人白細(xì)胞介素1IL1酶聯(lián)免疫分析ELISA

1、人白細(xì)胞介素1（IL-1）酶聯(lián)免疫分析（ELISA ）試劑盒使用說(shuō)明書本試劑僅供研究使用目的：本試劑盒用于測(cè)定人血清，血漿及相關(guān)液體樣本中白細(xì)胞介素1（ IL-1 ）的含量。實(shí)驗(yàn)原理：本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人白細(xì)胞介素-1（ IL-1 ）水平。用純化的人白細(xì)胞介素-1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白細(xì)胞介素-1，再與HRP標(biāo)記的羊抗人抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過(guò)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 淺和樣品中的白細(xì)胞介素-1（ IL-1 ）呈正相關(guān)。用酶標(biāo)儀在 450

2、nm波長(zhǎng)下測(cè)定吸光度（0D 值），通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中人白細(xì)胞介素-1（ IL-1 ）含量。試劑盒組成：試劑盒組成48孔配置96孔配置保存說(shuō)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 962-8 C保存標(biāo)準(zhǔn)品：180 ng/LX 1瓶X 1瓶2-8 C保存標(biāo)準(zhǔn)品稀釋液X 1瓶X 1瓶2-8 C保存酶標(biāo)試劑3 ml X 1 瓶6 ml X 1 瓶2-8 C保存樣品稀釋液3 ml X 1 瓶6 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

3、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/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)份）。仔細(xì)收集上 清，保存過(guò)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(guò)程中如有沉淀形成，應(yīng)該再次 離心。3. 尿液：用無(wú)菌管收集，離心20分鐘左右（2000-3000轉(zhuǎn)份）。仔細(xì)收集上清，保存過(guò)程中如有沉淀形成，

4、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(cè)分泌性的成份時(shí)，用無(wú)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(cè)細(xì)胞內(nèi)的成份時(shí)，用PBS （）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100萬(wàn)/ml左右。通過(guò)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（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。保存過(guò)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,。用液氮迅速冷凍保存?zhèn)溆?。?biāo)本融化后仍然保持 2-8 C的溫度。加入一定量的 PBS （）,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

5、細(xì)收集上清。分裝后一份待檢測(cè)，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 C保存，但應(yīng)避免反復(fù)凍融 .7. 不能檢測(cè)含NaN3的樣品，因NaN3抑制辣根過(guò)氧化物酶的（HRP）活性。操作步驟1. 標(biāo)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100 M，然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液50 d，混勻；然后從第一孔、第二孔中各取100 d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50 d,混勻；然后在第三孔和第四孔中先各取50 d棄掉，再各取50 d分別加到

6、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50 d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50 d，混勻后從第七、第八孔中分別取50 d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50 d，混勻后從第九第十孔中各取50 d棄掉。（稀釋后各孔加樣量都為 50 d,濃度分別為 120ng/L, 60ng/L , 30ng/L , 15ng/L , L）。、2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、待測(cè)樣品孔。在酶標(biāo)包被板上待測(cè)樣品孔中先加樣品稀釋液40 d,然后再加待測(cè)樣品10 d （

7、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 C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，靜置30秒后棄去，如此重復(fù)5次，拍干。6. 加酶：每孔加入酶標(biāo)試劑 50 d，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 A50 d，再加入顯色劑 B50 d，輕輕震蕩混勻，37 C避光顯色 15分鐘.10. 終止：每孔加終止液 50 d，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）

8、。11. 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。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(qǐng)每次測(cè)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(cè)物質(zhì)含量過(guò)高（樣本OD值大于標(biāo)準(zhǔn)品孔第一孔的 OD值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（ n

9、倍）后再測(cè)定，計(jì) 算時(shí)請(qǐng)最后乘以總稀釋倍數(shù)（Xn X 5）5. 封板膜只限一次性使用，以避免交叉污染。6. 底物請(qǐng)避光保存。7. 嚴(yán)格按照說(shuō)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) &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(hào)組分不得混用。10. 如與英文說(shuō)明書有異，以英文說(shuō)明書為準(zhǔn)。 計(jì)算：以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)， 0D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0D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 0D值計(jì)算出標(biāo) 準(zhǔn)曲線的直線回歸方程式，將樣品的0D值（此圖僅供參考）代入方程式，計(jì)算出樣品濃度，再乘以稀釋 倍數(shù)，即

10、為樣品的實(shí)際濃度。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值為以上。2批內(nèi)與批見應(yīng)分別小于 9%和11%檢測(cè)范圍：5n g/L -150 ng/L保存條件及有效期：1試劑盒保存：;2-8Co2 有效期：6個(gè)月RDHuma n In terleuk in 1Drug NamesGen eric Nam: Human In terleukin 1 (IL-1) ELISA Kit.PurposeThis kit allows for the determ in ati on of IL-1 concen trati ons in Huma n serum, blood plasma, and

11、 other biological fluids.Pr in ciple of the assayThe kit assay Human IL-1 level in the sampluse Purified Human IL-1antibody to coat microtiter plate wells, make solid-phase antibody, then add IL-1 to wells, Combined antibody which With HRP labeled goat an ti-Humanbecome an tibody- an tige n - en zym

12、e-a ntibody complex, after wash ing Completely, Add TMB substrate solutio n,TMB substrate becomes blue color At HRP en zyme-catalyzedeacti onis term in atedby the additi onof a sulphuricacid soluti on and the color cha nge is measured spectrophotometrically at a wavele ngth of 450 nm. The concen tra

13、ti on of IL-1 in the samples is the n determ ined by compari ng the . of the samples to the sta ndard curve.Materials provided with the kitMaterials provided with the kit48determ in ati ons96 determ in atio nsStorageUser manual1、1Closure plate membra ne2 2Sealed bags11Microelisa stripplate112-8 CSta

14、 ndard 180 ng/Lxi bottle>1 bottle2-8 CStan dard dilue ntxi bottle>1 bottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle '、2-8 CSample dilue nt3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti on B3ml 1 bottle6ml 1 bottle2-8 CStop Soluti

15、on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml x 20 fold)x 1bottle(20ml x 30 fold)x 1 bottle2-8 CSpecime n requireme nts1. serum- coagulation at room temperature 10-20，cieiistrifugation 20-min at the speed of 2000-3000 remove supernatant. If precipitation appeared, Centrifugal again.2. plasma-use

16、 suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation20-min at the speed of 2000-3000 remove supernatant,lf precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container,centrifugatior20-min at the speed of 2000-3000 remove supernata nt,If precipitati

17、 on appeared, Cen trifugalaga in. The Operatio n of Hydrothorax and cerebrosp inal fluid Refere nee to it.4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, centrifugation20-min at the speed of 2000-3000 remove supernatant,detecthe composition of cells, Dilut cell

18、suspension with )BCell concentration reached 1 million/ ml, repeated freeze-thaw cycles, damage cells and release of in tracellular comp onents, cen trifugatior2 0-min at the speed of 2000-3000 removesuper nata nt,If precipitati on appeared, Cen trifugal aga in.5. Tissue samples After cutting sample

19、s, check the weight,add(PB Rapidly frozen withliquid n itroge n, maintain samples at 2-8after melt in g,add PBS) , Homoge ni zed by hand or Grin ders, cen trifugati on 20-min at the speed of 2OOO-e00fiVe super nata nt.6. extract as soon as possibleafter Specime ncollecti on,an daccord in gto the rel

20、eva ntliterature,a nd shouldbe experime ntas soon as possibleafter the extractio n.lf it can'specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Can' t detect the sample which conaNS, because NaN3 inhibits HRP active.Assay procedureand add sample to Sta ndard:set 1

21、0 Sta ndardwells on the ELISA plates coated, addStandard 100 卩 l to the first and the second well, then add StandSOdidilurtittne first andthe second well, mix; take ok00 卩 l form the first and the second well then add it to the thirdand the forth well separatelythen add Standarddilution 50 卩 to the

22、third and the forthwell ,mix ; then take out 50卩 l from the third and the forth well discard, add 50the sixth well ,then add Standard dili50)p l to the fifth and the sixth well, mix ; take out 50from the fifth and the sixth well and add to the seve nth and the eighth well, the n add Sta ndard diluti

23、on50 卩 l to the seventh and the eighth well ,mix ; take out 50卩 l from the seveneighth well and add to the ninth and the tenth well, add Sta ndard5DI(utionD the ninth andthe ten th well, mix , take out 50 m the nin thpahdcthe ten th wescard(add Sample 50 卩 l toeach well after Dilut in g ,(de nsity:

24、120 ng/L,60 ng/L,30 ng/L,15 ng/L丄)sample Set bla nk wells separately (bla nk comparis on wells don' add sample andHRP-Conjugate reage nt, other each step operati on is same). test ing sample well. add Samplediluti on40 ytlo test in gsamplewel, the n add testi ng sample10 Qsamplefi nal diluti oni

25、s5-fold), add sample to weldon' t touch the well wall as far as poanibGently mix.:After closi ng plate with Closure plate membra ne ,in cubate for 30 nSin at 37liquid:30-fold(or 20-fold)washsolutio ndiluted30-fold(or 20-fold)with distilledwater and reserve.:Un cover Closure plate membra ne, disc

26、ard Liquid, dry by swing, add wash ing buffer to everywell, still for 30s then drain, repeat 5 times, dry by pat.enzyme Add HRP-Conjugate reagent l to each well, exceptank well.:Operation with 3.:Operation with 5.: Add Chromogen Solution A 50ul and Chromogen SolUBiOoeach well, evade the lightpreserv

27、ati on for 15 min att37the reaction Add Stop Solution) tbeach well, Stop the reaction(the blue color change to yellow color).:take bla nk well as zero , Read absorba nce at 450nm after Add ing Stop Soluti on and with in15mi n.Importa nt no tes1. The kit takes out from the refrigeration environment s

28、hould 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. washingbufferwill Crystallizatiorseparationjt can be heatedthe water helps dissolve whe n dilute . Wash ing does not affect the result.3. add Samplewi