大鼠(Rat)血管內(nèi)皮生長(zhǎng)因子(VEGF)-NEWA

1、本試劑盒只能用于科學(xué)研究,不得用于醫(yī)學(xué)診斷大鼠(大鼠(Rat Rat Rat血管內(nèi)皮生長(zhǎng)因子(血管內(nèi)皮生長(zhǎng)因子(VEGF VEGF VEGFELISA 檢測(cè)試劑盒使用說明書檢測(cè)原理試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)(ELISA。往預(yù)先包被血管內(nèi)皮生長(zhǎng)因子(VEGF抗體的包被微孔中,依次加入標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測(cè)抗體,經(jīng)過溫育并徹底洗滌。用底物TMB 顯色,TMB在過氧化物酶的催化下轉(zhuǎn)化成藍(lán)色,并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的血管內(nèi)皮生長(zhǎng)因子(VEGF呈正相關(guān)。用酶標(biāo)儀在450nm 波長(zhǎng)下測(cè)定吸光度(OD 值,計(jì)算樣品濃度。樣品收集、處理及保存方法1.血清:使

2、用不含熱原和內(nèi)毒素的試管,操作過程中避免任何細(xì)胞刺激,收集血液后,3000轉(zhuǎn)離心10分鐘將血清和紅細(xì)胞迅速小心地分離。2.血漿:EDTA、檸檬酸鹽或肝素抗凝。3000轉(zhuǎn)離心30分鐘取上清。3.細(xì)胞上清液:3000轉(zhuǎn)離心10分鐘去除顆粒和聚合物。4.組織勻漿:將組織加入適量生理鹽水搗碎。3000轉(zhuǎn)離心10分鐘取上清。5.保存:如果樣本收集后不及時(shí)檢測(cè),請(qǐng)按一次用量分裝,凍存于-20,避免反復(fù)凍融,在室溫下解凍并確保樣品均勻地充分解凍。自備物品1.酶標(biāo)儀(450nm2.高精度加樣器及槍頭:0.5-10uL、2-20uL、20-200uL、200-1000uL3.37恒溫箱操作注意事項(xiàng)1.試劑盒保存

3、在2-8,使用前室溫平衡20分鐘。從冰箱取出的濃縮洗滌液會(huì)有結(jié)晶,這屬于正常現(xiàn)象,水浴加熱使結(jié)晶完全溶解后再使用。2.實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中,密封(低溫干燥保存。3.濃度為0的S0號(hào)標(biāo)準(zhǔn)品即可視為陰性對(duì)照或者空白;按照說明書操作時(shí)樣本已經(jīng)稀釋5倍,最終結(jié)果乘以5才是樣本實(shí)際濃度。4.嚴(yán)格按照說明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。5.所有液體組分使用前充分搖勻。試劑盒組成名稱96孔配置48孔配置備注微孔酶標(biāo)板12孔×8條12孔×4條無樣本稀釋液6mL3mL無檢測(cè)抗體-HRP10mL5mL無20×洗滌緩沖液25mL15mL按說明書進(jìn)行稀釋底物A6m

4、L3mL無底物B6mL3mL無終止液6mL3mL無封板膜2張2張無說明書1份1份無自封袋1個(gè)1個(gè)無注:標(biāo)準(zhǔn)品(S0-S5濃度依次為:0、20、40、80、160、320pg/mL試劑的準(zhǔn)備20×洗滌緩沖液的稀釋:蒸餾水按1:20稀釋,即1份的20×洗滌緩沖液加19份的蒸餾水。洗板方法1.手工洗板:甩盡孔內(nèi)液體,每孔加滿洗滌液,靜置1min后甩盡孔內(nèi)液體,在吸水紙上拍干,如此洗板5次。2.自動(dòng)洗板機(jī):每孔注入洗液350L,浸泡1min,洗板5次。操作步驟1.從室溫平衡20min后的鋁箔袋中取出所需板條,剩余板條用自封袋密封放回4。2.設(shè)置標(biāo)準(zhǔn)品孔和樣本孔,標(biāo)準(zhǔn)品孔各加不同濃度

5、的標(biāo)準(zhǔn)品50L;3.樣本孔先加待測(cè)樣本10L,再加樣本稀釋液40L;空白孔不加。4.除空白孔外,標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶(HRP標(biāo)記的檢測(cè)抗體100L,用封板膜封住反應(yīng)孔,37水浴鍋或恒溫箱溫育60min。5.棄去液體,吸水紙上拍干,每孔加滿洗滌液,靜置1min,甩去洗滌液,吸水紙上拍干,如此重復(fù)洗板5次(也可用洗板機(jī)洗板。6.每孔加入底物A、B 各50L,37避光孵育15min。7.每孔加入終止液50L,15min 內(nèi),在450nm 波長(zhǎng)處測(cè)定各孔的OD 值。結(jié)果判斷繪制標(biāo)準(zhǔn)曲線:在Excel工作表中,以標(biāo)準(zhǔn)品濃度作橫坐標(biāo),對(duì)應(yīng)OD 值作縱坐標(biāo),繪制出標(biāo)準(zhǔn)品線性回歸曲線,按曲

6、線方程計(jì)算各樣本濃度值。試劑盒性能1.準(zhǔn)確性:標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R 值,大于等于0.9900。2.靈敏度:最低檢測(cè)濃度小于1.0pg/mL。3.特異性:不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。4.重復(fù)性:板內(nèi)、板間變異系數(shù)均小于15%。5.貯藏:2-8,避光防潮保存。6.有效期:6個(gè)月 免責(zé)聲明1.試劑盒僅供研究使用,不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn),否則所產(chǎn)生的一切后果,由實(shí)驗(yàn)者承擔(dān),本公司概不負(fù)責(zé)。2.嚴(yán)格按照說明書操作,實(shí)驗(yàn)者違反說明書操作,后果由實(shí)驗(yàn)者承擔(dān)。FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURESPROCED

7、URES.Rat Vascular Endothelial cell Growth Factor(VEGFELISA Kit instructionIntended useS ample collection and storagesSerum-Use a serum separator tube and allow samples to clot for30minutes before centrifugation for10minutes at approximately3000×g.Remove serum and assay immediately or aliquot an

8、d store samples at-20or-80.Avoid repeated freeze-thaw cyclesPlasma-Collect plasma using EDTA or heparin as an anticoagulant.Centrifuge samples for30minutes at3000×g at2-8within30minutes of collection.Store samples at-20or-80.Avoid repeated freeze-thaw cycles.Cell culture supernates and other bi

9、ological fluids-Remove particulates by centrifugation and assay immediately or aliquot and store samples at-20or -80.Avoid repeated freeze-thaw cycles.Note:The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.Materials required but not supplied1.Standard microplate r

10、eader(450nm2.Precision pipettes and Disposable pipette tips.3.37incubatorP recautions1.Do not substitute reagents from one kit to another.Standard,conjugate and microplates are matched for optimal performance.Use only the reagents supplied by manufacturer.2.Do not remove microplate from the storage

11、bag until needed.Unused strips should be stored at2-8°C in their pouch with the desiccant provided.3.Mix all reagents before using.Remove all kit reagents from refrigerator and allow them to reach room temperature (20-25°CMaterials suppliedName96determinations48determinations Microelisa st

12、ripplate12*8strips12*4stripsSample Diluent 6.0ml 3.0mlHRP-Conjugate reagent10.0ml 5.0ml20X Wash solution25ml15mlChromogen Solution A 6.0ml 3.0mlChromogen Solution B 6.0ml 3.0mlStop Solution 6.0ml 3.0mlClosure plate membrane22User manual11Sealed bags11Note:Standard(S0S5concentration was followed by:0

13、,20,40,80,160,320 pg/ml.Reagent preparation20×wash solution:Dilute with Distilled or deionized water1:20.A ssay procedure1.Prepare all re a g e n t s before starting assay procedure.It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.2.Add standar

14、d:Set Standard wells,testing sample wells.Add standard50l to standard well.3.Add Sample:Add testing sample10l then add Sample Diluent40l to testing sample well;Blank well doesnt add anyting.4.Add100l of HRP-conjugate reagent to each well,c over with an adhesive strip and incubate for60minutes at37&#

15、176;C.5.Aspirate each well and wash,repeating the process four times for a total of five washes.Wash by filling each well with Wash Solution(400lusing a squirt bottle, manifold dispenser or autowasher.Complete removal of liquid at each step is essential to good performance.After the last wash,remove

16、 any remaining Wash Solution by aspirating or decanting.Invert the plate and blot it against clean paper towels.6.Add chromogen solution A50l and chromogen solution B50l to each well. Gently mix and incubate for15minutes at37°C.Protect from lightlight.7.Add50l Stop Solution to each well.The col

17、or in the wells should change from blue to yellow.If the color in the wells is green or the color change does not appear uniform,gently tap the plate to ensure thorough mixing.C alculation of results1.This standard curve is used to determine the amount in an unknown sample.The standard curve is gene

18、rated by plotting the average O.D. (450 nm obtained for each of the six standard concentrations on the vertical (Y axis versus the corresponding concentration on the horizontal (X axis. 2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software. 3. To determine the amount in each samp