小鼠尿微量白蛋白ALB說(shuō)明書(shū)

1、小鼠尿微量白蛋白(ALB )酶聯(lián)免疫分析(ELISA )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)定小鼠血清，血漿，相關(guān)液體樣本尿微量白蛋白(ALB)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)本中小鼠尿微量蛋白(ALB)水平。用純化的小鼠尿微量蛋白(ALB)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尿微量蛋白(ALB)，再與HRP標(biāo)記的ALB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尿微量蛋白(ALB)呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定

2、吸光度(OD值)，通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中小鼠尿微量蛋白(ALB)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 9628C保存標(biāo)準(zhǔn)品：540/L0.5ml X 1 瓶0.5ml X 1 瓶28C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)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

4、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12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 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 用。標(biāo)本融化后仍然保持 將標(biāo)本勻漿充分。離心 檢測(cè)，其余冷凍備用。6. 標(biāo)本采集后盡早進(jìn)行提取， 進(jìn)行試驗(yàn)，可將標(biāo)本放于20分PBS, PH7.

5、4。用液氮迅速冷凍保存?zhèn)?-8 C的溫度。加入一定量的PBS （ PH7.4）,用手工或勻漿器20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。分裝后一份待提取按相關(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 -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1.2.3.4.5.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 d，然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液50也混勻；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

6、,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 360 dg/L , 240 /L , 120 /L, 60 dg/L , 30 dg/L ）。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）品孔。在酶

7、標(biāo)包被板上待測(cè)樣品孔中先加樣品稀釋液 品最終稀釋度為5倍）。加樣將樣品加于酶標(biāo)板孔底部， 勻。溫育：用封板膜封板后置 37 C溫育30分鐘。配液：將30 （48T的20倍）倍濃縮洗滌液用蒸餾水、待測(cè)樣40 M,然后再加待測(cè)樣品10 d （樣 盡量不觸及孔壁，輕輕晃動(dòng)混30（ 48T的20倍）倍稀釋后備用。30秒后棄去，如此洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置重復(fù)5次，拍干。加酶：每孔加入酶標(biāo)試劑50 d，空白孔除外。溫育：操作同3。 洗滌：操作同5。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，15分鐘.終止：每孔加終止液 50 d,l終止反應(yīng)

8、（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。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)：1.6.7.8.9.10.11.2.3.4.37 C避光顯色試劑盒從冷臧環(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。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。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。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

9、 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)（Xn X 5 ）。5.6.7.&9.（此圖僅供參考）封板膜只限一次性使用，以避免交叉污染。 底物請(qǐng)避光保存。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) 所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。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)。 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，

10、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:20 用/L -400 jXg/L 保存條件及有效期：1試劑盒保存：；2-8Co2 .有效期：6個(gè)月FOR RESEARCH USE ONLYMouse microalb unmin uriaDrug NamesGen eric Nam: Mouse microalbu nmin uria (ALB) ELISA KitPurposeThis kit allows for the determ in atioALB concen

11、trati ons in Mouse serum, blood p lasma.tissue and other biological fluids.P rinc iple of the assayThe kit assay Mouse ALB level in the sam plese Purified Mouse ALB an tibody to coat microtiterplate wells, make solid-p hasea ntibody,the n add ALB to wells, Comb in edALB an tibodywhichWith HRP labele

12、d, becomea ntibody- an tige n- en zyme-a ntibockyom plex, after wash ing Comp letely, Add TMB substrate solutio n,TMB substrate becomes blue color AtHRP en zyme-catalyzed, react ion is termi nated by the additi on of a sulp huric acid soluti on and the color cha nge is measureds pectro photometrical

13、Ot a wavele ngthof 450 nm. The concen trati on of ALB in the samples is the n determ ined by comparing the O.D. of the samp les to the sta ndard curve.Materials p rovided with the kitMaterials pro vided with the kit48determ in ati ons96 determ in atio nsStorageUser manual11Closure pl ate membra ne22

14、Sealed bags11Microelisa stri pp late112-8 CStandard 540 /L0.5ml 1Xbottle0.5ml Kbottle2-8 CStan dard dilue nt1.5ml Xbottle1.5ml Kbottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle2-8 CSample dilue nt3ml Xbottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml Xbottle6ml 1 bottle2-8 CChromoge n Soluti

15、on B3ml 1 bottle6ml 1 bottle2-8 CStop Soluti on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml X 20 fold)X 1bottle(20ml X 30 fold)X 1 bottle2-8 CSp ecimen requirements1.serum- coagulatio n at room temp erature 10-20 ,raeintrifugati on 20-min at the sp eed of2000-3000 r.p .m. remove supern ata nt. If

16、 precip itati on app eared, Cen trifugal aga in.2.pl asma-use suited EDTA or citrate or as an an ticoagula nt,mix 10-20 mi ns ,ce ntrifugatio n20-minat the sp eed of 2000-3000r. p.m .removes upern ata ntf precip itationa pp eared,Cen trifugal aga in.3.Urine collect sue a sterile container, cen trifu

17、gati on 20-min at the sp eed of 2000-3000 r.p.m.remove supern ata nt,lf precip itati onapp eared, Cen trifugalaga in. The Op eratio n ofHydrothorax and cerebros pinal fluid Refere nee to it.4.cell culture supern ata n-detect secretoryco mponen tscollect sue a sterile container,cen trifugatior2 0-min

18、 at the sp eed of 2000-3000r. p.m. removes upern ata nt,detechecompositionof cells, Dilut cell suspensionwith PBS (PH7.2-7.4 , Cell concentrationreached 1 million / ml, repeated freeze-thawcycles, damage cells and release ofin tracellular componen ts, cen trifugati on 20-min at the sp eed of 2000-30

19、00 r.p.m. removesupern ata nt, If precip itati on app eared, Cen trifugal aga in.5.Tissue samp les After cutt ing sampi es, check the weight,add (PBS7.2-7.4 , Rapi dlyfroze n with liquid n itroge n, mai ntain samp les af(2-8fter melt in g,add PBSP H7.4 ,Homoge ni zed by hand or Grin ders, cen trifug

20、ati on 20-min at the sp eed of 2000-3000 r.p.m.remove supern ata nt.6. extract as soon as p ossibleafter Sp ecime ncollecti on,an daccord in gto the releva ntliterature,a nd shouldbe exp erime ntas soon as p ossibleafter the extractio n.lf it can'sp ecime n can be kept in -20 to p reserve, Avoid

21、 rep eated freeze-thaw cycles.7. Can' t detect the sampie which coN)aN3, because NaN3 inhibits HRP active.Assay p rocedureI.Dilute and add sam pie to Sta ndard: set 10 Sta ndard wells on the ELISA p lates coated, addStandard 100 11 to the first and the second well, then add StandOdidiiurtittne f

22、irst andthe sec ond well, mix; take out 100i l form the first and the second well then add it to the thiand the forth well separatelythen add Standarddilution 50 卩 to the third and the forthwell ,mix ; the n take out 5011 from the third and the forth well discard, add 50the sixth well ,then add Stan

23、dard dilL50)B 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 ndarddilution50 卩 l to the seventh and the eighth well ,mix ; take out 5011 from the seveneighth well and add to the ninth and the tenth well

24、, add Sta ndard50liutionD thenth andthe tenth well, mix , take out 50卩 l from the ninth and the tenth well discard(add Sample 5each well after Diluting ,(density：/L, 240/L , 120 用/L, 60 用/L, 30 /L)2.add sampie Set blank wells separately(blank comparisorwells don' add sampleandHRP-Conjugate reage

25、 nt, other each ste p op eratio n is same). test ing sam pie well. add Samplediluti on40 jiHo test in gsa mp lewell the n add testi ng sampi e10 卩(sa mplefinal diluti onis5-fold), add sample to weldojn' t touch the well waa rass possible, and Gently mix.3.ln cubate: After closi ng plate with Clo

26、sure plate membra ne ,in cubate for 30(Bnin at 374.C on figurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilledwater and reserve.5.washi ng Un cover Closure p late membra ne, discard Liquid, dry by swing, add wash ing bufferto every well, still for 30s the n dr

27、ain, rep eat 5 times, dry by p at.6.add enzyme Add HRP-Conjugate reagent l to each well, exceptank well.7.incubate Operation with 3.8.washing Operation with 5.9.C0I0: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight p reservati on for 15 min at3710.Stopthe reaction

28、 Add Stop Solutio50 卩1 each well, Stop the react ion (th由lue colorcha nge to yellow color).11.assay take bla nk well as zero , Read absorba nee at 450nm after Add ing Stop Soluti on andwithin 15mi n.Impo rtant notes1.The kit takes out from the refrigerati on en vir onment should be bala need 15-30 minu tes inthe room temp erature, ELISA pl ates coated if has n ot use up after open ed, the p late shouldbe stored in Sealed bag.2.wash in gbufferwill Crystallizatiorse parati onjt can be heatedthe water helps dissolvewhe n dilute . Wash ing does not affect the result.3.add