大鼠膽固醇合成酶HMG-COA酶聯(lián)免疫分析ELISA

1、7大鼠膽固醇合成酶（HMG-COA酶聯(lián)免疫分析（ELISA）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)定大鼠血液，血清及相關(guān)液體樣本中膽固醇合成酶（HMG-COA水平實(shí)驗(yàn)原理：本試劑盒采用雙抗體夾心酶聯(lián)免疫法（ELISA）測(cè)定標(biāo)本中大鼠膽固醇合成酶 （HMG-COA ）。用純化的大鼠膽固醇合成酶（HMG-COA ）抗體包被微孔板，制成固相抗體，可與樣品中膽固醇合成酶（ HMG-COA相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗原和其他成分后 再與HRP標(biāo)記的膽固醇合成酶（HMG-COA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，

2、并在酸的作用下轉(zhuǎn) 化成最終的黃色。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），與CUTOFF值相比較，從而判定標(biāo)本中大鼠膽固醇合成酶（HMG-COA ）存在與否。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保存陰性對(duì)照0.5ml X 1 瓶0.5ml X 1 瓶2-8 C保存陽(yáng)性對(duì)照0.5ml X 1 瓶0.5ml 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

3、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 離心。3. 尿液

4、：用無菌管收集，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（PH7.2-7.4 ）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?/p>

5、。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抑制辣根過氧化物酶的（HRP）活性。操作步驟：1. 編號(hào)：將樣品對(duì)應(yīng)微孔按序編號(hào)，每板應(yīng)設(shè)陰性對(duì)照 2孔、陽(yáng)性對(duì)照 2孔、空白對(duì)照 1 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）2. 加樣：分別在

6、陰、陽(yáng)性對(duì)照孔中加入陰性對(duì)照、陽(yáng)性對(duì)照50卩。然后在待測(cè)樣品孔先加樣品稀釋液 40卩,然后再加待測(cè)樣品10卩。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）倍濃縮洗滌液加蒸餾水至600ml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卩,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 A 50 ，再加入顯色劑 B 50卩,輕輕震蕩混勻，37 C避光顯色 1

7、5分鐘10. 終止：每孔加終止液 50卩終止反應(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。結(jié)果判定：試驗(yàn)有效性：陽(yáng)性對(duì)照孔平均值羽.00；陰性對(duì)照平均值 切.10臨界值（CUT OFF ）計(jì)算：臨界值=陰性對(duì)照孔平均值+0.15 陰性判定：樣品 OD值 臨界值（CUT OFF）者為膽固醇合成酶（HMG-CC）陰性 陽(yáng)性判定：樣品 OD值臨界值（CUT OFF ）者為膽固醇合成酶（HMG-CC）陽(yáng)性 注意事項(xiàng) 1操作嚴(yán)格按照說明書進(jìn)行，本試劑不同批號(hào)組分不得混用。2試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡

8、 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。3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。 4 封板膜只限一次性使用，以避免交叉污染。630nm5底物請(qǐng)避光保存。 6試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)，使用雙波長(zhǎng)檢測(cè)時(shí)，參考波長(zhǎng)為2M 的硫酸，使用時(shí)必須7所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。終止液為注意安全。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 ONLYRat HMG-COADrug NamesGeneric Nam: RatHMG-COA ELISA Kit.Pu

9、rposeThis kit allows for the determ in ationof HMG-COA n Rat serum,a nd other biologicalfluids.Principle of the assayThe kit assay Rat HMG-COA level in the sa,plee Purified RdrfMG-COA an tibody toIdCOA to wells, Comb inedcoat microtiter plate wells, make solid-phase an tibody, then/With HMG-COA, aft

10、er wash ing and removing non-comb in ativea ntige n and other comp onents ,the n Comb ined HMG-COA an tibody which with HRP labeled become an tibody - an tige n - en zyme-a ntibody complex, after wash ing Completely, Add TMB substrate soluti on,TMB substrate becomes blue color At HRP en zyme-catalyz

11、ed, react ion is termi nated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavele ngthof 450 nm. Comparedwith the CUTOFFvalue,accord ingto this to judge HMG-COA exist in the sample or n ot.Materials provided with the kitMaterials provided w

12、ith the kit48determ in ati ons96 determ in atio nsStorageUser manual11Closure plate membra ne22Sealed bags11Microelisa stripplate112-8 CNegative con trol0.5ml 1Xbottle0.5ml Kbottle2-8 CPositive con trol0.5ml Xbottle0.5ml Kbottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle2-8 CSample dilue nt3m

13、l Xbottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti 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 CSpecimen requirements1. serum- coagulation at room temperatu

14、re 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 plasma as an anticoagulant,mix 10-20 mins ,cen trifugatior20-min at the speed of 2000-3000r.p.m. remove super nata nt,If precip

15、itation appeared, Centrifugal 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 supernata

16、n-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 million / ml, repeated freeze-thawcycles, damage cells and rel

17、ease ofin 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(PPBHS7.2-7.4) , Rapidlyfroze n with liquid n itroge n, mai ntain samples at2-af

18、ter melt in g,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,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction.If it c

19、ant, specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Cant detect the sample which coNnataNin3, because NaN3 inhibits HRP active.Assay procedure1. Number: to sample correspond microtitration well and Number Sequence, each plate should be set femininecomparison2 wells, m

20、asculinecomparison2 wells, blank comparison1 well(don t add sampHleRaPn-dConjugate reagent to blank comparison well, other each step the operation are same).2. add sample separately add Positive control and Negative co5tJoJ l to thPositive andNegative well . add Sample dilution to testing sample wel

21、l, thentesting sample 10 卩 l.add sample to the bottom oEfLISA plates coated welld,ont touch the well wall as far aspossible, and Gently mix.3.ln cubate: After closi ng plate with Closure plate membra ne ,in cubate for 30tmin at 374. Configurate liquid: 30-fold (or 20-fold)wash solution diluted until

22、 600ml,and reserve.5. washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. add enzym: Add HRP-Conjugate reagent lto each well, except tllanlbwell.7.incubate： Operation with 3.8. washing： Operat

23、ion with 5.9. color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight preservati on for 15 min at37lO.Stopthe reaction Add Stop Solutio50 卩 l each well, Stop the react ion (thdblue color change to yellow color).11. assaytake blank well as zero , Read absorbance at 4

24、50nm after Adding Stop Solution and within 15min.Determine the resultTest validity: the average of Positive cowtrtbl 1.00e average of Negative con trol well 0.10.Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.15.Negative control: sample OD Calculate Critical(CUT HMG -COA Positive control.Important notes1. Please according to use instruction strictly, Do not mix reagents with those from other lots.2. The kit takes out from the refrigeration environment should be balanced