人凝血酶TM酶聯(lián)免疫檢測(cè)

1、人凝血酶仃M)酶聯(lián)免疫檢測(cè)試劑盒使用說明書使用前仔細(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(cè)人凝血酶(TM),只能用于研究用途，不得用于醫(yī)學(xué)診斷。用 途：用于人血清、血漿及相關(guān)液體樣本中凝血酶 (TM)測(cè)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(ELISA)測(cè)定樣品中人凝血酶(TM)水平。向預(yù)先包被了人凝血酶(TM)單克隆抗體的酶標(biāo)孔中加入人凝 血酶(TM)，溫育；溫育后，加入生物素標(biāo)記的抗 TM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

2、淺與樣品中人凝血 酶(TM)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份圭寸板膜2 片(48)2 片(96)密封袋1個(gè)1個(gè)酶標(biāo)包被板1X 481x 9628C保存標(biāo)準(zhǔn)品1600U/L0.5ml X 1 瓶0.5ml x 1 瓶28C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 mlx 1 瓶6 ml x 1 瓶2-8 C保存生物素標(biāo)記的抗TM抗體0.5ml x 1 瓶1 ml x 1 瓶2-8 C保存顯色劑A液3 mlx 1 瓶6 ml x 1 瓶2-8 C保存顯色劑B液3 mlx 1 瓶6 ml x 1 瓶2-8 C保存

3、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(20ml x 30 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. 精密移液器及一次性吸頭4. 蒸餾水，5. 一次性試管6. 吸水紙注意事項(xiàng)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(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差3. 嚴(yán)格按照說明書的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)4. 為避免交叉污染，要避免重復(fù)使用手中的吸頭和

4、封板膜。5. 不用的其它試劑應(yīng)包裝好或蓋好。不同批號(hào)的試劑不要混用。保質(zhì)前使用。6. 底物B對(duì)光敏感，避免長(zhǎng)時(shí)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(shí)驗(yàn)臺(tái)上鋪墊幾層吸水紙，酶標(biāo)板朝下 用力拍幾次；將稀釋后的洗滌液至少 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(dòng)洗板：如果有自動(dòng)洗板機(jī)，應(yīng)在熟練使用后再用到正式實(shí)驗(yàn)過程中標(biāo)本要求1不能檢測(cè)含NaN3的樣品，因NaN3抑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(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，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)

5、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。）800U/L5號(hào)標(biāo)準(zhǔn)品120卩l(xiāng)的原倍標(biāo)準(zhǔn)品加入120卩l(xiāng)標(biāo)準(zhǔn)品稀釋液400U/L4號(hào)標(biāo)準(zhǔn)品120卩l(xiāng)的5號(hào)標(biāo)準(zhǔn)品加入120卩l(xiāng)標(biāo)準(zhǔn)品稀釋液200U/L3號(hào)標(biāo)準(zhǔn)品120卩l(xiāng)的4號(hào)標(biāo)準(zhǔn)品加入120卩l(xiāng)標(biāo)準(zhǔn)品稀釋液100U/L2號(hào)標(biāo)準(zhǔn)品120卩l(xiāng)的3號(hào)標(biāo)準(zhǔn)品加入120卩l(xiāng)標(biāo)準(zhǔn)品稀釋液50U/L1號(hào)標(biāo)準(zhǔn)品120卩l(xiāng)的2號(hào)標(biāo)準(zhǔn)品加入120卩l(xiāng)標(biāo)準(zhǔn)品稀釋液2. 根據(jù)代測(cè)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(gè)標(biāo)準(zhǔn)品和空白孔建議做復(fù)孔。每個(gè)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(duì)照孔

6、不加樣品，生物素標(biāo)記的抗 TM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-HRP50ul（標(biāo)準(zhǔn)品中已事先整合好生物素抗體， 故不加）;3）代測(cè)樣品孔：加入樣本40ul，然后各加入抗TM抗體10ul、鏈 霉親和素-HRP50ul，蓋上封板膜，輕輕震蕩混勻,37T溫育60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. 顯色：每孔先加入顯色劑A50ul，再加入顯色劑B50卩l(xiāng)，輕輕震蕩混勻，37C 避光顯色

7、10分鐘.7. 終止：每孔加終止液50卩l(xiāng)，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。8. 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10分鐘以內(nèi)進(jìn)行。9. 根據(jù)標(biāo)準(zhǔn)品的濃度及對(duì)應(yīng)的 0D值計(jì)算出標(biāo)準(zhǔn)曲線的直線回歸方程，再根據(jù) 樣品的0D值在回歸方程上計(jì)算出對(duì)應(yīng)的樣品濃度。也可以使用各種應(yīng)用軟 件來計(jì)算。操作程序總結(jié):檢測(cè)范圍：30U/L800U/L 保存：2-8 C。有效期：6個(gè)月(2-8 C)。Human Thrombin (TM) ELISA KitInstructionThis kit is only for scientific research, a

8、nd shall not be used as a clinical diagnosis of use.PurposeThis kit allows for the determ in ationof TM concen trati onSn Huma n serum, cell culture super nata nt, and other biological fluids.PrincipleThe kit assay HumairM level in the sampladd HumaiTM antibody to microtiter plate wells,after Incuba

9、ting,addBiotinylatedanti -TM -antibody, then CombinedStreptavidin-HRP, become complex, after In cubat ing and wash ing Completely, Add TMB substrate solutio n,TMB substrate becomes blue color, react ion is term in ated by the additi on of a sulphuric acid soluti on and the color cha ngeis measuredsp

10、ectrophotometricallyt a wavele ngthof 450 nm. The concentration of TM in the samples is then determined by comparing the O.D. of the samples to the sta ndard curve.Materials provided with the kitMaterials provided with th kite48determ in ati ons96 determ in atio nsStorageUser manual11Closure plate m

11、embra ne 22Sealed bags11Microelisa stripplate1128CSta ndard 1600U/L0.5ml 1Xbottle0.5ml Kbottle28CSta ndard dilue nt3ml Xbottle6ml 1 bottle2-8 CStreptavidi n-HRP3ml 1 bottle6ml 1 bottle2-8 CBioti ny lated anti -TM-a ntibody0.5ml Kbottle1ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 bottle6ml 1 bottle2-

12、8 CChromoge n Soluti on B3ml 1 bottle6ml 1 bottle2-8 CStop Soluti on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml X 20 fold)x 1bottle(20ml X 30 fold)x 1 bottle2-8 CMaterials required but not supplied1.37 C incubator2. Sta ndard microplate reader(45 Onm)3. Precision pipettes and Disposable pipette

13、tips.4. dei oni zed water.5. Disposable Test tube6 Absorbe nt paperImportant notes1. The kit takes out from the refrigeration environment should be balaneed 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 Samp

14、le with sampler Each step, And proofread its accuracy frequently, avoids the experime ntal error.3. Please accord ing to use in structi on strictly, The test result determ in ati on must take the microtiter plate reader as a sta ndard.4. Use new disposal plastic pipette tips and Closure plate membra

15、ne for each standard, in order to avoid cross con tam in atio n.5. Do not mix reagents with those from other lots.6. The substrate evade the light preservationSpecimen requirements1. extract as soon as possibleafter Specime ncollecti on,an daccord in gto the releva nt literature,a nd shouldbe experi

16、me ntas soon as possibleafter the extractio n.lf it can specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.2. Can t detect the sample which coNiaNS, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Diloteinai density Standard as follow table:800U/L5 S

17、ta ndard120 d Original density Standard120 M Standard diluent400U/L4 Sta ndard120 d 5 Standard120 d Standard diluent200U/L3 Sta ndard120 d 4 Standard120 d Standard diluent100U/L2 Sta ndard120 d 3 Standarc+120 d Standard diluent50U/L1 Sta ndard120 d 2 Standarc+120 d Standard diluentt,adldtsiampied an

18、tTM2. accord ing test ing Sample nu mberdeo ne how many wells n edd, Stan dard and bla nk suggested Do holes.3. add samp：1) bla nk wells(bla nk comparis on wells don -antibody and Streptavidin-HRP ,other each step operation is same); 2) Standard wells: addStandard 0 and Streptavidin-HRP 5； 3) testin

19、g Sample wells: add sampl鶴len addanti -TM -antibody01,l Streptavidin-HRF0念 closing plate with Closure platemembra ne ,in cubate for 60 min aE374. C on figurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5. washi ng Un cover Closure plate membra ne, discard Liquid, dry by swing, add wash ing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. colo【Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservati on for 10 min a