人谷胱甘肽（GSH）試劑盒說明書

本試劑(ji)僅供研究使(shi)用       

目的：本試劑盒用于測定人血清，血漿及相關液體樣本中谷(gu)胱甘肽(tai)（GSH）的含量。

實驗原理：

   本試劑盒應用雙抗體夾心法測定標本中人谷胱甘肽(tai)（GSH）水平。用純化的人谷胱甘(gan)肽（GSH）抗(kang)體(ti)包被微孔板，制成固相抗體，往包被單抗的微孔中加入谷胱甘(gan)肽（GSH），再與HRP標記的谷胱甘肽（GSH）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谷胱甘肽（GSH）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人谷(gu)胱甘肽（GSH）的含量。

試劑(ji)盒組成：

樣本(ben)處理及要(yao)求：

1. 血(xue)清：室溫血(xue)液自然凝固10-20分鐘，離心20分鐘左(zuo)右（2000-3000轉/分）。仔細收集上清，保存過程中如出現沉淀(dian)，應再次離心。

2. 血漿：應根據(ju)標本的要求選擇EDTA或檸檬酸鈉(na)作為抗凝(ning)劑，混合(he)10-20分(fen)鐘(zhong)后(hou)，離心20分(fen)鐘(zhong)左右（2000-3000轉(zhuan)/分(fen)）。仔(zi)細收(shou)集(ji)上清(qing)，保存過(guo)程(cheng)中如有沉淀形成(cheng)，應該(gai)再次離心。

3. 尿液：用(yong)無菌管收集，離心(xin)(xin)20分鐘(zhong)左右（2000-3000轉/分）。仔細(xi)收集上清，保(bao)存過程中如(ru)有沉淀形成，應(ying)再(zai)次離心(xin)(xin)。胸腹水、腦(nao)脊液參(can)照實行。

4. 細(xi)(xi)胞培(pei)養上(shang)清(qing)：檢測(ce)分泌性的成(cheng)(cheng)份(fen)時，用無菌管(guan)收集(ji)。離心(xin)20分鐘左(zuo)右（2000-3000轉/分）。仔(zi)細(xi)(xi)收集(ji)上(shang)清(qing)。檢測(ce)細(xi)(xi)胞內(nei)(nei)的成(cheng)(cheng)份(fen)時，用PBS（PH7.2-7.4）稀釋細(xi)(xi)胞懸液，細(xi)(xi)胞濃度(du)達到100萬/ml左(zuo)右。通過反復凍融，以(yi)使細(xi)(xi)胞破壞(huai)并放出細(xi)(xi)胞內(nei)(nei)成(cheng)(cheng)份(fen)。離心(xin)20分鐘左(zuo)右（2000-3000轉/分）。仔(zi)細(xi)(xi)收集(ji)上(shang)清(qing)。保存過程中如有沉(chen)淀形(xing)成(cheng)(cheng)，應(ying)再次離心(xin)。

5. 組(zu)織標本：切割標本后，稱取重量。加入(ru)一定量的PBS，PH7.4。用液氮(dan)迅速(su)冷凍保存(cun)備用。標本融化后仍然保持2-8℃的溫度。加入(ru)一定量的PBS（PH7.4），用手工(gong)或(huo)勻漿(jiang)器將標本勻漿(jiang)充分。離心20分鐘左右（2000-3000轉/分）。仔細(xi)收(shou)集上清。分裝后一份待檢(jian)測，其(qi)余冷凍備用。

6. 標本采集后(hou)盡早(zao)進行(xing)提(ti)(ti)取，提(ti)(ti)取按相關文(wen)獻進行(xing)，提(ti)(ti)取后(hou)應盡快(kuai)進行(xing)實驗。若不(bu)能(neng)馬上進行(xing)試(shi)驗，可將標本放(fang)于-20℃保存，但應避免(mian)反復凍融(rong).

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

操作步(bu)驟(zou)：

1. 標(biao)準(zhun)(zhun)(zhun)品(pin)的稀釋(shi)與加(jia)(jia)樣：在(zai)(zai)酶標(biao)包被板上設標(biao)準(zhun)(zhun)(zhun)品(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)，在(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)別(bie)加(jia)(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)100μl，然(ran)后在(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)稀釋(shi)液50μl，混勻(yun)；然(ran)后從(cong)*孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)(qu)(qu)100μl分(fen)別(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)，再(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)別(bie)加(jia)(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)稀釋(shi)液50μl，混勻(yun)；然(ran)后在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)三(san)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)先各(ge)取(qu)(qu)(qu)50μl棄掉(diao)，再(zai)各(ge)取(qu)(qu)(qu)50μl分(fen)別(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)別(bie)加(jia)(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)稀釋(shi)液50ul，混勻(yun)；混勻(yun)后從(cong)第(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)(qu)(qu)50μl分(fen)別(bie)加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)八(ba)(ba)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)八(ba)(ba)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)別(bie)加(jia)(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)稀釋(shi)液50μl，混勻(yun)后從(cong)第(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)八(ba)(ba)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)別(bie)取(qu)(qu)(qu)50μl加(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)九(jiu)、第(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)別(bie)加(jia)(jia)標(biao)準(zhun)(zhun)(zhun)品(pin)稀釋(shi)液50μl，混勻(yun)后從(cong)第(di)(di)(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)(qu)(qu)50μl棄掉(diao)。（稀釋(shi)后各(ge)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)樣量都(dou)為50μl，濃度分(fen)別(bie)為60 ng/L，40 ng/L，20 ng/L，10 ng/L，5 ng/L）。
2. 加(jia)(jia)樣：分別(bie)設(she)空(kong)(kong)白孔(kong)（空(kong)(kong)白對照孔(kong)不加(jia)(jia)樣品(pin)及酶標試劑，其余(yu)各步操(cao)作相同）、待測樣品(pin)孔(kong)。在酶標包被板(ban)上(shang)待測樣品(pin)孔(kong)中先加(jia)(jia)樣品(pin)稀釋液40μl，然后(hou)再加(jia)(jia)待測樣品(pin)10μl（樣品(pin)zui終稀釋度(du)為5倍）。加(jia)(jia)樣將(jiang)樣品(pin)加(jia)(jia)于酶標板(ban)孔(kong)底(di)部(bu)，盡量不觸及孔(kong)壁，輕(qing)輕(qing)晃動混勻。
3. 溫育：用封板膜(mo)封板后置37℃溫育30分鐘。
4. 配液(ye)：將30（48T的20倍）倍濃縮洗滌液(ye)用蒸餾水30（48T的20倍）倍稀釋后(hou)備用。
5. 洗(xi)滌：小心(xin)揭掉封板膜(mo)，棄(qi)去液體，甩干(gan)，每孔加滿洗(xi)滌液，靜置30秒(miao)后(hou)棄(qi)去，如此重復(fu)5次，拍干(gan)。
6. 加酶(mei)：每孔加入酶(mei)標(biao)試劑50μl，空白(bai)孔除外。
7. 溫育：操(cao)作(zuo)同3。
8. 洗滌(di)：操作同5。
9. 顯(xian)(xian)色：每孔(kong)先加(jia)入(ru)顯(xian)(xian)色劑A50μl，再加(jia)入(ru)顯(xian)(xian)色劑B50μl，輕輕震蕩混勻，37℃避光顯色15分(fen)鐘.
10. 終止：每孔加終止液(ye)50μl，終止反應（此時藍色(se)立轉黃色(se)）。
11. 測定：以空白空調零，450nm波長依(yi)序測量(liang)各孔的(de)吸光度（OD值）。 測定應在(zai)加終止液后15分鐘以內進行。

注意事項：

1. 試劑盒(he)從冷藏環境中取出應在室溫平(ping)衡15-30分鐘后方(fang)可(ke)使用，酶標(biao)包被板開(kai)封后如未用完，板條應裝入(ru)密封袋中保存(cun)。
2. 濃洗滌液可能會有結(jie)晶析出，稀(xi)釋時可在水浴中加溫助溶，洗滌時不影(ying)響結(jie)果。
3. 各步加樣(yang)均應使(shi)用加樣(yang)器，并經常校(xiao)對其準確性，以避免試驗誤差。一次(ci)加樣(yang)時間(jian)控制在(zai)5分鐘內，如標本數量多，推薦使(shi)用排槍加樣(yang)。
4. 請每(mei)次測定的(de)(de)同時做標(biao)準曲線，做復孔(kong)(kong)。如標(biao)本中待測物質(zhi)含量(liang)過高（樣本OD值(zhi)大于標(biao)準品(pin)孔(kong)(kong)*孔(kong)(kong)的(de)(de)OD值(zhi)），請先用樣品(pin)稀釋(shi)液稀釋(shi)一定倍數（n倍）后再測定，計算(suan)時請zui后乘以總(zong)稀釋(shi)倍數（×n×5）。
5. 封板(ban)膜只(zhi)限一次性使(shi)用(yong)，以(yi)避(bi)免(mian)交叉污染。
6. 底(di)物(wu)請(qing)避光保存。
7. 嚴格按照(zhao)說明書的操作進行，試驗結(jie)果判定必(bi)須以酶標(biao)儀(yi)讀數為(wei)準.
8. 所有樣(yang)品(pin)，洗滌(di)液和(he)各種廢棄(qi)物(wu)都應按(an)傳染物(wu)處理。
9. 本試劑不同批號組分不得混用。

10. 如與英(ying)文說(shuo)明書有異，以英(ying)文說(shuo)明書為準。

計算：

以標準物的濃度為橫坐標，OD值為縱坐標，   

在坐標紙上繪出標準(zhun)曲線，根據(ju)樣品的(de)OD     

值由標準曲線查(cha)出相應(ying)的(de)濃度；再(zai)乘以(yi)稀釋     

倍數；或用標準(zhun)物的濃度與OD值計(ji)算出標     

準曲(qu)線的直線回歸方程式，將樣品的OD值(zhi)     

代(dai)入方程式，計算(suan)出樣(yang)品濃度(du)，再乘以稀釋     

倍數，即為(wei)樣品的實際濃度。                 

                       ;                      

試(shi)劑盒性能：

1.樣品線性回歸與預(yu)期濃度相關系數(shu)R值為0.990以上。

2.批(pi)內與批(pi)見應分別小于9%和11%

檢測范圍：

0.78 ug/ml-50 ug/ml

靈(ling)敏度：

0.195 ug/ml

                                         ;                       

保存條(tiao)件及有效期：

1.試劑盒保存(cun)：；2-8℃。

2．有效期：6個(ge)月