大鼠核轉錄因子p65(NF-KB

1、大鼠核轉錄因子p65(NF-KB p65)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：本試劑盒用于測定大鼠血清，血漿及相關液體樣本中核轉錄因子p65的含量。實驗原理： 本試劑盒應用雙抗體夾心法測定標本中大鼠核轉錄因子p65的水平。用純化的大鼠核轉錄因子p65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核轉錄因子 p65，再與HRP標記的核轉錄因子p65抗體結合，形成抗體-抗原-酶標抗體復合物，經(jīng)過徹底洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸的作用下轉化成最終的黃色。顏色的深淺和樣品中的核轉錄因子p65呈正相關。用酶標儀在450nm波長下測定吸光度

2、（OD值），通過標準曲線計算樣品中大鼠核轉錄因子p65濃度。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片（48）2片（96）密封袋1個1個酶標包被板1481962-8保存標準品：1350ng/L0.5ml1瓶0.5ml1瓶2-8保存標準品稀釋液1.5ml1瓶1.5ml1瓶2-8保存酶標試劑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20倍濃縮洗滌液20ml1瓶30ml1瓶2-8保存樣本處理及要求：1. 血清：室溫

3、血液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如出現(xiàn)沉淀，應再次離心。2. 血漿：應根據(jù)標本的要求選擇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/分）。仔細收集上清，保存過程中如有沉淀形成，應該再次離心。3. 尿液：用無菌管收集，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。4. 細胞培養(yǎng)上清：檢測分泌性的成份時，用無菌管收集。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。檢測細胞內的成份時，用PBS（PH7

4、.2-7.4）稀釋細胞懸液，細胞濃度達到100萬/ml左右。通過反復凍融，以使細胞破壞并放出細胞內成份。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。保存過程中如有沉淀形成，應再次離心。5. 組織標本：切割標本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存?zhèn)溆?。標本融化后仍然保?-8的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標本勻漿充分。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。分裝后一份待檢測，其余冷凍備用。6. 標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上進行試驗，可將標本放于-20保存，但

5、應避免反復凍融.7. 不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。操作步驟：1. 標準品的稀釋與加樣：在酶標包被板上設標準品孔10孔，在第一、第二孔中分別加標準品100l，然后在第一、第二孔中加標準品稀釋液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、第四孔分別加標準品稀釋液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、第六孔中分別加標準品稀釋液50ul，混勻；混勻后從第五、第六孔中各取50l分別加到第七、第八孔中，再在第七、第八孔中分別加標準品稀釋液50l，混勻后從第七、第八

6、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準品稀釋液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900 ng/L，600ng/L ，300 ng/L，150 ng/L，75 ng/L）。2. 加樣：分別設空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、待測樣品孔。在酶標包被板上待測樣品孔中先加樣品稀釋液40l，然后再加待測樣品10l（樣品最終稀釋度為5倍）。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻。3. 溫育：用封板膜封板后置37溫育30分鐘。4. 配液：將20倍濃縮洗滌液用蒸餾水20倍稀釋后備用。5. 洗滌：小

7、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5次，拍干。6. 加酶：每孔加入酶標試劑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（此時藍色立轉黃色）。11. 測定：以空白空調零，450nm波長依序測量各孔的吸光度（OD值）。 測定應在加終止液后15分鐘以內進行。注意事項：1 試劑盒從冷藏環(huán)境中取出應在室溫平衡15-30分鐘后方可使用，酶標包被板開封后如未用完，板條應裝入密封袋中保存。2 濃洗滌液可能會有結晶析

8、出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。3 各步加樣均應使用加樣器，并經(jīng)常校對其準確性，以避免試驗誤差。一次加樣時間最好控制在5分鐘內，如標本數(shù)量多，推薦使用排槍加樣。4 請每次測定的同時做標準曲線，最好做復孔。如標本中待測物質含量過高（樣本OD值大于標準品孔第一孔的OD值），請先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測定，計算時請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 底物請避光保存。7 嚴格按照說明書的操作進行，試驗結果判定必須以酶標儀讀數(shù)為準.8 所有樣品，洗滌液和各種廢棄物都應按傳染物處理。9 本試劑不同批號組分不得混用。10. 如與英文說明書有

9、異，以英文說明書為準。計算：以標準物的濃度為橫坐標，OD值為縱坐標， 在坐標紙上繪出標準曲線，根據(jù)樣品的OD 值由標準曲線查出相應的濃度；再乘以稀釋 倍數(shù)；或用標準物的濃度與OD值計算出標 準曲線的直線回歸方程式，將樣品的OD值 代入方程式，計算出樣品濃度，再乘以稀釋 倍數(shù)，即為樣品的實際濃度。 （此圖僅供參考）試劑盒性能：1.樣品線性回歸與預期濃度相關系數(shù)R值為0.990以上。2.批內與批見應分別小于9%和11%檢測范圍： 50 ng/L -1000 ng/L 保存條件及有效期：1.試劑盒保存：；2-8。2有效期：6個月Rat NF-B p65FOR RESEARCH USE ONLYDru

10、g NamesGeneric Name：Rat NF-B p65 ELISA Kit.PurposeThis kit allows for the determination of NF-B p65 concentrations in Rat serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay Rat NF-B p65 level in the sample，use Purified Rat NF-B p65 antibody to coat microtiter plate

11、wells, make solid-phase antibody, then add NF-B p65 to wells, Combined NF-B p65 antibody which With HRP labeled , 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

12、 by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of NF-B p65 in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kitMaterials provide

13、d with the kit48determinations96 determinationsStorageUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8Standard：1350ng/L0.5ml1 bottle0.5ml1 bottle2-8Standard diluent1.5ml1 bottle1.5ml1 bottle2-8HRP-Conjugate reagent3ml1 bottle6ml1 bottle2-8Sample diluent3ml1 bottle6ml1 bot

14、tle2-8Chromogen Solution A3ml1 bottle6ml1 bottle2-8Chromogen Solution B3ml1 bottle6ml1 bottle2-8Stop Solution3ml1 bottle6ml1 bottle2-8wash solution20ml1 bottle30ml1 bottle2-8Specimen requirements1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of 2000-3000 r.p.

15、m. remove supernatant, If precipitation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile c

16、ontainer, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.4. cell culture supernatant-detect secretory components, collect sue a sterile container, centrifuga

17、tion 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed

18、 of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain samples at 2-8 after melting,add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugat

19、ion 20-min at the speed of 2000-3000 r.p.m. remove 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, specimen can be kept in -20 to preserve, Avoid repeated fre

20、eze-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 well, then add Standard dilution 50l to the first and the

21、 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 well discard, add 50l to the fifth and the sixth well ,then

22、 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 from the seventh and the eighth well and add to the ninth

23、 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: 900 ng/L,600ng/L ,300 ng/L,150 ng/L,75 ng/L)2.add sample：Set blank wells separately (blank comparison wel

24、ls 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 the well wall as far as possible, and Gently mix.3.In

25、cubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37.4.Configurate liquid: wash solution diluted 20-fold with distilled water and reserve.5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain,

26、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 Solution B to each well, evade the light preservation for 15 min at 3710.Stop the reaction

27、：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. The kit takes out from the refrigeration environment should be balanced 15-30 minutes

28、 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 dilute . Washing does not affect the result.3. add Sample with sampler Each step, A

29、nd 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 only limits the disposable use, to avoid c