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• 人纖溶酶原激活物YZ因子1(PAI-1)ELISA試劑盒

  人纖溶酶原激活物YZ因子1(PAI-1)ELISA試劑盒[詳細]

  2013-12-06 00:00

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• 人纖溶酶原激活物YZ因子1(PAI-1)elisa試劑盒說明書

  人纖溶酶原激活物YZ因子1(PAI-1)elisa試劑盒說明書[詳細]

  2024-10-08 05:28

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• 人纖溶酶原激活物YZ因子1(PAI-1)elisa試劑盒使用說明書

  人纖溶酶原激活物YZ因子1(PAI-1)elisa試劑盒使用說明書[詳細]

  2024-10-08 05:25

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• 大鼠纖溶酶原激活物YZ因子1(PAI-1)ELISA試劑盒

  大鼠纖溶酶原激活物YZ因子1(PAI-1)ELISA試劑盒[詳細]

  2024-09-19 05:37

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• 豬纖溶酶原激活物YZ因子1(PAI-1)ELISA試劑盒

  豬纖溶酶原激活物YZ因子1(PAI-1)ELISA試劑盒[詳細]

  2024-09-28 00:35

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• 人纖溶酶原激活物YZ劑1（PAI-1）ELISA試劑盒

  人纖溶酶原激活物YZ劑1(PAI-1)ELISA試劑盒操作步驟：1.標準品的稀釋與加樣：在酶標包被板上設標準品孔10孔，在**、第二孔中分別加標準品100μl，然后在**、第二孔中加標準品稀釋液50μl，混勻；然后從**孔、第二孔中各取100μl分別加到第三孔和第四孔，再在第三、第四孔分別加標準品稀釋液50μl，混勻；然后在第三孔和第四孔中先各取50μl棄掉，再各取50μl分別加到第五、第六孔中，再在第五、第六孔中分別加標準品稀釋液50ul，混勻；混勻后從第五、第六孔中各取50μl分別加到第七、第八孔中，再在第七、第八孔中分別加標準品稀釋液50μl，混勻后從第七、第八孔中分別取50μl加到第九、第十孔中，再在第九第十孔分別加標準品稀釋液50μl，混勻后從第九第十孔中各取50μl棄掉。（稀釋后各孔加樣量都為50μl，濃度分別為600ng/L，400ng/L，200ng/L，100ng/L，50ng/L）。2.加樣：分別設空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、待測樣品孔。在酶標包被板上待測樣品孔中先加樣品稀釋液40μl，然后再加待測樣品10μl（樣品Z終稀釋度為5倍）。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻。3.溫育：用封板膜封板后置37℃溫育30分鐘。4.配液：將30（48T的20倍）倍濃縮洗滌液用蒸餾水30（48T的20倍）倍稀釋后備用。5.洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復5次，拍干。6.加酶：每孔加入酶標試劑50μl，空白孔除外。7.溫育：操作同3。8.洗滌：操作同5。9.顯色：每孔先加入顯色劑A50μl，再加入顯色劑B50μl，輕輕震蕩混勻，37℃避光顯色15分鐘.10.終止：每孔加終止液50μl，終止反應（此時藍色立轉(zhuǎn)黃色）。11.測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。測定應在加終止液后15分鐘以內(nèi)進行。[詳細]

  2018-11-05 10:00

  產(chǎn)品樣冊

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• 人纖溶酶原激活物YZ因子1（PAI-1）試劑盒實驗原理

  人纖溶酶原激活物YZ因子1（PAI-1）酶聯(lián)免疫分析（ELISA）試劑盒僅供研究使用，用于測定人血清，血漿及相關液體樣本中纖溶酶原激活物YZ因子1（PAI-1）的含量，應用雙抗體夾心法測定標本中人纖溶酶原激活物YZ物-1（PAI-1）水平。用純化的人纖溶酶原激活物YZ物-1抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入PAI-1，再與HRP標記的PAI-1抗體結合，形成抗體-抗原-酶標抗體復合物，經(jīng)過徹底洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍色，并在酸的作用下轉(zhuǎn)化成Z終的黃色。顏色的深淺和樣品中的纖溶酶原激活物YZ物-1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纖溶酶原激活物YZ物-1（PAI-1）濃度。試劑盒保存：；2-8℃。有效期：6個月注：信息僅供參考，具體產(chǎn)品信息以隨貨說明書為準上海華壹生物科技有限公司ShanghaiHuaYiBioTechnologyCo.,Ltd咨詢電話：021-24209195,13661674336[詳細]

  2018-11-15 10:00

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• 兔子纖溶酶原激活物YZ因子1（PAI-1）elisa試劑盒使用說明書

  兔子纖溶酶原激活物YZ因子1（PAI-1）elisa試劑盒使用說明書[詳細]

  2014-03-03 00:00

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• 人纖溶酶原激活物YZ因子(PAI)ELISA試劑盒

  人纖溶酶原激活物YZ因子(PAI)ELISA試劑盒[詳細]

  2013-12-06 00:00

  實驗操作

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• 人纖溶酶原激活物YZ劑1(PAI-1)ELISA試劑盒正確使用說

  人纖溶酶原激活物YZ劑1(PAI-1)ELISA試劑盒正確使用說[詳細]

  2014-07-14 00:00

  期刊論文

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• 纖溶酶原激活物YZ因子1分析測試

  纖溶酶原激活物YZ因子1分析測試目的：本試劑盒用于測定大鼠血清，血漿及相關液體樣本中纖溶酶原激活物YZ因子1(PAI-1)的含量。實驗原理：本試劑盒應用雙抗體夾心法測定標本中大鼠纖溶酶原激活物YZ因子1(PAI-1)水平。用純化的大鼠纖溶酶原激活物YZ因子1(PAI-1)抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入PAI-1，再與HRP標記的羊抗鼠抗體結合，形成抗體-抗原-酶標抗體復合物，經(jīng)過徹底洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍色，并在酸的作用下轉(zhuǎn)化成Z終的黃色。顏色的深淺和樣品中的PAI-1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大鼠纖溶酶原激活物YZ因子1(PAI-1)濃度。纖溶酶原激活物YZ因子1分析測試纖溶酶原激活物YZ因子1分析測試大鼠組織多肽抗原(TPA)ELISA試劑盒人凝血酶受體(TR)ELISA試劑盒人巨噬細胞趨化因子(MCF)ELISA試劑盒小鼠心肌轉(zhuǎn)錄因子GATA4ELISA試劑盒大鼠低氧誘導因子1α(HIF-1α)ELISA試劑盒大鼠前列腺素E2(PGE2)ELISA試劑盒雞血管內(nèi)皮細胞生長因子(VEGF)ELISA試劑盒大鼠胰島素樣生長因子2(IGF-2)ELISA試劑盒人類乳頭狀瘤病毒抗體牛乙酰乙酸檢測(ACAC)ELISA試劑盒雞白介素4(IL-4)ELISA試劑盒克氏雙糖鐵瓊脂（下層）250gBRV5tag標簽抗體明膠25g培養(yǎng)基蛋白質(zhì)原料單位：元貨號產(chǎn)品名稱規(guī)格價格備注BS7001胰蛋白胨250g32ARBS7002...人脊髓灰質(zhì)炎病毒IgG(PV-IgG)ELISA試劑盒人神經(jīng)趨化蛋白(fractalkine/CX3CL1)ELISA試劑盒人?；碳さ鞍?ASP)ELISA試劑盒小鼠胰島素自身抗體(IAA)ELISA試劑盒4-甲基傘形酮葡萄糖酸苷MUG培養(yǎng)基2000藥典25gBR魚雌激素（E）ELISA試劑盒大鼠可溶性腫瘤壞死因子相關凋亡誘導配體(sTRAIL)ELISA試劑盒豬載脂蛋白A1(apo-A1)ELISA試劑盒P53凋亡刺激蛋白2抗體纖溶酶原激活物YZ因子1分析測試[詳細]

  2018-10-23 10:30

  產(chǎn)品樣冊

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• 人纖溶酶原激活劑YZ物-1 （PAI-1）ELISA試劑盒說明書

  電話：021-6533363955229872網(wǎng)址：http://www.westang.com人纖溶酶原激活劑YZ物-1（PAI-1）ELISA試劑盒（用于血清、血漿、細胞培養(yǎng)上清液和其它生物體液內(nèi)）原理本實驗采用雙抗體夾心ABC-ELISA法。用抗人PAI-1單抗包被于酶標板上，標準品和樣品中的PAI-1與單抗結合，加入生物素化的抗人PAI-1，形成免疫復合物連接在板上，辣根過氧化物酶標記的Streptavidin與生物素結合，加入底物工作液顯藍色，Z后加終止液硫酸，在450nm處測OD值，PAI-1濃度與OD值成正比，可通過繪制標準曲線求出標本中PAI-1濃度。試劑盒組成（2-8℃保存）酶標板（CoatedWells）96孔酶標抗體工作液（EnzymeConjugate）12ml10×標本稀釋液（SampleBuffer）12ml20×濃縮洗滌液（WashBuffer）50ml標準品（Standards）：72ng/瓶2瓶底物工作液（TMBSolution）12ml**抗體工作液（BiotinylatedAntibody）12ml終止液（StopSolution）12ml準備試劑與收集血樣1.收集標本：血清、血漿（EDTA、檸檬酸鹽、肝素抗凝）、細胞培養(yǎng)上清液、組織勻漿等盡早檢測，2-8℃保存48小時；更長時間須冷凍（-20℃或-70℃）保存。2.標準品液配制：使用前加入0.3ml蒸餾水混勻，配成240ng/ml的溶液。設標準管8管，每管加入標本稀釋液300ul。在**管中加入240ng/ml的標準品溶液300ul混勻后用加樣器吸出300ul，移至第二管。如此反復作對倍稀釋，從第七管中吸出300ul棄去。第八管為空白對照。3.10×標本稀釋液用蒸餾水作1:10倍稀釋（示例：1ml濃稀釋液+9ml蒸餾水）。4.洗滌液：用重蒸水1:20稀釋（示例：1ml濃縮洗滌液加入19ml的重蒸水）檢測程序1.加樣：每孔各加入標準品或待測樣品100ul，將反應板充分混勻后置37℃120分鐘。2.洗板：用洗滌液將反應板充分洗滌4-6次，向濾紙上印干。3.每孔中加入**抗體工作液100ul。將反應板充分混勻后置37℃60分鐘。4.洗板：同前。5.每孔加酶標抗體工作液100ul。將反應板置37℃30分鐘。6.洗板：同前。7.每孔加入底物工作液100ul，置37℃暗處反應15分鐘。8.每孔加入100ul終止液混勻。9.30分鐘內(nèi)用酶標儀在450nm處測吸光值。結果計算與判斷1.所有OD值都應減除空白值后再行計算。2.以標準品120、60、30、15、7.5、3.75、1.875、0ng/ml為橫坐標，OD值為縱坐標，在坐標紙上作圖，畫出標準曲線。3.根據(jù)樣品OD值在該曲線圖上查出相應PAI-1含量即可。試劑盒性能1.靈敏度：Z小的PAI-1檢測濃度小于1ng/ml。2.特異性：可同時檢測重組或天然的人PAI-1。不與人其它細胞因子有交叉反應。3.重復性：板內(nèi)、板見變異系數(shù)均小于10％。注意事項1.以上標準孔及待測樣品均建議做復孔，每次測定應同時做標準曲線。2.洗滌過程很關鍵。洗滌不充分將導致極ng確度誤差及OD值錯誤地升高。3.板條開封后剩余板條要再封好，保持板條干燥。4.本試劑盒宜置4oC冰箱保存。5.本試劑盒僅用于科研，不能用于臨床診斷！[詳細]

  2018-09-13 10:01

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• 豬纖溶酶原激活物YZ因子(PAI)ELISA試劑盒

  豬纖溶酶原激活物YZ因子(PAI)ELISA試劑盒[詳細]

  2024-09-17 11:02

  應用文章

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• 大鼠纖溶酶原激活物YZ因子(PAI)ELISA試劑盒

  大鼠纖溶酶原激活物YZ因子(PAI)ELISA試劑盒[詳細]

  2024-09-29 00:38

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• 大鼠纖溶酶原激活劑YZ物-1 （PAI-1）ELISA試劑盒

  大鼠纖溶酶原激活劑YZ物-1（PAI-1）ELISA試劑盒（用于血清、血漿、細胞培養(yǎng)上清液和其它生物體液內(nèi)）原理本實驗采用雙抗體夾心ABC-ELISA法。用抗大鼠PAI-1單抗包被于酶標板上，標準品和樣品中的PAI-1與單抗結合，加入生物素化的抗大鼠PAI-1，形成免疫復合物連接在板上，辣根過氧化物酶標記的Streptavidin與生物素結合，加入底物工作液顯藍色，Z后加終止液硫酸，在450nm處測OD值，PAI-1濃度與OD值成正比，可通過繪制標準曲線求出標本中PAI-1濃度。試劑盒組成（2-8℃保存）酶標板（CoatedWells）96孔酶標抗體工作液（EnzymeConjugate）12ml10×標本稀釋液（SampleBuffer）12ml20×濃縮洗滌液（WashBuffer）50ml標準品（Standards）：600ng/瓶2瓶底物工作液（TMBSolution）12ml**抗體工作液（BiotinylatedAntibody）12ml終止液（StopSolution）12ml準備試劑與收集血樣1.收集標本：血清、血漿（EDTA、檸檬酸鹽、肝素抗凝）、細胞培養(yǎng)上清液、組織勻漿等盡早檢測，2-8℃保存48小時；更長時間須冷凍（-20℃或-70℃）保存，避免反復凍融。血清、血漿、細胞培養(yǎng)上清可能需要稀釋,請先做預實驗,以確定稀釋倍數(shù)。2.標準品液配制：使用前加入0.5ml蒸餾水混勻，配成1200ng/ml的溶液。設標準管8管，**管加標本稀釋液900ul，第二至第八管加入標本稀釋液500ul。在**管中加入1200ng/ml的標準品溶液100ul混勻后用加樣器吸出500ul，移至第二管。如此反復作對倍稀釋，從第七管中吸出500ul棄去。第八管為空白對照。3.10×標本稀釋液用蒸餾水作1:10倍稀釋（示例：1ml濃稀釋液+9ml蒸餾水）。4.洗滌液：用重蒸水1:20稀釋（示例：1ml濃縮洗滌液加入19ml的重蒸水）檢測程序1.加樣：每孔各加入標準品或待測樣品100ul，將反應板充分混勻后置37℃120分鐘。2.洗板：用洗滌液將反應板充分洗滌4-6次，向濾紙上印干。3.每孔中加入**抗體工作液100ul。將反應板充分混勻后置37℃60分鐘。4.洗板：同前。5.每孔加酶標抗體工作液100ul。將反應板置37℃30分鐘。6.洗板：同前。7.每孔加入底物工作液100ul，置37℃暗處反應15分鐘。8.每孔加入100ul終止液混勻。9.30分鐘內(nèi)用酶標儀在450nm處測吸光值。結果計算與判斷1.所有OD值都應減除空白值后再行計算。2.以標準品120、60、30、15、7.5、3.75、1.875、0ng/ml為橫坐標，OD值為縱坐標，在坐標紙上作圖，畫出標準曲線。3.根據(jù)樣品OD值在該曲線圖上查出相應PAI-1含量。試劑盒性能1.靈敏度：Z小的PAI-1檢測濃度小于1ng/ml。2.特異性：可同時檢測重組或天然的大鼠PAI-1。不與大鼠其它細胞因子有交叉反應。3.重復性：板內(nèi)、板見變異系數(shù)均小于10％。注意事項1.以上標準孔及待測樣品均建議做復孔，每次測定應同時做標準曲線。2.洗滌過程很關鍵。洗滌不充分將導致極ng確度誤差及OD值錯誤地升高。3.板條開封后剩余板條要再封好，保持板條干燥。4.本試劑盒宜置4oC冰箱保存。5.本試劑盒僅用于科研，不能用于臨床診斷！[詳細]

  2018-09-13 10:00

  產(chǎn)品樣冊

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• 纖溶酶原激活物YZ物

  纖溶酶原激活物YZ物[詳細]

  2024-09-28 01:25

  應用文章

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• 大鼠纖溶酶原激活劑YZ物(PAI－1)檢測試劑盒

  大鼠纖溶酶原激活劑YZ物(PAI－1)檢測試劑盒[詳細]

  2014-09-29 00:00

  標準

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• 人纖溶酶原（Plasminogen）ELISA試劑盒說明書

  電話：021-6533363955229872網(wǎng)址：http://www.westang.com人纖溶酶原（Plasminogen）ELISA試劑盒（用于血清、血漿、細胞培養(yǎng)上清液和尿液生物體液內(nèi)）原理本實驗采用雙抗體夾心ABC-ELISA法。用抗人Plasminogen單抗包被于酶標板上，標準品和樣品中的Plasminogen與單抗結合，加入生物素化的抗人Plasminogen，形成免疫復合物連接在板上，辣根過氧化物酶標記的Streptavidin與生物素結合，加入底物工作液顯藍色，Z后加終止液硫酸，在450nm處測OD值，Plasminogen濃度與OD值成正比，可通過繪制標準曲線求出標本中Plasminogen濃度。試劑盒組成（2-8℃保存）酶標板（CoatedWells）96孔酶標抗體工作液（EnzymeConjugate）12ml10×標本稀釋液（SampleBuffer）12ml20×濃縮洗滌液（WashBuffer）50ml標準品（Standards）：2000ng/瓶2瓶底物工作液（TMBSolution）12ml**抗體工作液（BiotinylatedAntibody）12ml終止液（StopSolution）12ml準備試劑與收集血樣1.收集標本：血清、血漿、細胞培養(yǎng)上清液等盡早檢測，2-8℃保存48小時；更長時間須冷凍（-20℃或-70℃）保存，避免反復凍融。血清、血漿、細胞培養(yǎng)上清至少作1：1000-10000倍稀釋（取10ul，加標本稀釋液990ul,稀釋100倍，然后再取前液10ul,加標本稀釋液990ul，此時一共稀釋了10000倍）。尿液不用稀釋直接檢測。2.標準品液配制：使用前加入1ml蒸餾水混勻，配成2000ng/ml的溶液。設標準管8管，**管加標本稀釋液900ul，第二至第八管加入標本稀釋液500ul。在**管中加入2000ng/ml的標準品溶液100ul混勻后用加樣器吸出500ul，移至第二管。如此反復作對倍稀釋，從第七管中吸出500ul棄去。第八管為空白對照。3.10×標本稀釋液用蒸餾水作1:10倍稀釋（示例：1ml濃稀釋液+9ml蒸餾水）。4.洗滌液：用重蒸水1:20稀釋（示例：1ml濃縮洗滌液加入19ml的重蒸水）檢測程序1.加樣：每孔各加入標準品或待測樣品100ul，將反應板充分混勻后置37℃120分鐘。2.洗板：用洗滌液將反應板充分洗滌4-6次，向濾紙上印干。3.每孔中加入**抗體工作液100ul。將反應板充分混勻后置37℃60分鐘。4.洗板：同前。5.每孔加酶標抗體工作液100ul。將反應板置37℃30分鐘。6.洗板：同前。7.每孔加入底物工作液100ul，置37℃暗處反應15分鐘。8.每孔加入100ul終止液混勻。9.30分鐘內(nèi)用酶標儀在450nm處測吸光值。結果計算與判斷1.所有OD值都應減除空白值后再行計算。2.以標準品200、100、50、25、12.5、6.25、3.12、0ng/ml為橫坐標，OD值為縱坐標，在坐標紙上作圖，畫出標準曲線。3.根據(jù)樣品OD值在該曲線圖上查出相應Plasminogen含量，再乘上稀釋倍數(shù)即可。試劑盒性能1.靈敏度：Z小的Plasminogen檢測濃度小于1.5ng/ml。2.特異性：可同時檢測重組或天然的人Plasminogen。不與人其它細胞因子有交叉反應。3.重復性：板內(nèi)、板見變異系數(shù)均小于10％。注意事項1.以上標準孔及待測樣品均建議做復孔，每次測定應同時做標準曲線。2.洗滌過程很關鍵。洗滌不充分將導致極ng確度誤差及OD值錯誤地升高。3.板條開封后剩余板條要再封好，保持板條干燥。4.本試劑盒宜置4oC冰箱保存。5.本試劑盒僅用于科研，不能用于臨床診斷！[詳細]

  2018-09-13 10:01

  產(chǎn)品樣冊

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• 人尿激酶型纖溶酶原激活物受體（PLAUR/uPAR）Elisa試劑盒說明書

  FORINVITROUSEANDRESEARCHUSEONLYNOTFORUSEINDIAGNOSTICORTHERAPEUTICPROCEDURES7thEdition（RevisedinNovember,2011）[INTENDEDUSE]ThekitisasandwichenzymeimmunoassayforinvitroquantitativemeasurementofuPARinhumanserum,plasmaandotherbiologicalfluids.[REAGENTSANDMATERIALSPROVIDED]ReagentsQuantityReagentsQuantityPre-coated,readytouse96-wellstripplate1Platesealerfor96wells4Standard（lyophilized）2StandardDiluent1×20mLDetectionReagentA（green）1×120μLAssayDiluentA（2×concentrate）1×6mLDetectionReagentB（red）1×120μLAssayDiluentB（2×concentrate）1×6mLTMBSubstrate1×9mLStopSolution1×6mLWashBuffer（30×concentrate）1×20mLInstructionmanual1[MATERIALSREQUIREDBUTNOTSUPPLIED]1.Microplatereaderwith450±10nmfilter.2.Precisionsingleormulti-channelpipettesanddisposabletips.3.EppendorfTubesfordilutingsamples.4.Deionizedordistilledwater.5.Absorbentpaperforblottingthemicrotiterplate.6.ContainerforWashSolution[STORAGEOFTHEKITS]1.Forunopenedkit:Allthereagents首ldbekeptaccordingtothelabelsonvials.TheStandard,DetectionReagentA,DetectionReagentBandthe96-wellstripplate首ldbestoredat-20oCuponreceiptwhiletheothers首ldbeat4oC.2.Foropenedkit:Whenthekitisopened,theremainingreagentsstillneedtobestoredaccordingtotheabovestoragecondition.Besides,pleasereturntheunusedwellstothefoilpouchcontainingthedesiccantpack,andresealalongentireedgeofzip-seal.Note:Itishighlyrecommendedtousetheremainingreagentswithin1monthprovidedthisiswithintheexpirationdateofthekit.[SAMPLECOLLECTIONANDSTORAGE]Serum-Allowsamplestoclotfortwohoursatroomtemperatureorovernightat4oCbeforecentrifugationfor20minutesatapproximately1000×g.Assayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Plasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gwithin30minutesofcollection.Removeplasmaandassayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Otherbiologicalfluids-Centrifugesamplesfor20minutesat1000×g.Removeparticulatesandassayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Note:1.Samplestobeusedwithin5daysmaybestoredat4oC,otherwisesamplesmustbestoredat-20oC（1month）or-80oC（2months）toavoidlossofbioactivityandcontamination.2.Samplehemolysiswillinfluencetheresult,sohemolyticspecimencannotbedetected.3.Whenperformingtheassay,bringsamplestoroomtemperature.[REAGENTPREPARATION]1.Bringallkitcomponentsandsamplestoroomtemperature（18-25oC）beforeuse.2.Standard-ReconstitutetheStandardwith1.0mLofStandardDiluent,keptfor10minutesatroomtemperature,shakegently（nottofoam）.Theconcentrationofthestandardinthestocksolutionis200ng/mL.Pleasefirstlydilutethestocksolutionto20ng/mLandthedilutedstandardservesasthehigheststandard（20ng/mL）.Thenprepare7tubescontaining0.5mLStandardDiluentandusethedilutedstandardtoproduceadoubledilutionseriesaccordingtothepictureshownbelow.Mixeachtubethoroughlybeforethenexttransfer.Setup7pointsofdilutedstandardsuchas20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL,andthelastEPtubeswithStandardDiluentistheblankas0ng/mL.3.AssayDiluentAandAssayDiluentB-Dilute6mLofAssayDiluentAorBConcentrate（2×）with6mLofdeionizedordistilledwatertoprepare12mLofAssayDiluentAorB.（Infact,morethan6mLAssayDiluentAandAssayDiluentBarecontainedinthebottles.Therefore,ineverytest,pleasepreciselypipetterequiredamountofDiluentandmakedoubledilutioninanewcontainer.Thepreparedworkingdilutioncan'tbefrozen.）Tube123456789ng/mL200201052.51.250.6250.31204.DetectionReagentAandDetectionReagentB-BrieflyspinorcentrifugethestockDetectionAandDetectionBbeforeuse.DilutetotheworkingconcentrationwithworkingAssayDiluentAorB,respectively（1:100）.5.WashSolution-Dilute20mLofWashSolutionconcentrate（30×）with580mLofdeionizedordistilledwatertoprepare600mLofWashSolution（1×）.6.TMBsubstrate-Aspiratetheneededdosageofthesolutionwithsterilizedtipsanddonotdumptheresidualsolutionintothevialagain.Note:1.Makingserialdilutioninthewellsdirectlyisnotpermitted.2.Preparestandardwithin15minutesbeforeassay.Pleasedonotdissolvethereagentsat37oCdirectly.3.PleasecarefullyreconstituteStandardsorworkingDetectionReagentAandBaccordingtotheinstruction,andavoidfoamingandmixgentlyuntilthecrystalsarecompletelydissolved.Tominimizeimprecisioncausedbypipetting,usesmallvolumesandensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10μLforoncepipetting.4.ThereconstitutedStandards,DetectionReagentAandDetectionReagentBcanbeusedonlyonce.5.IfcrystalshaveformedintheWashSolutionconcentrate（30×）,warmtoroomtemperatureandmixgentlyuntilthecrystalsarecompletelydissolved.6.Contaminatedwaterorcontainerforreagentpreparationwillinfluencethedetectionresult.[SAMPLEPREPARATION]1.Uscn,Inc.isonlyresponsibleforthekititself,butnotforthesamplesconsumedduringtheassay.Theuser首ldcalculatethepossibleamountofthesamplesusedinthewholetest.Pleasereservesufficientsamplesinadvance.2.Pleasepredicttheconcentrationbeforeassaying.Ifvaluesforthesearenotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsampledilutionsfortheirparticularexperiments.3.Ifthesamplesarenotindicatedinthemanual,apreliminaryexperimenttodeterminethevalidityofthekitisnecessary.4.TissueorcellextractionsamplespreparedbychemicallysisbuffermaycauseunexpectedELISAresultsduetotheimpactsfromcertainchemicals.5.Duetothepossibilityofmismatchingbetweenantigenfromotheroriginandantibodyusedinourkits（e.g.,antibodytargetsconformationalepitoperatherthanlinearepitope）,somenativeorrecombinantproteinsfromothermanufacturersmaynotberecognizedbyourproducts.6.Influencedbythefactorsincludingcellviability,cellnumberorsamp領time,samplesfromcellculturesupernatantmaynotbedetectedbythekit.7.Freshsampleswithoutlongtimestorageisrecommendedforthetest.Otherwise,proteindegradationanddenaturalizationmayoccurinthosesamplesandfinallyleadtowrongresults.[ASSAYPROCEDURE]1.Determinewellsfordilutedstandard,blankandsample.Prepare7wellsforstandard,1wellforblank.Add100μLeachofdilutionsofstandard（readReagentPreparation）,blankandsamplesintotheappropriatewells.CoverwiththePlatesealer.Incubatefor2hoursat37oC.2.Removetheliquidofeachwell,don’twash.3.Add100μLofDetectionReagentAworkingsolutiontoeachwell.Incubatefor1hourat37oCaftercoveringitwiththePlatesealer.4.Aspiratethesolutionandwashwith350μLof1×WashSolutiontoeachwellusingasquirtbottle,multi-channelpipette,manifolddispenserorautowasher,andletitsitfor1~2minutes.Removetheremainingliquidfromallwellscompletelybysnappingtheplateontoabsorbentpaper.Totallywash3times.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstabsorbentpaper.5.Add100μLofDetectionReagentBworkingsolutiontoeachwell.Incubatefor30minutesat37oCaftercoveringitwiththePlatesealer.6.Repeattheaspiration/washprocessfortotal5timesasconductedinstep4.7.Add90μLofSubstrateSolutiontoeachwell.CoverwithanewPlatesealer.Incubatefor15-25minutesat37oC（Don'texceed30minutes）.Protectfromlight.TheliquidwillturnbluebytheadditionofSubstrateSolution.8.Add50μLofStopSolutiontoeachwell.TheliquidwillturnyellowbytheadditionofStopsolution.Mixtheliquidbytappingthesideoftheplate.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.9.Removeanydropofwaterandfingerprintonthebottomoftheplateandconfirmthereisnobubbleonthesurfaceoftheliquid.Then,runthemicroplatereaderandconductmeasurementat450nmimmediately.Note:1.Assaypreparation:Keepappropriatenumbersofwellsfor1experimentandremoveextrawellsfrommicroplate.Restwells首ldberesealedandstoredat-20oC.2.SamplesorreagentsadditionPleaseusethefreshlypreparedStandard.Pleasecarefullyaddsamplestowellsandmixgentlytoavoidfoaming.Donottouchthewellwall.Foreachstepintheprocedure,totaldispensingtimeforadditionofreagentsorsamplestotheassayplate首ldnotexceed10minutes.Thiswillensureequalelapsedtimeforeachpipettingstep,withoutinterruption.Duplicationofallstandardsandspecimens,althoughnotrequired,isrecommended.Toavoidcross-contamination,changepipettetipsbetweenadditionsofstandards,samples,andreagents.Also,useseparatedreservoirsforeachreagent.3.Incubation:Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.Donotallowwellstosituncoveredforextendedperiodsbetweenincubationsteps.Oncereagentsareaddedtothewellstrips,DONOTletthestripsDRYatanytimeduringtheassay.Incubationtimeandtemperaturemustbecontrolled.4.Washing:Thewashprocedureiscritical.Completeremovalofliquidateachstepisessentialforgoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecantingandremoveanydropofwaterandfingerprintonthebottomoftheplate.Insufficientwashingwillresultinpoorprecisionandfalseelevatedabsorbancereading.5.Control領ofreactiontime:ObservethechangeofcolorafteraddingTMBSubstrate（e.g.observationonceevery10minutes）,ifthecoloristoodeep,addStopSolutioninadvancetoavoidexcessivelystrongreactionwhichwillresultininaccurateabsorbancereading.6.TMBSubstrateiseasilycontaminated.Pleaseprotectitfromlight.7.Theenvironmenthumiditywhichislessthan60%mighthavesomeeffectsonthefinalperformance,therefore,ahumidifierisrecommendedtobeusedatthatcondition.[TESTPRINCIPLE]Themicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictouPAR.Standardsorsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedantibodypreparationspecificforuPAR.Next,AvidinconjugatedtoHorseradishPeroxidase（HRP）isaddedtoeachmicroplatewellandincubated.AfterTMBsubstratesolutionisadded,onlythosewellsthatcontainuPAR,biotin-conjugatedantibodyandenzyme-conjugatedAvidinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofsulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm±10nm.TheconcentrationofuPARinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.[CALCULATIONOFRESULTS]Averagetheduplicatereadingsforeachstandard,control,andsamplesandsubtracttheaveragezerostandardopticaldensity.Createastandardcurveonlog-loggraphpaper,withuPARconcentrationonthey-axisandabsorbanceonthex-axis.Drawthebestfitstraightlinethroughthestandardpointsanditcanbedeterminedbyregressionanalysis.Usingsomeplotsoftware,forinstance,curveexpert1.30,isalsorecommended.Ifsampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.[TYPICALDATA]Inordertomakethecalculationeasier,weplottheO.D.valueofthestandard（X-axis）againsttheknownconcentrationofthestandard（Y-axis）,althoughconcentrationistheindependentvariableandO.D.valueisthedependentvariable.However,theO.D.valuesofthestandardcurvemayvaryaccordingtotheconditionsofassayperformance（e.g.operator,pipettingtechnique,washingtechniqueortemperatureeffects）,plottinglogofthedatatoestablishstandardcurveforeachtestisrecommended.Typicalstandardcurvebelowisprovidedforreferenceonly.TypicalStandardCurveforHumanuPARELISA.[DETECTIONRANGE]0.312-20ng/mL.ThestandardcurveconcentrationsusedfortheELISA’swere20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL.[SENSITIVITY]TheminimumdetectabledoseofhumanuPARistypicallylessthan0.123ng/mL.Thesensitivityofthisassay,orLowerLimitofDetection（LLD）wasdefinedasthelowestproteinconcentrationthatcouldbedifferentiatedfromzero.Itwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.[SPECIFICITY]ThisassayhashighsensitivityandexcellentspecificityfordetectionofhumanuPAR.Nosignificantcross-reactivityorinterferencebetweenhumanuPARandanalogueswasobserved.Note:Limitedbycurrentskillsandknowledge,itisimpossibleforustocompletethecross-reactivitydetectionbetweenhumanuPARandalltheanalogues,therefore,crossreactionmaystillexist.[RECOVERY]MatriceslistedbelowwerespikedwithcertainlevelofrecombinanthumanuPARandtherecoveryrateswerecalculatedbycomparingthemeasuredvaluetotheexpectedamountofuPARinsamples.MatrixRecoveryrange（%）Average（%）humanserum（n=5）91-10597humanEDTAplasma（n=5）95-10498humanheparinplasma（n=5）80-9688[LINEARITY]ThelinearityofthekitwasassayedbytestingsamplesspikedwithappropriateconcentrationofhumanuPARandtheirserialdilutions.Theresultsweredemonstratedbythepercentageofcalculatedconcentrationtotheexpected.Sample121418116humanserum（n=5）95-109%80-91%91-107%81-95%humanEDTAplasma（n=5）93-101%76-99%90-99%83-93%humanheparinplasma（n=5）78-96%94-107%101-106%89-98%[PRECISION]Intra-assayPrecision（Precisionwithinanassay）:3sampleswithlow,middleandhighlevelhumanuPARweretested20timesononeplate,respectively.Inter-assayPrecision（Precisionbetweenassays）:3sampleswithlow,middleandhighlevelhumanuPARweretestedon3differentplates,8replicatesineachplate.CV（%）=SD/meanX100Intra-Assay:CV<10%Inter-Assay:CV<12%[STABILITY]ThestabilityofELISAkitisdeterminedbythelossrateofactivity.Thelossrateofthiskitislessthan5%withintheexpirationdateunderappropriatestoragecondition.Thelossratewasdeterminedbyacceleratedthermaldegradationtest.Keepthekitat37oCfor3days,andcompareO.D.valuesofthekitkeptat37oCwiththatofatrecommendedtemperature.（referringfromChinaBiologicalProductsStandard,whichwascalculatedbytheArrheniusequation.ForELISAkit,1daystorageat37oCcanbeconsideredas2monthsat4oC,whichmeans3daysat37oCequa領6monthsat4oC）.Note:Tominimizeextrainfluenceontheperformance,operationproceduresandlabconditions,especiallyroomtemperature,airhumidity,incubatortemperature首ldbestrictlycontrolled.Itisalsostronglysuggestedthatthewholeassayisperformedbythesameoperatorfromthebeginningtotheend.[ASSAYPROCEDURESUMMARY]1.Prepareallreagents,samplesandstandards;2.Add100μLstandardorsampletoeachwell.Incubate2hoursat37oC;3.Add100μLpreparedDetectionReagentA.Incubate1hourat37oC;4.Aspirateandwash3times;5.Add100μLpreparedDetectionReagentB.Incubate30minutesat37oC;6.Aspirateandwash5times;7.Add90μLSubstrateSolution.Incubate15-25minutesat37oC;8.Add50μLStopSolution.Readat450nmimmediately.[IMPORTANTNOTE]1.Limitedbythecurrentconditionandscientifictechnology,wecan'tcompletelyconductthecomprehensiveidentificationandanalysisontherawmaterialprovidedbysuppliers.Sotheremightbesomequalitativeandtechnicalriskstousethekit.2.Thefinalexperimentalresultswillbecloselyrelatedtovalidityoftheproducts,operationskillsoftheendusersandtheexperimentalenvironments.Pleasemakesurethatsufficientsamplesareavailable.3.Kitsfromdifferentbatchesmaybealittledifferentindetectionrange,sensitivityandcolordevelopingtime.Pleaseperformtheexperimentexactlyaccordingtotheinstructionattachedinkitwhileelectroniconesfromourwebsite（www.uscnk.us;www.uscnk.cn;www.uscnk.com）isonlyforinformation.4.Donotmixorsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.5.Protectallreagentsfromstronglightduringstorageandincubation.Allthebottlecapsofreagents首ldbecoveredtightlytopreventtheevaporationandcontaminationofmicroorganism.6.Theremaybesomefoggysubstanceinthewellswhentheplateisopenedatthefirsttime.Itwillnothaveanyeffectonthefinalassayresults.Donotremovemicrotiterplatefromthestoragebaguntilneeded.7.Wrongoperationsduringthereagentspreparationandloading,aswellasincorrectparametersettingfortheplatereadermayleadtoincorrectresults.Amicroplateplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3O.D.orgreaterat450±10nmwavelengthisacceptableforuseinabsorbancemeasurement.Pleasereadtheinstructioncarefullyandadjusttheinstrumentpriortotheexperiment.Formoreinformation,pleaserefertotheoperationvideo（http://www.uscnk.com/homepage/operate-elisa.htm）.8.Eventhesameoperatormightgetdifferentresultsintwoseparateexperiments.Inordertogetbetterreproducibleresults,theoperationofeverystepintheassay首ldbecontrolled.Furthermore,apreliminaryexperimentbeforeassayforeachbatchisrecommended.9.EachkithasbeenstrictlypassedQ.Ctest.However,resultsfromendusersmightbeinconsistentwithourin-housedataduetosomeunexpectedtransportationconditionsordifferentlabequipments.Intra-assayvarianceamongkitsfromdifferentbatchesmightarisefromabovefactors,too.10.Kitsfromdifferentmanufacturerswiththesameitemmightproducedifferentresults,sincewehaven’tcomparedourproductswithothermanufacturers.11.Validperiod:sixmonths.12.Theinstructionmanualalsosuitsforthekitof48T,butallreagentsof48Tkitarereducedbyhalf.[PRECAUTION]TheStopSolutionsuggestedforusewiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.[詳細]

  2018-10-01 10:01

  產(chǎn)品樣冊

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• （原裝進口）人尿激酶型纖溶酶原激活物受體（uPAR）Elisa試劑盒說明書

  QuantikineHumanuPARImmunoassayCatalogNumberDUP00Forthequantitativedeterminationofhumanurokinase-typeplasminogenactivatorreceptor（uPAR）concentrationsincellculturesupernates,serum,plasma,andurine.Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.TABLEOFCONTENTSContentsPageINTRODUCTION2PRINCIPLEOFTHEASSAY..................................3LIMITATIONSOFTHEPROCEDURE3MATERIALSPROVIDED....................................3STORAGE4OTHERSUPPLIESREQUIRED.................................4PRECAUTION4SAMPLECOLLECTIONANDSTORAGE............................5SAMPLEPREPARATION5REAGENTPREPARATION...................................6ASSAYPROCEDURE7ASSAYPROCEDURESUMMARY...............................8CALCULATIONOFRESULTS9TYPICALDATA.........................................9TECHNICALHINTS10PRECISION...........................................10LINEARITY11SENSITIVITY..........................................11CALIBRATION11SAMPLEVALUES.......................................12SPECIFICITY13REFERENCES.........................................14PLATELAYOUT15MANUFACTUREDANDDISTRIBUTEDBY:R&DSystems,Inc.TELEPHONE:（800）343-7475614McKinleyPlaceNE（612）379-2956Minneapolis,MN55413FAX:（612）656-4400UnitedStatesofAmericaE-MAIL:info@RnDSystems.comDISTRIBUTEDBY:R&DSystemsEurope,Ltd.19BartonLaneTELEPHONE:+44（0）1235529449AbingdonScienceParkFAX:+44（0）1235533420Abingdon,OX143NBE-MAIL:info@RnDSystems.co.ukUnitedKingdomR&DSystemsChinaCo.Ltd.24A1HuaMinEmpirePlazaTELEPHONE:+86（21）52380373726WestYanAnRoadFAX:+86（21）52371001ShanghaiPRC200050E-MAIL:info@RnDSystemsChina.com.cnINTRODUCTIONUrokinase-typeplasminogenactivatorreceptor（uPAR,CD87）isacellsurfacereceptorthatbindsurokinase-typeplasminogenactivator（uPA）withhighaffinity,therebyfacilitatingthepericellularactivationofplasminogen（seereferences1and2forreviews）.uPAR,uPAandplasminogenactivatorinhibitor-1（PAI-1）,formatriadwithmultiplefunctions,includingregulationofcellattachment,migration,proliferationanddifferentiation,bybothproteolyticandnonproteolyticmechanisms（2）.uPARisanchoredtoextracellularsurfacesthroughaglycosylphosphatidylinositol（GPI）linkage,withnotransmembranedomain（3）.uPARissynthesizedasa335-amino-acidpolypeptidethatincludesa22-residuesignalpeptide（4）.A30-residuepeptideiscleavedfromtheC-terminusduringadditionoftheGPIanchor（3）.WithlossofthesignalpeptideandtheC-terminalpeptide,thematureproteinhas283aminoacids.Itisvariablyglycosylated,increasingitsmassfromabout31kDafortheproteinbackbonetoasmuchas55kDaforthematureglycoprotein（5）.Pro-uPA,asingle-chainprotein,isactivatedtoadisulfide-linked,two-chainproteinbyproteolyticcleavagebyplasmin（1,2,6）.Eitherpro-uPAortheactivetwo-chainuPAbindwithhighaffinitytouPAR.Thus,tracesofplasminactivatepro-uPAtouPA,leadingtoincreasinggenerationofplasmininapositivefeedbackloopthatisamplifiedbyuPAR.Whiletheinitiatingeventisnotclear,theeffectisthegenerationofplasminatthecellsurface,whereitdegradestheextracellularmatrixbyactivatingmatrixmetalloproteinases.Thisappearstobeakeyeventintumorinvasivenessandmetastasisandinmigrationofcellsingeneral（1,2,7）.ThefunctionsoftheuPA/uPARsystemare,however,moreextensivethanmediationofplasminformation,andtheyincludenon-proteolyticfunctions（1,2）.uPA/uPARislocalizedtofocalcontactpointsofcellswithinthesubstratum.Thesesitesincludeintracellularvinculinandtheextracellularadhesionmoleculevitronectin,suggestingdirectadhesivefunctionsandintracellularsignal領functionsforuPAR.uPARbindstointegrins,anditcontainschemotacticactivityinitssingleprotease-sensitiveregion.uPARhasbeenmeasuredinhumanplasma（7-9）.SolubleuPARisgeneratedbyremovaloftheGPIanchorbyanendogenousphospholipaseD,freeinguPARofitssurfaceattachment（10）.uPARiselevatedinplasmaofpatientswithparoxysmalnocturnalhemoglobinuria（7,8）,amanifestationofaninabilitytoaddGPIanchorstoproteins.IthasbeenpostulatedthattherealsomaybesolubleuPARduetoalternativesplicingoftheprimarytranscript（1）,ashasbeendemonstratedformouseuPAR（11）.uPARhasbeenidentifiedinurine,wherethelevelisaconsistentfractionofcreatinineconcentration（12）.TheQuantikineHumanuPARImmunoassayisa4.5hoursolid-phaseELISAdesignedtomeasurehumanuPARincellculturesupernates,serum,plasma,andurine.ItcontainsNS0-expressedrecombinanthumanuPARandantibodiesraisedagainsttherecombinantfactor.Ithasbeenshowntoaccuratelyquantitatetherecombinantfactor.ResultsobtainedusingnaturalhumanuPARshowedlinearcurvesthatwereparalleltothestandardcurvesobtainedusingtheQuantikinekitstandards.TheseresultsindicatethatthiskitcanbeusedtodeterminerelativemassvaluesofnaturalhumanuPAR.2PRINCIPLEOFTHEASSAYThisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.AmonoclonalantibodyspecificforuPARhasbeenpre-coatedontoamicroplate.StandardsandsamplesarepipettedintothewellsandanyuPARpresentisboundbytheimmobilizedantibody.Afterwashingawayanyunboundsubstances,anenzyme-linkedpolyclonalantibodyspecificforuPARisaddedtothewells.Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolutionisaddedtothewellsandcolordevelopsinproportiontotheamountofuPARboundintheinitialstep.Thecolordevelopmentisstoppedandtheintensityofthecolorismeasured.LIMITATIONSOFTHEPROCEDUREFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Thekit首ldnotbeusedbeyondtheexpirationdateonthekitlabel.Donotmixorsubstitutereagentswithotherlotsorsources.Ifsamplesfalloutsidethedynamicrangeoftheassay,furtherdilutethesampleswithCalibratorDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.Thisassayisdesignedtoeliminateinterferencebyligandsandotherproteinspresentinbiologicalsamples.UntilallfactorshavebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.MATERIALSPROVIDEDuPARMicroplate（Part890714）-96wellpolystyrenemicroplate（12stripsof8wells）coatedwithamousemonoclonalantibodyagainstuPAR.uPARConjugate（Part890715）-21mLofpolyclonalantibodyagainstuPARconjugatedtohorseradishperoxidasewithpreservatives.uPARStandard（Part890716）-40ngofrecombinanthumanuPARinabufferedproteinbasewithpreservatives;lyophilized.AssayDiluentRD1W（Part895117）-11mLofabufferedproteinbasewithpreservatives.CalibratorDiluentRD6P（Part895118）-21mLofanimalserumwithpreservatives.WashBufferConcentrate（Part895003）-21mLofa25-foldconcentratedsolutionofbufferedsurfactantwithpreservatives.ColorReagentA（Part895000）-12.5mLofstabilizedhydrogenperoxide.ColorReagentB（Part895001）-12.5mLofstabilizedchromogen（tetramethylbenzidine）.StopSolution（Part895032）-6mLof2Nsulfuricacid.PlateCovers-4adhesivestrips.3STORAGEUnopenedKitStoreat2-8°C.Donotusepastkitexpirationdate.Opened/ReconstitutedReagentsDilutedWashBufferMaybestoredforupto1monthat2-8°C.*StopSolutionAssayDiluentRD1WCalibratorDiluentRD6PConjugateUnmixedColorReagentAUnmixedColorReagentBStandardMicroplateWellsReturnunusedwellstothefoilpouchcontainingthedesiccantpack,resealalongentireedgeofzip-seal.Maybestoredforupto1monthat2-8°C.**Providedthisiswithintheexpirationdateofthekit.OTHERSUPPLIESREQUIREDMicroplatereadercapableofmeasuringabsorbanceat450nm,withthecorrectionwavelengthsetat540nmor570nm.Pipettesandpipettetips.Deionizedordistilledwater.Squirtbottle,manifolddispenser,orautomatedmicroplatewasher.500mLgraduatedcylinder.Testtubesfordilution.HumanuPARControls（optional;availablefromR&DSystems）.PRECAUTIONTheStopSolutionprovidedwiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.4SAMPLECOLLECTIONANDSTORAGECellCultureSupernates-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Serum-Useaserumseparatortube（SST）andallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesat1000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Plasma-CollectplasmausingheparinorEDTAasananticoagulant.Centrifugeat1000xgwithin30minutesofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Note:Citrateplasmahasnotbeenvalidatedforuseinthisassay.LipemicsamplesandsamplescontaininghighlevelsofhumanserumalbuminarenotsuitableforthemeasurementofhumanuPARwiththisassay.Urine-Asepticallycollectthefirsturineoftheday（mid-stream）,voideddirectlyintoasterilecontainer.Centrifugetoremoveparticulatematter.Assayimmediatelyoraliquotandstoreat-20°C.Avoidrepeatedfreeze-thawcycles.Formoreinformationoncollectingandhand領urinespecimens,refertotheNationalCommitteeforClinicalLaboratoryStandards（NCCLS）publicationGP16-T.SAMPLEPREPARATIONCellculturesupernates,serum,plasma,andurinesamplesrequireatleasta5-folddilution.Asuggested5-folddilutionis25Lofsample+100LofCalibratorDiluentRD6P.5REAGENTPREPARATIONBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalshaveformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalshavecompletelydissolved.Dilute20mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare500mLofWashBuffer.SubstrateSolution-ColorReagentsAandB首ldbemixedtogetherinequalvolumeswithin15minutesofuse.Protectfromlight.200Loftheresultantmixtureisrequiredperwell.uPARStandard-ReconstitutetheuPARStandardwith1.0mLofdeionizedordistilledwater.Thisreconstitutionproducesastocksolutionof40,000pg/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingdilutions.Pipette450LofCalibratorDiluentRD6Pintothe4000pg/mLtube.Pipette200LofCalibratorDiluentRD6Pintotheremainingtubes.Usethestocksolutiontoproduceadilutionseries（below）.Mixeachtubethoroughlybeforethenexttransfer.The4000pg/mLstandardservesasthehighstandard.CalibratorDiluentRD6Pservesasthezerostandard（0pg/mL）.6ASSAYPROCEDUREBringallreagentsandsamplestoroomtemperaturebeforeuse.Itisrecommendedthatallsamples,standards,andcontrolsbeassayedinduplicate.1.Prepareallreagents,workingstandards,andsamplesasdirectedintheprevioussections.2.Removeexcessmicroplatestripsfromtheplateframe,returnthemtothefoilpouchcontainingthedesiccantpack,andreseal.3.Add100LofAssayDiluentRD1Wtoeachwell.4.Add50LofStandard,control,orsample*perwell.Coverwiththeadhesivestripprovided.Incubatefor2hoursatroomtemperature.Aplatelayoutisprovidedtorecordstandardsandsamplesassayed.5.Aspirateeachwellandwash,repeatingtheprocessthreetimesforatotaloffourwashes.Washbyfil領eachwellwithWashBuffer（400L）usingasquirtbottle,manifolddispenser,orautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.6.Add200LofuPARConjugatetoeachwell.Coverwithanewadhesivestrip.Incubatefor2hoursatroomtemperature.7.Repeattheaspiration/washasinstep5.8.Add200LofSubstrateSolutiontoeachwell.Incubatefor30minutesatroomtemperature.Protectfromlight.9.Add50LofStopSolutiontoeachwell.Thecolorinthewells首ldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorifthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.10.Determinetheopticaldensityofeachwellwithin30minutes,usingamicroplatereadersetto450nm.Ifwavelengthcorrectionisavailable,setto540nmor570nm.Ifwavelengthcorrectionisnotavailable,subtractreadingsat540nmor570nmfromthereadingsat450nm.Thissubtractionwillcorrectforopticalimperfectionsintheplate.Readingsmadedirectlyat450nmwithoutcorrectionmaybehigherandlessaccurate.*Samplesrequiredilution.SeeSamplePreparationsection.7ASSAYPROCEDURESUMMARY8CALCULATIONOFRESULTSAveragetheduplicatereadingsforeachstandard,control,andsampleandsubtracttheaveragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingalog/logcurve-fit.Asanalternative,constructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainsttheconcentrationonthex-axisanddrawabestfitcurvethroughthepointsonalog/loggraph.ThedatamaybelinearizedbyplottingthelogoftheuPARconcentrationsversusthelogoftheO.D.onalinearscale,andthebestfitlinecanbedeterminedbyregressionanalysis.Ifthesampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.TYPICALDATAThisstandardcurveisprovidedfordemonstrationonly.Astandardcurve首ldbegeneratedforeachsetofsamplesassayed.9pg/mL062.5125250500100020004000O.D.0.0460.0440.0720.0730.1050.1050.1700.1710.3000.3040.5650.5451.0661.0752.1222.076Average0.0450.0720.1050.1700.3020.5551.0702.099Corrected___0.0270.0600.1250.2570.5101.0252.054TECHNICALHINTSWhenmixingorreconstitutingproteinsolutions,alwaysavoidfoaming.Toavoidcross-contamination,changepipettetipsbetweenadditionsofeachstandardlevel,betweensampleadditions,andbetweenreagentadditions.Also,useseparatereservoirsforeachreagent.Whenusinganautomatedplatewasher,addinga30secondsoakperiodfollowingtheadditionofwashbuffer,and/orrotatingtheplate180degreesbetweenwashstepsmayimproveassayprecision.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.SubstrateSolution首ldremaincolorlessuntiladdedtotheplate.KeeptheSubstrateSolutionprotectedfromlight.SubstrateSolution首ldchangefromcolorlesstogradationsofblue.StopSolution首ldbeaddedtotheplateinthesameorderastheSubstrateSolution.ThecolordevelopedinthewellswillturnfrombluetoyellowuponadditionoftheStopSolution.WellsthataregreenincolorindicatethattheStopSolutionhasnotmixedthoroughlywiththeSubstrateSolution.PRECISIONIntra-assayPrecision（Precisionwithinanassay）Threesamplesofknownconcentrationweretestedtwentytimesononeplatetoassessintra-assayprecision.Inter-assayPrecision（Precisionbetweenassays）Threesamplesofknownconcentrationweretestedinfortyseparateassaystoassessinter-assayprecision.Intra-assayPrecisionInter-assayPrecisionSample123123n202020404040Mean（pg/mL）8361593241279615462300Standarddeviation17.365.418144.378.9136CV（%）2.14.17.55.65.15.910LINEARITYToassessthelinearityoftheassay,samplesspikedwithhighconcentrationsofuPARweredilutedwithCalibratorDiluentRD6Ptoproducesampleswithvalueswithinthedynamicrangeoftheassay.Cellculturemedia*（n=4）Serum*（n=5）Heparinplasma*（n=5）EDTAplasma*（n=5）Urine*（n=5）1:2Average%ofExpected10396-112104101-105104101-10610194-105102Range（%）99-1051:4Average%ofExpected109101-115106103-111107104-110105101-108104Range（%）95-1121:8Average%ofExpected109101-111106103-112106102-110106103-113108Range（%）104-1131:16Average%ofExpected10999-11410599-11110598-11010498-111108Range（%）103-112*Sampleswerediluted5-foldpriortoassayasdirectedinSamplePreparation.SENSITIVITYTheminimumdetectabledose（MDD）ofuPARistypicallylessthan33pg/mL.TheMDDwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.CALIBRATIONThisimmunoassayiscalibratedagainstahighlypurifiedNS0-expressedrecombinanthumanuPARproducedatR&DSystems.11SAMPLEVALUESSerum/Plasma/Urine-SamplesfromapparentlyhealthyvolunteerswereevaluatedforthepresenceofuPARinthisassay.Nomedicalhistorieswereavailableforthedonorsusedinthisstudy.SampleTypeMean（pg/mL）Range（pg/mL）Serum（n=60）23701195-4415Heparinplasma（n=35）2165977-5347EDTAplasma（n=35）1871864-3829Urine（n=35）2975691-6098CellCultureSupernates-Humanperipheralbloodmononuclearcells（5x106cells/mL）wereculturedinRPMIsupplementedwith5%fetalcalfserum,50M-mercaptoethanol,2mML-glutamine,100U/mLpenicillin,and100g/mLstreptomycinsulfate.Thecellswereculturedunstimulatedorstimulatedwith10g/mLPHA.AliquotsofthecellculturesupernateswereremovedandassayedforlevelsofnaturaluPAR.ConditionDay1（pg/mL）Day5（pg/mL）Unstimulated12282123Stimulated10,005689512SPECIFICITYThisassayrecognizesrecombinantandnaturalhumanuPAR.Thefactorslistedbelowwerepreparedat50ng/mLinCalibratorDiluentRD6Pandassayedforcross-reactivity.Preparationsofthefollowingfactorsat50ng/mLinamid-rangerecombinanthumanuPARcontrolwereassayedforinterference.Nosignificantcross-reactivityorinterferencewasobserved.RecombinanthumanuPAdoesnotcross-reactinthisassaybutdoesinterfereatconcentrationsgreaterthan3000pg/mL.13Recombinanthuman:ANGARCNTF-ECGFEGFEpoFGFacidicFGFbasicFGF-4FGF-5FGF-6Flt-3LigandG-CSFGM-CSFsgp130GROGROGROHB-EGFHGFIFN-IGF-IIGF-IIIL-1IL-1IL-1raIL-1sRIIL-1sRIIIL-2IL-2sRIL-3IL-3sRIL-4IL-4sRIL-5IL-5sRIL-5sRIL-6IL-6sRIL-7IL-8IL-9IL-10IL-11IL-12IL-13KGF（FGF-7）LAP（TGF-1）LIFM-CSFMCP-1MIP-1MIP-1-NGFOSMPD-ECGFPDGF-AAPDGF-ABPDGF-BBPTNRANTESSCFSLPITGF-TGF-1TGF-2TGF-3TGF-sRIITNF-TNF-sTNFRIsTNFRIIVEGFRecombinantmouse:GM-CSFIL-1IL-1IL-3IL-4IL-5IL-6IL-7IL-9IL-10IL-13LIFMIP-1MIP-1SCFTNF-uPARRecombinantamphibian:TGF-5Naturalproteins:bovineFGFacidicbovineFGFbasichumanPDGFporcinePDGFhumanTGF-1porcineTGF-1REFERENCES1.Dear,A.E.andR.L.Medcalf（1998）Eur.J.Biochem.252:185.2.Blasi,F.（1997）ImmunlogyToday18:415.3.Ploug,M.etal.（1991）J.Biol.Chem.266:1926.4.Roldan,A.L.（1990）EMBOJ.9:467.5.Behrendt,N.（1990）J.Biol.Chem.265:6453.6.Blasi,F.etal.（1987）J.CellBiol.104:801.7.Ronne,E.etal.（1995）Br.J.Haematol.89:576.8.Ploug,M.etal.（1992）Blood79:1447.9.Stephens,R.W.etal.（1999）J.Natl.CancerInst.91:869.10.Wilhelm,O.G.etal.（1999）J.CellularPhysiol.180:225.11.Kristensen,P.etal.（1991）J.CellBiol.115:1763.12.Sier,C.F.M.etal.（1999）Lab.Invest.79:717.14PLATELAYOUTUsethisplatelayouttorecordstandardsandsamplesassayed.2010R&DSystems,Inc.12.99750440.57/10[詳細]

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