5-HT大(da)鼠5-羥色胺Elisa試劑(ji)盒說明書

規格：96T

檢測范圍：5pg/ml -180 pg/ml

靈敏(min)度：95%

特異性98%

目的(de)：本(ben)試劑盒用于測定大鼠(shu)血清，血漿及(ji)相關液體(ti)樣本(ben)中(zhong)5-羥色(se)胺(5-HT)的(de)含量。

運輸方式：2-8°C

有(you)效期(qi)：6個月(yue)

樣本處理及(ji)要求：

1. 血清：室(shi)溫血液(ye)自然凝(ning)固(gu)10-20分(fen)(fen)鐘(zhong)，離心(xin)20分(fen)(fen)鐘(zhong)左(zuo)右(you)（2000-3000轉/分(fen)(fen)）。仔細收集上清，保存過程中如出現沉(chen)淀(dian)，應再次(ci)離心(xin)。

2. 血漿：應根(gen)據標(biao)本的要求選擇(ze)EDTA或(huo)檸檬(meng)酸鈉作為抗凝劑，混合10-20分鐘(zhong)后(hou)，離心(xin)20分鐘(zhong)左右（2000-3000轉/分）。仔細收集上清，保存(cun)過(guo)程(cheng)中如有沉淀(dian)形(xing)成，應該再次離心(xin)。

3. 尿液：用無菌(jun)管收集，離心20分鐘左(zuo)右（2000-3000轉/分）。仔細收集上清(qing)，保存過程中如(ru)有沉淀形成，應(ying)再次離心。胸腹水、腦脊液參照實行(xing)。

4. 細(xi)胞培養上(shang)清(qing)：檢測分泌性的成份時(shi)，用無菌管收集。離(li)心20分鐘(zhong)左(zuo)右（2000-3000轉/分）。仔(zi)細(xi)收集上(shang)清(qing)。檢測細(xi)胞內的成份時(shi)，用PBS（PH7.2-7.4）稀釋細(xi)胞懸液，細(xi)胞濃(nong)度達到(dao)100萬/ml左(zuo)右。通過反復凍融，以使細(xi)胞破壞并放出(chu)細(xi)胞內成份。離(li)心20分鐘(zhong)左(zuo)右（2000-3000轉/分）。仔(zi)細(xi)收集上(shang)清(qing)。保存(cun)過程中如有沉淀形成，應再次離(li)心。

5. 組織(zhi)標本(ben)：切割(ge)標本(ben)后(hou)，稱取重量。加(jia)入一(yi)定量的PBS，PH7.4。用(yong)(yong)液氮迅速(su)冷凍保存備用(yong)(yong)。標本(ben)融化(hua)后(hou)仍然保持2-8℃的溫度。加(jia)入一(yi)定量的PBS（PH7.4），用(yong)(yong)手工(gong)或(huo)勻漿(jiang)器將(jiang)標本(ben)勻漿(jiang)充分(fen)。離心20分(fen)鐘左右（2000-3000轉(zhuan)/分(fen)）。仔細收集上(shang)清。分(fen)裝后(hou)一(yi)份(fen)待(dai)檢測，其(qi)余冷凍備用(yong)(yong)。

6. 標本采集(ji)后(hou)盡(jin)早(zao)進行提(ti)(ti)(ti)取，提(ti)(ti)(ti)取按相關文獻進行，提(ti)(ti)(ti)取后(hou)應盡(jin)快進行實(shi)驗。若不能馬(ma)上進行試驗，可將(jiang)標本放于-20℃保存(cun)，但(dan)應避免反復凍融.

7. 不(bu)能(neng)檢(jian)測含(han)NaN3的(de)樣品(pin)，因NaN3抑(yi)制辣(la)根過氧化(hua)物酶(mei)的（HRP）活性。

實(shi)驗(yan)原理：本(ben)試劑(ji)盒應用(yong)(yong)(yong)雙抗(kang)(kang)體(ti)(ti)(ti)夾(jia)心法(fa)測(ce)定(ding)標本(ben)中大(da)鼠(shu)(shu)(shu)5-羥(qian)(qian)色(se)(se)(se)胺(5-HT)水平。用(yong)(yong)(yong)純化(hua)的(de)(de)(de)大(da)鼠(shu)(shu)(shu)5-羥(qian)(qian)色(se)(se)(se)胺(5-HT)抗(kang)(kang)體(ti)(ti)(ti)包(bao)被微(wei)孔板，制(zhi)成(cheng)固相(xiang)抗(kang)(kang)體(ti)(ti)(ti)，往包(bao)被單抗(kang)(kang)的(de)(de)(de)微(wei)孔中依次加(jia)入5-羥(qian)(qian)色(se)(se)(se)胺(5-HT)再(zai)與HRP標記的(de)(de)(de)ET-1抗(kang)(kang)體(ti)(ti)(ti)結合，形(xing)成(cheng)抗(kang)(kang)體(ti)(ti)(ti)-抗(kang)(kang)原-酶標抗(kang)(kang)體(ti)(ti)(ti)復合物，經過*洗滌后加(jia)底(di)物TMB顯色(se)(se)(se)。TMB在(zai)HRP酶的(de)(de)(de)催(cui)化(hua)下(xia)轉化(hua)成(cheng)藍色(se)(se)(se)，并(bing)在(zai)酸(suan)的(de)(de)(de)作用(yong)(yong)(yong)下(xia)轉化(hua)成(cheng)zui終的(de)(de)(de)黃色(se)(se)(se)。顏色(se)(se)(se)的(de)(de)(de)深(shen)淺和(he)樣品中的(de)(de)(de)大(da)鼠(shu)(shu)(shu)5-羥(qian)(qian)色(se)(se)(se)胺(5-HT)呈正相(xiang)關。用(yong)(yong)(yong)酶標儀在(zai)450nm波長下(xia)測(ce)定(ding)吸(xi)光度（OD值），通過標準曲線計算樣品中大(da)鼠(shu)(shu)(shu)5-羥(qian)(qian)色(se)(se)(se)胺(5-HT)濃度。

試劑(ji)盒組(zu)成：

操作(zuo)步(bu)驟(zou)：

1. 標(biao)(biao)準(zhun)(zhun)品(pin)(pin)的(de)稀釋(shi)與加(jia)(jia)(jia)樣(yang)：在(zai)(zai)酶(mei)標(biao)(biao)包被板上(shang)設標(biao)(biao)準(zhun)(zhun)品(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)(kong)，在(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)100μl，然(ran)后(hou)(hou)(hou)在(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀釋(shi)液50μl，混(hun)勻(yun)；然(ran)后(hou)(hou)(hou)從*孔(kong)(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取(qu)(qu)(qu)100μl分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)(kong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀釋(shi)液50μl，混(hun)勻(yun)；然(ran)后(hou)(hou)(hou)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)先各(ge)(ge)取(qu)(qu)(qu)50μl棄掉，再(zai)(zai)(zai)各(ge)(ge)取(qu)(qu)(qu)50μl分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀釋(shi)液50ul，混(hun)勻(yun)；混(hun)勻(yun)后(hou)(hou)(hou)從第(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取(qu)(qu)(qu)50μl分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀釋(shi)液50μl，混(hun)勻(yun)后(hou)(hou)(hou)從第(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)取(qu)(qu)(qu)50μl加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)九、第(di)(di)(di)(di)(di)(di)(di)十(shi)(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)(zai)(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)十(shi)(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀釋(shi)液50μl，混(hun)勻(yun)后(hou)(hou)(hou)從第(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)十(shi)(shi)(shi)孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各(ge)(ge)取(qu)(qu)(qu)50μl棄掉。（稀釋(shi)后(hou)(hou)(hou)各(ge)(ge)孔(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)樣(yang)量都為50μl，濃度分(fen)(fen)別(bie)(bie)為150 pg/ml，100 pg/ml ，50 pg/ml，25 pg/ml，12.5 pg/ml）。
2. 加(jia)樣(yang)(yang)(yang)：分別設空白孔(kong)（空白對照(zhao)孔(kong)不(bu)(bu)加(jia)樣(yang)(yang)(yang)品(pin)及(ji)酶標試劑(ji)，其余各步操(cao)作(zuo)相(xiang)同）、待(dai)測樣(yang)(yang)(yang)品(pin)孔(kong)。在酶標包被板上待(dai)測樣(yang)(yang)(yang)品(pin)孔(kong)中(zhong)先加(jia)樣(yang)(yang)(yang)品(pin)稀釋(shi)液40μl，然(ran)后再加(jia)待(dai)測樣(yang)(yang)(yang)品(pin)10μl（樣(yang)(yang)(yang)品(pin)zui終稀釋(shi)度為5倍）。加(jia)樣(yang)(yang)(yang)將樣(yang)(yang)(yang)品(pin)加(jia)于酶標板孔(kong)底部，盡量不(bu)(bu)觸及(ji)孔(kong)壁，輕(qing)輕(qing)晃動混勻。
3. 溫育：用封(feng)板(ban)膜(mo)封(feng)板(ban)后置37℃溫(wen)育30分鐘(zhong)。
4. 配液：將30（48T的20倍）倍濃(nong)縮洗滌液用(yong)蒸(zheng)餾水30（48T的20倍）倍稀(xi)釋后備用(yong)。
5. 洗(xi)滌(di)(di)：小(xiao)心(xin)揭掉封板膜(mo)，棄(qi)去液(ye)體，甩干，每孔加滿洗(xi)滌(di)(di)液(ye)，靜置(zhi)30秒后棄(qi)去，如此(ci)重復5次，拍干。
6. 加酶(mei)：每(mei)孔加入酶(mei)標試劑(ji)50μl，空(kong)白(bai)孔除外。
7. 溫育：操作同3。
8. 洗滌：操作同(tong)5。
9. 顯(xian)色(se)：每孔先加入顯(xian)色(se)劑A50μl，再加入顯(xian)色(se)劑B50μl，輕輕震(zhen)蕩混勻，37℃避光顯(xian)色15分鐘(zhong).
10. 終(zhong)止(zhi)：每(mei)孔加終(zhong)止(zhi)液(ye)50μl，終(zhong)止(zhi)反應(ying)（此(ci)時藍色立轉黃色）。
11. 測(ce)定：以(yi)空白空調零，450nm波長依序(xu)測(ce)量各(ge)孔的(de)吸光度（OD值(zhi)）。 測(ce)定應在(zai)加終止液后(hou)15分鐘(zhong)以(yi)內進行。

注意(yi)事項：

1. 試劑盒(he)從冷藏環境中取出(chu)應(ying)在室溫平衡(heng)15-30分鐘后(hou)方可使用(yong)，酶標包被板(ban)開封后(hou)如未用(yong)完(wan)，板(ban)條應(ying)裝入密封袋中保存(cun)。
2. 濃洗滌(di)液可能(neng)會有結晶析出，稀釋時可在水浴中加溫助(zhu)溶(rong)，洗滌(di)時不影響結果(guo)。
3. 各步加(jia)樣(yang)均(jun)應使(shi)用加(jia)樣(yang)器(qi)，并經常(chang)校對其準確性，以避免試驗(yan)誤差。一次加(jia)樣(yang)時間控制在5分鐘內，如標本數量多，推薦使(shi)用排(pai)槍加(jia)樣(yang)。
4. 請(qing)(qing)每次測定的(de)同時做標(biao)準曲線，做復(fu)孔。如標(biao)本中待測物質含量(liang)過高（樣本OD值大于標(biao)準品(pin)(pin)孔*孔的(de)OD值），請(qing)(qing)先(xian)用樣品(pin)(pin)稀(xi)釋(shi)(shi)液(ye)稀(xi)釋(shi)(shi)一定倍數（n倍）后(hou)(hou)再(zai)測定，計算時請(qing)(qing)zui后(hou)(hou)乘以總稀(xi)釋(shi)(shi)倍數（×n×5）。
5. 封板膜只限一次性使用，以避免交叉污(wu)染。
6. 底物請(qing)避光保存。
7. 嚴(yan)格按照說明(ming)書(shu)的操作進行，試驗結果(guo)判定必須以酶(mei)標儀讀數(shu)為準.
8. 所有樣品，洗滌液(ye)和(he)各種廢棄物都應按傳(chuan)染物處理。
9. 本試劑不同批號組分不得混用。

10. 如與英文(wen)說明書有異，以英文(wen)說明書為(wei)準。

計算：

以標(biao)準(zhun)(zhun)物(wu)(wu)的(de)(de)濃(nong)(nong)度為(wei)橫坐(zuo)(zuo)標(biao)，OD值為(wei)縱坐(zuo)(zuo)標(biao)，在(zai)坐(zuo)(zuo)標(biao)紙上繪出標(biao)準(zhun)(zhun)曲(qu)線，根(gen)據樣(yang)(yang)品的(de)(de)OD值由標(biao)準(zhun)(zhun)曲(qu)線查出相應的(de)(de)濃(nong)(nong)度；再乘以稀釋倍數；或用(yong)標(biao)準(zhun)(zhun)物(wu)(wu)的(de)(de)濃(nong)(nong)度與OD值計算出標(biao)準(zhun)(zhun)曲(qu)線的(de)(de)直線回歸方程式，將樣(yang)(yang)品的(de)(de)OD值代入方程式，計算出樣(yang)(yang)品濃(nong)(nong)度，再乘以稀釋倍數，即為(wei)樣(yang)(yang)品的(de)(de)實(shi)際濃(nong)(nong)度。