小鼠GATA結(jié)合蛋白GATAELISA試劑盒說明書

1、小鼠GATA結(jié)合蛋白3 (GATA3)酶聯(lián)免疫吸附測定試劑盒使用說明書產(chǎn)品編號：E-EL-M0545 （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，采用專業(yè)ELISA kit生產(chǎn)技術(shù)制造。適用于體外定量檢測小鼠血清、血漿、組織勻漿或細胞培養(yǎng)上清液中天然和重組GATA3濃度。使用前請仔細閱讀說明書并檢查試劑組分！如有疑問，請及時聯(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酶標板Micro ELISA Plate 8×12 / 8×6*4凍干標準品Referenc

2、e Standard 2/ 1支*4標準品&樣品稀釋液Reference Standard & Sample Diluent 1瓶 20mL/12mL*4濃縮生物素化抗體 Concentrated Biotinylated Detection Ab 1支 120L/70L*4生物素化抗體稀釋液 Diluent for Biotinylated Detection Ab 1瓶 10mL/6mL*4濃縮HRP酶結(jié)合物 Concentrated HRP Conjugate 1支 120L/70L*4(避光)酶結(jié)合物稀釋液 Diluent for HRP Conjugate 1瓶 10

3、mL/6mL* 4濃縮洗滌液（25×） Concentrated Wash Buffer （25×）1瓶 30mL/16mL*4底物溶液（TMB） Substrate Reagent 1瓶 10mL/6mL*4(避光)反應(yīng)終止液 Stop Solution 1瓶 10mL/6mL*4封板覆膜Plate Sealer5/3張*產(chǎn)品說明書Product Description1份*: 96T/48T（打開包裝后請及時檢查所有物品是否齊全完整）檢測原理:本試劑盒采用雙抗體夾心ELISA法。用抗小鼠GATA3抗體包被于酶標板上，實驗時標本或標準品中的GATA3會與包被抗體結(jié)合，游離

4、的成分被洗去。依次加入生物素化的抗小鼠GATA3抗體和辣根過氧化物酶標記的親和素?？剐∈驡ATA3抗體與結(jié)合在包被抗體上的小鼠GATA3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在辣根過氧化物酶的催化下現(xiàn)藍色，加終止液后變黃。用酶標儀在450nm波長處測OD值，GATA3濃度與OD450值之間呈正比，通過繪制標準曲線求出標本中GATA3的濃度。標本收集:1. 血清：全血標本于室溫放置2小時或4過夜后于1000×g離心20分鐘，取上清即可檢測，收集血液的試管應(yīng)為一次性的無熱原，無內(nèi)毒素試管。2. 血漿：抗凝劑推薦使用EDTA.Na2

5、，標本采集后30分鐘內(nèi)于1000×g離心15分鐘，取上清即可檢測。避免使用溶血，高血脂標本。3. 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（勻漿中裂解的紅細胞會影響測量結(jié)果），稱重后將組織剪碎。將剪碎的組織與對應(yīng)體積的PBS（一般按1:9的重量體積比，比如1g的組織樣本對應(yīng)9mL的PBS，具體體積可根據(jù)實驗需要適當調(diào)整，并做好記錄。推薦在PBS中加入蛋白酶抑制劑）加入玻璃勻漿器中，于冰上充分研磨。為了進一步裂解組織細胞，可以對勻漿液進行超聲破碎，或反復(fù)凍融。最后將勻漿液于5000×g 離心510分鐘，取上清檢測。4. 細胞培養(yǎng)上清：取

6、細胞培養(yǎng)上清于1000×g離心20分鐘，除去雜質(zhì)及細胞碎片。取上清檢測。5. 其它生物標本：1000×g離心20分鐘，取上清即可檢測（具體處理方法可參考： ）6. 標本應(yīng)清澈透明，懸浮物應(yīng)離心去除。7. 標本收集后若不及時檢測，請按一次使用量分裝，凍存于-20/-80冰箱內(nèi)，避免反復(fù)凍融，1-6月內(nèi)檢測,4保存的應(yīng)在1周內(nèi)進行檢測。 8. 如果您的樣本中檢測物濃度高于標準品最高值，請根據(jù)實際情況，做適當倍數(shù)稀釋(建議先做預(yù)實驗，以確定稀釋倍數(shù))。試驗所需自備物品:1. 酶標儀(450nm波長濾光片)2. 高精度移液器，EP管及一次性吸頭：0.5-10L, 2-20L, 20

7、-200L, 200-1000L3. 37恒溫箱, 雙蒸水或去離子水4. 吸水紙檢測前準備工作:1. 請?zhí)崆?0分鐘從冰箱中取出試劑盒，平衡至室溫。2. 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可能有結(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50，使用時洗滌液應(yīng)為室溫）。3. 標準品: 加入標準品&樣品稀釋液1.0mL至凍干標準品中，靜置10分鐘，待其充分溶解后，輕輕混勻(濃度為1000pg/ml)。然后根據(jù)需要進行倍比稀釋（注：不要直接在反應(yīng)孔中進行倍比稀釋）。建議配制成以下濃度： 1000、500、25

8、0、125、62.5、31.25、15.63、0 pg/ml ，樣品稀釋液直接作為空白孔0pg/ml。如配制500pg/ml標準品：取0.5mL 1000pg/ml的上述標準品加入含有0.5mL樣品稀釋液的EP管中，混勻即可，其余濃度依此類推。4. 生物素化抗體工作液：實驗前計算當次實驗所需用量（以100L/孔計），實際配制時應(yīng)多配制100-200L。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。當日使用。5. 酶結(jié)合物工作液：實驗前計算當次實驗所需用量（以100L/孔計），實際配制時應(yīng)多配制 100-200L。使用前15分鐘，以酶結(jié)合物稀釋液稀釋濃縮HRP

9、酶結(jié)合物(1:100)成工作濃度。當日使用。標準品稀釋方法圖例：(以500L/管為例，也可根據(jù)實際用量來稀釋，如200L/管)1000 500 250 125 62.531.2515.630pg/ml洗滌方法:1. 自動洗板機：每孔加入洗滌液350L，注入與吸出間隔60秒。2. 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350L，浸泡1-2分鐘，吸去（不可觸及板壁）或甩掉酶標板內(nèi)的液體，在厚的吸水紙上拍干。操作步驟:實驗開始前，各試劑均應(yīng)平衡至室溫；試劑或樣品配制時，均需充分混勻，并盡量避免起泡。1. 加樣：分別設(shè)空白孔、標準孔、待測樣品孔?？瞻卓准訕悠废♂屢?00L，余孔分別

10、加標準品或待測樣品100L，注意不要有氣泡，加樣時將樣品加于酶標板底部，盡量不觸及孔壁，輕輕晃動混勻。給酶標板覆膜，37孵育90分鐘。為保證實驗結(jié)果有效性，每次實驗請使用新的標準品溶液。2. 棄去液體，甩干，不用洗滌。每個孔中加入生物素化抗體工作液100L（在使用前15分鐘內(nèi)配制），酶標板加上覆膜，37溫育1小時。3. 棄去孔內(nèi)液體，甩干，洗板 3次，每次浸泡1-2分鐘，大約350L/每孔，甩干并在吸水紙上輕拍將孔內(nèi)液體拍干。 4. 每孔加酶結(jié)合物工作液（臨用前15分鐘內(nèi)配制）100L，加上覆膜，37溫育30分鐘。5. 棄去孔內(nèi)液體，甩干，洗板5次，方法同步驟3。6. 每孔加底物溶液(TMB)

11、100L，酶標板加上覆膜37避光孵育15分鐘左右（根據(jù)實際顯色情況酌情縮短或延長，但不可超過30分鐘。當標準孔出現(xiàn)明顯梯度時，即可終止）。7. 每孔加終止液50L，終止反應(yīng)，此時藍色立轉(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。8. 立即用酶標儀在450nm波長測量各孔的光密度（OD值）。應(yīng)提前打開酶標儀電源，預(yù)熱儀器，設(shè)置好檢測程序。9. 實驗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：1. 保存：試劑盒中各試劑請按說明書提示合理存放。在儲存及溫育過程中避免將試劑暴露在強光中。所有試劑瓶蓋須旋緊以防止蒸發(fā)和微生物的污染，否則可能會出現(xiàn)錯誤的結(jié)果。2. 酶標板：剛開

12、啟的酶標板孔中可能會有少許水樣物質(zhì)，此為正?，F(xiàn)象，不會對實驗結(jié)果造成任何影響。3. 加樣：加樣或加試劑時，第一個孔與最后一個孔的加樣時間間隔如果太大，將會導(dǎo)致不同的 “預(yù)溫育”時間，從而明顯地影響到測量值的準確性及重復(fù)性。每次的加樣時間最好控制在10分鐘內(nèi)。推薦設(shè)置復(fù)孔。4. 溫育：為防止樣品蒸發(fā)，實驗時必須給酶標板覆膜；洗板后應(yīng)盡快進行下步操作，避免酶標板處于干燥狀態(tài)；嚴格遵守給定的溫育時間和溫度。5. 洗滌：洗滌過程中反應(yīng)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應(yīng)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，以免影響酶標儀讀數(shù)。6. 試劑配制：Concentrated B

13、iotinylated Detection Ab及Concentrated HRP Conjugate體積較小，運輸過程會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，請用移液器小心吹打4-5次使溶液混勻。標準品、生物素化抗體工作液、酶結(jié)合物工作液請根據(jù)所需用量配制，并使用相應(yīng)的稀釋液配制，不能混淆。請精確配制標準品及工作液，盡量不要微量配制（如吸取Concentrated Biotinylated Detection Ab時，一次不要小于10L），以避免由于不準確稀釋而造成濃度誤差；請勿重復(fù)使用已稀釋過的標準品、生物素化抗體工作液、酶

14、結(jié)合物工作液。若需要分次使用標準品應(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。7. 顯色時間的控制：加入底物后請定時觀察反應(yīng)孔的顏色變化（比如每隔5分鐘），如梯度已很明顯，請?zhí)崆凹尤虢K止液終止反應(yīng)，避免顏色過深影響酶標儀讀數(shù)。8. 底物：底物請避光保存，在儲存和溫育時避免強光直接照射。9. 混勻：充分輕微混勻?qū)Ψ磻?yīng)結(jié)果尤為重要，最好使用微量振蕩器(使用最低頻率)，如無微量振蕩器，可在反應(yīng)前手工輕輕敲擊酶標板框混勻。10. 安全：試驗中請穿著實驗服并帶乳膠手套做好防護工作。特別是檢測血液或者其他體液標本時，請按國家生物試驗室安全防護條例執(zhí)行。11. 不同批號的試劑盒組份不能混用

15、（洗滌液和反應(yīng)終止液除外）12. 試驗中所用的EP管和吸頭均為一次性使用，嚴禁混用，否則將影響試驗結(jié)果！結(jié)果判斷:1. 每個標準品的OD值減去空白孔的OD值后作圖，如設(shè)置復(fù)孔，則應(yīng)取其平均值計算。以標準品的濃度為橫坐標，OD值為縱坐標，繪出標準曲線。亦可以O(shè)D值為橫坐標，標準品的濃度為縱坐標，繪出標準曲線。 2. 推薦使用專業(yè)的曲線制作軟件，如curve expert 1.3，在軟件界面既可根據(jù)樣品OD值，由標準曲線查出相應(yīng)的濃度，乘以稀釋倍數(shù)；亦可將樣品的OD值代入標準曲線的擬合方程式，計算出樣品濃度，再乘以稀釋倍數(shù)，即為樣品的實際濃度。3. 若標本OD值高于標準曲線上限，應(yīng)適當稀釋后重測，

16、計算濃度時應(yīng)乘以稀釋倍數(shù)。靈敏度、檢測范圍、特異性和重復(fù)性: 靈敏度：最小可測9.38pg/ml。 檢測范圍：15.63 1000pg/ml。 特異性：可檢測重組或天然的小鼠GATA3，且與其它相關(guān)蛋白無交叉反應(yīng)。 重復(fù)性：板內(nèi)，板間變異系數(shù)均<10%。問題分析問題描述可能原因相應(yīng)對策標準曲線梯度差吸液或加液不準檢查移液器及吸頭標準品稀釋不正確溶解標準品時稍微旋轉(zhuǎn)瓶身，輕輕混勻使粉末完全溶解洗滌不完全保證洗滌時間和洗滌次數(shù)及每孔的加液量顯色很弱或無色孵育時間太短保證充足的孵育時間實驗溫度不正確使用推薦的實驗溫度試劑體積不夠或漏加檢查吸液及加液過程，保證所有試劑按順序足量添加稀釋不正確讀數(shù)

17、數(shù)值低酶標儀設(shè)置不正確在酶標儀上檢查波長及濾光片設(shè)置提前打開酶標儀預(yù)熱曲線偏差大加液不正確檢查加液情況背景值高檢測抗體的工作濃度過高使用推薦的稀釋倍數(shù)酶標板洗滌不完全重新閱讀操作手冊，保證清洗完全；如果用自動洗板機，請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試劑盒保存不當按說明書要求保存相關(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 說明 1. 限于現(xiàn)有條件及科學(xué)技術(shù)水平，尚不能對所有原料進行全面的鑒定分析，本產(chǎn)品可能存在一定的質(zhì)量技術(shù)風險。2. 最終的實驗結(jié)果與試劑的有效性、實驗者的相關(guān)操作以及當時的實驗環(huán)境密切相關(guān)，請務(wù)必準備充足的待測樣品。3. 只有全部使用

18、ElabTM試劑才能保證檢測效果，不能混用其他制造商的產(chǎn)品。只有嚴格遵守ElabTM試劑的實驗說明才會得到最佳的檢測結(jié)果。4. 有效期：6個月。5. 本操作說明同樣適用于48T試劑盒。Mouse GATA3 (GATA Binding Protein 3) ELISA Kit Product Description Catalog No: E-EL-M0545（FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGONOSIS !）Dear customer, Thank you for choosing our products. This

19、 product is produced using raw materials from world-renowned manufacturer, and professional manufacturing technology of ELISA kits. Please read the instructions carefully before use and check all the reagent compositions! If in doubt, please contact Elabscience Biotechnology Co., Ltd.Intended useThi

20、s immunoassay kit allows for the in vitro quantitative determination of Mouse GATA3 concentrations in serum, plasma and other biological fluids.Kit Components：ItemSpecificationsStorage Micro ELISA Plate 8×12 or 8×6 *4°CReference Standard 2/ 1vial *4°CReference Standard & Samp

21、le Diluent 1vial 20mL/12mL *4°CConcentrated Biotinylated Detection Ab 1via l 120L /70L *4°CDiluent for Biotinylated Detection Ab 1vial 10mL/6mL*4°CConcentrated HRP Conjugate 1vial 120L /70L *4°C (shading light)Diluent for HRP Conjugate 1vial 10mL/6mL * 4°CConcentrated Wash B

22、uffer （25×）1vial 30mL/16mL *4°CSubstrate Reagent 1vial 10mL/6mL *4°C (shading light)Stop Solution 1vial 10mL/6mL *4°CPlate Sealer5/3pieces *Product Description1 copy*: 96T/48TTest principleThis ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this ki

23、t has been pre-coated with an antibody specific to GATA3 Standards or samples are then added to the appropriate micro ELISA plate wells and combined to the specific antibody. Then a biotinylated detection antibody specific for GATA3 and Avidin-Horseradish Peroxidase (HRP) conjugate is added to each

24、micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain GATA3, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sul

25、phuric acid solution and the color turn yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of GATA3. You can calculate the concentration of GATA3 in the samples by comparing the O.D. of the samp

26、les to the standard curve.Sample collection and storageSerum - Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000×g. Collect the supernatant and carry out the assay immediately. Blood collection tubes should

27、be disposable, non-pyrogenic, and non-endotoxin.Plasma - Collect plasma using EDTA.Na2 or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high

28、cholesterol samples.Tissue homogenates: For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PB

29、S (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces.Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subje

30、ct it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.Cell culture supernate Centrifuge supernate for 20 minutes to remove insoluble impurity and cell debris at 1000×g at 2 - 8°C. Collect the clear supernate and carry out the a

31、ssay immediately.Other biological fluids Centrifuge samples for 20 minutes at 1000×g at 2 - 8°C. Collect the supernatant and carry out the assay immediately. (You can refer to our website for detailed processing method: )Sample preparation Samples should be clear and transparent and be cen

32、trifuged to remove suspended solids.Note: Serum and plasma to be used within 7 days when stored at 2-8°C, otherwise samples must be divided and stored at -20°C (1 month) or -80°C (6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the a

33、ssay slowly bring samples to room temperature. If the sample concentration is higher than the maximum standard value, please dilute it with appropriate factor according to the actual situation. (A pre-test is recommended to determine the dilute factor) Other supplies requiredMicroplate reader with 4

34、50nm wavelength filter High-precision transferpettor, EP tubes and disposable pipette tips37°C Incubator, Deionized or distilled water. Absorbent paperReagent preparationBring all reagents to room temperature before use. Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash

35、Buffer with deionized or distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely dissolved. The solution should

36、be cooled to room temperature before use.Standard - Reconstitute the Standard with 1.0mL of Sample Diluent, let it stand for 10minutes until it dissolved fully. This reconstitution produces a stock solution of 1000pg/ml. Then make serial dilutions as needed (Making serial dilution in the wells direc

37、tly is not permitted). The recommended concentrations are as follows: 1000、500、250、125、62.5、31.25、15.63、0pg/ml . As if you want to make standard solution at the concentration of 500pg/ml, you can take 0.5mL the standard at 1000pg/ml, add it to an EP tube with 0.5mL sample dilution, and mix it. The p

38、rocedures of making the remaining concentrations are all the same. The undiluted standard serves as the highest standard (1000pg/ml). The Sample Diluent serves as the zero (0pg/ml).(500L/tube，for example. Can also be diluted according to the actual amount，such as 200L/tube)100050025012562.531.2515.6

39、30pg/mlBiotinylated Detection Ab Calculate the required amount before experiment (100L /well). In actual preparation you should prepare 100200L more. Dilute the concentrated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100).Concentrated HRP Co

40、njugate Calculate the required amount before experiment (100L/well). In actual preparation you should prepare 100200L more. Dilute the Concentrated HRP Conjugate to the working concentration using Diluent for Concentrated HRP Conjugate (1:100).Washing Procedure:1. Automated washer: add 350L wash buf

41、fer into each well, the interval between injection and suction should be set about 60s.2. Manual wash: add 350L wash buffer into each well, soak it for 12minutes, suck(no inside wall touching) or get rid of liquid within the micro ELISA plate and pat it dry on thick clean absorbent paper.Assay proce

42、dureAllow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming.1. Add Sample: Add 100L of Standard, Blank, or Sample per well. The blank well is added with sample diluent. Solutions are added to the bottom of micro ELIS

43、A plate well, avoid inside wall touching and foaming to the best of your ability. Mix it gently. Cover the plate with sealer we provided. Incubate for 90 minutes at 37°C.2. Biotinylated Detection Ab: Remove the liquid of each well, dont wash. Immediately add 100L of Biotinylated Detection Ab wo

44、rking solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. 3. Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350L) using a squirt bottle,

45、multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.4. HRP Conjugate:

46、 Add 100L of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 minutes at 37°C.5. Wash: Repeat the wash process for five times as conducted in step 3.6. Substrate: Add 100L of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for ab

47、out 15 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.7. Stop: Add 50L of Stop Solution to each wel

48、l. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.8. OD Measurement: Determine the optical density (OD value) of each well at once, using a microplate reader set to 450nm. You should open the microplate reader ahead, preheat the in

49、strument, and set the testing parameters.9. After experiment, put all the unused reagents back into the refrigerator according to the specified storage temperature respectively until their expiry.Important Note:1. Storage: All the reagents in the kit should be stored following the instructions. Expo

50、sure of reagents to strong light should be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contamination, or erroneous results may occur.2. ELISA Plate: Little water-like substance may appear in the ELISA Plate just

51、opened, this is normal and will not have any impact on the experiment results.3. Add Sample: The interval of sample adding between the first well and the last well should not be too long, otherwise will cause different pre-incubation time, which will significantly affect the experiments accuracy and

52、 repeatability. The interval controlled within 10minutes is good. Parallel measurement is recommended.4. Incubation: To prevent evaporation, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Do no

53、t let the strips dry at any time during the assay. Strict compliance with the given incubation time and temperature.5. Washing: The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings Residual liquid in the reaction wells should be

54、pat dry against absorbent paper in the washing process. But dont put absorbent paper into reaction wells directly. Note that clear the residual liquid and fingerprint in the bottom before measurement, so as not to affect the microtiter plate reader.6. Reagent Preparation: As the volume of Concentrat

55、ed Biotinylated Detection Ab and Concentrated HRP Conjugate is very small, liquid may adhere to the tube wall or tube cap when being transported. You better hand-throw it or centrifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please carefully reconstit

56、ute Standards, working solutions of Biotinylated Detection Ab and HRP Conjugate according to the instructions. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10L for once pipetting. Do not reuse standard solution, working soluti

57、on of Biotinylated Detection Ab and HRP Conjugate, which have been diluted. If you need to use standard repeatedly, you can divide the standard into small pack according to the amount of each assay, keep them at -20-80°C and avoid repeated freezing and thawing.7. Reaction Time Control: Please c

58、ontrol reaction time strictly following this product description!8. Substrate: Substrate Solution is easily contaminated. Please protect it from light.9. Mixing: Youd better use microoscillator at the lowest frequency, as sufficient and gentle mixing is particularly important to reaction result. If there is no microsocillator available, you can knock the ELISA plate frame gently with