(yong)雙抗體(ti)夾心法(fa)測(ce)定標本中 小鼠(shu)組織(zhi)因子(TF)水平。用(yong)純化(hua)的(de)小鼠(shu)組織(zhi)因子(TF)抗體包(bao)(bao)被(bei)微孔(kong)板，制(zhi)成固相抗(kang)(kang)(kang)體(ti)，往(wang)包(bao)(bao)被(bei)單抗(kang)(kang)(kang)的(de)(de)微孔(kong)中加(jia)入組(zu)(zu)織(zhi)因子(zi)(zi)(zi)(TF)，再與HRP標(biao)(biao)記(ji)的(de)(de)組(zu)(zu)織(zhi)因子(zi)(zi)(zi)(TF)抗(kang)(kang)(kang)體(ti)結合，形(xing)成抗(kang)(kang)(kang)體(ti)-抗(kang)(kang)(kang)原-酶(mei)標(biao)(biao)抗(kang)(kang)(kang)體(ti)復合物(wu)，經過*洗滌后(hou)加(jia)底物(wu)TMB顯色(se)。TMB在(zai)(zai)HRP酶(mei)的(de)(de)催化(hua)下轉化(hua)成藍色(se)，并在(zai)(zai)酸(suan)的(de)(de)作用下轉化(hua)成zui終(zhong)的(de)(de)黃色(se)。顏色(se)的(de)(de)深淺和(he)樣品(pin)中的(de)(de)組(zu)(zu)織(zhi)因子(zi)(zi)(zi)(TF)呈正相關。用酶(mei)標(biao)(biao)儀在(zai)(zai)450nm波長下測定(ding)吸(xi)光度（OD值），通過標(biao)(biao)準曲線計算樣品(pin)中小鼠組(zu)(zu)織(zhi)因子(zi)(zi)(zi)(TF)的(de)(de)濃(nong)度。

試劑盒組(zu)成(cheng)：

樣本處理(li)及要求：

1. 血清：室溫(wen)血液(ye)自然凝固(gu)10-20分鐘，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中(zhong)如出現沉淀，應再次離心。

2. 血漿：應(ying)根據標本的(de)要求選(xuan)擇EDTA或檸檬(meng)酸鈉作為抗凝劑(ji)，混合10-20分鐘(zhong)后，離(li)心20分鐘(zhong)左右（2000-3000轉/分）。仔細收集上清(qing)，保(bao)存過程中如(ru)有沉淀形(xing)成，應(ying)該再次離(li)心。

3. 尿液(ye)：用(yong)無菌管收集，離心20分(fen)鐘(zhong)左右(you)（2000-3000轉/分(fen)）。仔(zi)細收集上(shang)清，保存過(guo)程(cheng)中如有沉淀形成，應(ying)再(zai)次(ci)離心。胸(xiong)腹水、腦脊液(ye)參(can)照(zhao)實行。

4. 細(xi)胞(bao)(bao)培養上(shang)清：檢測分泌性的成(cheng)份時，用無菌管收(shou)集(ji)。離(li)心(xin)20分鐘左(zuo)右（2000-3000轉(zhuan)(zhuan)/分）。仔(zi)細(xi)收(shou)集(ji)上(shang)清。檢測細(xi)胞(bao)(bao)內(nei)的成(cheng)份時，用PBS（PH7.2-7.4）稀釋細(xi)胞(bao)(bao)懸液，細(xi)胞(bao)(bao)濃度達到100萬/ml左(zuo)右。通過反復凍融，以使細(xi)胞(bao)(bao)破壞(huai)并放出細(xi)胞(bao)(bao)內(nei)成(cheng)份。離(li)心(xin)20分鐘左(zuo)右（2000-3000轉(zhuan)(zhuan)/分）。仔(zi)細(xi)收(shou)集(ji)上(shang)清。保存過程中(zhong)如有(you)沉淀形成(cheng)，應(ying)再次離(li)心(xin)。

5. 組織標本：切割標本后(hou)，稱取(qu)重(zhong)量。加入(ru)(ru)一(yi)定量的(de)PBS，PH7.4。用(yong)(yong)液氮(dan)迅(xun)速冷凍保(bao)存備用(yong)(yong)。標本融化后(hou)仍(reng)然保(bao)持2-8℃的(de)溫度。加入(ru)(ru)一(yi)定量的(de)PBS（PH7.4），用(yong)(yong)手工或勻(yun)漿(jiang)器將(jiang)標本勻(yun)漿(jiang)充(chong)分。離心20分鐘左(zuo)右（2000-3000轉/分）。仔細(xi)收集上(shang)清。分裝后(hou)一(yi)份(fen)待(dai)檢測(ce)，其余冷凍備用(yong)(yong)。

6. 標本(ben)采集后盡(jin)早進(jin)行提取(qu)(qu)，提取(qu)(qu)按相關文獻(xian)進(jin)行，提取(qu)(qu)后應(ying)盡(jin)快進(jin)行實驗。若(ruo)不(bu)能(neng)馬上進(jin)行試驗，可(ke)將標本(ben)放于-20℃保存(cun)，但應避免反復凍融.

7. 不能檢測(ce)含NaN3的樣品，因NaN3抑制(zhi)辣根(gen)過氧化物酶的（HRP）活性。

操(cao)作步驟：

1. 標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)的稀釋(shi)與加(jia)樣：在(zai)酶標(biao)包(bao)被板上設(she)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)，在(zai)*、第(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)加(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)100μl，然后在(zai)*、第(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)加(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀釋(shi)液(ye)(ye)50μl，混勻(yun)；然后從*孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)100μl分(fen)(fen)別(bie)(bie)(bie)加(jia)到第(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)，再(zai)在(zai)第(di)(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)(bie)加(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀釋(shi)液(ye)(ye)50μl，混勻(yun)；然后在(zai)第(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)先(xian)各(ge)取(qu)(qu)50μl棄掉(diao)，再(zai)各(ge)取(qu)(qu)50μl分(fen)(fen)別(bie)(bie)(bie)加(jia)到第(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再(zai)在(zai)第(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)加(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀釋(shi)液(ye)(ye)50ul，混勻(yun)；混勻(yun)后從第(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)50μl分(fen)(fen)別(bie)(bie)(bie)加(jia)到第(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再(zai)在(zai)第(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)加(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀釋(shi)液(ye)(ye)50μl，混勻(yun)后從第(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)取(qu)(qu)50μl加(jia)到第(di)(di)(di)(di)(di)九(jiu)、第(di)(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再(zai)在(zai)第(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)(bie)加(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀釋(shi)液(ye)(ye)50μl，混勻(yun)后從第(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)50μl棄掉(diao)。（稀釋(shi)后各(ge)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)加(jia)樣量都為(wei)50μl，濃(nong)度分(fen)(fen)別(bie)(bie)(bie)為(wei)120 ng/L ，80 ng/L，40 ng/L，20 ng/L，10 ng/L）。
2. 加(jia)樣：分別設空白孔(kong)（空白對照(zhao)孔(kong)不(bu)加(jia)樣品(pin)(pin)及(ji)酶標試劑，其余(yu)各步操(cao)作相(xiang)同）、待(dai)測樣品(pin)(pin)孔(kong)。在酶標包被板(ban)上待(dai)測樣品(pin)(pin)孔(kong)中先加(jia)樣品(pin)(pin)稀(xi)釋液(ye)40μl，然后(hou)再加(jia)待(dai)測樣品(pin)(pin)10μl（樣品(pin)(pin)zui終稀(xi)釋度為5倍）。加(jia)樣將樣品(pin)(pin)加(jia)于酶標板(ban)孔(kong)底部，盡量不(bu)觸(chu)及(ji)孔(kong)壁，輕(qing)輕(qing)晃(huang)動混(hun)勻。
3. 溫育：用封板(ban)膜封板(ban)后置(zhi)37℃溫育30分鐘。
4. 配液：將30（48T的20倍(bei)(bei)）倍(bei)(bei)濃縮(suo)洗滌液用蒸餾水30（48T的20倍(bei)(bei)）倍(bei)(bei)稀(xi)釋(shi)后備(bei)用。
5. 洗滌(di)：小心揭(jie)掉封板膜(mo)，棄去(qu)(qu)液體(ti)，甩干，每孔加滿洗滌(di)液，靜置30秒后棄去(qu)(qu)，如此重(zhong)復5次(ci)，拍干。
6. 加酶：每孔(kong)加入酶標試劑50μl，空(kong)白孔(kong)除(chu)外。
7. 溫育：操作(zuo)同3。
8. 洗滌(di)：操作同5。
9. 顯(xian)色：每孔(kong)先加入(ru)顯(xian)色劑A50μl，再加入(ru)顯(xian)色劑B50μl，輕(qing)輕(qing)震蕩混勻，37℃避光(guang)顯色(se)15分鐘.
10. 終(zhong)止：每孔加終(zhong)止液(ye)50μl，終(zhong)止反(fan)應（此時(shi)藍色(se)立轉(zhuan)黃色(se)）。
11. 測定(ding)：以(yi)空白空調零，450nm波長依序測量各(ge)孔的(de)吸光(guang)度（OD值）。 測定(ding)應在加(jia)終止液后15分鐘以(yi)內進行。

注(zhu)意事項：

1. 試劑盒從冷藏環境中取出應(ying)在室溫(wen)平衡15-30分(fen)鐘后(hou)方可使(shi)用，酶標包被(bei)板開封后(hou)如未用完(wan)，板條應(ying)裝入密(mi)封袋中保存(cun)。
2. 濃洗滌液可能會(hui)有結(jie)晶析出，稀(xi)釋時可在水浴(yu)中加溫助溶，洗滌時不影(ying)響結(jie)果。
3. 各步(bu)加(jia)樣(yang)均應使用加(jia)樣(yang)器，并(bing)經(jing)常校對(dui)其準確性，以避免試驗誤差(cha)。一次(ci)加(jia)樣(yang)時間(jian)控制在5分鐘(zhong)內，如標本數量多，推薦使用排槍加(jia)樣(yang)。
4. 請每次測(ce)(ce)定(ding)的同時做(zuo)標準(zhun)曲線，做(zuo)復孔。如(ru)標本(ben)中待測(ce)(ce)物質含量過高（樣(yang)(yang)本(ben)OD值大于(yu)標準(zhun)品(pin)孔*孔的OD值），請先用樣(yang)(yang)品(pin)稀釋液稀釋一(yi)定(ding)倍數（n倍）后再(zai)測(ce)(ce)定(ding)，計算(suan)時請zui后乘以總稀釋倍數（×n×5）。
5. 封板膜(mo)只限一(yi)次性(xing)使(shi)用，以避免交叉(cha)污染(ran)。
6. 底物請(qing)避光保存(cun)。
7. 嚴格(ge)按照(zhao)說明書(shu)的操作進行，試驗結果判(pan)定必須以酶(mei)標儀讀數為準.
8. 所有樣品，洗滌液和各種(zhong)廢棄物都應按傳(chuan)染物處理(li)。
9. 本試劑不同批號組(zu)分不得混用。

10. 如與英文說(shuo)明書(shu)有異，以英文說(shuo)明書(shu)為準。

計算：

以(yi)標(biao)(biao)準物(wu)的(de)濃度(du)為橫(heng)坐(zuo)(zuo)標(biao)(biao)，OD值(zhi)為縱坐(zuo)(zuo)標(biao)(biao)，在坐(zuo)(zuo)標(biao)(biao)紙上(shang)繪出標(biao)(biao)準曲線，根據樣(yang)品(pin)的(de)OD值(zhi)由(you)標(biao)(biao)準曲線查出相應的(de)濃度(du)；再乘(cheng)以(yi)稀釋(shi)倍數；或用(yong)標(biao)(biao)準物(wu)的(de)濃度(du)與OD值(zhi)計(ji)算出標(biao)(biao)準曲線的(de)直線回歸方程式(shi)，將樣(yang)品(pin)的(de)OD值(zhi)代入方程式(shi)，計(ji)算出樣(yang)品(pin)濃度(du)，再乘(cheng)以(yi)稀釋(shi)倍數，即為樣(yang)品(pin)的(de)實(shi)際(ji)濃度(du)。                 

 試劑盒性(xing)能：

1.樣品線(xian)性回歸與預期(qi)濃度(du)相關系數R值為0.92以上。

2.批(pi)內與批(pi)見應分(fen)別小(xiao)于(yu)9%和15%。

檢(jian)測范圍(wei)： 3ng/L-120ng/L 

靈敏度(du)：大于(yu)92%  

特異性：大于95%                ;                   

保存條件及有效期：

1.試(shi)劑盒保(bao)存：2-8℃。

2．有效期(qi)：6個月(yue)