小鼠ⅰ型膠原n末端肽ntxelisa試劑盒說(shuō)明書(shū)

MERGEFIELD"種屬中文"小鼠MERGEFIELD"指標(biāo)中文"Ⅰ型膠原N末端肽(MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX)酶聯(lián)免疫吸附測(cè)定試劑盒使用說(shuō)明書(shū) 產(chǎn)品編號(hào)：MERGEFIELD"編號(hào)"E-EL-M0360 （本試劑盒僅供體外研究使用、不用于臨床診斷!）聲明：尊敬的客戶，感謝您選用本公司的產(chǎn)品。本產(chǎn)品選用世界著名生產(chǎn)廠家的原料，采用專(zhuān)業(yè)ELISAkit生產(chǎn)技術(shù)制造。合用于體外定量檢測(cè)MERGEFIELD"種屬中文"小鼠血清、血漿、組織勻漿或細(xì)胞培養(yǎng)上清液中天然和重組MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX濃度。使用前請(qǐng)仔細(xì)閱讀說(shuō)明書(shū)并檢查試劑組分！如有疑問(wèn)，請(qǐng)及時(shí)聯(lián)系伊萊瑞特生物科技有限公司。試劑盒組成：名稱-中文名稱-英文規(guī)格保存條件ELISA酶標(biāo)板MicroELISAPlate8×12/8×6*4℃凍干標(biāo)準(zhǔn)品ReferenceStandard2/1支*4℃標(biāo)準(zhǔn)品&樣品稀釋液ReferenceStandard&SampleDiluent1瓶20mL/12mL*4℃濃縮生物素化抗體ConcentratedBiotinylatedDetectionAb1支120μL/70μL*4℃生物素化抗體稀釋液DiluentforBiotinylatedDetectionAb1瓶10mL/6mL*4℃濃縮HRP酶結(jié)合物ConcentratedHRPConjugate1支120μL/70μL*4℃(避光)酶結(jié)合物稀釋液DiluentforHRPConjugate1瓶10mL/6mL*4℃濃縮洗滌液（25×）ConcentratedWashBuffer（25×）1瓶30mL/16mL*4℃底物溶液（TMB）SubstrateReagent1瓶10mL/6mL*4℃(避光)反映終止液StopSolution1瓶10mL/6mL*4℃封板覆膜PlateSealer5/3張*產(chǎn)品說(shuō)明書(shū)ProductDescription1份*:[96T/48T]（打開(kāi)包裝后請(qǐng)及時(shí)檢查所有物品是否齊全完整）檢測(cè)原理:本試劑盒采用雙抗體夾心ELISA法。用抗MERGEFIELD"種屬中文"小鼠MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX抗體包被于酶標(biāo)板上，實(shí)驗(yàn)時(shí)標(biāo)本或標(biāo)準(zhǔn)品中的MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX會(huì)與包被抗體結(jié)合，游離的成分被洗去。依次加入生物素化的抗MERGEFIELD"種屬中文"小鼠MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX抗體和辣根過(guò)氧化物酶標(biāo)記的親和素。抗MERGEFIELD"種屬中文"小鼠MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX抗體與結(jié)合在包被抗體上的MERGEFIELD"種屬中文"小鼠MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX結(jié)合、生物素與親和素特異性結(jié)合而形成免疫復(fù)合物，游離的成分被洗去。加入顯色底物(TMB)，TMB在辣根過(guò)氧化物酶的催化下現(xiàn)藍(lán)色，加終止液后變黃。用酶標(biāo)儀在450nm波長(zhǎng)處測(cè)OD值，MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX濃度與OD450值之間呈正比，通過(guò)繪制標(biāo)準(zhǔn)曲線求出標(biāo)本中MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX的濃度。標(biāo)本收集:血清：全血標(biāo)本于室溫放置2小時(shí)或4℃過(guò)夜后于1000×g離心20分鐘，取上清即可檢測(cè)，收集血液的試管應(yīng)為一次性的無(wú)熱原，無(wú)內(nèi)毒素試管。血漿：抗凝劑推薦使用EDTA.Na2，標(biāo)本采集后30分鐘內(nèi)于1000×g離心15分鐘，取上清即可檢測(cè)。避免使用溶血，高血脂標(biāo)本。組織勻漿：用預(yù)冷的PBS(0.01M,pH=7.4)沖洗組織，以去除殘留血液（勻漿中裂解的紅細(xì)胞會(huì)影響測(cè)量結(jié)果），稱重后將組織剪碎。將剪碎的組織與相應(yīng)體積的PBS（一般按1:9的重量體積比，比如1g的組織樣本相應(yīng)9mL的PBS，具體體積可根據(jù)實(shí)驗(yàn)需要適當(dāng)調(diào)整，并做好記錄。推薦在PBS中加入蛋白酶克制劑）加入玻璃勻漿器中，于冰上充足研磨。為了進(jìn)一步裂解組織細(xì)胞，可以對(duì)勻漿液進(jìn)行超聲破碎，或反復(fù)凍融。最后將勻漿液于5000×g離心5~10分鐘，取上清檢測(cè)。細(xì)胞培養(yǎng)上清：取細(xì)胞培養(yǎng)上清于1000×g離心20分鐘，除去雜質(zhì)及細(xì)胞碎片。取上清檢測(cè)。其它生物標(biāo)本：1000×g離心20分鐘，取上清即可檢測(cè)（具體解決方法可參考：）標(biāo)本應(yīng)清澈透明，懸浮物應(yīng)離心去除。標(biāo)本收集后若不及時(shí)檢測(cè)，請(qǐng)按一次使用量分裝，凍存于-20℃/-80℃冰箱內(nèi)，避免反復(fù)凍融，1-6月內(nèi)檢測(cè),4℃保存的應(yīng)在1周內(nèi)進(jìn)行檢測(cè)。假如您的樣本中檢測(cè)物濃度高于標(biāo)準(zhǔn)品最高值，請(qǐng)根據(jù)實(shí)際情況，做適當(dāng)倍數(shù)稀釋(建議先做預(yù)實(shí)驗(yàn)，以擬定稀釋倍數(shù))。實(shí)驗(yàn)所需自備物品:酶標(biāo)儀(450nm波長(zhǎng)濾光片)高精度移液器，EP管及一次性吸頭：0.5-10μL,2-20μL,20-200μL,200-1000μL37℃恒溫箱,雙蒸水或去離子水吸水紙檢測(cè)前準(zhǔn)備工作:請(qǐng)?zhí)崆?0分鐘從冰箱中取出試劑盒，平衡至室溫。將濃縮洗滌液用雙蒸水稀釋(1:25)。未用完的放回4℃。從冰箱中取出的濃縮洗滌液也許有結(jié)晶，屬于正?，F(xiàn)象，可用40℃水浴微加熱使結(jié)晶完全溶解后再配制洗滌液（加熱溫度不要超過(guò)50℃，使用時(shí)洗滌液應(yīng)為室溫）。標(biāo)準(zhǔn)品:加入標(biāo)準(zhǔn)品&樣品稀釋液1.0mL至凍干標(biāo)準(zhǔn)品中，靜置10分鐘，待其充足溶解后，輕輕混勻(濃度為MERGEFIELD"最大值"200MERGEFIELD"單位"ng/mL)。然后根據(jù)需要進(jìn)行倍比稀釋?zhuān)ㄗⅲ翰灰苯釉诜从晨字羞M(jìn)行倍比稀釋?zhuān)?。建議配制成以下濃度：MERGEFIELD"最大值"200、MERGEFIELD"M_2"100、MERGEFIELD"M_3"50、MERGEFIELD"M_4"25、MERGEFIELD"M_5"12.5、MERGEFIELD"M_6"6.25、MERGEFIELD"M_7"3.13、0MERGEFIELD"單位"ng/mL，樣品稀釋液直接作為空白孔0MERGEFIELD"單位"ng/mL。如配制MERGEFIELD"M_2"100MERGEFIELD"單位"ng/mL標(biāo)準(zhǔn)品：取0.5mLMERGEFIELD"最大值"200MERGEFIELD"單位"ng/mL的上述標(biāo)準(zhǔn)品加入具有0.5mL樣品稀釋液的EP管中，混勻即可，其余濃度依此類(lèi)推。生物素化抗體工作液：實(shí)驗(yàn)前計(jì)算當(dāng)次實(shí)驗(yàn)所需用量（以100μL/孔計(jì)），實(shí)際配制時(shí)應(yīng)多配制100-200μL。使用前15分鐘，以生物素化抗體稀釋液稀釋濃縮生物素化抗體(1:100)成工作濃度。當(dāng)天使用。酶結(jié)合物工作液：實(shí)驗(yàn)前計(jì)算當(dāng)次實(shí)驗(yàn)所需用量（以100μL/孔計(jì)），實(shí)際配制時(shí)應(yīng)多配制100-200μL。使用前15分鐘，以酶結(jié)合物稀釋液稀釋濃縮HRP酶結(jié)合物(1:100)成工作濃度。當(dāng)天使用。標(biāo)準(zhǔn)品稀釋方法圖例：(以500μL/管為例，也可根據(jù)實(shí)際用量來(lái)稀釋?zhuān)?00μL/管)MERGEFIELD"最大值"200MERGEFIELD"M_2"100MERGEFIELD"M_3"50MERGEFIELD"M_4"25MERGEFIELD"M_5"12.5MERGEFIELD"M_6"6.25MERGEFIELD"M_7"3.130MERGEFIELD"單位"ng/mL洗滌方法:自動(dòng)洗板機(jī)：每孔加入洗滌液350μL，注入與吸出間隔60秒。手工洗板：甩盡孔內(nèi)液體，在潔凈的吸水紙上拍干，每孔加洗滌液350μL，浸泡1-2分鐘，吸去（不可觸及板壁）或甩掉酶標(biāo)板內(nèi)的液體，在厚的吸水紙上拍干。操作環(huán)節(jié):實(shí)驗(yàn)開(kāi)始前，各試劑均應(yīng)平衡至室溫；試劑或樣品配制時(shí)，均需充足混勻，并盡量避免起泡。加樣：分別設(shè)空白孔、標(biāo)準(zhǔn)孔、待測(cè)樣品孔?？瞻卓准訕悠废♂屢?00μL，余孔分別加標(biāo)準(zhǔn)品或待測(cè)樣品100μL，注意不要有氣泡，加樣時(shí)將樣品加于酶標(biāo)板底部，盡量不觸及孔壁，輕輕晃動(dòng)混勻。給酶標(biāo)板覆膜，37℃孵育90分鐘。為保證實(shí)驗(yàn)結(jié)果有效性，每次實(shí)驗(yàn)請(qǐng)使用新的標(biāo)準(zhǔn)品溶液。棄去液體，甩干，不用洗滌。每個(gè)孔中加入生物素化抗體工作液100μL（在使用前15分鐘內(nèi)配制），酶標(biāo)板加上覆膜，37℃溫育1小時(shí)。棄去孔內(nèi)液體，甩干，洗板3次，每次浸泡1-2分鐘，大約350μL/每孔，甩干并在吸水紙上輕拍將孔內(nèi)液體拍干。每孔加酶結(jié)合物工作液（臨用前15分鐘內(nèi)配制）100μL，加上覆膜，37℃溫育30分鐘。棄去孔內(nèi)液體，甩干，洗板5次，方法同環(huán)節(jié)3。每孔加底物溶液(TMB)100μL，酶標(biāo)板加上覆膜37℃避光孵育15分鐘左右（根據(jù)實(shí)際顯色情況酌情縮短或延長(zhǎng)，但不可超過(guò)30分鐘。當(dāng)標(biāo)準(zhǔn)孔出現(xiàn)明顯梯度時(shí)，即可終止）。每孔加終止液50μL，終止反映，此時(shí)藍(lán)色立轉(zhuǎn)黃色。終止液的加入順序應(yīng)盡量與底物溶液的加入順序相同。立即用酶標(biāo)儀在450nm波長(zhǎng)測(cè)量各孔的光密度（OD值）。應(yīng)提前打開(kāi)酶標(biāo)儀電源，預(yù)熱儀器，設(shè)立好檢測(cè)程序。實(shí)驗(yàn)完畢后將未用完的試劑按規(guī)定的保存溫度放回冰箱保存。注意事項(xiàng)：保存：試劑盒中各試劑請(qǐng)按說(shuō)明書(shū)提醒合理存放。在儲(chǔ)存及溫育過(guò)程中避免將試劑暴露在強(qiáng)光中。所有試劑瓶蓋須旋緊以防止蒸發(fā)和微生物的污染，否則也許會(huì)出現(xiàn)錯(cuò)誤的結(jié)果。酶標(biāo)板：剛啟動(dòng)的酶標(biāo)板孔中也許會(huì)有少許水樣物質(zhì)，此為正?，F(xiàn)象，不會(huì)對(duì)實(shí)驗(yàn)結(jié)果導(dǎo)致任何影響。加樣：加樣或加試劑時(shí)，第一個(gè)孔與最后一個(gè)孔的加樣時(shí)間間隔假如太大，將會(huì)導(dǎo)致不同的“預(yù)溫育”時(shí)間，從而明顯地影響到測(cè)量值的準(zhǔn)確性及反復(fù)性。每次的加樣時(shí)間最佳控制在10分鐘內(nèi)。推薦設(shè)立復(fù)孔。溫育：為防止樣品蒸發(fā)，實(shí)驗(yàn)時(shí)必須給酶標(biāo)板覆膜；洗板后應(yīng)盡快進(jìn)行下步操作，避免酶標(biāo)板處在干燥狀態(tài)；嚴(yán)格遵守給定的溫育時(shí)間和溫度。洗滌：洗滌過(guò)程中反映孔中殘留的洗滌液應(yīng)在吸水紙上拍干，勿將濾紙直接放入反映孔中吸水。在讀數(shù)前要注意清除底部殘留的液體和手指印，以免影響酶標(biāo)儀讀數(shù)。試劑配制：ConcentratedBiotinylatedDetectionAb及ConcentratedHRPConjugate體積較小，運(yùn)送過(guò)程會(huì)使液體沾到管壁或瓶蓋，因此使用前1000轉(zhuǎn)/分離心1min，以使附著管壁或瓶蓋的液體沉積到管底。取用前，請(qǐng)用移液器小心吹打4-5次使溶液混勻。標(biāo)準(zhǔn)品、生物素化抗體工作液、酶結(jié)合物工作液請(qǐng)根據(jù)所需用量配制，并使用相應(yīng)的稀釋液配制，不能混淆。請(qǐng)精確配制標(biāo)準(zhǔn)品及工作液，盡量不要微量配制（如吸取ConcentratedBiotinylatedDetectionAb時(shí)，一次不要小于10μL），以避免由于不準(zhǔn)確稀釋而導(dǎo)致濃度誤差；請(qǐng)勿反復(fù)使用已稀釋過(guò)的標(biāo)準(zhǔn)品、生物素化抗體工作液、酶結(jié)合物工作液。若需要分次使用標(biāo)準(zhǔn)品應(yīng)按照每一次用量分裝，將其放在-20～-80℃貯存。避免反復(fù)凍融。顯色時(shí)間的控制：加入底物后請(qǐng)定期觀測(cè)反映孔的顏色變化（比如每隔5分鐘），如梯度已很明顯，請(qǐng)?zhí)崆凹尤虢K止液終止反映，避免顏色過(guò)深影響酶標(biāo)儀讀數(shù)。底物：底物請(qǐng)避光保存，在儲(chǔ)存和溫育時(shí)避免強(qiáng)光直接照射。混勻：充足輕微混勻?qū)Ψ从辰Y(jié)果尤為重要，最佳使用微量振蕩器(使用最低頻率)，如無(wú)微量振蕩器，可在反映前手工輕輕敲擊酶標(biāo)板框混勻。安全：實(shí)驗(yàn)中請(qǐng)穿著實(shí)驗(yàn)服并帶乳膠手套做好防護(hù)工作。特別是檢測(cè)血液或者其他體液標(biāo)本時(shí)，請(qǐng)按國(guó)家生物實(shí)驗(yàn)室安全防護(hù)條例執(zhí)行。不同批號(hào)的試劑盒組份不能混用（洗滌液和反映終止液除外）實(shí)驗(yàn)中所用的EP管和吸頭均為一次性使用，嚴(yán)禁混用，否則將影響實(shí)驗(yàn)結(jié)果！結(jié)果判斷:每個(gè)標(biāo)準(zhǔn)品的OD值減去空白孔的OD值后作圖，如設(shè)立復(fù)孔，則應(yīng)取其平均值計(jì)算。以標(biāo)準(zhǔn)品的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)，繪出標(biāo)準(zhǔn)曲線。亦可以O(shè)D值為橫坐標(biāo)，標(biāo)準(zhǔn)品的濃度為縱坐標(biāo)，繪出標(biāo)準(zhǔn)曲線。推薦使用專(zhuān)業(yè)的曲線制作軟件，如curveexpert1.3，在軟件界面既可根據(jù)樣品OD值，由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度，乘以稀釋倍數(shù)；亦可將樣品的OD值代入標(biāo)準(zhǔn)曲線的擬合方程式，計(jì)算出樣品濃度，再乘以稀釋倍數(shù)，即為樣品的實(shí)際濃度。若標(biāo)本OD值高于標(biāo)準(zhǔn)曲線上限，應(yīng)適當(dāng)稀釋后重測(cè)，計(jì)算濃度時(shí)應(yīng)乘以稀釋倍數(shù)。靈敏度、檢測(cè)范圍、特異性和反復(fù)性:●靈敏度：最小可測(cè)MERGEFIELD"靈敏度"1.88MERGEFIELD"單位"ng/mL。●檢測(cè)范圍：MERGEFIELD"M_7"3.13–MERGEFIELD"最大值"200MERGEFIELD"單位"ng/mL。●特異性：可檢測(cè)重組或天然的MERGEFIELD"種屬中文"小鼠MERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX，且與其它相關(guān)蛋白無(wú)交叉反映?！穹磸?fù)性：板內(nèi)，板間變異系數(shù)均<10%。問(wèn)題分析問(wèn)題描述也許因素相應(yīng)對(duì)策標(biāo)準(zhǔn)曲線梯度差吸液或加液不準(zhǔn)檢查移液器及吸頭標(biāo)準(zhǔn)品稀釋不對(duì)的溶解標(biāo)準(zhǔn)品時(shí)稍微旋轉(zhuǎn)瓶身，輕輕混勻使粉末完全溶解洗滌不完全保證洗滌時(shí)間和洗滌次數(shù)及每孔的加液量顯色很弱或無(wú)色孵育時(shí)間太短保證充足的孵育時(shí)間實(shí)驗(yàn)溫度不對(duì)的使用推薦的實(shí)驗(yàn)溫度試劑體積不夠或漏加檢查吸液及加液過(guò)程，保證所有試劑按順序足量添加稀釋不對(duì)的讀數(shù)數(shù)值低酶標(biāo)儀設(shè)立不對(duì)的在酶標(biāo)儀上檢查波長(zhǎng)及濾光片設(shè)立提前打開(kāi)酶標(biāo)儀預(yù)熱曲線偏差大加液不對(duì)的檢查加液情況背景值高檢測(cè)抗體的工作濃度過(guò)高使用推薦的稀釋倍數(shù)酶標(biāo)板洗滌不完全重新閱讀操作手冊(cè)，保證清洗完全；假如用自動(dòng)洗板機(jī)，請(qǐng)檢查所有的出口是否有堵塞洗液有污染配制新鮮的洗液靈敏度低ELISA試劑盒保存不妥按說(shuō)明書(shū)規(guī)定保存相關(guān)試劑讀數(shù)前未終止OD讀數(shù)前應(yīng)在每孔中加入終止液說(shuō)明限于現(xiàn)有條件及科學(xué)技術(shù)水平，尚不能對(duì)所有原料進(jìn)行全面的鑒定分析，本產(chǎn)品也許存在一定的質(zhì)量技術(shù)風(fēng)險(xiǎn)。最終的實(shí)驗(yàn)結(jié)果與試劑的有效性、實(shí)驗(yàn)者的相關(guān)操作以及當(dāng)時(shí)的實(shí)驗(yàn)環(huán)境密切相關(guān)，請(qǐng)務(wù)必準(zhǔn)備充足的待測(cè)樣品。只有所有使用ElabTM試劑才干保證檢測(cè)效果，不能混用其他制造商的產(chǎn)品。只有嚴(yán)格遵守ElabTM試劑的實(shí)驗(yàn)說(shuō)明才會(huì)得到最佳的檢測(cè)結(jié)果。有效期：6個(gè)月。本操作說(shuō)明同樣合用于48T試劑盒。MERGEFIELD"種屬英文"MouseMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX(MERGEFIELD"指標(biāo)英文"crosslinkedN-telopeptideoftypeⅠcollagen)ELISAKitProductDescriptionCatalogNo:MERGEFIELD"編號(hào)"E-EL-M0360（FORRESEARCHUSEONLY.DONOTUSEITINCLINICALDIAGONOSIS!）Dearcustomer,Thankyouforchoosingourproducts.Thisproductisproducedusingrawmaterialsfromworld-renownedmanufacturer,andprofessionalmanufacturingtechnologyofELISAkits.Pleasereadtheinstructionscarefullybeforeuseandcheckallthereagentcompositions!Ifindoubt,pleasecontactElabscienceBiotechnologyCo.,Ltd.IntendeduseThisimmunoassaykitallowsfortheinvitroquantitativedeterminationofMERGEFIELD"種屬英文"MouseMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTXconcentrationsinserum,plasmaandotherbiologicalfluids.KitComponents：ItemSpecificationsStorageMicroELISAPlate8×12or8×6*4°CReferenceStandard2/1vial*4°CReferenceStandard&SampleDiluent1vial20mL/12mL*4°CConcentratedBiotinylatedDetectionAb1vial120μL/70μL*4°CDiluentforBiotinylatedDetectionAb1vial10mL/6mL*4°CConcentratedHRPConjugate1vial120μL/70μL*4°C(shadinglight)DiluentforHRPConjugate1vial10mL/6mL*4°CConcentratedWashBuffer（25×）1vial30mL/16mL*4°CSubstrateReagent1vial10mL/6mL*4°C(shadinglight)StopSolution1vial10mL/6mL*4°CPlateSealer5/3pieces*ProductDescription1copy*:[96T/48T]TestprincipleThisELISAkitusesSandwich-ELISAasthemethod.ThemicroELISAplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTXStandardsorsamplesarethenaddedtotheappropriatemicroELISAplatewellsandcombinedtothespecificantibody.ThenabiotinylateddetectionantibodyspecificforMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTXandAvidin-HorseradishPeroxidase(HRP)conjugateisaddedtoeachmicroplatewellandincubated.Freecomponentsarewashedaway.Thesubstratesolutionisaddedtoeachwell.OnlythosewellsthatcontainMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX,biotinylateddetectionantibodyandAvidin-HRPconjugatewillappearblueincolor.Theenzyme-substratereactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorturnyellow.Theopticaldensity(OD)ismeasuredspectrophotometricallyatawavelengthof450nm±2nm.TheODvalueisproportionaltotheconcentrationofMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTX.YoucancalculatetheconcentrationofMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTXinthesamplesbycomparingtheO.D.ofthesamplestothestandardcurve.SamplecollectionandstorageSerum-Allowsamplestoclotfor2hoursatroomtemperatureorovernightat4°Cbeforecentrifugationfor20minutesatapproximately1000×g.Collectthesupernatantandcarryouttheassayimmediately.Bloodcollectiontubesshouldbedisposable,non-pyrogenic,andnon-endotoxin.Plasma-CollectplasmausingEDTA.Na2orheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gat2-8°Cwithin30minutesofcollection.Collectthesupernatantandcarryouttheassayimmediately.Avoidhemolysis,highcholesterolsamples.Tissuehomogenates:Forgeneralinformation,hemolysisbloodmayaffecttheresult,soyoushouldrinsethetissueswithice-coldPBS(0.01M,pH=7.4)toremoveexcessbloodthoroughly.TissuepiecesshouldbeweighedandthenmincedtosmallpieceswhichwillbehomogenizedinPBS(thevolumedependsontheweightofthetissue.9mLPBSwouldbeappropriateto1gramtissuepieces.SomeproteaseinhibitorisrecommendedtoaddintothePBS.)withaglasshomogenizeronice.Tofurtherbreakthecells,youcansonicatethesuspensionwithanultrasoniccelldisrupterorsubjectittofreeze-thawcycles.Thehomogenatesarethencentrifugatedfor5minutesat5000×gtogetthesupernate.Cellculturesupernate–Centrifugesupernatefor20minutestoremoveinsolubleimpurityandcelldebrisat1000×gat2-8°C.Collecttheclearsupernateandcarryouttheassayimmediately.Otherbiologicalfluids–Centrifugesamplesfor20minutesat1000×gat2-8°C.Collectthesupernatantandcarryouttheassayimmediately.(Youcanrefertoourwebsitefordetailedprocessingmethod:)Samplepreparation–Samplesshouldbeclearandtransparentandbecentrifugedtoremovesuspendedsolids.Note:Serumandplasmatobeusedwithin7dayswhenstoredat2-8°C,otherwisesamplesmustbedividedandstoredat-20°C(≤1month)or-80°C(≤6months)toavoidlossofbioactivityandcontamination.Avoidfreeze-thawcycles.Whenperformingtheassayslowlybringsamplestoroomtemperature.Ifthesampleconcentrationishigherthanthemaximumstandardvalue,pleasediluteitwithappropriatefactoraccordingtotheactualsituation.(Apre-testisrecommendedtodeterminethedilutefactor)OthersuppliesrequiredMicroplatereaderwith450nmwavelengthfilterHigh-precisiontransferpettor,EPtubesanddisposablepipettetips37°CIncubator,Deionizedordistilledwater.AbsorbentpaperReagentpreparationBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Dilute30mLofConcentratedWashBufferinto750mLofWashBufferwithdeionizedordistilledwater.Putunusedsolutionbackat4°C.Ifcrystalshaveformedintheconcentrate,youcanwarmitwith40°Cwaterbath(Heatingtemperatureshouldnotexceed50°C)andmixitgentlyuntilthecrystalshavecompletelydissolved.Thesolutionshouldbecooledtoroomtemperaturebeforeuse.Standard-ReconstitutetheStandardwith1.0mLofSampleDiluent,letitstandfor10minutesuntilitdissolvedfully.ThisreconstitutionproducesastocksolutionofMERGEFIELD"最大值"200MERGEFIELD"單位"ng/mL.Thenmakeserialdilutionsasneeded(Makingserialdilutioninthewellsdirectlyisnotpermitted).Therecommendedconcentrationsareasfollows:MERGEFIELD"最大值"200、MERGEFIELD"M_2"100、MERGEFIELD"M_3"50、MERGEFIELD"M_4"25、MERGEFIELD"M_5"12.5、MERGEFIELD"M_6"6.25、MERGEFIELD"M_7"3.13、0MERGEFIELD"單位"ng/mL.AsifyouwanttomakestandardsolutionattheconcentrationofMERGEFIELD"M_2"100MERGEFIELD"單位"ng/mL,youcantake0.5mLthestandardatMERGEFIELD"最大值"200MERGEFIELD"單位"ng/mL,addittoanEPtubewith0.5mLsampledilution,andmixit.Theproceduresofmakingtheremainingconcentrationsareallthesame.Theundilutedstandardservesasthehigheststandard(MERGEFIELD"最大值"200MERGEFIELD"單位"ng/mL).TheSampleDiluentservesasthezero(0MERGEFIELD"單位"ng/mL).(500μL/tube，forexample.Canalsobedilutedaccordingtotheactualamount，suchas200μL/tube)MERGEFIELD"最大值"200MERGEFIELD"M_2"100MERGEFIELD"M_3"50MERGEFIELD"M_4"25MERGEFIELD"M_5"12.5MERGEFIELD"M_6"6.25MERGEFIELD"M_7"3.130MERGEFIELD"單位"ng/mLBiotinylatedDetectionAb—Calculatetherequiredamountbeforeexperiment(100μL/well).Inactualpreparationyoushouldprepare100~200μLmore.DilutetheconcentratedBiotinylatedDetectionAbtotheworkingconcentrationusingDiluentforBiotinylatedDetectionAb(1:100).ConcentratedHRPConjugate—Calculatetherequiredamountbeforeexperiment(100μL/well).Inactualpreparationyoushouldprepare100~200μLmore.DilutetheConcentratedHRPConjugatetotheworkingconcentrationusingDiluentforConcentratedHRPConjugate(1:100).WashingProcedure:1.Automatedwasher:add350μLwashbufferintoeachwell,theintervalbetweeninjectionandsuctionshouldbesetabout60s.2.Manualwash:add350μLwashbufferintoeachwell,soakitfor1~2minutes,suck(noinsidewalltouching)orgetridofliquidwithinthemicroELISAplateandpatitdryonthickcleanabsorbentpaper.AssayprocedureAllowallreagentstoreachroomtemperatureAllthereagentsshouldbemixedthoroughlybygentlyswirlingbeforepipetting.Avoidfoaming.AddSample:Add100μLofStandard,Blank,orSampleperwell.Theblankwellisaddedwithsamplediluent.SolutionsareaddedtothebottomofmicroELISAplatewell,avoidinsidewalltouchingandfoamingtothebestofyourability.Mixitgently.Covertheplatewithsealerweprovided.Incubatefor90minutesat37°C.BiotinylatedDetectionAb:Removetheliquidofeachwell,don’twash.Immediatelyadd100μLofBiotinylatedDetectionAbworkingsolutiontoeachwell.CoverwiththePlatesealer.Gentlytaptheplatetoensurethoroughmixing.Incubatefor1hourat37°C.Wash:Aspirateeachwellandwash,repeatingtheprocessthreetimes.WashbyfillingeachwellwithWashBuffer(approximately350μL)usingasquirtbottle,multi-channelpipette,manifolddispenserorautomatedwasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandpatitagainstthickcleanabsorbentpaper.4.HRPConjugate:Add100μLofHRPConjugateworkingsolutiontoeachwell.CoverwiththePlatesealer.Incubatefor30minutesat37°C.5.Wash:Repeatthewashprocessforfivetimesasconductedinstep3.6.Substrate:Add100μLofSubstrateSolutiontoeachwell.CoverwithanewPlatesealer.Incubateforabout15minutesat37°C.Protecttheplatefromlight.Thereactiontimecanbeshortenedorextendedaccordingtotheactualcolorchange,butnotmorethan30minutes.Whenapparentgradientappearedinstandardwells,youcanterminatethereaction.7.Stop:Add50μLofStopSolutiontoeachwell.Colorturntoyellowimmediately.Theaddingorderofstopsolutionshouldbeasthesameasthesubstratesolution.8.ODMeasurement:Determinetheopticaldensity(ODvalue)ofeachwellatonce,usingamicroplatereadersetto450nm.Youshouldopenthemicroplatereaderahead,preheattheinstrument,andsetthetestingparameters.9.Afterexperiment,putalltheunusedreagentsbackintotherefrigeratoraccordingtothespecifiedstoragetemperaturerespectivelyuntiltheirexpiry.ImportantNote:1.Storage:Allthereagentsinthekitshouldbestoredfollowingtheinstructions.Exposureofreagentstostronglightshouldbeavoidedintheprocessofincubationandstorage.Allthetapsofreagentsshouldbetightenedtopreventevaporationandmicrobialcontamination,orerroneousresultsmayoccur.2.ELISAPlate:Littlewater-likesubstancemayappearintheELISAPlatejustopened,thisisnormalandwillnothaveanyimpactontheexperimentresults.3.AddSample:Theintervalofsampleaddingbetweenthefirstwellandthelastwellshouldnotbetoolong,otherwisewillcausedifferentpre-incubationtime,whichwillsignificantlyaffecttheexperiment’saccuracyandrepeatability.Theintervalcontrolledwithin10minutesisgood.Parallelmeasurementisrecommended.Incubation:Topreventevaporation,properadhesionofplatesealersduringincubationstepsisnecessary.Donotallowwellstosituncoveredforextendedperiodsbetweenincubationsteps.Donotletthestripsdryatanytimeduringtheassay.Strictcompliancewiththegivenincubationtimeandtemperature.Washing:Thewashprocedureiscritical.InsufficientwashingwillresultinpoorprecisionandfalselyelevatedabsorbancereadingsResidualliquidinthereactionwellsshouldbepatdryagainstabsorbentpaperinthewashingprocess.Butdon’tputabsorbentpaperintoreactionwellsdirectly.Notethatcleartheresidualliquidandfingerprintinthebottombeforemeasurement,soasnottoaffectthemicrotiterplatereader.ReagentPreparation:AsthevolumeofConcentratedBiotinylatedDetectionAbandConcentratedHRPConjugateisverysmall,liquidmayadheretothetubewallortubecapwhenbeingtransported.Youbetterhand-throwitorcentrifugalitfor1minuteat1000rpm.Pleasepipettethesolutionfor4-5timesbeforepippeting.PleasecarefullyreconstituteStandards,workingsolutionsofBiotinylatedDetectionAbandHRPConjugateaccordingtotheinstructions.Tominimizeimprecisioncausedbypipetting,ensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10μLforoncepipetting.Donotreusestandardsolution,workingsolutionofBiotinylatedDetectionAbandHRPConjugate,whichhavebeendiluted.Ifyouneedtousestandardrepeatedly,youcandividethestandardintosmallpackaccordingtotheamountofeachassay,keepthemat-20～-80°Candavoidrepeatedfreezingandthawing.ReactionTimeControl:Pleasecontrolreactiontimestrictlyfollowingthisproductdescription!Substrate:SubstrateSolutioniseasilycontaminated.Pleaseprotectitfromlight.Mixing:You’dbetterusemicrooscillatoratthelowestfrequency,assufficientandgentlemixingisparticularlyimportanttoreactionresult.Ifthereisnomicrosocillatoravailable,youcanknocktheELISAplateframegentlywithyourfingerbeforereaction.Security:Pleasewearlabcoatsandlatexglovesforprotection.Especiallydetectingsamplesofbloodorotherbodyfluid,pleaseperformfollowingthenationalsecuritycolumnsofbiologicallaboratories.Donotusecomponentfromdifferentbatchesofkit(washingbufferandstopsolutioncanbeanexception)Toavoidcross-contamination,changepipettetipsbetweenaddingofeachstandardlevel,betweensampleadding,andbetweenreagentadding.Also,useseparatereservoirsforeachreagent.Otherwise,theresultswillbeinaccurate!CalculationofresultsAveragetheduplicatereadingsforeachstandardandsamplesandsubtracttheaveragezerostandardopticaldensity.CreateastandardcurvebyplottingthemeanODvalueforeachstandardonthey-axisorx-axisagainsttheconcentrationonthex-axisory-axisanddrawabestfitcurvethroughthepointsonthegraph.Itisrecommendedtousesomeprofessionalsoftwaretodothiscalculation,suchascurveexpert1.3.Inthesoftwareinterface,abestfittingequationofstandardcurvewillbecalculatedusingODvaluesandconcentrationsofstandardsample.ThesoftwarewillcalculatetheconcentrationofsamplesafterenteringtheODvalueofsamples.Also,youcanenterthecorrespondingfittingequationandODvalueofsamplesintoExceltogettheconcentrationofsamples.Ifsampleshavebeendiluted,theconcentrationcalculatedfromthestandardcurvemustbemultipliedbythedilutionfactor.IftheODofthesamplesurpassestheupperlimitofthestandardcurve,youshouldre-testitafterappropriatedilution.Theactualconcentrationisthecalculatedconcentrationmultiplieddilutionfactor.SensitivityTheminimumdetectabledoseofMERGEFIELD"種屬英文"MouseMERGEFIELD"指標(biāo)簡(jiǎn)寫(xiě)"NTXisMERGEFIELD"靈敏度"1.88MERGEFIELD"單位"ng/mL(Thesensitivityofthisassay,orLowerLimitofDetection(LLD)wasdefinedasthe