兔子線粒體呼吸鏈復(fù)合物III輔酶Q-細(xì)胞色素C還原酶

1、1兔子線粒體呼吸鏈復(fù)合物兔子線粒體呼吸鏈復(fù)合物 III(III(輔酶輔酶 Q-Q-細(xì)胞色素細(xì)胞色素 C C 還原酶還原酶) )酶聯(lián)免疫分析試劑盒使用說明書酶聯(lián)免疫分析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(cè)定兔子血清，血漿，細(xì)胞上清及相關(guān)液體樣本中線粒體呼吸鏈復(fù)合物 III(輔酶 Q-細(xì)胞色素 C 還原酶)的活性。實(shí)驗(yàn)原理：實(shí)驗(yàn)原理： 本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中兔子線粒體呼吸鏈復(fù)合物 III(輔酶 Q-細(xì)胞色素C 還原酶)水平。用純化的兔子線粒體呼吸鏈復(fù)合物 III(輔酶 Q-細(xì)胞色素 C 還原酶)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線粒體呼吸鏈復(fù)合

2、物 III(輔酶 Q-細(xì)胞色素 C 還原酶)，再與 HRP 標(biāo)記的線粒體呼吸鏈復(fù)合物 III(輔酶 Q-細(xì)胞色素 C 還原酶)抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)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(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線粒體呼吸鏈復(fù)合物 III(輔酶 Q-細(xì)胞色素 C 還原酶)呈正相關(guān)。用酶標(biāo)儀在 450nm 波長(zhǎng)下測(cè)定吸光度（OD 值） ，通過標(biāo)準(zhǔn)曲線計(jì)算樣品中兔子線粒體呼吸鏈復(fù)合物 III(輔酶 Q-細(xì)胞色素 C 還原酶)濃度。試劑盒組成試劑盒組成：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

3、 片（48）2 片（96）密封袋1 個(gè)1 個(gè)酶標(biāo)包被板1481962-8保存標(biāo)準(zhǔn)品：135U/mL0.5ml1 瓶0.5ml1 瓶2-8保存標(biāo)準(zhǔn)品稀釋液1.5ml1 瓶1.5ml1 瓶2-8保存酶標(biāo)試劑3 ml1 瓶6 ml1 瓶2-8保存樣品稀釋液3 ml1 瓶6 ml1 瓶2-8保存顯色劑 A 液3 ml1 瓶6 ml1 瓶2-8保存顯色劑 B 液3 ml1 瓶6 ml1 瓶2-8保存終止液3ml1 瓶6ml1 瓶2-8保存濃縮洗滌液（20ml20 倍）1 瓶（20ml30 倍）1 瓶2-8保存樣本處理及要求樣本處理及要求：1. 血清：室溫血液自然凝固 10-20 分鐘，離心 20 分鐘左

4、右（2000-3000 轉(zhuǎn)/分） 。仔細(xì)收集上清，保存過程中如出現(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，保存過程中如有沉淀形成，應(yīng)該再次2離心。3. 尿液：用無菌管收集，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

5、-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到 100 萬/ml 左右。通過反復(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 2-8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

6、將標(biāo)本放于-20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 抑制辣根過氧化物酶的（HRP）活性。操作步驟：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 100l，然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液 50l，混勻；然后從第一孔、第二孔中各取 100l 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50l，混勻；然后在第三孔和第四孔中先各取 50l 棄掉，再各取 50l 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 50l 分別加到第七、第八孔

7、中，再在第七、第八孔中分別加標(biāo)準(zhǔn)品稀釋液50l，混勻后從第七、第八孔中分別取 50l 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 50l，混勻后從第九第十孔中各取 50l 棄掉。 （稀釋后各孔加樣量都為 50l，濃度分別為 90U/mL，60U/mL ，30U/mL，15U/mL，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 40l，然后再加待測(cè)樣品10l（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溫育

8、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 50l，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 A50l，再加入顯色劑 B50l，輕輕震蕩混勻，37避光顯色15 分鐘. 10. 終止：每孔加終止液 50l，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)

9、進(jìn)行。3注意事項(xiàng)：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，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)（n5） 。5 封板膜只限一次性使用，以避免交叉污染。6

10、 底物請(qǐng)避光保存。7 嚴(yán)格按照說明書的操作進(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. 如與英文說明書有異，以英文說明書為準(zhǔn)。計(jì)算：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，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：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 0.95

11、以上。2.批內(nèi)與批見應(yīng)分別小于 9%和 11%檢測(cè)范圍：檢測(cè)范圍： 3U/mL - 100U/mL 保存條件及有效期：保存條件及有效期：1.試劑盒保存：；2-8。2有效期：6 個(gè)月4Rabbit complex IIIFOR RESEARCH USE ONLYDrug NamesGeneric Name：Rabbit complex III ELISA Kit.PurposeThis kit allows for the determination of complex III concentrations in Rabbit serum, blood plasma, cell culture

12、 supernatant and other biological fluids.Principle of the assayThe kit assay Rabbit complex III level in the sample，use Purified Rabbit complex III antibody to coat microtiter plate wells, make solid-phase antibody, then add complex III to wells, Combined complex III antibody which With HRP labeled

13、,become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavele

14、ngth of 450 nm. The concentration of complex III in the samples is then determined by comparing the O.D. of the samples to the standard curve.RD5Materials provided with the kitMaterials provided with the kit48determinations96 determinationsStorageUser manual11Closure plate membrane22Sealed bags11Mic

15、roelisa stripplate112-8Standard：135U/mL0.5ml1 bottle0.5ml1 bottle2-8Standard diluent1.5ml1 bottle1.5ml1 bottle2-8HRP-Conjugate reagent3ml1 bottle6ml1 bottle2-8Sample diluent3ml1 bottle6ml1 bottle2-8Chromogen Solution A3ml1 bottle6ml1 bottle2-8Chromogen Solution B3ml1 bottle6ml1 bottle2-8Stop Solutio

16、n3ml1 bottle6ml1 bottle2-8wash solution（20ml20 fold）1bottle（20ml30 fold）1bottle2-8Specimen requirements1.serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of supernatant, If precipitation appeared, Centrifugal again.2.plasma-use suited EDTA or citrate plasma as an

17、anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of supernatant, If precipitation appeared, Centrifugal again.3.Urine-collect sue a sterile container, centrifugation 20-min at the speed of supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cer

18、ebrospinal fluid Reference to it.4.cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of supernatant,detect the composition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeated

19、freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of supernatant, If precipitation appeared, Centrifugal again.65.Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain sampl

20、es at 2-8 after melting,add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugation 20-min at the speed of supernatant.6.extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it cant,

21、specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7.Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100l to the first and the second

22、 well, then add Standard dilution 50l to the first and the second well, mix; take out 100l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50l to the third and the forth well ,mix ; then take out 50l from the third and the forth w

23、ell discard, add 50l to the fifth and the sixth well ,then add Standard dilution 50l to the fifth and the sixth well, mix ; take out 50l from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50l to the seventh and the eighth well ,mix ; take out 50l

24、 from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50l to the ninth and the tenth well, mix , take out 50l from the ninth and the tenth well discard(add Sample 50l to each well after Diluting ,(density: 90U/mL，60U/mL ，30U/mL，15U/mL，7.5U/mL)2.add samp

25、le：Set blank wells separately (blank comparison wells dont add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well, then add testing sample 10l (sample final dilution is 5-fold), add sample to wells , dont touch th

26、e well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37.4.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, discar

27、d Liquid, dry by swing, add washing 7buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzyme：Add HRP-Conjugate reagent 50l to each well, except blank well. 7.incubate：Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solut

28、ion B to each well, evade the light preservation for 15 min at 3710.Stop the reaction：Add Stop Solution50l to each well, Stop the reaction(the blue color change to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Important notes1.T

29、he 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.washing buffer will Crystallization separation, it can be heated the water helps dissolve when

30、dilute . Washing does not affect the result.3.add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 mins, if the number of sample is much , recommend to use Volley .4.if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.（n5）.5.Closure plate membrane