大鼠睪(gao)酮(T)酶聯免疫分析（ELISA）

試劑盒使用說明書

本(ben)試劑僅供研究使(shi)用      

 目的：本(ben)(ben)試(shi)劑盒用于測定(ding)大鼠血(xue)清，血(xue)漿及相關液體樣本(ben)(ben)中(zhong)睪酮(T)含量(liang)。

實驗(yan)原理：

    本試(shi)劑盒應(ying)用雙(shuang)抗(kang)(kang)(kang)體(ti)夾(jia)心法測(ce)定標(biao)(biao)(biao)本中(zhong)大(da)(da)鼠睪(gao)酮(T)水平。用純化(hua)的(de)大(da)(da)鼠睪(gao)酮(T)抗(kang)(kang)(kang)體(ti)包被(bei)微(wei)孔(kong)板，制成固相抗(kang)(kang)(kang)體(ti)，往(wang)包被(bei)單抗(kang)(kang)(kang)的(de)微(wei)孔(kong)中(zhong)依次加入睪(gao)酮(T)，再與(yu)HRP標(biao)(biao)(biao)記的(de)睪(gao)酮(T)抗(kang)(kang)(kang)體(ti)結(jie)合，形成抗(kang)(kang)(kang)體(ti)-抗(kang)(kang)(kang)原(yuan)-酶標(biao)(biao)(biao)抗(kang)(kang)(kang)體(ti)復(fu)合物，經過*洗滌后加底(di)物TMB顯色。TMB在HRP酶的(de)催化(hua)下(xia)轉化(hua)成藍色，并在酸的(de)作用下(xia)轉化(hua)成zui終的(de)黃色。顏色的(de)深淺和(he)樣品中(zhong)的(de)睪(gao)酮(T)呈正(zheng)相關。用酶標(biao)(biao)(biao)儀(yi)在450nm波長下(xia)測(ce)定吸光(guang)度(du)（OD值），通過標(biao)(biao)(biao)準曲線計算樣品中(zhong)大(da)(da)鼠睪(gao)酮(T)濃度(du)。

試劑盒組(zu)成：

樣本(ben)處(chu)理及要求：

1.血(xue)清：室溫(wen)血(xue)液自然凝固(gu)10-20分(fen)鐘(zhong)(zhong)，離(li)(li)心20分(fen)鐘(zhong)(zhong)左右（2000-3000轉/分(fen)）。仔細收(shou)集上清，保存過程(cheng)中如出現沉淀(dian)，應再次(ci)離(li)(li)心。

2.血漿：應根據(ju)標本(ben)的(de)要求(qiu)選擇EDTA、者檸檬酸鈉或肝素(su)作(zuo)為抗凝劑，混合10-20分(fen)鐘(zhong)后，離(li)心20分(fen)鐘(zhong)左右（2000-3000轉/分(fen)）。仔細收集(ji)上(shang)清，保存過(guo)程中如有沉淀形成，應該再(zai)次離(li)心。

3.尿液(ye)：用無菌(jun)管(guan)收(shou)集，離心20分(fen)鐘左右（2000-3000轉/分(fen)）。仔細收(shou)集上清，保存過程中(zhong)如有沉淀形成，應再次離心。胸腹水、腦脊液(ye)參照實(shi)行。

4.細(xi)(xi)胞培養上清(qing)：檢測(ce)分(fen)(fen)泌性的成(cheng)份(fen)時，用無菌管(guan)收集。離心(xin)(xin)(xin)20分(fen)(fen)鐘左右(you)（2000-3000轉(zhuan)(zhuan)/分(fen)(fen)）。仔(zi)(zi)細(xi)(xi)收集上清(qing)。檢測(ce)細(xi)(xi)胞內(nei)的成(cheng)份(fen)時，用PBS（PH7.2-7.4）稀釋細(xi)(xi)胞懸液，細(xi)(xi)胞濃度達到(dao)100萬/ml左右(you)。通過(guo)反復凍融，以使細(xi)(xi)胞破壞并放出細(xi)(xi)胞內(nei)成(cheng)份(fen)。離心(xin)(xin)(xin)20分(fen)(fen)鐘左右(you)（2000-3000轉(zhuan)(zhuan)/分(fen)(fen)）。仔(zi)(zi)細(xi)(xi)收集上清(qing)。保存(cun)過(guo)程中(zhong)如有沉淀形成(cheng)，應再次離心(xin)(xin)(xin)。

5.組織標本(ben)(ben)(ben)：切割標本(ben)(ben)(ben)后(hou)(hou)，稱取重量。加入一(yi)(yi)(yi)定量的PBS，PH7.4。用液氮迅速冷(leng)凍保存備用。標本(ben)(ben)(ben)融化后(hou)(hou)仍然保持2-8℃的溫度。加入一(yi)(yi)(yi)定量的PBS（PH7.4），用手工或(huo)勻(yun)漿器將標本(ben)(ben)(ben)勻(yun)漿充(chong)分(fen)。離(li)心20分(fen)鐘(zhong)左右（2000-3000轉/分(fen)）。仔細(xi)收集上(shang)清(qing)。分(fen)裝(zhuang)后(hou)(hou)一(yi)(yi)(yi)份(fen)待檢測，其余冷(leng)凍備用。

操作步驟

1. 標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)的稀釋(shi)與加(jia)(jia)樣(yang)：在酶(mei)標(biao)(biao)(biao)包被板上設(she)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)，在*、第(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)100μl，然后(hou)(hou)在*、第(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)加(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)稀釋(shi)液(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)(yun)(yun)；然后(hou)(hou)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)別(bie)(bie)加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)，再在第(di)(di)(di)(di)(di)三、第(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)稀釋(shi)液(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)(yun)(yun)；然后(hou)(hou)在第(di)(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)先(xian)各(ge)取(qu)(qu)50μl棄(qi)掉(diao)，再各(ge)取(qu)(qu)50μl分(fen)別(bie)(bie)加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)五(wu)(wu)、第(di)(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再在第(di)(di)(di)(di)(di)五(wu)(wu)、第(di)(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)稀釋(shi)液(ye)50ul，混(hun)(hun)(hun)勻(yun)(yun)(yun)(yun)；混(hun)(hun)(hun)勻(yun)(yun)(yun)(yun)后(hou)(hou)從第(di)(di)(di)(di)(di)五(wu)(wu)、第(di)(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)50μl分(fen)別(bie)(bie)加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再在第(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)稀釋(shi)液(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)(yun)(yun)后(hou)(hou)從第(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)別(bie)(bie)取(qu)(qu)50μl加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)九、第(di)(di)(di)(di)(di)十(shi)(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再在第(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)十(shi)(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)別(bie)(bie)加(jia)(jia)標(biao)(biao)(biao)準(zhun)品(pin)(pin)(pin)(pin)稀釋(shi)液(ye)50μl，混(hun)(hun)(hun)勻(yun)(yun)(yun)(yun)后(hou)(hou)從第(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)十(shi)(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取(qu)(qu)50μl棄(qi)掉(diao)。（稀釋(shi)后(hou)(hou)各(ge)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)樣(yang)量都為(wei)50μl，濃(nong)度分(fen)別(bie)(bie)為(wei)120 nmol/L，80nmol/L ，40 nmol/L，20 nmol/L，10 nmol/L）。
2. 加(jia)(jia)樣(yang)：分別設空白(bai)孔(kong)(kong)(kong)（空白(bai)對照孔(kong)(kong)(kong)不(bu)(bu)加(jia)(jia)樣(yang)品及(ji)酶標(biao)試(shi)劑，其(qi)余各(ge)步操作(zuo)相同）、待(dai)測樣(yang)品孔(kong)(kong)(kong)。在酶標(biao)包被板(ban)上(shang)待(dai)測樣(yang)品孔(kong)(kong)(kong)中先加(jia)(jia)樣(yang)品稀釋液40μl，然(ran)后(hou)再加(jia)(jia)待(dai)測樣(yang)品10μl（樣(yang)品zui終稀釋度為5倍）。加(jia)(jia)樣(yang)將樣(yang)品加(jia)(jia)于酶標(biao)板(ban)孔(kong)(kong)(kong)底部，盡量不(bu)(bu)觸及(ji)孔(kong)(kong)(kong)壁，輕輕晃動混勻。
3. 溫育：用封(feng)(feng)板膜封(feng)(feng)板后置(zhi)37℃溫育30分(fen)鐘。
4. 配液：將30（48T的20倍）倍濃縮(suo)洗滌液用蒸餾水(shui)30（48T的20倍）倍稀釋后備用。
5. 洗(xi)滌(di)：小心揭(jie)掉封板膜(mo)，棄去(qu)液(ye)體(ti)，甩干，每孔(kong)加(jia)滿洗(xi)滌(di)液(ye)，靜置30秒后棄去(qu)，如(ru)此重復5次，拍干。
6. 加酶：每孔(kong)加入酶標試劑50μl，空白(bai)孔(kong)除外。
7. 溫育：操作同3。
8. 洗(xi)滌：操作同5。
9. 顯色(se)：每孔先加入(ru)顯色(se)劑(ji)A50μl，再加入(ru)顯色(se)劑(ji)B50μl，輕(qing)輕(qing)震蕩混勻，37℃避(bi)光顯色15分鐘.
10. 終(zhong)止：每(mei)孔加終(zhong)止液(ye)50μl，終(zhong)止反應（此(ci)時藍(lan)色立轉黃色）。
11. 測(ce)定：以(yi)空(kong)白空(kong)調(diao)零，450nm波長依序測(ce)量各(ge)孔(kong)的(de)吸光度（OD值）。 測(ce)定應在(zai)加(jia)終止液(ye)后(hou)15分鐘(zhong)以(yi)內進行。

注意事項：

1. 試劑盒從冷藏環境中取出應(ying)在室溫(wen)平衡15-30分(fen)鐘后方可使用，酶(mei)標(biao)包被板(ban)開封后如未用完，板(ban)條應(ying)裝入密封袋中保存(cun)。
2. 濃洗滌液可(ke)能會有結晶析出，稀釋時可(ke)在水浴中加溫助溶，洗滌時不影響結果。
3. 各步(bu)加(jia)(jia)樣(yang)均(jun)應使(shi)用加(jia)(jia)樣(yang)器，并經(jing)常(chang)校對其準確性，以避免(mian)試驗誤差。一(yi)次(ci)加(jia)(jia)樣(yang)時間(jian)控(kong)制(zhi)在(zai)5分鐘內，如標本數量多，推薦使(shi)用排槍(qiang)加(jia)(jia)樣(yang)。
4. 請(qing)每次測定(ding)的(de)同(tong)時(shi)做標準(zhun)曲線，做復孔(kong)。如標本(ben)中待測物質含量過高（樣(yang)本(ben)OD值大于標準(zhun)品孔(kong)*孔(kong)的(de)OD值），請(qing)先用(yong)樣(yang)品稀釋液稀釋一(yi)定(ding)倍(bei)數(shu)(shu)（n倍(bei)）后再(zai)測定(ding)，計算(suan)時(shi)請(qing)zui后乘以總稀釋倍(bei)數(shu)(shu)（×n×5）。
5. 封板膜只限一(yi)次性使用，以避免(mian)交(jiao)叉污染。
6. 底物(wu)請避光保(bao)存。
7. 嚴(yan)格按(an)照說明書的操(cao)作進(jin)行，試(shi)驗結果判定必須以酶標儀讀數為(wei)準.
8. 所有樣品，洗滌液和各種廢(fei)棄物都應按傳染物處理。
9. 本試(shi)劑不同批(pi)號組(zu)分不得(de)混用。

10. 如與英文說明書有異，以英文說明書為準(zhun)。

計算：

以標準物的濃度為橫(heng)坐標，OD值為縱坐標，   

在坐標紙上繪(hui)出標準曲線，根(gen)據樣品的OD     

值由標準曲線(xian)查出相應的濃度；再(zai)乘以稀釋     

倍數；或用標準物的濃度與OD值計(ji)算(suan)出(chu)標     

準曲線(xian)的直線(xian)回歸方程式，將樣品的OD值     

代入方程式，計(ji)算(suan)出樣品濃度，再乘以稀釋   ;  

倍數，即(ji)為樣品的實(shi)際(ji)濃度。                 

        ;                                     

試(shi)劑盒性能：

1.樣品(pin)線性(xing)回歸與預(yu)期濃度相關系數R值為0.95以上。

2.批內與批見(jian)應分(fen)別小于9%和11%

檢測范圍：

0.13 ng/ml-25.6 ng/ml

靈(ling)敏度：

0.06 ng/ml

保存(cun)條件及有效期：

1.試劑盒保(bao)存(cun)：；2-8℃。

2．有效期：6個(ge)月