人白細(xì)胞介素10IL-10酶聯(lián)免疫分析ELISA

1、兔子白細(xì)胞介素4 （il-4 ）酶聯(lián)免疫分析試劑盒使用說(shuō)明書(shū)本試劑僅供研究使用目的：本試劑盒用于測(cè)定兔子血清，血漿，細(xì)胞上清及相關(guān)液體樣本中白細(xì)胞介素4 （il-4）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)本中兔子白細(xì)胞介素4 （il-4）水平。用純化的兔子白細(xì)胞介素4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4,再與hrp標(biāo)記的il-4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il-4呈正相關(guān)。用酶標(biāo)儀在 450nm波長(zhǎng)下測(cè)定吸光度（od值

2、），通過(guò) 標(biāo)準(zhǔn)曲線(xiàn)計(jì)算樣品中兔子白細(xì)胞介素4 （il-4）濃度。試劑盒組成試劑盒組成48孔配置96孔配置保存說(shuō)明書(shū)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1x481x 962-8 c保存標(biāo)準(zhǔn)品：135ng/l0.5ml x 1 瓶0.5ml x 1 瓶2-8 c保存標(biāo)準(zhǔn)品稀釋液1.5ml x 1 瓶1.5ml 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

3、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(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)/分）。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)/分）。仔細(xì)收集上清，保

4、存過(guò)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(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 （ ph7.2-7.4 ）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(chēng)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 ）

5、 ，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(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出然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液50 口，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

6、小混勻；然后在第三孔和第四孔中先各取50m棄掉，再各取50 m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50 m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出混勻后從第七、第八孔中分別取50 m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 口，混勻后從第九第十孔中各取50 m棄掉。（稀釋后各孔加樣量都為50小濃度分別為 90 ng/l， 60ng/l ， 30 ng/l ， 15ng/l， 7.5 ng/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。

7、在酶標(biāo)包被板上待測(cè)樣品孔中先加樣品稀釋液40小 然后再加待測(cè)樣品10 u （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(mǎn)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 口，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a50u,再加入顯色劑 b50m,輕輕震

8、蕩混勻，37c避光顯色15 分鐘 .10 .終止：每孔加終止液50 終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。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?xún)?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(kāi)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。84 .請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線(xiàn)，最好

9、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倍）后再測(cè)定，計(jì) 算時(shí)請(qǐng)最后乘以總稀釋倍數(shù)（x n 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(shū)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(hào)組分不得混用。10 .如與英文說(shuō)明書(shū)有異，以英文說(shuō)明書(shū)為準(zhǔn)。（此圖僅供參考）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(xiàn)，根據(jù)樣品的od值由標(biāo)準(zhǔn)曲線(xiàn)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

10、與 od值計(jì)算出標(biāo) 準(zhǔn)曲線(xiàn)的直線(xiàn)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。試劑盒性能：1 .樣品線(xiàn)性回歸與預(yù)期濃度相關(guān)系數(shù)r值為0.95以上。2 .批內(nèi)與批見(jiàn)應(yīng)分別小于9%和11%檢測(cè)范圍：5ng/l-100ng/l保存條件及有效期：1 .試劑盒保存：；2-8 c。2 .有效期：6個(gè)月rabbit interleukin 4for research use onlydrug namesgeneric name rabbit interleukin4 (il-4) elisa kit.purposethis kit allows for the

11、determination of il-4 concentrations in rabbit serum, blood plasma, cell culture supernatant and other biological fluids.principle of the assaythe kit assay rabbit il-4 level in the sam pluse purified rabbit il-4 antibody to coat microtiterplate wells, make solid-phaseantibody,then add il-4 to wells

12、, combinedil-4 antibody which with hrp labeled ,become antibody - antigen - enzyme-antibody complex, after washing completely, add tmb substrate solution,tmb substrate becomes blue color at hrp enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change

13、 is measured spectrophotometricallyit a wavelength of 450 nm. the concentration of il-4 in the samples is then determined by comparing the o.d. of the samples to the standard curve.materials provided with the kitmaterials provided with the kit48determinations96 determinationsstorageuser manual11clos

14、ure plate membrane22sealed bags11microelisa stripplate112-8 cstandard 135ng/l0.5ml 1xbottle0.5ml 1xbottle2-8 cstandard diluent1.5ml 1xbottle1.5ml 1xbottle2-8 chrp-conjugate reagent 3ml 1 bottle6ml 1 bottle2-8 csample diluent3ml 1 bottle6ml 1 bottle2-8 cchromogen solution a3ml 1 bottle6ml 1 bottle2-8

15、 cchromogen solution b3ml 1 bottle6ml 1 bottle2-8 cstop solution3ml 1 bottle6ml 1 bottle2-8 cwash solution(20ml x 20 folcd x 1bottle(20mlx 30 folcd x 1 bottle2-8 cspecimen requirements1. serum- coagulation at room temperature 10-20 geistrifugation 20-min at the speed of 2000-3000 r.p.m. remove super

16、natant, if precipitation appeared, centrifugal again.2. plasma-use suited edta or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugatior20-min at the speed of 2000-3000r.p.m. remove supernatant,if precipitation appeared, centrifugal again.3. urine-collect sue a sterile container, centrifu

17、gation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,if precipitationappeared, centrifugalagain. the operation of hydrothorax and cerebrospinal fluid reference to it.4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, centrifugatior20-min at the speed o

18、f 2000-3000r.p.m. removesupernatant,detedttie compositionof cells, dilut cell suspensionwith pbs (ph7.2-7.4 , cell concentration reached 1 million / ml, repeated freeze-thawcycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove sup

19、ernatant, if precipitation appeared, centrifugal again.5. tissue samples- after cutting samples, check the weight,add(ppbhs7.2-7.4) , rapidly frozen with liquid nitrogen, maintain samples at 2-8after melting,add pb(sph7.4) , homogenized by hand or grinders, centrifugation 20-min at the speed of 2000

20、-3000 r.p.m. remove supernatant.6. extract as soon as possibleafter specimencollection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction.if it can t, specimen can be kept in -20 to preserve, avoid repeated freeze-thaw cycles.7. can t detect the sam

21、ple which connatanin3, because nan3 inhibits hrp active.assay procedure1 .dilute and add sample to standard: set 10 standard wells on the elisa plates coated, add standard 100 仙 l to the first and the second well, then add standoodidiltotithe first andthe second well, mix; take out 100仙 l form the f

22、irst and the second well then add it to the thiand the forth well separatelythen add standarddilution 50 仙 to the third and the forth well ,mix ; then take out 50仙 l from the third and the forth well discard, add 50the sixth well ,then add standard dili50)n l to the fifth and the sixth well, mix ; t

23、ake out 50from the fifth and the sixth well and add to the seventh and the eighth well, then add standard dilution50 仙 l to the seventh and the eighth well ,mix ; take out 50仙 l from the seveneighth well and add to the ninth and the tenth well, add standard50l utioto the ninth andthe tenth well, mix

24、 , take out 50 m the ninth 11ahddhe tenth wescard(add sample 50 仙 l to each well after diluting ,(dens9i0tyn:g/l ， 60ng/l ， 30 ng/l ， 15ng/l ， 7.5 ng/l )2 .add sample： set blank wells separately(blank compairson wells don atdd sampleand hrp-conjugate reagent, other each step operation is same). test

25、ing sample well. add sample dilution40(ito testingsamplewell then add testing sample10 仙 qsamplefinal dilutionis 5-fold), add sample to welldso,n t tcohuthe well wall as far as possible, and gently mix.3 .incubate: after closing plate with closure plate membrane ,incubate for 30 m.in at 374.configur

26、ate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5 .washin：g 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 .add enzyme add hrp-conjugate

27、 reag50t l to each well, excebtank well.7.incubat：e operation with 3.8.washin：g operation with 5.9.colo：r add chromogen solution a 50ul and chromogen solution b to each well, evade the light preservation for 15 min at 3710.stopthe reaction add stop solutio50(ito each well, stop the reaction(thdolue

28、colorchange to yellow color).11.assa：y take blank well as zero , read absorbance at 450nm after adding stop solution and within 15min.important notes1. the kit takes out from 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. washingbufferwill crystallizationseparation,it can be heatedthe water helps dissolve when dilute . washing d