小鼠膽囊收縮素受體CCKR酶聯(lián)免疫分析ELISA

1、如有你有幫助，請購買下載，謝謝!4頁小鼠膽囊收縮素受體(CCKR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：本試劑盒用于測定小鼠血清，血漿及相關(guān)液體樣本中膽囊收縮素受體(CCKR的含量。實驗原理：本試劑盒應(yīng)用雙抗體夾心法測定標本中小鼠膽囊收縮素受體(CCKR)水平。用純化的小鼠膽囊收縮素受體(CCKR)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膽 囊收縮素受體(CCKR)，再與HRP標記的膽囊收縮素受體(CCKR)抗體結(jié)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(zhuǎn)化成藍色， 并在酸的作用下轉(zhuǎn)化成最終的黃色

2、。顏色的深淺和樣品中的膽囊收縮素受體(CCKR)呈正相關(guān)。用酶標儀在 450nm波長下測定吸光度(0D值)，通過標準曲線計算樣品中小鼠膽囊收 縮素受體(CCKR)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1X 481X 9628C保存標準品：360 pg/ml0.5ml X 1 瓶0.5ml X 1 瓶28C保存標準品稀釋液1.5ml X 1 瓶1.5ml X 1 瓶2-8 C保存酶標試劑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

3、液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20倍濃縮洗滌液20ml X 1 瓶30ml 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：用無

4、菌管收集，離心20分鐘左右(2000-3000轉(zhuǎn)份)。仔細收集上清，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(zhuǎn)/分)。仔細收集上清。檢測細胞內(nèi)的成份時，用PBS( PH7.2-7.4 )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(nèi)成份。離心20分鐘左右（ 2000-3000 轉(zhuǎn)/分）。仔細收集上清。保存過程中如有沉淀形成，應(yīng)再次離心。5. 組織標本：切割標本后，稱取重量。加入一定量的 用。標本融化后仍然保持 將標本勻漿充分。離心 檢測，其余冷凍備

5、用。6. 標本采集后盡早進行提取，進行試驗，可將標本放于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.PBS， PH7.4 。用液氮迅速冷凍保存?zhèn)?-8 C的溫度。加入一定量的PBS （ PH7.4）,用手工或勻漿器20 分鐘左右（ 2000-3000 轉(zhuǎn) /分）。仔細收集上清。分裝后一份待提取按相關(guān)文獻進行，提取后應(yīng)盡快進行實驗。若不能馬上-20 C保存，但應(yīng)避免反復凍融.2.3.4.5.6.7.8.9.10.11.標準品的稀釋與加樣：在酶標包被板上設(shè)標準品孔 10 孔，在第一、第二孔中分別加標 準品100 M，然后在第一、第二孔中加標準品稀釋液5

6、0 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、第四孔分別加標準品稀釋液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、第六孔中分別加標準品稀釋液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、第八孔中分別加標準品稀釋液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準品稀釋液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 240 pg/ml ， 160 pg/ml ， 80

7、 pg/ml ， 40 pg/ml ， 20 pg/ml ）。 加樣：分別設(shè)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40 d,然后再加待測樣品10 d （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。溫育：用封板膜封板后置 37 C溫育30分鐘。 配液：將 20 倍濃縮洗滌液用蒸餾水 20 倍稀釋后備用。 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置 重復 5 次，拍干。加酶：每孔加入酶標試劑 50 dl ，空白孔除外。 溫育：操作同 3。 洗滌：操作同 5。37 C避光

8、顯色顯色：每孔先加入顯色劑 A50 d，再加入顯色劑 B50 d，輕輕震蕩混勻，15分鐘.終止：每孔加終止液 50 d，終止反應(yīng)（此時藍色立轉(zhuǎn)黃色）。測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（ OD值）。測定應(yīng)在加終止 液后 15 分鐘以內(nèi)進行。注意事項：30 秒后棄去，如此試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡 15-30 分鐘后方可使用，酶標包被板開封后如未 用完，板條應(yīng)裝入密封袋中保存。濃洗滌液可能會有結(jié)晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結(jié)果。 各步加樣均應(yīng)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(nèi)，如標本數(shù)量多，推薦使用排

9、槍加樣。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(zhì)含量過高（樣本OD 值大于標準品孔第一孔的 0D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)（X n X 5 ）。封板膜只限一次性使用，以避免交叉污染。234516.7.&9.底物請避光保存。嚴格按照說明書的操作進行，試驗結(jié)果判定必須以酶標儀讀數(shù)為準 所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。本試劑不同批號組分不得混用。10. 如與英文說明書有異，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(yīng)的濃度；再乘以稀釋 倍數(shù)

10、；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(guān)系數(shù)R值為0.990以上。2. 批內(nèi)與批見應(yīng)分別小于 9%和11% 檢測范圍：10 pg/ml -300 pg/ml保存條件及有效期：1試劑盒保存：；2-8Co2 .有效期：6個月Mouse Cholecystok inin Acce ptorFOR RESEARCH USE ONL YDrug NamesGen eric Nam: Mouse Cholecystok inin Acce pto

11、r (CCKR) ELISA Kit.PurposeThis kit allows for the determ in ationof CCKR concen trati onin Mouse serum, blood pl asma, and other biological fluids.P rinc iple of the assayThe kit assayMouse CCKR level in the samp lejse P urified Mouse CCKR an tibody to coat microtiterplate wells, make solid-p hasea

12、ntibody,the n add CCKRto wells,Combi nedCCKR which With HRP labeled , become an tibody - an tige n - en zyme-a ntibody comp lex, after wash ing Comp letely, Add TMB substrate solutio n,TMB substrate becomes blue color At HRP en zyme-catalyzed, reacti on is term in ated by the additi on of a sulp hur

13、ic acid soluti on and the如有你有幫助，請購買下載，謝謝!4.6頁color change is measured spectrophotometricallgt a wavelength of 450 nm. Theconcen trati on of CCK in the samp les is the n determ ined by comparing the O.D. of the samplesto the sta ndard curve.Materials pro vided with the kit48determ in ati ons96 determ

14、 in atio nsStorageUser manual11Closure pl ate membra ne22Sealed bags11Microelisa stri pp late112-8 CStandard 360 pg/ml0.5ml 1Xbottle0.5ml Kbottle2-8 CStan dard dilue nt1.5ml Xbottle1.5ml Kbottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle2-8 CSample dilue nt3ml 1 bottle6ml 1 bottle2-8 CChromog

15、e 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 solution20ml 1 bottle30ml 1 bottle2-8 CMaterials p rovided with the kitSp ecimen requirements1.serum- coagulatio n at room temp erature 10-20 ,nceinitrifugat

16、i on 20-min at the sp eed of2000-3000 remove supern ata nt. If precip itati on app eared, Cen trifugal aga in.2.plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20mins ,centrifugation20-min at the speed of 2000-3000 remove supernatant,lfprecip itati on app eared, Cen trifugal aga

17、in.3.Urin e-collect sue a sterile contain er,ce ntrifugatior2 0-min at the sp eed of 2000-3000remove supern ata nt,lf precip itati onapp eared, Cen trifugalaga in. The Op eratio n ofHydrothorax and cerebros pinal fluid Refere nee to it.cell culture supern ata n-detect secretoryco mponen tscollect su

18、e a sterile container.centrifugation20-min at the speed of 2000-3000 remove supernatant,detecthecompositionof cells, Dilut cell suspensionwith PBS (PH7.2-7.4 , Cell concentrationreached 1 million / ml, repeated freeze-thawcycles, damage cells and release ofintracellularcomponents,centrifugationZ0-mi

19、n at the speed of 2000-3000 remove如有你有幫助，請購買下載，謝謝!11頁5.6.7.supernatant, If precipitation appeared, Centrifugal again.Tissue samples- After cutting samples, check the weight,add(PPBHS7.2-7.4) , Rapidlyfroze n with liquid n itroge n, mai ntain samp les af(2-8fter melt in g,add PBSP H7.4 ,Homogenizedby

20、 hand or Grinders,centrifugation20-min at the speed of 2000-3000remove supernatant.extract as soon as possibleafter Specimencollection,andaccordingto the relevantliterature,and shouldbe experimentas soon as possibleafter the extraction.If it cant,sp ecime n can be kept in -20 to p reserve, Avoid rep

21、 eated freeze-thaw cycles.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 Stand50didilurtittnB first andthe

22、second well, mix; take out 100卩 l form the first anchtheadcontd wellthirdand 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 ,then add Standard dilL50)p l to the fifth an

23、d 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 the tenth well, add Sta ndard50liutionD the nin

24、th andthe tenth well, mix , take out 50 m the nin thpahdcthe ten th wescard(add Samp le 50卩 l toeach well after Diluting ,(dens2i4ty0: pg/ml ， 160 pg/ml ，80 pg/ml ，40 pg/ml ， 20 pg/ml )2.add sample：Set blank wells separately(blank comparisonwells donatdd sampleandHRP-Conjugate reagent, other each st

25、ep operation is same). testing sample well. add Samplediluti on40 ytlo test in gsa mp lewell the n add testi ng sampi e10 (sa mplefinal diluti onis5-fold), add sample to welldso,nt touch the well wall as far as poasnsidblGe,ently mix.3.ln cubate: After closi ng p late with Closure plate membra ne ,i

26、n cubate for 30(min at 374.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 bufferto every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzym: Add HRP-Conjugate r

27、eagent l to each Wlxcept 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 p reservati on for 15 min at3710.Stopthe reaction Add Stop Solutio50 卩1 each well, Stop the react ion (th由lue color

28、change to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Important notes1.The kit takes out from the refrigeration environment should be balanced 15-30 minutes inthe room temperature, ELISA plates coated if has not use up after op

29、ened, the plate shouldbe stored in Sealed bag.2.washingbufferwill Crystallizationseparation,it can be heatedthe water helps dissolvewhen dilute . Washing does not affect the result.3.add Samplewith samplerEach step, And proofreadits accuracyfrequently,avoidstheexperimentalerror. 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 firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied