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· 資料簡(jiǎn)介： 小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）酶聯(lián)免疫分析試劑盒使用說(shuō)明書(shū)本試劑盒僅供研究使用。檢測(cè)范圍： 96T15ng/L -350ng/L使用目的：本試劑盒用于測(cè)定小鼠血清、血漿及相關(guān)液體樣本中血管內(nèi)皮生長(zhǎng)因子 （VEGF）含量。實(shí)驗(yàn)原理本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）水平。用純化的小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）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血管內(nèi)皮生長(zhǎng)因子 （VEGF），再與HRP 標(biāo)記的血管內(nèi)皮生長(zhǎng)因子 （VEGF）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過(guò)徹底洗滌后加底物TMB 顯色。TMB 在HRP 酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成*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），通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）濃度。試劑盒組成1 30 倍濃縮洗滌液 20ml×1 瓶 7 終止液 6ml×1 瓶2 酶標(biāo)試劑 6ml×1 瓶 8 標(biāo)準(zhǔn)品（640ng/L） 0.5ml×1 瓶3 酶標(biāo)包被板 12 孔×8 條 9 標(biāo)準(zhǔn)品稀釋液 1.5ml×1 瓶4 樣品稀釋液 6ml×1 瓶 10 說(shuō)明書(shū) 1 份5 顯色劑A 液 6ml×1 瓶 11 封板膜 2 張6 顯色劑B 液 6ml×1/瓶 12 密封袋 1 個(gè)標(biāo)本要求1．標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20℃保存，但應(yīng)避免反復(fù)凍融2．不能檢測(cè)含NaN3 的樣品，因NaN3 抑制辣根過(guò)氧化物酶的（HRP）活性。 小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）酶聯(lián)免疫分析試劑盒操作步驟1. 標(biāo)準(zhǔn)品的稀釋：本試劑盒提供原倍標(biāo)準(zhǔn)品一支，用戶可按照下列圖表在小試管中進(jìn)行稀釋。320ng/L 5 號(hào)標(biāo)準(zhǔn)品 150μl 的原倍標(biāo)準(zhǔn)品加入150μl 標(biāo)準(zhǔn)品稀釋液160ng/L 4 號(hào)標(biāo)準(zhǔn)品 150μl 的5 號(hào)標(biāo)準(zhǔn)品加入150μl 標(biāo)準(zhǔn)品稀釋液80ng/L 3 號(hào)標(biāo)準(zhǔn)品 150μl 的4 號(hào)標(biāo)準(zhǔn)品加入150μl 標(biāo)準(zhǔn)品稀釋液40ng/L 2 號(hào)標(biāo)準(zhǔn)品 150μl 的3 號(hào)標(biāo)準(zhǔn)品加入150μl 標(biāo)準(zhǔn)品稀釋液20ng/L 1 號(hào)標(biāo)準(zhǔn)品 150μl 的2 號(hào)標(biāo)準(zhǔn)品加入150μl 標(biāo)準(zhǔn)品稀釋液2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、標(biāo)準(zhǔn)孔、待測(cè)樣品孔。在酶標(biāo)包被板上標(biāo)準(zhǔn)品準(zhǔn)確加樣50μl，待測(cè)樣品孔中先加樣品稀釋液40μl，然后再加待測(cè)樣品10μl（樣品*終稀釋度為5 倍）。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動(dòng)混勻。3. 溫育：用封板膜封板后置37℃溫育30 分鐘。4. 配液：將30 倍濃縮洗滌液用蒸餾水30 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(fù)5 次，拍干。6. 加酶：每孔加入酶標(biāo)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(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。11. 測(cè)定：以空白空調(diào)零，450nm 波長(zhǎng)依序測(cè)量各孔的吸光度（OD 值）。 測(cè)定應(yīng)在加終止液后15 分鐘以內(nèi)進(jìn)行。操作程序總結(jié)： 小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）酶聯(lián)免疫分析試劑盒計(jì)算以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD 值為縱坐標(biāo)，在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的OD 值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；再乘以稀釋倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與OD 值計(jì)算出標(biāo)準(zhǔn)曲線的直線回歸方程式，將樣品的OD 值代入方程式，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。注意事項(xiàng)1．試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30 分鐘后方可使用，酶標(biāo)包被板開(kāi)封后如未用完，板條應(yīng)裝入密封袋中保存。2．濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。3．各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)間控制在5 分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。4． 請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線，做復(fù)孔。如標(biāo)本中待測(cè)物質(zhì)含量過(guò)高（樣本OD 值大于標(biāo)準(zhǔn)品孔孔的OD 值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（n 倍）后再測(cè)定，計(jì)算時(shí)請(qǐng)*后乘以總稀釋倍數(shù)（×n×5）。5． 封板膜只限一次性使用，以避免交叉污染。6．底物請(qǐng)避光保存。7．嚴(yán)格按照說(shuō)明書(shū)的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).8．所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。9．本試劑不同批號(hào)組分不得混用。10. 如與英文說(shuō)明書(shū)有異，以英文說(shuō)明書(shū)為準(zhǔn)。保存條件及有效期1．試劑盒保存：；2-8℃。2．有效期：6 個(gè)月 Mouse VEGFFOR RESEARCH USE ONLYAssay range：15ng/L -350ng/L 96 determinationsPurposeThis kit allows for the determination of VEGF concentrations in Mouse serum, cellculture supernates ａnd other biological fluidsPrinciple of the assayThe kit assay Mouse VEGF level in the sample，use Purified Mouse VEGF antibody tocoat microtiter plate wells, make solid-phase antibody, then add VEGF to wells, CombinedVEGF antibody which With HRP labeled,become antibody - antigen - enzyme-antibodycomplex, after washing Completely, Add TMB substrate solution,TMB substrate becomes bluecolor At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acidsolution ａnd the color change is measured spectrophotometrically at a wavelength of 450 nm.The concentration of Mouse VEGF in the samples is then determined by comparing the O.D. ofthe samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stopp Solution 6ml×1 bottle2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard（640ng/L） 0.5ml×1 bottle3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle4 Sample diluent 6ml×1 bottle 10 Instruction 15 Chromogen Solution A 6ml×1 bottle 11Closure platemembrane26 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevantRDliterature, ａnd should be experiment as soon as possible after the extraction. If it can’t,specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute ａnd add sample:Dilute Original density Standard as follow table:320ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent160ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent80ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent40ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent20ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent2.add sample：Set blank wells separately （blank comparison wells don’t add sample andHRP-Conjugate reagent, other each step operation is same）. testing sample well. add Sampledilution 40μl to testing sample well, then add testing sample 10μl （sample final dilution is5-fold）, add sample to wells , don’t touch the well wall as far as possible, ａnd Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.4.Configurate liquid: 30-fold（or 20-fold） wash solution diluted 30-fold （or 20-fold） with distilledwater ａnd reserve.5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing bufferto every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.7.incubate：Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul ａnd Chromogen Solution B to each well, evade thelight preservation for 15 min at 37℃10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue colorchange to yellow color）.11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Steps descriptionStandard, Sample diluentAdd Standard, Sample diluent, incubate for 30 min at 37℃.Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.Wash 5 times,Add Chromogen Solution A ａnd B, incubate for 30 min at 37℃.Add Stopp SolutionRead absorbance at 450nm within 15 mincalculateCalculateTake the standard density as the horizontal, the OD value for the vertical ,draw thestandard curve on graph paper, Find out the corresponding density according to the sampleOD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight lineregression equation of the standard curve with the standard density ａnd the OD value ,with thesample OD value in the equation, calculate the sample density, multiplied by the dilution factor,the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes inthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental error. add sample within 5 min, if the number of sample is much , recommendto use Volley .4. if the testing material content is excessively higher （The sample OD is bigger than the firststandard well ）,please dilute Sample （n-fold）, Please diluente ａnd multiplied by the dilutionfactor.（×n×5）.5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take themicrotiter plate reader as a standard.8. All samples, washing buffer ａnd each kind of reject should according to infective materialprocess.9. Do not mix reagents with those from other lots.Storage ａnd validity1．Storage： 2-8℃. 小鼠血管內(nèi)皮生長(zhǎng)因子 （VEGF）酶聯(lián)免疫分析試劑盒2．validity： six months
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