小鼠白介素10(IL-10)ELISAKit

1、AMEKOTEL:+86-656775338FAXE-mail: lianshuo試劑盒使用說明書AE90224MUAMEK6Onlv use for reM&rrb For life srience小鼠白細(xì)胞介素10(IL-10)幅聯(lián)免疫檢測使用前仔細(xì)閱讀本說明書。本酶聯(lián)免疫試劑盒是基于雙抗體夾心技術(shù)原理, 來檢測小鼠白細(xì)胞介素10(IL-10),只能用于研究用途，不得用于醫(yī)學(xué)診斷。用 途：用于小鼠血清、血漿及相關(guān)液體樣本中白細(xì)胞介素10(IL-10)測定。工作原理本試劑盒采用的是生物素雙抗體夾心酶聯(lián)免疫吸附法(ELIS

2、A)測定樣品中小鼠白細(xì)胞介素10(IL-10)水平。向預(yù)先包被了小鼠白細(xì)胞介素10(IL-10)單克隆抗體的酶標(biāo)孔中加入白細(xì)胞介素 10(IL-10),溫育；溫育后，加入生物素標(biāo)記 的抗IL-10抗體。再與鏈霉親和素-HRP結(jié)合，形成免疫復(fù)合物，再經(jīng)過溫育和 洗滌，去除未結(jié)合的酶，然后加入底物A、B,產(chǎn)生藍(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺與樣品中小鼠白細(xì)胞介素10(IL-10)的濃度呈正相關(guān)。試劑盒組成試劑盒組成48孔配置96孔配置保存說明書1份1份封板膜2 片（48）2 片（96）密封袋1個1個酶標(biāo)包被板1X481X962-8 C保存標(biāo)準(zhǔn)品400 ng/L0.5ml X 1 瓶

3、0.5ml X 1 瓶2-8 C保存標(biāo)準(zhǔn)品稀釋液3ml x 1 瓶6ml x 1 瓶2-8 C保存鏈霉親和素-HRP3 ml x 1 瓶6 ml x 1 瓶2-8C保存生物素標(biāo)記的抗IL-10抗體0.5ml X 1 瓶1 ml x 1 瓶2-8 C保存顯色劑A液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存顯色劑B液3 ml x 1 瓶6 ml x 1 瓶2-8 C保存終止液3ml x 1 瓶6ml x 1 瓶2-8 C保存濃縮洗滌液（20ml X 20 倍）X 1 瓶（20mlX30倍）X 1 瓶2-8 C保存需要而未提供的試劑和器材1 . 37 c恒溫箱。2 .標(biāo)準(zhǔn)規(guī)格酶標(biāo)儀。3

4、.精密移液器及一次性吸頭4 .蒸儲水，5 . 一次性試管6 .吸水紙注意事項1.從2-8 C取出的試劑盒，在開啟試劑盒之前要室溫平衡至少 30分鐘。酶標(biāo)包 被板開封后如未用完，板條應(yīng)裝入密封袋中保存。2,各步加樣均應(yīng)使用加樣器，并經(jīng)常校對其準(zhǔn)確性，以避免試驗誤差3 .嚴(yán)格按照說明書的操作進(jìn)行，試驗結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).4 .為避免交叉污染，要避免重復(fù)使用手中的吸頭和封板膜。5 .不用的其它試劑應(yīng)包裝好或蓋好。不同批號的試劑不要混用。保質(zhì)前使用。6 .底物B對光敏感，避免長時間暴露于光下。洗板方法手工洗板方法：甩掉酶標(biāo)板內(nèi)的液體；在實驗臺上鋪墊幾層吸水紙，酶標(biāo)板朝下 用力拍幾次；將稀釋后

5、的洗滌液至少 0.35ml注入孔內(nèi)，浸泡1-2分鐘。根據(jù)需 要，重復(fù)此過程數(shù)次。自動洗板：如果有自動洗板機(jī)，應(yīng)在熟練使用后再用到正式實驗過程中標(biāo)本要求1 .不能檢測含NaN3勺樣品，因NaN3卬制辣根過氧化物酶的（HRP活性。2 .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)行實驗。若不能馬上進(jìn)行試驗，可將標(biāo)本放于-20 C保存，但應(yīng)避免反復(fù)凍融。操作程序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。）200 ng/L5號標(biāo)準(zhǔn)品120 l的原倍標(biāo)準(zhǔn)品加入120仙l標(biāo)準(zhǔn)品稀釋液100 ng/L4號標(biāo)準(zhǔn)品120仙l的5號標(biāo)準(zhǔn)品加入120仙l標(biāo)

6、準(zhǔn)品稀釋液50 ng/L3號標(biāo)準(zhǔn)品1201的4號標(biāo)準(zhǔn)品加入120（i l標(biāo)準(zhǔn)品稀釋液25ng/L2號標(biāo)準(zhǔn)品1201的3號標(biāo)準(zhǔn)品加入120（i l標(biāo)準(zhǔn)品稀釋液12.5 ng/L1號標(biāo)準(zhǔn)品1201的2號標(biāo)準(zhǔn)品加入120（i l標(biāo)準(zhǔn)品稀釋液2 .根據(jù)待測樣品數(shù)量加上標(biāo)準(zhǔn)品的數(shù)量決定所需的板條數(shù)。每個標(biāo)準(zhǔn)品和空白孔建議做復(fù)孔。每個樣品根據(jù)自己的數(shù)量來定，能使用復(fù)孔的盡量做復(fù)孔。3 .加樣：1）空白孔，空白對照孔不加樣品，生物素標(biāo)記的抗 IL-4抗體，鏈霉 親和素-HRP,只加顯色劑A&B和終止液，其余各步操作相同；2）標(biāo)準(zhǔn)品孔： 加入標(biāo)準(zhǔn)品50ul ,鏈霉親和素-HRP50U1（標(biāo)準(zhǔn)品中已事先

7、整合好生物素抗體， 故不加）:3）待測樣品孔：加入 樣本40ul、然后各加入 抗IL-4 抗體10ul、 鏈霉親和素-HRP50U1,蓋上封板膜，車5輕震蕩混勻，37c溫育60分鐘。4 .配液：將30倍濃縮洗滌液用蒸儲水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 .顯色：每孔先加入顯色劑 A50ul,再加入顯色劑B50p 1,輕輕震蕩混勻，37c 避光顯色15分鐘.7 .終止：每孔加終止液50口，終止反應(yīng)（此時藍(lán)色立轉(zhuǎn)黃色）。8 .測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度(ODfi)。測定應(yīng) 在加終止

8、液后10分鐘以內(nèi)進(jìn)行。9 .根據(jù)標(biāo)準(zhǔn)品的濃度及對應(yīng)的 OD值計算出標(biāo)準(zhǔn)曲線的直線回歸方程，再根據(jù) 樣品的OD值在回歸方程上計算出對應(yīng)的樣品濃度。也可以使用各種應(yīng)用軟 件來計算。操作程序總結(jié):檢測范圍：6ng/L-200 ng/L。保存：2-8 C。有效期：6個月(2-8 C)。Mouse IL-10 ELISA KitInstructionThis kit is only for scientific research, and shall not be used as a clinical diagnosis of use.PurposeThis kit allows for the det

9、erminationof IL-10 concentrationsn Mouseserum, cell culture supernatant, and other biological fluids.PrincipleThe kit assay Mouse-10 level in the samp leadd MousdL-10 antibody to microtiter platewells, after Incubating,add Biotinylated ant1 0-antibody , then Combined Streptavidin-HRP, become complex

10、, after Incubating and washing Completely, Add TMB substrate solution,TMB substrate becomes blue color, reaction is terminated by the addition of a sulphuric acid solution and the color changeis measuredspectrophotometricallyt a wavelengthof 450 nm. The concentration of IL-10 in the samples is then

11、determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kitMaterials provided with th kite48determinations96 determinationsStorageUser manual11Closure plate membrane!22Sealed bags11Microelisa stripplate112-8 CStandard 400 ng/L0.5ml 1Xbottle0.5ml 1Xbottle2-8

12、CStandard diluent3ml 1 bottle6ml 1 bottle2-8 CStreptavidin-HRP3ml 1 bottle6ml 1 bottle2-8 CBiotinylated anti TL-10-antibody0.5ml 1Xbottle1ml 1 bottle2-8 CChromogen Solution A3ml 1 bottle6ml 1 bottle2-8 CChromogen Solution B3ml 1 bottle6ml 1 bottle2-8 CStop Solution3ml 1 bottle6ml 1 bottle2-8 Cwash s

13、olution(20ml x 20 folcD x 1bottle(20mlx 30 folcD x 1 bottle2-8 CMaterials required but not supplied1.37 C incubator2. Standard microplate reader(450nm)3. Precision pipettes and Disposable pipette tips.4. deionized water.5. Disposable Test tube6. Absorbent paperImportant notes1. The kit takes out fro

14、m 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. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error.3. Please

15、according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.4. Use new disposal plastic pipette tips and Closure plate membrane for each standard, in order to avoid cross contamination.5. Do not mix reagents with those from other lots.6. T

16、he substrate evade the light preservationSpecimen requirements1 . extract as soon as possibleafter Specimencollection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction .If it cant, specimen can be kept in - 20 to preserve, Avoid repeated freeze-tha

17、w cycles.2 . Can' t detect the sample which conaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Diloteinal density Standard as follow table:200 ng/L5 Standard120 dOriginal density Standard120Standard diluent100 ng/L4 Standard120 d5 Standard120 d Standard diluent50 ng

18、/L3 Standard120 d4 Standard120 日Standard diluent25 ng/L2 Standard120 d3 Standarc+120 口Standard diluent12.5 ng/L1 Standard120 d2 Standard+120Standard diluent3 .according testing Sample numberSetOne how many wells nedd, Standard and blank suggested Do holes.4 .add sample 1) blank wells(blank compariso

19、n wells don ' t addtsnymped anti-IL-4 -antibody and Streptavidin-HRP ,other each step operation is same); 2) Standard wells:add Standard05仙 and Streptavidin-HRP) 5 3) testing Sample wells: add sample,thenadd antHL-4 -antibody 1(i,lStreptavidin-HRF05L Closing plate with Closure platemembrane ,inc

20、ubate for 60 min aC374.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 .color Add Chromogen Solution