大鼠極低密度脂蛋白(VLDL)酶聯(lián)免疫分析.doc

大鼠極低密度脂蛋白 大鼠極低密度脂蛋白 VLDL 酶聯(lián)免疫分析 酶聯(lián)免疫分析 試劑盒使用說(shuō)明書(shū)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 檢測(cè)范圍 96T 3 g ml 120 g ml 使用目的 使用目的 本試劑盒用于測(cè)定大鼠血清 血漿及相關(guān)液體樣本中極低密度脂蛋白 VLDL 含量 實(shí)驗(yàn)原理實(shí)驗(yàn)原理 本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中大鼠極低密度脂蛋白 VLDL 水平 用純化的 大鼠極低密度脂蛋白 VLDL 抗體包被微孔板 制成固相抗體 往包被單抗的微孔中依次 加入極低密度脂蛋白 VLDL 再與 HRP 標(biāo)記的極低密度脂蛋白 VLDL 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)化成最終的黃色 顏色的深淺和樣品中的極低密度脂蛋白 VLDL 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(xiàn)計(jì)算樣 品中大鼠極低密度脂蛋白 VLDL 濃度 試劑盒組成試劑盒組成 130 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 240 g ml 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 操作步驟操作步驟 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(hù)可按照下列圖表在小試管中進(jìn)行 稀釋 120 g ml5 號(hào)標(biāo)準(zhǔn)品150 l 的原倍標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液 60 g ml4 號(hào)標(biāo)準(zhǔn)品150 l 的 5 號(hào)標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液 30 g ml3 號(hào)標(biāo)準(zhǔn)品150 l 的 4 號(hào)標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液 15 g ml2 號(hào)標(biāo)準(zhǔn)品150 l 的 3 號(hào)標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液 7 5 g ml1 號(hào)標(biāo)準(zhǔn)品150 l 的 2 號(hào)標(biāo)準(zhǔn)品加入 150 l 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 50 l 待測(cè)樣品孔中先加樣品稀釋液 40 l 然后再加待測(cè)樣品 10 l 樣品最終稀釋度為 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(mǎn)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 50 l 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 A50 l 再加入顯色劑 B50 l 輕輕震蕩混勻 37 避光顯色 15 分鐘 10 終止 每孔加終止液 50 l 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?xún)?nèi)進(jìn)行 操作程序總結(jié) 操作程序總結(jié) 計(jì)算計(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(xiàn) 根據(jù)樣品的 OD 值由標(biāo)準(zhǔn)曲線(xiàn)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(xiàn)的直線(xiàn)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)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)間最好 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(xiàn) 最好做復(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)品孔第一孔的 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)最后乘以總稀釋倍數(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) 保存條件及有效期保存條件及有效期 1 試劑盒保存 2 8 2 有效期 6 個(gè)月 Rat VLDL FOR RESEARCH USE ONLY Assay range 3 g mL 120 g mL 96 determinations Purpose This kit allows for the determination of VLDL concentrations in Rat serum cell culture supernates and other biological fluids Principle of the assay The kit assay Rat VLDL level in the sample use Purified Rat VLDL antibody to coat microtiter plate wells make solid phase antibody then add VLDL to wells Combined VLDL antibody which With HRP labeled become antibody antigen enzyme antibody complex after washing Completely Add TMB substrate solution TMB substrate becomes blue color At HRP enzyme catalyzed reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm The concentration of Rat VLDL in the samples is then determined by comparing the O D of the samples to the standard curve Materials provided with the kit 1wash solution20ml 1bottle7Stopp Solution6ml 1 bottle 2HRP Conjugate reagent6ml 1 bottle8 Standard 240 g ml 0 5ml 1 bottle 3Microelisa stripplate12well 8strips9Standard diluent1 5ml 1bottle 4Sample diluent6ml 1 bottle10Instruction1 5Chromogen Solution A6ml 1 bottle11 Closure plate membrane 2 6Chromogen Solution B6ml 1 bottle12Sealed bags1 Specimen requirements 1 extract as soon as possible after Specimen collection and according to the relevant RD literature 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 procedure 1 Dilute and add sample Dilute Original density Standard as follow table 120 g ml 5 Standard150 l Original density Standard 150 l Standard diluent 80 g mL 4 Standard150 l 5 Standard 150 l Standard diluent 40 g mL 3 Standard150 l 4 Standard 150 l Standard diluent 20 g mL 2 Standard150 l 3 Standard 150 l Standard diluent 10 g mL 1 Standard150 l 2 Standard 150 l Standard diluent 2 add sample Set blank wells separately blank comparison wells don t add sample and HRP Conjugate reagent other each step operation is same testing sample well add Sample dilution 40 l to testing sample well then add testing sample 10 l sample final dilution is 5 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 distilled water and reserve 5 washing Uncover Closure plate membrane discard Liquid dry by swing add washing buffer to every well still for 30s then drain repeat 5 times dry by pat 6 add enzyme Add HRP Conjugate reagent 50 l 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 the light preservation for 15 min at 37 10 Stop the reaction Add Stop Solution50 l to each well Stop the reaction the blue color change to yellow color 11 assay take blank well as zero Read absorbance at 450nm after Adding Stop Solution and within 15min Steps description Standard Sample diluent Add 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 Solution Read absorbance at 450nm within 15 min calculate Calculate Take the standard density as the horizontal the OD value for the vertical draw the standard curve on graph paper Find out the corresponding density according to the sample OD value by the Sample curve multiplied by the dilution multiple or calculate the straight line regression equation of the standard curve with the standard density and the OD value with the sample OD value in the equation calculate the sample density multiplied by the dilution factor the result is the sample actual density Important notes 1 The kit takes out from the refrigeration environment should be balanced 15 30 minutes in the room temperature ELISA plates coated if has not use up after opened the plate should be stored in Sealed bag 2 washing buffer will Crystallization separation it can be heated the water helps dissolve when dilute Washing does not affect the result 3 add Sample with sampler Each step And proofread its accuracy frequently avoids the experimental error add sample within 5