本(ben)(ben)(ben)(ben)試劑(ji)盒(he)只能用(yong)(yong)于(yu)(yu)科(ke)學(xue)研究(jiu)，不(bu)(bu)得(de)用(yong)(yong)于(yu)(yu)醫學(xue)診斷(duan) 細(xi)菌(jun)(jun)（Bacteria）葉酸(suan)（FA）ELISA 檢(jian)測(ce)(ce)(ce)試劑(ji)盒(he) 使(shi)(shi)用(yong)(yong)說明書(shu)(shu)(shu) 檢(jian)測(ce)(ce)(ce)原理(li) 試劑(ji)盒(he)采用(yong)(yong)雙(shuang)抗(kang)體(ti)(ti)(ti)(ti)一(yi)步夾心法(fa)酶聯免疫(yi)吸(xi)附試驗（ELISA）。往 預(yu)先(xian)(xian)包(bao)被(bei)葉酸(suan)（FA）抗(kang)體(ti)(ti)(ti)(ti)的(de)(de)包(bao)被(bei)微孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，依次加(jia)(jia)(jia)入(ru)(ru)標(biao)本(ben)(ben)(ben)(ben)、 標(biao)準(zhun)(zhun)品(pin)(pin)(pin)、HRP標(biao)記(ji)的(de)(de)檢(jian)測(ce)(ce)(ce)抗(kang)體(ti)(ti)(ti)(ti)，經過(guo)溫(wen)(wen)(wen)育(yu)并(bing)徹底(di)(di)洗(xi)(xi)滌(di)。用(yong)(yong)底(di)(di)物(wu)TMB顯(xian) 色(se)，TMB在(zai)(zai)過(guo)氧化(hua)物(wu)酶的(de)(de)催(cui)化(hua)下轉(zhuan)化(hua)成(cheng)藍色(se)，并(bing)在(zai)(zai)酸(suan)的(de)(de)作用(yong)(yong)下轉(zhuan)化(hua)成(cheng) ＊終(zhong)的(de)(de)黃色(se)。顏色(se)的(de)(de)深淺和(he)(he)樣(yang)(yang)(yang)(yang)品(pin)(pin)(pin)中(zhong)(zhong)(zhong)(zhong)的(de)(de)葉酸(suan)（FA）呈正相關。 用(yong)(yong)酶標(biao)儀在(zai)(zai)450nm 波長下測(ce)(ce)(ce)定吸(xi)光度(du)(du)(du)(du)（OD 值），計算樣(yang)(yang)(yang)(yang)品(pin)(pin)(pin)濃(nong)(nong)(nong)度(du)(du)(du)(du)。樣(yang)(yang)(yang)(yang)品(pin)(pin)(pin)收集、處理(li)及(ji)(ji)保(bao)存方(fang)法(fa)1. 血(xue)(xue)清(qing)(qing)(qing)：使(shi)(shi)用(yong)(yong)不(bu)(bu)含熱原和(he)(he)內(nei)毒素的(de)(de)試管，操(cao)作過(guo)程中(zhong)(zhong)(zhong)(zhong)避免任何細(xi) 胞刺激，收集血(xue)(xue)液(ye)(ye)后(hou)(hou)(hou)，3000 轉(zhuan)離(li)心 10 分(fen)(fen)鐘將血(xue)(xue)清(qing)(qing)(qing)和(he)(he)紅細(xi)胞迅速小心 地(di)分(fen)(fen)離(li)。 2. 血(xue)(xue)漿：EDTA、檸檬酸(suan)鹽(yan)或肝(gan)素抗(kang)凝。3000 轉(zhuan)離(li)心 30 分(fen)(fen)鐘取(qu)(qu)上(shang)(shang)清(qing)(qing)(qing)。3. 細(xi)胞上(shang)(shang)清(qing)(qing)(qing)液(ye)(ye)：3000 轉(zhuan)離(li)心 10 分(fen)(fen)鐘去除顆粒和(he)(he)聚(ju)合物(wu)。 4. 組織勻(yun)漿：將組織加(jia)(jia)(jia)入(ru)(ru)適量生(sheng)理(li)鹽(yan)水(shui)搗(dao)碎。3000 轉(zhuan)離(li)心 10 分(fen)(fen)鐘 取(qu)(qu)上(shang)(shang)清(qing)(qing)(qing)。 5. 保(bao)存：如果樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)收集后(hou)(hou)(hou)不(bu)(bu)及(ji)(ji)時檢(jian)測(ce)(ce)(ce)，請按(an)一(yi)次用(yong)(yong)量分(fen)(fen)裝，凍存 于(yu)(yu)-20℃，避免反復(fu)凍融(rong)，在(zai)(zai)室溫(wen)(wen)(wen)下解凍并(bing)確保(bao)樣(yang)(yang)(yang)(yang)品(pin)(pin)(pin)均(jun)勻(yun)地(di)充(chong)分(fen)(fen)解凍。 自(zi)(zi)(zi)(zi)備物(wu)品(pin)(pin)(pin) 1. 酶標(biao)儀（450nm）2. 高精度(du)(du)(du)(du)加(jia)(jia)(jia)樣(yang)(yang)(yang)(yang)器及(ji)(ji)槍頭(tou)：0.5-10uL、2-20uL、20-200uL、200-1000uL 3. 37℃恒溫(wen)(wen)(wen)箱(xiang) 操(cao)作注意事項 1. 試劑(ji)盒(he)保(bao)存在(zai)(zai) 2-8℃，使(shi)(shi)用(yong)(yong)前室溫(wen)(wen)(wen)平衡(heng) 20 分(fen)(fen)鐘。從冰箱(xiang)取(qu)(qu)出的(de)(de) 濃(nong)(nong)(nong)縮(suo)洗(xi)(xi)滌(di)液(ye)(ye)會(hui)有(you)(you)結晶(jing)，這屬于(yu)(yu)正常現(xian)象，水(shui)浴加(jia)(jia)(jia)熱使(shi)(shi)結晶(jing)完全溶(rong)(rong)解 后(hou)(hou)(hou)再使(shi)(shi)用(yong)(yong)。2. 實(shi)(shi)驗中(zhong)(zhong)(zhong)(zhong)不(bu)(bu)用(yong)(yong)的(de)(de)板(ban)(ban)(ban)(ban)條(tiao)(tiao)應(ying)立即(ji)放(fang)(fang)回(hui)自(zi)(zi)(zi)(zi)封(feng)(feng)(feng)袋中(zhong)(zhong)(zhong)(zhong)，密封(feng)(feng)(feng)（低溫(wen)(wen)(wen)干(gan)燥）保(bao) 存。 3. 濃(nong)(nong)(nong)度(du)(du)(du)(du)為(wei) 0 的(de)(de) S0 號標(biao)準(zhun)(zhun)品(pin)(pin)(pin)即(ji)可視為(wei)陰性(xing)(xing)對照或者空白；按(an)照說明 書(shu)(shu)(shu)操(cao)作時樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)已(yi)經稀(xi)釋(shi) 5 倍，＊終(zhong)結果乘(cheng)以 5 才(cai)是樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)實(shi)(shi)際(ji)濃(nong)(nong)(nong)度(du)(du)(du)(du)。4. 嚴格按(an)照說明書(shu)(shu)(shu)中(zhong)(zhong)(zhong)(zhong)標(biao)明的(de)(de)時間、加(jia)(jia)(jia)液(ye)(ye)量及(ji)(ji)順(shun)序進(jin)行溫(wen)(wen)(wen)育(yu)操(cao)作。 5. 所(suo)有(you)(you)液(ye)(ye)體(ti)(ti)(ti)(ti)組分(fen)(fen)使(shi)(shi)用(yong)(yong)前充(chong)分(fen)(fen)搖勻(yun)。試劑(ji)盒(he)組成(cheng)96孔(kong)(kong)(kong)(kong)(kong)(kong)配置(zhi)48孔(kong)(kong)(kong)(kong)(kong)(kong)配置(zhi)備注微孔(kong)(kong)(kong)(kong)(kong)(kong)酶標(biao)版(ban)12孔(kong)(kong)(kong)(kong)(kong)(kong)*8條(tiao)(tiao)12孔(kong)(kong)(kong)(kong)(kong)(kong)*4條(tiao)(tiao)無(wu)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)0.3ml*6管0.3ml*6管無(wu)樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)稀(xi)釋(shi)液(ye)(ye)6ml3ml無(wu)檢(jian)測(ce)(ce)(ce)抗(kang)體(ti)(ti)(ti)(ti)-HRP10ml5ml無(wu)20*洗(xi)(xi)滌(di)緩沖液(ye)(ye)25ml15ml按(an)說明書(shu)(shu)(shu)進(jin)行稀(xi)釋(shi)底(di)(di)物(wu)A6ml3ml無(wu)底(di)(di)物(wu)B6ml3ml無(wu)終(zhong)止(zhi)液(ye)(ye)6ml3ml無(wu)封(feng)(feng)(feng)板(ban)(ban)(ban)(ban)膜2張2張無(wu)說明書(shu)(shu)(shu)1份(fen)1份(fen)無(wu)自(zi)(zi)(zi)(zi)封(feng)(feng)(feng)袋1個(ge)(ge)1個(ge)(ge)無(wu)注：標(biao)準(zhun)(zhun)品(pin)(pin)(pin)（S0-S5）濃(nong)(nong)(nong)度(du)(du)(du)(du)依次為(wei)：0、10、20、40、80、160 ng/ml 試劑(ji)的(de)(de)準(zhun)(zhun)備 20×洗(xi)(xi)滌(di)緩沖液(ye)(ye)的(de)(de)稀(xi)釋(shi)：蒸餾水(shui)按(an) 1：20 稀(xi)釋(shi)，即(ji) 1 份(fen)的(de)(de) 20×洗(xi)(xi)滌(di) 緩沖液(ye)(ye)加(jia)(jia)(jia) 19 份(fen)的(de)(de)蒸餾水(shui)。 洗(xi)(xi)板(ban)(ban)(ban)(ban)方(fang)法(fa) 1. 手工洗(xi)(xi)板(ban)(ban)(ban)(ban)：甩(shuai)盡(jin)(jin)孔(kong)(kong)(kong)(kong)(kong)(kong)內(nei)液(ye)(ye)體(ti)(ti)(ti)(ti)，每(mei)孔(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)滿(man)(man)洗(xi)(xi)滌(di)液(ye)(ye)，靜置(zhi) 1min 后(hou)(hou)(hou)甩(shuai)盡(jin)(jin) 孔(kong)(kong)(kong)(kong)(kong)(kong)內(nei)液(ye)(ye)體(ti)(ti)(ti)(ti)，在(zai)(zai)吸(xi)水(shui)紙(zhi)(zhi)上(shang)(shang)拍(pai)干(gan)，如此(ci)洗(xi)(xi)板(ban)(ban)(ban)(ban) 5 次。 2. 自(zi)(zi)(zi)(zi)動洗(xi)(xi)板(ban)(ban)(ban)(ban)機：每(mei)孔(kong)(kong)(kong)(kong)(kong)(kong)注入(ru)(ru)洗(xi)(xi)液(ye)(ye) 350μL，浸泡 1min，洗(xi)(xi)板(ban)(ban)(ban)(ban) 5 次。 操(cao)作步驟 1. 從室溫(wen)(wen)(wen)平衡(heng) 20min 后(hou)(hou)(hou)的(de)(de)鋁箔袋中(zhong)(zhong)(zhong)(zhong)取(qu)(qu)出所(suo)需板(ban)(ban)(ban)(ban)條(tiao)(tiao)，剩余板(ban)(ban)(ban)(ban)條(tiao)(tiao)用(yong)(yong)自(zi)(zi)(zi)(zi) 封(feng)(feng)(feng)袋密封(feng)(feng)(feng)放(fang)(fang)回(hui) 4℃。 2. 設置(zhi)標(biao)準(zhun)(zhun)品(pin)(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)和(he)(he)樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)孔(kong)(kong)(kong)(kong)(kong)(kong)，標(biao)準(zhun)(zhun)品(pin)(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)各加(jia)(jia)(jia)不(bu)(bu)同(tong)濃(nong)(nong)(nong)度(du)(du)(du)(du)的(de)(de)標(biao)準(zhun)(zhun)品(pin)(pin)(pin) 50μ L；3. 樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)孔(kong)(kong)(kong)(kong)(kong)(kong)先(xian)(xian)加(jia)(jia)(jia)待測(ce)(ce)(ce)樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben) 10μL，再加(jia)(jia)(jia)樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)稀(xi)釋(shi)液(ye)(ye) 40μL(即(ji)樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)稀(xi) 釋(shi) 5 倍)；空白孔(kong)(kong)(kong)(kong)(kong)(kong)不(bu)(bu)加(jia)(jia)(jia)。4. 除空白孔(kong)(kong)(kong)(kong)(kong)(kong)外，標(biao)準(zhun)(zhun)品(pin)(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)和(he)(he)樣(yang)(yang)(yang)(yang)本(ben)(ben)(ben)(ben)孔(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)(zhong)每(mei)孔(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)入(ru)(ru)辣根(gen)過(guo)氧化(hua)物(wu)酶 （HRP）標(biao)記(ji)的(de)(de)檢(jian)測(ce)(ce)(ce)抗(kang)體(ti)(ti)(ti)(ti) 100μL，用(yong)(yong)封(feng)(feng)(feng)板(ban)(ban)(ban)(ban)膜封(feng)(feng)(feng)住反應(ying)孔(kong)(kong)(kong)(kong)(kong)(kong)，37℃水(shui)浴鍋(guo) 或恒溫(wen)(wen)(wen)箱(xiang)溫(wen)(wen)(wen)育(yu) 60min。 5. 棄去液(ye)(ye)體(ti)(ti)(ti)(ti)，吸(xi)水(shui)紙(zhi)(zhi)上(shang)(shang)拍(pai)干(gan)，每(mei)孔(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)滿(man)(man)洗(xi)(xi)滌(di)液(ye)(ye)，靜置(zhi) 1min，甩(shuai)去 洗(xi)(xi)滌(di)液(ye)(ye)，吸(xi)水(shui)紙(zhi)(zhi)上(shang)(shang)拍(pai)干(gan)，如此(ci)重(zhong)復(fu)洗(xi)(xi)板(ban)(ban)(ban)(ban) 5 次（也可用(yong)(yong)洗(xi)(xi)板(ban)(ban)(ban)(ban)機洗(xi)(xi)板(ban)(ban)(ban)(ban)）。6. 每(mei)孔(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)入(ru)(ru)底(di)(di)物(wu) A、B 各 50μL，37℃避光孵育(yu) 15min。7. 每(mei)孔(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)入(ru)(ru)終(zhong)止(zhi)液(ye)(ye) 50μL，15min 內(nei)，在(zai)(zai) 450nm 波長處測(ce)(ce)(ce)定各孔(kong)(kong)(kong)(kong)(kong)(kong)的(de)(de) OD 值。結果判斷(duan)繪制(zhi)標(biao)準(zhun)(zhun)曲線：在(zai)(zai) Excel 工作表中(zhong)(zhong)(zhong)(zhong)，以標(biao)準(zhun)(zhun)品(pin)(pin)(pin)濃(nong)(nong)(nong)度(du)(du)(du)(du)作橫坐標(biao)，對應(ying) OD 值作縱坐標(biao)，繪制(zhi)出標(biao)準(zhun)(zhun)品(pin)(pin)(pin)線性(xing)(xing)回(hui)歸曲線，按(an)曲線方(fang)程計算各樣(yang)(yang)(yang)(yang) 本(ben)(ben)(ben)(ben)濃(nong)(nong)(nong)度(du)(du)(du)(du)值。試劑(ji)盒(he)性(xing)(xing)能 1. 準(zhun)(zhun)確性(xing)(xing)：標(biao)準(zhun)(zhun)品(pin)(pin)(pin)線性(xing)(xing)回(hui)歸與預(yu)期濃(nong)(nong)(nong)度(du)(du)(du)(du)相關系(xi)數(shu) R 值，大(da)于(yu)(yu)等(deng)于(yu)(yu) 0.9900。2. 靈敏度(du)(du)(du)(du)：＊低檢(jian)測(ce)(ce)(ce)濃(nong)(nong)(nong)度(du)(du)(du)(du)小于(yu)(yu) 1.0 ng/ml。 3. 特異(yi)性(xing)(xing)：不(bu)(bu)與其它可溶(rong)(rong)性(xing)(xing)結構類似物(wu)交叉反應(ying)。4. 重(zhong)復(fu)性(xing)(xing)：板(ban)(ban)(ban)(ban)內(nei)、板(ban)(ban)(ban)(ban)間變(bian)異(yi)系(xi)數(shu)均(jun)小于(yu)(yu) 15%。 5. 貯(zhu)藏：2-8℃，避光防潮(chao)保(bao)存。6. 有(you)(you)效期：6 個(ge)(ge)月 免責(ze)聲(sheng)明 1. 試劑(ji)盒(he)僅供研究(jiu)使(shi)(shi)用(yong)(yong)，不(bu)(bu)得(de)用(yong)(yong)于(yu)(yu)臨(lin)床實(shi)(shi)驗或人體(ti)(ti)(ti)(ti)實(shi)(shi)驗，否則所(suo) 產生(sheng)的(de)(de)一(yi)切后(hou)(hou)(hou)果，由(you)實(shi)(shi)驗者承擔，本(ben)(ben)(ben)(ben)公司(si)概不(bu)(bu)負責(ze)。 2. 嚴格按(an)照說明書(shu)(shu)(shu)操(cao)作，實(shi)(shi)驗者違反說明書(shu)(shu)(shu)操(cao)作，后(hou)(hou)(hou)果由(you)實(shi)(shi)驗者承擔細(xi)菌(jun)(jun)（Bacteria）葉酸(suan)（FA）ELISA 檢(jian)測(ce)(ce)(ce)試劑(ji)盒(he)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 Washfllygs.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 curvefllygs.com Storage： 2-8℃. validity： six months. FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
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