牛脂多糖結(jié)合蛋白LBP酶聯(lián)免疫分析ELISA

1、牛脂多糖結(jié)合蛋白（LBP）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)定牛血清，血漿及相關(guān)液體樣本中脂多糖結(jié)合蛋白（LBP）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)本中牛脂多糖結(jié)合蛋白（LBP）水平。用純化的牛脂多糖結(jié)合蛋白（LBP）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脂多糖結(jié)合 蛋白（LBP），再與HRP標(biāo)記的脂多糖結(jié)合蛋白（LBP）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脂多糖結(jié)合蛋白（LBP）呈正相關(guān)。

2、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)算樣品中牛脂多糖結(jié)合蛋白（LBP）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 962-8 C保存標(biāo)準(zhǔn)品：45 ug/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

3、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

4、-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2-8C的溫度。加入一定量的PBS

5、 （ 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

6、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50dl 棄掉，再各取 50dl 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50dl 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 dl ，混勻后從第九第十孔中各取50dl 棄掉。（稀釋后各孔加樣量都為 50dl，濃度分別為 30ug/ml， 20 ug/ml， 10 ug/ml， 5 ug/ml， 2.5 ug/ml）。2. 加樣：分別設(shè)空白

7、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 dl ，空白孔除外。7. 溫育：操作同 3。8. 洗滌：操作同 5。9. 顯色：每孔

8、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 d，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 分鐘以內(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

9、，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)（XnX 5）O5. 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) &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)。 計(jì)算：以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)， 0D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0D值由標(biāo)準(zhǔn)曲線查出相應(yīng)的

10、濃度；再乘以稀釋 倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與 0D值計(jì)算出標(biāo) 準(zhǔn)曲線的直線回歸方程式，將樣品的0D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。（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)與批間應(yīng)分別小于9%和15%檢測(cè)范圍：1ug/ml -40ug/ml保存條件及有效期：1.試劑盒保存：;2-8Co2 有效期：6個(gè)月Bov ine lipolysaccharide binding prote inFOR RESEARCH USE ONLYDrug NamesGen eric Nam: Bovine lipolysaccharide

11、 bi ndi ng protein (LBP) ELISA Kit.PurposeThis kit allows for the determ in ati on of LBP concen tratio ns in Bovine serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay Bovine LBP level in the samplese Purified Bovine LBP antibody to coat microtiter plate wells, make

12、 solid-phase an tibody, the n add LBP to wells, Comb ined LBP which With HRP labeled, becomea ntibody- an tige n - en zyme-a ntibodyomplex,after wash ing Completely,Add TMB substrate solution,TMBsubstrate becomes blue color At HRP en zyme-catalyzed, reacti on is term in ated by the additi on of a su

13、lphuric acid soluti on and the color change is measured spectrophotometricallyit a wavelengthof 450 nm. The concentration of LBP in the samples is then determined by comparing the O.D. of the samples to the sta ndard curve.Materials provided with the kitMaterials provided wit the kith48determ in ati

14、 ons96 determ inationsStorageUser manual11Closure plate membra ne22Sealed bags11Microelisa stripplate112-8 CStandard 45 ug/ml0.5ml 1Xbottle0.5ml Xbottle2-8 CStan dard dilue nt1.5ml Xbottle1.5ml Xbottle2-8 CHRP-Co njugate reagent 3ml X bottle6ml X bottle2-8 CSample dilue nt3ml X bottle6ml X bottle2-8

15、 CChromoge n Soluti on A3ml X bottle6ml X bottle2-8 CChromoge n Soluti on BI3ml X bottle6ml X bottle2-8 CStop Soluti on3ml X bottle6ml X bottle2-8 Cwash solution(20ml X 20 foldx 1 bottle(20ml X 30 foldx 1 bottle2-8 CSpecimen requirements1. serum- coagulation at room temperature 10-20 ,meintrifugatio

16、n 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 plasma as an anticoagulant,mix 10-20 mins ,cen trifugatior20-min at the speed of 2000-3000r.p.m. remove super nata nt,If precipitation appeared, Centri

17、fugal again.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 cerebrosp inal fluid Refere nee to it.4. cell culture supernatan-detect secretorycompon

18、entscollect 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 million / ml, repeated freeze-thawcycles, damage cells and release ofin tracellular co

19、mp 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 , Rapidlyfroze n with liquid n itroge n, mai ntain samples atQ-8fter melt in g,add PBSPH7.4

20、,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,andaccordingto the relevantliterature,and shouldbe experimentas soon as possibleafter the extraction.If it cant,specime n can be kept in

21、-20 to preserve, Avoid repeated freeze-thaw cycles.7. Cant 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 卩 l to the first and the second well, then add Stand

22、50didilUrtithe first andthe second well, mix; take out 100卩 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 ; then take out 50卩 l from the third and the forth well discard, add 50the sixth well

23、 ,then add Standard dilU50)p l to the 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 Standarddilution50 卩 l to the seventh and the eighth well ,mix ; take out 50卩 l from the seveneighth well and add to the ninth and t

24、he tenth well, add Sta ndard50l(utioni)the ninth andthe ten th well, mix , take out 50 m the nin thpahdcthe ten th wescard(add Sample 50 卩 l toeach well after Diluting ,(dens3i0tyu:g/ml，20 ug/ml ， 10 ug/ml ， 5 ug/ml ， 2.5 ug/ml)2. add sample：Set blank wells separately(blank comparisonwells donatdd s

25、ample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample diluti on40 ytlo test in gsamplewel, the n add testi ng sample10 y(samplefi nal diluti onis5-fold), add sample to welldso,nt touch the well wall as far as poasnsidblGe,ently mix.3.ln cubate: After cl

26、osi ng plate with Closure plate membra ne ,in cubate for 30CBnin 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, dry by swing, add washing bufferto every well, still

27、 for 30s then drain, repeat 5 times, dry by pat.6. add enzym: Add HRP-Conjugate reagent l to each well, ept blank well.7.incubate： Operation with 3.8. washing：Operation with 5.9. color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight preservati on for 15 min at37lO

28、.Stopthe reaction Add Stop Solutio50 卩1 each well, Stop the react ion (theolue color change to yellow color).11.assay：take olank well as zero , Read aosoroance at 450nm after Adding Stop Solution and within 15min.Important notes1. The kit takes out from the refrigeration environment should oe oalanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should oe stored in Sealed oag.2. washingoufferwill Crystallizationseparation,it can oe heatedthe water helps dissolve when dilute . Washing does not affect the result.3. add Sample