【豬(zhu)透明質酸(HA) elisa試劑(ji)盒】本試劑(ji)僅供研(yan)究使用

計算：

以標(biao)(biao)準(zhun)物的(de)濃(nong)(nong)度為(wei)橫坐標(biao)(biao)，OD值為(wei)縱坐標(biao)(biao)，在坐標(biao)(biao)紙上繪出(chu)標(biao)(biao)準(zhun)曲(qu)線，根據樣(yang)品(pin)(pin)的(de)OD值由(you)標(biao)(biao)準(zhun)曲(qu)線查出(chu)相(xiang)應的(de)濃(nong)(nong)度；再(zai)乘以稀釋(shi)倍數(shu)；或用標(biao)(biao)準(zhun)物的(de)濃(nong)(nong)度與OD值計算出(chu)標(biao)(biao)準(zhun)曲(qu)線的(de)直線回歸方(fang)程式，將樣(yang)品(pin)(pin)的(de)OD值代入方(fang)程式，計算出(chu)樣(yang)品(pin)(pin)濃(nong)(nong)度，再(zai)乘以稀釋(shi)倍數(shu)，即為(wei)樣(yang)品(pin)(pin)的(de)實際濃(nong)(nong)度。

【豬透明質酸(HA) elisa試(shi)劑(ji)盒】試(shi)劑(ji)盒組成：

封板(ban)膜:2片（48）/2片（96）

說明書:1份

密封袋:1個

標準品：   2700ng/L 0.5ml×1瓶 0.5ml×1瓶  2-8℃保存

酶標(biao)包被板:  1×48     1×96         2-8℃保存

樣品稀釋(shi)液: 3ml×1瓶(ping)  6 ml×1瓶(ping)     2-8℃保存

顯色劑A液: 3ml×1瓶 6 ml×1瓶     2-8℃保存

顯色劑B液(ye): 3ml×1瓶(ping) 6 ml×1瓶(ping)     2-8℃保存(cun)

終止液(ye):     3ml×1瓶 6ml×1瓶     2-8℃保存

濃縮洗(xi)滌液:（20ml×20倍）×1瓶 （20ml×30倍）×1瓶   2-8℃保存

實驗原理：

本試劑盒應用(yong)雙抗(kang)體夾(jia)心(xin)法測定(ding)標(biao)本中(zhong)豬(zhu)透(tou)(tou)明(ming)質酸(suan)(suan)(HA) 水平。用(yong)純化(hua)(hua)的(de)(de)(de)(de)豬(zhu)透(tou)(tou)明(ming)質酸(suan)(suan)(HA) 抗(kang)體包(bao)被微(wei)孔(kong)板(ban)，制成(cheng)固相(xiang)抗(kang)體，往包(bao)被單抗(kang)的(de)(de)(de)(de)微(wei)孔(kong)中(zhong)依次加入(ru)豬(zhu)透(tou)(tou)明(ming)質酸(suan)(suan)(HA) ，再與HRP標(biao)記(ji)的(de)(de)(de)(de)豬(zhu)透(tou)(tou)明(ming)質酸(suan)(suan)(HA) 抗(kang)體結合(he)，形成(cheng)抗(kang)體-抗(kang)原-酶(mei)標(biao)抗(kang)體復(fu)合(he)物(wu)(wu)，經過(guo)*洗滌(di)后加底物(wu)(wu)TMB顯色。TMB在HRP酶(mei)的(de)(de)(de)(de)催(cui)化(hua)(hua)下(xia)(xia)轉(zhuan)化(hua)(hua)成(cheng)藍色，并在酸(suan)(suan)的(de)(de)(de)(de)作用(yong)下(xia)(xia)轉(zhuan)化(hua)(hua)成(cheng)zui終的(de)(de)(de)(de)。顏色的(de)(de)(de)(de)深(shen)淺和樣(yang)品中(zhong)的(de)(de)(de)(de)豬(zhu)透(tou)(tou)明(ming)質酸(suan)(suan)(HA) 呈正相(xiang)關(guan)。用(yong)酶(mei)標(biao)儀在450nm波長下(xia)(xia)測定(ding)吸光度（OD值），通過(guo)標(biao)準(zhun)曲線(xian)計算樣(yang)品中(zhong)豬(zhu)透(tou)(tou)明(ming)質酸(suan)(suan)(HA) 濃度。

目(mu)的：本試劑盒用于(yu)測定(ding)豬血(xue)清，血(xue)漿(jiang)及(ji)相關液體樣本中豬透明質酸(HA) 的含量。

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保存條件及有效期：

試(shi)劑盒保(bao)存(cun)：2-8℃| 有(you)效期(qi)：6個月

【豬透明(ming)質酸(HA) elisa試(shi)劑(ji)盒】樣本處理(li)及(ji)要求：

1. 血清：室溫血液自然凝固10-20分鐘(zhong)，離(li)心20分鐘(zhong)左右（2000-3000轉/分）。仔細收集上清，保存(cun)過程中如出現(xian)沉淀，應(ying)再次離(li)心。

2. 血漿：應(ying)(ying)根(gen)據(ju)標本的要(yao)求選擇EDTA或檸檬(meng)酸鈉作為(wei)抗凝劑，混合10-20分(fen)鐘后，離心(xin)20分(fen)鐘左右(you)（2000-3000轉/分(fen)）。仔(zi)細收集上清，保存過程中如(ru)有沉(chen)淀形成(cheng)，應(ying)(ying)該再次(ci)離心(xin)。

3. 尿液(ye)：用無菌管收(shou)集，離(li)心(xin)20分(fen)鐘左右（2000-3000轉/分(fen)）。仔(zi)細收(shou)集上清，保存過程中如有(you)沉(chen)淀形成，應再次離(li)心(xin)。胸腹水(shui)、腦脊液(ye)參(can)照(zhao)實(shi)行。

4. 細(xi)(xi)胞(bao)培養上清(qing)：檢測分(fen)泌性的成份時，用(yong)(yong)無菌管收集(ji)。離心(xin)20分(fen)鐘左右（2000-3000轉/分(fen)）。仔細(xi)(xi)收集(ji)上清(qing)。檢測細(xi)(xi)胞(bao)內的成份時，用(yong)(yong)PBS（PH7.2-7.4）稀釋細(xi)(xi)胞(bao)懸液，細(xi)(xi)胞(bao)濃度達到100萬/ml左右。通過(guo)反(fan)復凍融，以使(shi)細(xi)(xi)胞(bao)破(po)壞并放(fang)出(chu)細(xi)(xi)胞(bao)內成份。離心(xin)20分(fen)鐘左右（2000-3000轉/分(fen)）。仔細(xi)(xi)收集(ji)上清(qing)。保存過(guo)程中如有(you)沉淀形成，應再次離心(xin)。

5. 組織標本(ben)：切割標本(ben)后(hou)，稱取重量。加(jia)入(ru)一(yi)(yi)定(ding)量的PBS，PH7.4。用液氮迅(xun)速冷凍保存備(bei)用。標本(ben)融化后(hou)仍(reng)然保持2-8℃的溫度。加(jia)入(ru)一(yi)(yi)定(ding)量的PBS（PH7.4），用手工或勻漿器將(jiang)標本(ben)勻漿充分。離(li)心(xin)20分鐘左右(you)（2000-3000轉(zhuan)/分）。仔細收集上清。分裝(zhuang)后(hou)一(yi)(yi)份待檢測，其余冷凍備(bei)用。

6. 標本采集后盡(jin)(jin)早進(jin)行(xing)(xing)提(ti)取，提(ti)取按相關(guan)文獻進(jin)行(xing)(xing)，提(ti)取后應盡(jin)(jin)快(kuai)進(jin)行(xing)(xing)實(shi)驗(yan)。若不能馬(ma)上(shang)進(jin)行(xing)(xing)試驗(yan)，可將標本放于-20℃保存，但應避免反(fan)復(fu)凍融(rong).

7. 不能(neng)檢測含(han)NaN3的樣品，因NaN3抑(yi)制辣根(gen)過(guo)氧(yang)化物酶的（HRP）活性(xing)。

【豬(zhu)透明質酸(HA) elisa試劑盒】操作步驟(zou)：

1. 標(biao)準(zhun)(zhun)品(pin)(pin)(pin)的稀(xi)(xi)釋與加(jia)(jia)(jia)樣(yang)：在(zai)(zai)(zai)酶標(biao)包(bao)被板上設標(biao)準(zhun)(zhun)品(pin)(pin)(pin)孔(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)，在(zai)(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)100μl，然后(hou)在(zai)(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混(hun)(hun)勻(yun)(yun)；然后(hou)從(cong)*孔(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各取(qu)100μl分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)三(san)(san)孔(kong)(kong)(kong)(kong)和(he)(he)第(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)，再在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)三(san)(san)、第(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混(hun)(hun)勻(yun)(yun)；然后(hou)在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)三(san)(san)孔(kong)(kong)(kong)(kong)和(he)(he)第(di)(di)(di)(di)(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)先各取(qu)50μl棄掉，再各取(qu)50μl分(fen)(fen)別(bie)(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)中(zhong)(zhong)(zhong)(zhong)，再在(zai)(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)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50ul，混(hun)(hun)勻(yun)(yun)；混(hun)(hun)勻(yun)(yun)后(hou)從(cong)第(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)六孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各取(qu)50μl分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混(hun)(hun)勻(yun)(yun)后(hou)從(cong)第(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)(bie)取(qu)50μl加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)(di)(di)(di)九(jiu)、第(di)(di)(di)(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)分(fen)(fen)別(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)稀(xi)(xi)釋液(ye)(ye)50μl，混(hun)(hun)勻(yun)(yun)后(hou)從(cong)第(di)(di)(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)(di)(di)十(shi)孔(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各取(qu)50μl棄掉。（稀(xi)(xi)釋后(hou)各孔(kong)(kong)(kong)(kong)加(jia)(jia)(jia)樣(yang)量(liang)都為50μl，濃(nong)度分(fen)(fen)別(bie)(bie)(bie)為1800ng/L，1200ng/L ，600ng/L，300ng/L， 150ng/L）。

2. 加(jia)樣(yang)：分別設(she)空(kong)白(bai)孔(kong)（空(kong)白(bai)對照孔(kong)不(bu)加(jia)樣(yang)品(pin)(pin)及酶標試劑，其(qi)余(yu)各步(bu)操作相(xiang)同）、待測樣(yang)品(pin)(pin)孔(kong)。在酶標包被板(ban)上(shang)待測樣(yang)品(pin)(pin)孔(kong)中先加(jia)樣(yang)品(pin)(pin)稀(xi)釋液40μl，然(ran)后(hou)再加(jia)待測樣(yang)品(pin)(pin)10μl（樣(yang)品(pin)(pin)zui終稀(xi)釋度為5倍）。加(jia)樣(yang)將樣(yang)品(pin)(pin)加(jia)于(yu)酶標板(ban)孔(kong)底部，盡量不(bu)觸及孔(kong)壁，輕(qing)輕(qing)晃動混(hun)勻。

3. 溫育：用封板膜(mo)封板后置37℃溫育30分鐘(zhong)。

4. 配液：將30（48T的20倍(bei)(bei)(bei)）倍(bei)(bei)(bei)濃縮洗滌液用(yong)蒸餾水30（48T的20倍(bei)(bei)(bei)）倍(bei)(bei)(bei)稀釋后備(bei)用(yong)。

5. 洗滌(di)：小心揭掉封(feng)板膜，棄去液體，甩干，每孔(kong)加滿(man)洗滌(di)液，靜置30秒(miao)后棄去，如此重復5次(ci)，拍(pai)干。

6. 加酶(mei)：每孔(kong)加入酶(mei)標試(shi)劑(ji)50μl，空白孔(kong)除外(wai)。

7. 溫育：操作同3。

8. 洗滌：操作同5。

9. 顯(xian)(xian)(xian)色(se)：每孔先加(jia)入(ru)顯(xian)(xian)(xian)色(se)劑(ji)A50μl，再(zai)加(jia)入(ru)顯(xian)(xian)(xian)色(se)劑(ji)B50μl，輕輕震(zhen)蕩混(hun)勻(yun)，37℃避光顯(xian)(xian)(xian)色(se)15分鐘.

10. 終(zhong)(zhong)止(zhi)：每(mei)孔加終(zhong)(zhong)止(zhi)液50μl，終(zhong)(zhong)止(zhi)反(fan)應(ying)（此時藍色立轉(zhuan)）。