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 48 1X 96 2-8 C保存 標(biāo)準(zhǔn)品：540舊/L 0.5ml 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

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)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

4、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，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P

5、BS, 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。加入一定量的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)。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

6、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 , 2

7、40 /L , 120 /L, 60 d/L , 30 dg/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 （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(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

8、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卩,1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

9、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(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) 2-8Co 2 有效期：6個(gè)月 FOR RESEARCH USE ONLY Mouse microalb unmin uria

10、 Drug Names Gen eric Nam: Mouse microalbu nmin uria (ALB) ELISA Kit Purpose This kit allows for the determ in atioALB concen trati ons in Mouse serum, blood plasma, tissue and other biological fluids. Principle of the assay The kit assay Mouse ALB level in the samplese Purified Mouse ALB antibody to

11、 coat microtiterplate wells, make solid-phasea ntibody,the n add ALB to wells, Comb in edALB an tibodywhichWith HRP labeled, becomea ntibody- an tige n- en zyme-a ntibockyomplex, after wash ing Completely, Add TMB substrate solutio n,TMB substrate becomes blue color At HRP en zyme-catalyzed, react i

12、on is termi nated by the additi on of a sulphuric acid soluti on and the color cha nge is measuredspectrophotometricaHyt a wavele ngthof 450 nm. The concentration of ALB in the samples is then determined by comparing the O.D. of the samples to the sta ndard curve. Materials provided with the kit Mat

13、erials provided with the kit 48determ in ati ons 96 determ in atio ns Storage User manual 1 1 Closure plate membra n e2 2 Sealed bags 1 1 Microelisa stripplate 1 1 2-8 C Standard 540 舊/L 0.5ml 1Xbottle 0.5ml Kbottle 2-8 C Stan dard dilue nt 1.5ml Xbottle 1.5ml Kbottle 2-8 C HRP-Co njugate reagen t3m

14、l Xbottle 6ml 1 bottle 2-8 C Sample dilue nt 3ml Xbottle 6ml 1 bottle 2-8 C Chromoge n Soluti on A 3ml Xbottle 6ml 1 bottle 2-8 C Chromoge n Soluti on B 3ml 1 bottle 6ml 1 bottle 2-8 C Stop Soluti on 3ml 1 bottle 6ml 1 bottle 2-8 C wash solution (20ml X 20 fold) x 1bottle (20ml X 30 fold) x 1 bottle

15、 2-8 C Specimen requirements 1. serum- coagulation at room temperature 10-20 ,meintrifugation 20-min at the speed of 2000-3000 r.p.m. remove super nata nt. If precipitati on appeared, Cen trifugal aga in. 2. plasma-use suited EDTA or citrate or as an anticoagulant,mix 10-20 mins ,centrifugation 20-m

16、inat the speed of 2000-3000r.p.m.removesuper nata ntf precipitationappeared, Cen trifugal aga in. 3. Urine collect sue a sterile container, cen trifugati on 20-min at the speed of 2000-3000 r.p.m. remove supernata nt,If precipitati on appeared, Cen trifugalaga in. The Operatio n of Hydrothorax and c

17、erebrosp inal fluid Refere nee to it. 4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, cen trifugatior2 0-min at the speed of 2000-3000r.p.m. removesuper nata nt,detectie compositionof cells, Dilut cell suspensiorwith PBS (PH7.2-7.4 , Cell concentration reached 1

18、 million / ml, repeated freeze-thawcycles, damage cells and release of in tracellular comp onen ts, cen trifugati on 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 (PBS7.2-7.4 , R

19、apidly froze n with liquid n itroge n, mai ntain samples atQ-8fter melt in g,add PBSPH7.4 , Homoge ni zed by hand or Grin ders, cen trifugati on 20-min at the speed of 2000-3000 r.p.m. remove super nata nt. 6. extract as soon as possibleafter Specime ncollecti on,an daccord in gto the releva nt lite

20、rature,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 conaN3, because NaN3 inhibits HRP active. Assay procedure 1. Dilute and add sample to Sta ndard: set 10 Sta

21、 ndard wells on the ELISA plates coated, add Standard 100 卩 l to the first and the second well, then add Stand50didilUrtittne first and the second well, mix; take out 100卩 l form the first and the second well then add it to the thi and the forth well separatelythen add Standarddilution 50 卩 to the t

22、hird and the forth well ,mix ; then take out 50卩 l from the third and the forth well discard, add 50 the sixth well ,then add Standard dilL50)p l to the fifth and the sixth well, mix ; take out 50 from the fifth and the sixth well and add to the seve nth and the eighth well, the n add Sta ndard dilu

23、tion50 卩 l to the seventh and the eighth well ,mix ; take out 50卩 l from the seven eighth well and add to the ninth and the tenth well, add Sta ndard50liutionD thenth and the ten th well, mix , take out 50卩 l from the nin th and the ten th well discard(add Sample 5 each well after Diluting ,(density

24、：舊/L , 240 曲/L , 120 用/L, 60 用/L, 30 /L) 2. add sample Set blank wells separately(blank comparisorwells don add sampleand HRP-Conjugate reage nt, other each step operati on is same). test ing sample well. add Sample diluti on40 卩 to test in gsamplewel, the n add testi ng sample10 y(samplefi nal dilu

25、ti onis 5-fold), add sample to weldon t touch the well waa rass possible, and Gently mix. 3.ln cubate: After closi ng plate with Closure plate membra ne ,in cubate for 30CBnin at 37 4. C on figurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reser

26、ve. 5. washi ng Un cover Closure plate membra ne, discard Liquid, dry by swing, add wash ing buffer to every well, still for 30s the n drain, repeat 5 times, dry by pat. 6. add enzyme Add HRP-Conjugate reagenit l to each well, exceptank well. 7.incubate Operation with 3. 8. washing Operation with 5.

27、 9. colo: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservati on for 15 min at37 10.Stopthe reaction Add Stop Solutio50 卩1 each well, Stop the react ion (th由lue color cha nge to yellow color). 11.assay take bla nk well as zero , Read absorba nee at 450nm a

28、fter Add ing Stop Soluti on and within 15mi n. Important notes 1. The kit takes out from the refrigeration environment should be balaneed 15-30 minutes in the room temperature, ELISA plates coated if has n ot use up after ope ned, 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. a