大鼠白細胞介素12IL-12酶聯(lián)免疫分析ELISA

1、大鼠白細胞介素12 (IL-12 )酶聯(lián)免疫分析(ELISA)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，細胞上清及相關液體樣本中白細胞介素 12 (IL-12)的含量。 實驗原理：本試劑盒應用雙抗體夾心法測定標本中大鼠白細胞介素12 (IL-12)水平。用純化的大鼠白細胞介素12 (IL-12)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 細胞介素12,再與HRP標記的羊抗鼠抗體Z合，形成抗體-抗原-酶標抗體復合物，經(jīng)過徹底洗滌后加底物 TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸的作用下轉化成 最終的黃色。顏色的深淺和樣品中的白細胞介素1

2、2呈正相關。用酶標儀在450nm波長下測定吸光度(OD值)，通過標準曲線計算樣品中大鼠白細胞介素12 (IL-12)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1個酶標包被板1X481X 962-8 C保存標準品：54ng/L0.5ml X 1 瓶0.5ml X 1 瓶2-8 C保存標準品稀釋液1.5ml X 1 瓶1.5ml X 1 瓶2-8 C保存酶標試劑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-

3、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(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/分)。仔細收集上 清，保存過程中如出現(xiàn)沉淀，應再次離心。2 .血漿：應根據(jù)標本的要求選擇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/分)。仔細收集上清，保存過程中如有沉淀形成，應該再次 離心。3 .尿液：用無菌管收集，離心

4、20分鐘左右(2000-3000轉/分)。仔細收集上清，保存過程 中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。4 .細胞培養(yǎng)上清：檢測分泌性的成份時，用無菌管收集。離心20分鐘左右(2000-3000轉/分)。仔細收集上清。檢測細胞內(nèi)的成份時，用 PBS (PH7.2-7.4)稀釋細胞懸液，細胞濃度達到100萬/ml左右。通過反復凍融，以使細胞破壞并放出細胞內(nèi)成份。離心 20分 鐘左右（ 2000-3000 轉/分） 。仔細收集上清。保存過程中如有沉淀形成，應再次離心。5 . 組織標本：切割標本后，稱取重量。加入一定量的 PBS， PH7.4 。用液氮迅速冷凍保存?zhèn)溆谩吮救诨笕匀槐?/p>

5、持2-8的溫度。加入一定量的PBS（ PH7.4 ） ，用手工或勻漿器將標本勻漿充分。離心20 分鐘左右（ 2000-3000 轉/分） 。仔細收集上清。分裝后一份待檢測，其余冷凍備用。6 . 標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上進行試驗，可將標本放于-20保存，但應避免反復凍融.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. 標準品的稀釋與加樣：在酶標包被板上設標準品孔10 孔，在第一、第二孔中分別加標準品100出然后在第一、第二孔中加標準品稀釋液50 口，混勻；然后從第一孔、第二孔中各取100 d分別加到第

6、三孔和第四孔，再在第三、第四孔分別加標準品稀釋液50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、第六孔中分別加標準品稀釋液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、第八孔中分別加標準品稀釋液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準品稀釋液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 36ng/L， 24ng/L ， 12 ng/L ， 6 ng/L， 3 ng/L） 。2. 加樣：分別設空白孔（空白對照孔不加樣品

7、及酶標試劑，其余各步操作相同） 、待測樣品孔。在酶標包被板上待測樣品孔中先加樣品稀釋液40小 然后再加待測樣品10 U （樣品最終稀釋度為 5 倍） 。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻。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，靜置 30 秒后棄去，如此 重復 5 次，拍干。6. 加酶：每孔加入酶標試劑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

8、劑A50U,再加入顯色劑 B50M,輕輕震蕩混勻，37c避光顯色15 分鐘 .10 .終止：每孔加終止液50終止反應（此時藍色立轉黃色）。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） 。 測定應在加終止 液后 15 分鐘以內(nèi)進行。注意事項：1 試劑盒從冷藏環(huán)境中取出應在室溫平衡15-30 分鐘后方可使用，酶標包被板開封后如未用完，板條應裝入密封袋中保存。2 濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。3 各步加樣均應使用加樣器，并經(jīng)常校對其準確性，以避免試驗誤差。一次加樣時間最好控制在 5 分鐘內(nèi)，如標本數(shù)量多，推薦使用排槍加樣。

9、4 請每次測定的同時做標準曲線，最好做復孔。如標本中待測物質含量過高（樣本OD 值大于標準品孔第一孔的 OD 值） ，請先用樣品稀釋液稀釋一定倍數(shù)（ n 倍）后再測定，計算時請最后乘以總稀釋倍數(shù)（X nX5）o5 .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 .嚴格按照說明書的操作進行，試驗結果判定必須以酶標儀讀數(shù)為準 8.所有樣品，洗滌液和各種廢棄物都應按傳染物處理。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。 計算：以標準物的濃度為橫坐標，OD值為縱坐標，在坐標紙上繪出標準曲線，根據(jù)樣品的OD值由標準曲線查出相應的濃度；再乘以稀釋 倍

10、數(shù)；或用標準物的濃度與 OD值計算出標 準曲線的直線回歸方程式，將樣品的OD值仃4"才日自 COrlEifetEticU (X.T（此圖僅供參考）代入方程式，計算出樣品濃度，再乘以稀釋 倍數(shù)，即為樣品的實際濃度。試劑盒性能：1 .樣品線性回歸與預期濃度相關系數(shù)R值為0.95以上。2 .批內(nèi)與批見應分別小于9%和11%檢測范圍：2ng/L -40 ng/L保存條件及有效期：1 .試劑盒保存：；2-8 C。2 .有效期：6個月RDRat Interleukin 12FOR RESEARCH USE ONLYDrug NamesGeneric Name Rat Interleukin 12

11、 (IL-12) ELISA Kit.PurposeThis kit allows for the determination of IL-12 concentrations in Rat serum, blood plasma, cell culture supernatant and other biological fluids.Principle of the assayThe kit assayRat IL-12 levelin the sample use PurifiedRat IL-12 antibodyto coat microtiter plate wells, make

12、solid-phase antibody, then add IL-12 to wells, Combined antibody which With HRP labeled goat anti-Mouse,becomeantibody- antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzedeactionis terminatedby the additiono

13、f a sulphuricacid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration ofL-12in the samplesis then determinecby comparing theO.D. of the samples to the standard curve.Materials provided with the kitMaterials provided with the kit48determinatio

14、ns96 determinationsStorageUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8 CStandard 54ng/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 CChrom

15、ogen Solution A3ml 1 bottle6ml 1 bottle2-8 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

16、the speed of 2000-3000 r.p.m. remove supernatant, 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

17、-collect sue a sterile container, centrifugation 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 cont

18、ainer, centrifugatior20-min at the speed of 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 a

19、t the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.5. Tissue samples After cutting samples, check the weight,add PPHS7.2-7.4 , Rapidly frozen with liquid nitrogen, maintain samples atC2-8fter melting,add PBSPH7.4 ,Homogenized by hand or Grinders, centrif

20、ugation 20-min at the speed of 2000-3000 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-

21、thaw cycles.7. Can t detect the sample which coNnataNin3, because NaN3 inhibits HRP active.Assay procedure1 .Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, addStandard 100 仙 l to the first and the second well, then add StandOOdidiltotithe first andthe second wel

22、l, mix; take out 100仙 l form the first and the second well then add it to the thiand the forth well separatelythen add Standarddilution 50 仙 to the third and the forthwell ,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

23、fifth and the sixth well, mix ; take 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

24、 the ninth andthe tenth well, mix , take out 50 m the ninth 11ahddhe tenth wescard(add Sample 50 仙 l toeach well after Diluting ,(density: 36ng/L,24ng/L ,12 ng/L,6 ng/L,3 ng/L)2 .add sample： Set blank wells separately(blank comparisonwells don atdd sample and HRP-Conjugate reagent, other each step o

25、peration is same). testing sample well. add Sample dilution40(ito testingsamplewell then add testing sample10 仙 Qsamplefinal dilutionis5-fold), add sample to welldso,n t touch the well wall as far as poasnsidblGe,ently mix.3 .Incubate: After closing plate with Closure plate membrane ,incubate for 30

26、 m.in at 374.Configurate 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 enz

27、yme Add HRP-Conjugate reagent仙 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 thelight preservation for 15 min at 3710.Stopthe reaction Add Stop Solutio50 仙 to each well, Stop

28、 the reaction(thdblue 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 does no