大鼠抗心肌抗體(AMA)ELISA試劑盒產(chǎn)品規(guī)格：96T/48T
產(chǎn)品庫存：現(xiàn)貨
產(chǎn)品價格：詢價
使用范圍：僅供科研檢測，不得用于臨床。
保存條件：2-8℃
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大鼠抗心肌抗體(AMA)ELISA試劑盒說明書:說明書隨貨發(fā)送，您也可以直接我司在線銷售人員索取。全國：
代理多種品牌進口原裝、分裝“ELISA試劑盒”。齊一生物科技有限公司作為酶聯(lián)免疫供應商，憑借*的技術，*的售前，售中，售后免費代測服務，共同鑄就高品質的產(chǎn)品。多年專業(yè)酶免服務，質量保證，可免費提供代測服務，一周內出結果。咨詢訂購。
ELISA試劑盒操作步驟
ELISA試劑盒使用前，將所有試劑充分混勻。不要使液體產(chǎn)生大量的泡沫，以免加樣時加入大量的氣泡，產(chǎn)生加樣上的誤差。
根據(jù)待測樣品數(shù)量加上標準品的數(shù)量決定所需的板條數(shù)。每個標準品和空白孔建議做復孔。每個樣品根據(jù)自己的數(shù)量來定，能使用復孔的盡量做復孔。標本用標本稀釋液1：1稀釋后加入50ul于反應孔內。
加入稀釋好后的標準品50ul于反應孔、加入待測樣品50ul于反應孔內。立即加入50ul的生物素標記的抗體。蓋上膜板，輕輕振蕩混勻，37℃溫育1小時。
甩去孔內液體，每孔加滿洗滌液，振蕩30秒，甩去洗滌液，用吸水紙拍干。重復此操作3次。如果用洗板機洗滌，洗滌次數(shù)增加一次。
每孔加入80ul的親和鏈酶素-HRP，輕輕振蕩混勻，37℃溫育30分鐘。
甩去孔內液體，每孔加滿洗滌液，振蕩30秒，甩去洗滌液，用吸水紙拍干。重復此操作3次。如果用洗板機洗滌，洗滌次數(shù)增加一次。
每孔加入底物A、B各50ul，輕輕振蕩混勻，37℃溫育10分鐘。避免光照。
取出酶標板，迅速加入50ul終止液，加入終止液后應立即測定結果。
在450nm波長處測定各孔的OD值。
ELISA操作步驟
酶聯(lián)免疫吸附實驗材料，試劑，溶液   
1．酶標板，100μltip頭，1ml Ep管，濕盒
2．包被稀釋液（0.05mol/L碳酸鈉-碳酸氫鈉buffer,pH 9.6）
  碳酸鈉0.15g,碳酸氫鈉0.29g,疊氮鈉0.02g,加雙蒸水至100ml,調至pH     9.6
3.  封閉液(5%小牛血清/PBS溶液)
    小牛血清5ml, 1*PBS(pH 7.4)95ml
4.  洗滌液(PBST, pH 7.4)
    NaCl 0.8g, KH2PO40.02g,Na2HPO4.12H2O 0.29g, KCl 0.02g,Tween20 0.05ml,疊氮鈉0.01g,加雙蒸水至100ml,調至pH 7.4
5.  樣本稀釋液(PBS, pH 7.4)
     NaCl 0.8g, KH2PO40.02g,Na2HPO4.12H2O 0.29g, KCl 0.02g,疊氮鈉0.01g,加雙蒸水至100ml,調至pH 7.4
6.  酶標第2抗體(羊抗兔)稀釋范圍1:5,000-1:100,000
7.  底物液(TMB-過氧化氫尿素溶液)
① 底物液A:TMB20mg,無水乙醇10ml,加雙蒸水至100ml.
②底物液B(0.1mol/L檸檬酸-0.2mol/L磷酸二氫鈉緩沖液, pH 5.0-5.4)
Na2HPO41.46g, 檸檬酸0.933g, 0.75%過氧化氫尿素0.64ml, 加三蒸水至100ml,調至pH 5.0-5.4
③底物A和B按1:1混合即成TMB-過氧化氫尿素溶液.
8. 終止液(2mol/LH2SO4溶液)雙蒸水200ml,濃硫酸34ml,(緩慢滴加并不斷攪拌),加雙蒸水至300ml
9. 0.9%生理鹽水

大鼠抗心肌抗體(AMA)ELISA試劑盒操作步驟: 
1.     包被過程(注意設置空白對照,陰性對照):
將所用抗原用包被稀釋液稀釋到適當濃度(一般所需抗原包被量為每孔20-200μg),每孔抗原加入100μl, 置37℃,4h,或4℃,24h;棄去孔中液體.(為避免蒸發(fā),板上應加蓋或將板平放在底部有濕紗布的金屬濕盒中)
2.     封閉酶標反應孔:
5%小牛血清置37℃封閉40min.封閉時將封閉液加滿各反應孔,并去除各孔中的氣泡,封閉結束后用洗滌液滿孔洗滌3遍,每遍3min.
洗滌方法:吸干孔內反應液,將洗滌液注滿板孔,放置2min略作搖動,吸干孔內液,傾去液體后在吸水紙上拍干         
        洗滌次數(shù)3次
3.     加入待檢測樣品(建立合適的濃度梯度):
檢測時一般采用1:50-1:400的稀釋度, 應采用較大稀釋體積進行,一般保證樣品吸取量>20μl.
將稀釋好的樣品加入酶標反應孔中,每樣品至少加雙孔, 每孔100μl,置于37℃,40-60min.用洗滌液滿孔洗滌3遍,每遍3min.
4.     加入酶標抗體:
酶標抗體: 根據(jù)酶結合物提供商提供的參考工作稀釋度進行. 37℃,30-60min之間.短于30min往往結果不穩(wěn)定.每孔加100μl.洗滌同前。
5.     加入底物液(現(xiàn)用現(xiàn)配):
*TMB-過氧化氫尿素溶液,OPD-過氧化氫底物液系統(tǒng)次之.
底物加入量:每孔100μl,置37℃避光放置3-5分鐘,加入終止液顯色.
6.     終止反應:
每孔加入終止液50μl終止反應,于20min內測定實驗結果.
7.     結果判斷:
OPD顯色后采用492nm波長,TMB反應產(chǎn)物檢測需要450nm波長.檢測時一定要首*行空白孔系統(tǒng)調零.用測定標本孔的吸收值與一組陰性標本測定孔平均值的比值(P/N)表示,當P/N大于2時作為抗體的效價.(數(shù)值的大小依具體檢測要求而定.)

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