本試劑盒隻能用于科學研究，不得用于醫學診斷 細菌（Bacteria）葉酸（FA）ELISA 檢測試劑盒           使用說明書 檢測原理 試劑盒采用雙抗體一步夾心法酶聯免疫吸附試驗（ELISA）。往 預先包被葉酸（FA）抗體的包被微孔中，依次加入标本、 标準品、HRP标記的檢測抗體，經過溫育并徹底洗滌。用底物TMB顯 色，TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用下轉化成 ＊終的黃色。顔色的深淺和樣品中的葉酸（FA）呈正相關。 用酶标儀在450nm 波長下測定吸光度（OD 值），計算樣品濃度。樣品收集、處理及保存方法1. 血清：使用不含熱原和内毒素的試管，操作過程中避免任何細 胞刺激，收集血液後，3000 轉離心 10 分鍾将血清和紅細胞迅速小心 地分離。 2. 血漿：EDTA、檸檬酸鹽或肝素抗凝。3000 轉離心 30 分鍾取上清。3. 細胞上清液：3000 轉離心 10 分鍾去除顆粒和聚合物。 4. 組織勻漿：将組織加入适量生理鹽水搗碎。3000 轉離心 10 分鍾 取上清。 5. 保存：如果樣本收集後不及時檢測，請按一次用量分裝，凍存 于-20℃，避免反複凍融，在室溫下解凍并确保樣品均勻地充分解凍。 自備物品 1. 酶标儀（450nm）2. 高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL 3. 37℃恒溫箱 操作注意事項 1. 試劑盒保存在 2-8℃，使用前室溫平衡 20 分鍾。從冰箱取出的 濃縮洗滌液會有結晶，這屬于正常現象，水浴加熱使結晶完全溶解 後再使用。2. 實驗中不用的闆條應立即放回自封袋中，密封（低溫幹燥）保 存。 3. 濃度爲 0 的 S0 号标準品即可視爲陰性對照或者空白；按照說明 書操作時樣本已經稀釋 5 倍，＊終結果乘以 5 才是樣本實際濃度。4. 嚴格按照說明書中标明的時間、加液量及順序進行溫育操作。 5. 所有液體組分使用前充分搖勻。試劑盒組成96孔配置48孔配置備注微孔酶标版12孔*8條12孔*4條無标準品0.3ml*6管0.3ml*6管無樣本稀釋液6ml3ml無檢測抗體-HRP10ml5ml無20*洗滌緩沖液25ml15ml按說明書進行稀釋底物A6ml3ml無底物B6ml3ml無終止液6ml3ml無封闆膜2張2張無說明書1份1份無自封袋1個1個無注：标準品（S0-S5）濃度依次爲：0、10、20、40、80、160 ng/ml 試劑的準備 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。 洗闆方法 1. 手工洗闆：甩盡孔内液體，每孔加滿洗滌液，靜置 1min 後甩盡 孔内液體，在吸水紙上拍幹，如此洗闆 5 次。 2. 自動洗闆機：每孔注入洗液 350μL，浸泡 1min，洗闆 5 次。 操作步驟 1. 從室溫平衡 20min 後的鋁箔袋中取出所需闆條，剩餘闆條用自 封袋密封放回 4℃。 2. 設置标準品孔和樣本孔，标準品孔各加不同濃度的标準品 50μ L；3. 樣本孔先加待測樣本 10μL，再加樣本稀釋液 40μL(即樣本稀 釋 5 倍)；空白孔不加。4. 除空白孔外，标準品孔和樣本孔中每孔加入辣根過氧化物酶 （HRP）标記的檢測抗體 100μL，用封闆膜封住反應孔，37℃水浴鍋 或恒溫箱溫育 60min。 5. 棄去液體，吸水紙上拍幹，每孔加滿洗滌液，靜置 1min，甩去 洗滌液，吸水紙上拍幹，如此重複洗闆 5 次（也可用洗闆機洗闆）。6. 每孔加入底物 A、B 各 50μL，37℃避光孵育 15min。7. 每孔加入終止液 50μL，15min 内，在 450nm 波長處測定各孔的 OD 值。結果判斷繪制标準曲線：在 Excel 工作表中，以标準品濃度作橫坐标，對應 OD 值作縱坐标，繪制出标準品線性回歸曲線，按曲線方程計算各樣 本濃度值。試劑盒性能 1. 準确性：标準品線性回歸與預期濃度相關系數 R 值，大于等于 0.9900。2. 靈敏度：＊低檢測濃度小于 1.0 ng/ml。 3. 特異性：不與其它可溶性結構類似物交叉反應。4. 重複性：闆内、闆間變異系數均小于 15%。 5. 貯藏：2-8℃，避光防潮保存。6. 有效期：6 個月 免責聲明 1. 試劑盒僅供研究使用，不得用于臨床實驗或人體實驗，否則所 産生的一切後果，由實驗者承擔，本公司概不負責。 2. 嚴格按照說明書操作，實驗者違反說明書操作，後果由實驗者承擔細菌（Bacteria）葉酸（FA）ELISA 檢測試劑盒FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.  urobilin ELISA Kit instruction Intended use This urobilin ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of urobilin in the sample, this urobilin ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus urobilin concentration. The concentration of urobilin in the samples is then determined by comparing the O.D. of the samples to the standard curve. Sample collection and storages Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles. Note: The samples shoule be centrifugated dequately and no hemolysis or granule was allowed. Materials required but not supplied 1. Standard microplate reader(450nm) 2. Precision pipettes and Disposable pipette tips. 3. 37 ℃ incubator Precautions1. Do not substitute reagents from one kit to another. Standard, conjugate  microplates are matched for optimal performance. Use only the reagents supplied by manufacturer. 2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°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°C) Materials supplied Name 96 determinations 48 determinations Microelisa stripplate 12*8strips 12*4strips Standard 0.3ml*6tubes 0.3ml*6tubes Sample Diluent 6.0ml 3.0ml HRP-Conjugate reagent 10.0ml 5.0ml 20X Wash solution 25ml 15ml Chromogen Solution A 6.0ml 3.0ml Chromogen Solution B 6.0ml 3.0ml Stop Solution 6.0ml 3.0ml Closure plate membrane 2 2 User manual 1 1 Sealed bags 1 1Note: Standard (S0 → S5) concentration was followed by:0,10,20,40,80,160 ng/ml Reagent preparation 20×wash solution:Dilute with Distilled or deionized water 1:20.Assay procedure1. Prepare all r e a g e n ts before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well. 3. Add Sample: Add testing sample 10μl then add 40μl of Sample Diluent to testing sample well; Blank well doesn’t add anyting. 4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°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 (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Washwww.dumabio.com  Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels. 6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light. 7. Add 50μl Stop Solution to each well. The color 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. 8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes. Calculation of results1. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated 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 sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration. 4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve. 5. The sensitivity by this assay is 1.0 ng/ml 6. Standard curvewww.dumabio.com Storage： 2-8℃. validity： six months. FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!