大鼠T淋巴細(xì)胞亞群CD3CD4CD8酶聯(lián)免疫分析

1、大鼠T淋巴細(xì)胞亞群CD3,CD4,CD8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)定大鼠血清,血漿及相關(guān)液體樣本中T淋巴細(xì)胞亞群CD3,CD4,CD8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)本中大鼠T淋巴細(xì)胞亞群CD3,CD4,CD8水平.用純化的大鼠T淋巴細(xì)胞亞群CD3,CD4,CD8抗體包被微孔板,制成固相抗體,往包被單抗的 微孔中依次參加 T淋巴細(xì)胞亞群CD3,CD4,CD8 ,再與 HRP標(biāo)記的 T淋巴細(xì)胞亞群CD3,CD4,CD8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

2、的作用下轉(zhuǎn)化成最終的黃色.顏色的深 淺和樣品中的T淋巴細(xì)胞亞群CD3,CD4,CD呈正相關(guān).用酶標(biāo)儀在 450nm波長(zhǎng)下測(cè)定吸 光度OD值,通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中大鼠T淋巴細(xì)胞亞群CD3,CD4,CD8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：135U/ml0.5ml 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

3、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)

4、收集上清,保存過(guò)程中如有沉淀形成,應(yīng)該再次 離,°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 濃度到達(dá)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車t/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.參

5、加一定量的 PBS, PH7.4.用液氮迅速冷凍保存?zhèn)?用.標(biāo)本融化后仍然保持2-8C的溫度.參加一定量的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(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).假設(shè)不能馬上 進(jìn)行試驗(yàn),可將標(biāo)本放于 -20 C保存,但應(yīng)預(yù)防反復(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

6、釋液50 4,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 90U/ml , 60U/ml , 30U/ml , 15U/ml

7、, 7.5U/ml.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小 然后再加待測(cè)樣品10 口樣品最終稀釋度為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儲(chǔ)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：每孔參加酶標(biāo)試劑 50 4,空白孔除外.7 .溫育:操作同3.8 .洗滌：操作同5.9

8、. 顯色：每孔先參加顯色劑 A50U,再參加顯色劑 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此 終止反響此時(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分鐘以內(nèi)進(jìn)行.本卷須知：1 .試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30分鐘前方可使用,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,以預(yù)防試驗(yàn)誤差.一次加樣時(shí)間最好 限制在5分鐘內(nèi),如標(biāo)本數(shù)量多,推薦使用排

9、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倍后再測(cè)定,計(jì) 算時(shí)請(qǐng)最后乘以總稀釋倍數(shù)XnX5o5 .封板膜只限一次性使用,以預(yù)防交叉污染.6 .底物請(qǐng)避光保存.7 .嚴(yán)格根據(jù)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) 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),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)

10、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值仃4"才日自 COrlEifetEticU (X.T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0.92以上.2 .批內(nèi)與批見(jiàn)應(yīng)分別小于 9%和15%檢測(cè)范圍：4U/ml -100U/ml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FOR RESEARCH USE ONLYRat T-cell subsets(CD3,CD4,CD8)Drug NamesGeneric Name Rat T-cell su

11、bsets(CD3,CD4,CD8) ELISA Kit.PurposeThis kit allows for thedeterminationof T-cellsubsets(CD3,CD4,CD8O ncentrationsn Rat serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay Rat T-cell subsets(CD3,CD4,CDeVel in the sample use Purified Rat T-cell subsets(CD3,CD4,CD8)ant

12、ibody to coat microtiterplate wells, make solid-phase antibody, then add T-cell subsets(CD3,CD4,CD8)to wells, Combined T-cell subsets(CD3,CD4,CD8i)ntibody which With HRP labeled, become antibody - antigen - enzyme-antibodycomplex, after washing Completely,Add TMB substrate solution,TMB substrate bec

13、omes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuricacid solutionand the color changeis measuredspectrophotometricallyt a wavelength of 450 nm. The concentration of T-cell subsets(CD3,CD4jCthe) samples is then determined by comparing the O.D. of the samples

14、to the standard curve.Materials provided with the kitMaterials provided with the kit48determinations96 determinationsStorageUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8 CStandard 135U/ml0.5ml 1Xbottle0.5ml 1Xbottle2-8 CStandard diluent1.5ml 1Xbottle1.5ml 1Xbottle2-8 C

15、HRP-Conjugate reagent 3ml 1 bottle6ml 1 bottle2-8 CSample diluent3ml 1 bottle6ml 1 bottle2-8 CChromogen 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 fold) x 1bottle(20ml x 30 fold) x 1 bottle

16、2-8 CSpecimen requirements1. serum- coagulation at room temperature 10-20 geistrifugation 20-min at 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 t

17、he speed of 2000-3000r.p.m. remove supernatant,If precipitation 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 precipitationappeared, Centrifugalagain. The Operation of Hydrothorax and cerebrospinal f

18、luid Reference to it.4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, centrifugatior20-min at the speed of 2000-3000r.p.m. removesupernatant,detedthe compositionof cells, Dilut cell suspensionwith PBS (PH7.2-7.4 , Cell concentration reached 1 million / ml, repeat

19、ed freeze-thawcycles, damage cells and release of intracellular components, centrifugation 20-min at 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 n

20、itrogen, maintain samples atC2-8fter melting,add PBSPH7.4 , Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m.remove supernatant.6. extract as soon as possibleafter Specimencollection,ancaccordingto the relevant literature,and shouldbe experimentas soon as possib

21、leafter the extraction .If it cant, specimen can be kept in - 20 to preserve, Avoid repeated freeze-thaw cycles.7. Can' t detect the sample which conaN3, because NaN3 inhibits HRP active.Assay procedure1 .Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add St

22、andard 00 仙 l to the first and the second well, then add Standard50il(uti加i the first and the second well, 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 forth well ,mix ; then take out 50

23、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 ; take out 50 from 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

24、; take out 50仙 l from the seveneighth well and add to the ninth and the tenth well, add Standard5©lutionD the ninth and thetenth well, mix , take out 50 m the ninthl annd the tenth wescard(add Sample 50 仙 l to each well after Diluting ,(densityj/ml , 60U/ml , 30U/ml, i5U/ml , 7.5U/ml)2 .add sam

25、ple Set blank wells separately(blank comparisorwells don' atdd sampleand HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution40 仙1.testingsamplewel, then add testing sample10 仙 Qsamplefinal dilutionis 5-fold), add sample to weldon' t touch the w

26、ell wall as far as poasd)Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30Cmin at 374 .Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5 .washing Uncover Closure plate membrane, discard Liquid,

27、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 reagent 仙 l to each well, excebtank well.7.incubate Operation with 3.8.washing Operation with 5.9.color Add Chromogen Solution A 50ul and Chromogen Solution B to each

28、well, evade the light preservation for 15 min a t23710.Stopthe reaction Add Stop Solutio50 ptb each well, Stop the reaction(thdolue colorchange to yellow color).Il.assay take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Important notes1. The kit takes out

29、 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 Crystallizationseparationjt can be heatedthe water helps dissolve when dilute . Washing does not affect the result.3. add Samplewith samplerEach step, And proofreadits accuracyfrequentlyavoidsthe experimentalerror. add sample within 5 mins, if the number of sample