人凋亡相關(guān)因子配體(FASL)酶聯(lián)免疫分析試劑盒使用說明書

1、人凋亡相關(guān)因子配體（FASL）酶聯(lián)免疫分析試劑盒使用說明書本試劑僅供研究使用 目的：本試劑盒用于測定大鼠血清，血漿及相關(guān)液體樣本中凋亡相關(guān)因子配體（FASL）的含量。實(shí)驗(yàn)原理：本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中人凋亡相關(guān)因子配體（FASL）水平。用純化的人凋亡相關(guān)因子配體（FASL）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凋亡相關(guān)因子配體（FASL），再與HRP 標(biāo)記的凋亡相關(guān)因子配體（FASL）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過徹底洗滌后加底物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。顏色的深淺和樣品的凋亡相關(guān)因子配體（

2、FASL）呈正相關(guān)。用酶標(biāo)儀在450nm 波長下測定吸光度（OD 值），通過標(biāo)準(zhǔn)曲線計(jì)算樣品中人凋亡相關(guān)因子配體（FASL）濃度。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(gè) 1 個(gè)酶標(biāo)包被板 1×48 1×96 2-8保存標(biāo)準(zhǔn)品：45ng/L 0.5ml×1 瓶 0.5ml×1 瓶 2-8保存標(biāo)準(zhǔn)品稀釋液 1.5ml×1 瓶 1.5ml×1 瓶 2-8保存酶標(biāo)試劑 3 ml×1 瓶 6 ml×1 瓶 2-8保存樣品稀釋液 3 m

3、l×1 瓶 6 ml×1 瓶 2-8保存顯色劑A 液 3 ml×1 瓶 6 ml×1 瓶 2-8保存顯色劑B 液 3 ml×1 瓶 6 ml×1 瓶 2-8保存終止液 3ml×1 瓶 6ml×1 瓶 2-8保存濃縮洗滌液 （20ml×20 倍）×1 瓶 （20ml×30 倍）×1 瓶 2-8保存樣本處理及要求：1. 血清：室溫血液自然凝固10-20 分鐘，離心20 分鐘左右（2000-3000 轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如出現(xiàn)沉淀，應(yīng)再次離心。2. 血漿：應(yīng)根據(jù)標(biāo)本的

4、要求選擇EDTA 或檸檬酸鈉作為抗凝劑，混合10-20 分鐘后，離心20 分鐘左右（2000-3000 轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)該再次離心。3. 尿液：用無菌管收集，離心20 分鐘左右（2000-3000 轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。4. 細(xì)胞培養(yǎng)上清：檢測分泌性的成份時(shí)，用無菌管收集。離心20 分鐘左右（2000-3000 轉(zhuǎn)/分）。仔細(xì)收集上清。檢測細(xì)胞內(nèi)的成份時(shí)，用PBS（PH7.2-7.4）稀釋細(xì)胞懸液，細(xì)胞2濃度達(dá)到100 萬/ml 左右。通過反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20 分鐘左右（

5、2000-3000 轉(zhuǎn)/分）。仔細(xì)收集上清。保存過程中如有沉淀形成，應(yīng)再次離心。5. 組織標(biāo)本：切割標(biāo)本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存?zhèn)溆?。?biāo)本融化后仍然保持2-8的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標(biāo)本勻漿充分。離心20 分鐘左右（2000-3000 轉(zhuǎn)/分）。仔細(xì)收集上清。分裝后一份待檢測，其余冷凍備用。6. 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.7. 不能檢測含NaN3 的樣品，因NaN3 抑制辣根過氧化物酶的（HRP）活性。操作步驟：1. 標(biāo)

6、準(zhǔn)品的稀釋與加樣：在酶標(biāo)包被板上設(shè)標(biāo)準(zhǔn)品孔10 孔，在第一、第二孔中分別加標(biāo)準(zhǔn)品100l，然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液50l，混勻；然后從第一孔、第二孔中各取100l 分別加到第三孔和第四孔，再在第三、第四孔分別加標(biāo)準(zhǔn)品稀釋液50l，混勻；然后在第三孔和第四孔中先各取50l 棄掉，再各取50l 分別加到第五、第六孔中，再在第五、第六孔中分別加標(biāo)準(zhǔn)品稀釋液50ul，混勻；混勻后從第五、第六孔中各取50l 分別加到第七、第八孔中，再在第七、第八孔中分別加標(biāo)準(zhǔn)品稀釋液50l，混勻后從第七、第八孔中分別取50l 加到第九、第十孔中，再在第九第十孔分別加標(biāo)準(zhǔn)品稀釋液50l，混勻后從第九第十孔中各取

7、50l 棄掉。（稀釋后各孔加樣量都為50l，濃度分別為30ng/L，20ng/L ，10 ng/L，5ng/L，2.5 ng/L）。2. 加樣：分別設(shè)空白孔（空白對照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、待測樣品孔。在酶標(biāo)包被板上待測樣品孔中先加樣品稀釋液40l，然后再加待測樣品10l（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（48T 的20 倍）倍濃縮洗滌液用蒸餾水30（48T 的20 倍）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，置30 秒后棄去

8、，如此重復(fù)5 次，拍干。6. 加酶：每孔加入酶標(biāo)試劑50l，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A50l，再加入顯色劑B50l，輕輕蕩混勻，37避光顯色15 分鐘.10. 終止：每孔加終止液50l，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。11. 測定：以空白空調(diào)零，450nm 波長依序測量各孔的吸光度（OD 值）。 測定應(yīng)在加終止液后15 分鐘以內(nèi)進(jìn)行。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

9、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(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 請每次測定的同時(shí)做標(biāo)準(zhǔn)曲線，最好做復(fù)孔。如標(biāo)本中待測物質(zhì)含量過高（樣本OD 值3大于標(biāo)準(zhǔn)品孔第一孔的OD 值），請先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測定，計(jì)算時(shí)請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 底物請避光保存。7 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).8 所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。9 本試劑不同批號組分不得混用。10. 如與英文說明書有異，以英文說明書為準(zhǔn)。

10、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，根據(jù)樣品的OD值由標(biāo)準(zhǔn)曲線查出相應(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)曲線的直線回歸方程式，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。（此圖僅供參考）試劑盒性能：1.樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R 值為0.990 以上。2.批內(nèi)與批見應(yīng)分別小于9%和11%檢測范圍：2ng/L -40ng/L保存條件及有效期：1.試劑盒保存：；2-8。2有效期：6 個(gè)月如果次技術(shù)資料，請加我扣扣835041457 或mobile call 一五一二一零七二二五三 Rat int

11、erleukin 1FOR RESEARCH USE ONLYDrug NamesGeneric Name：Rat interleukin1(FASL) ELISA Kit.PurposeThis kit allows for the determination of FASL concentrations in Rat serum, blood plasma,and other biological fluids.Principle of the assayThe kit assay Rat FASL level in the sample, use Purified Rat FASL an

12、tibody to coatmicrotiter plate wells, make solid-phase antibody, then add FASL to wells, Combined FASLantibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, afterwashing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRPenzyme-catalyzed, rea

13、ction is terminated by the addition of a sulphuric acid solution and thecolor change is measured spectrophotometrically at a wavelength of 450 nm. Theconcentration of FASL in the samples is then determined by comparing the O.D. of the samplesto the standard curve.5Materials provided with the kitMate

14、rials provided withthe kit48determinations 96 determinations StorageUser manual 1 1Closure plate membrane 2 2Sealed bags 1 1Microelisa stripplate 1 1 2-8Standard：45ng/L 0.5ml×1 bottle 0.5ml×1 bottle 2-8Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8HRP-Conjugate reagent 3ml

15、15;1 bottle 6ml×1 bottle 2-8Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8Chromogen Solution A 3ml×1 bottle 6ml×1 bottle 2-8Chromogen Solution B 3ml×1 bottle 6ml×1 bottle 2-8Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8wash solution（20ml×20 fold）×1b

16、ottle（20ml×30 fold）×1bottle2-8Specimen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix

17、 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, Ifprecipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugal again. The Ope

18、ration ofHydrothorax and cerebrospinal fluid Reference to it.4. cell culture supernatant-detect secretory components, collect sue a sterile container,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect thecomposition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, C

19、ell concentrationreached 1 million / ml, repeated freeze-thaw cycles, damage cells and release ofintracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. removesupernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight

20、,add PBS（PH7.2-7.4）, Rapidlyfrozen with liquid nitrogen, maintain samples at 2-8 after melting,add PBS（PH7.4）,6Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant.6. extract as soon as possible after Specimen collection,and according to the relev

21、antliterature, and should be experiment as soon as possible after the extraction. If it cant,specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1.Dilute and add sample to Standard: s

22、et 10 Standard wells on the ELISA plates coated, addStandard 100l to the first and the second well, then add Standard dilution 50l to the first andthe second well, mix; take out 100l form the first and the second well then add it to the thirdand the forth well separately. then add Standard dilution

23、50l to the third and the forthwell ,mix ; then take out 50l from the third and the forth well discard, add 50l to the fifth andthe sixth well ,then add Standard dilution 50l to the fifth and the sixth well, mix ; take out 50lfrom the fifth and the sixth well and add to the seventh and the eighth wel

24、l, then add Standarddilution 50l to the seventh and the eighth well ,mix ; take out 50l from the seventh and theeighth well and add to the ninth and the tenth well, add Standard dilution 50l to the ninth andthe tenth well, mix , take out 50l from the ninth and the tenth well discard(add Sample 50l t

25、oeach well after Diluting ,(density: 30ng/L,20ng/L ,10 ng/L,5ng/L,2.5 ng/L)2.add sample：Set blank wells separately (blank comparison wells dont add sample andHRP-Conjugate reagent, other each step operation is same). testing sample well. add Sampledilution 40l to testing sample well, then add testin

26、g sample 10l (sample final dilution is5-fold), add sample to wells , dont 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)

27、 with distilledwater and reserve.5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing bufferto every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzyme：Add HRP-Conjugate reagent 50l to each well, except blank well.7.incubate：Operation with 3.78.was

28、hing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight preservation for 15 min at 3710.Stop the reaction：Add Stop Solution50l to each well, Stop the reaction(the blue colorchange to yellow color).11.assay：take blank well as zero , Read absor

29、bance at 450nm after Adding Stop Solution andwithin 15min.important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes inthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.2. washing buffer will

30、 Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental error. add sample within 5 mins, if the number of sample is much ,recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilutionfactor.（×n×5）.5. Closu