小鼠胰島素Insulin說明書

1、小鼠胰島素(Insulin)說明書小鼠胰島素(Insulin)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)定小鼠血清，血漿及相關(guān)液體樣本中 胰島素(Insulin)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)本中小鼠胰島素(Insulin)水平。用純化的小鼠胰島素(Insulin)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胰 島素(Insulin),再與HRP標(biāo)記的胰島素(Insulin)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

2、樣品中的胰島素(Insulin)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)算樣品中小鼠 胰島素(Insulin)含量。試劑盒組成：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)/ 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、檸檬酸鈉或肝素作為抗凝劑，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:用無菌管收集，離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細(xì)收集上消 保

3、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稀釋細(xì)月fi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,。用液氮迅速 冷凍保存?zhèn)溆?。?biāo)本融化后仍然保持2-8 C的溫度。加入一定量的PBS,用手工或勻漿器 將標(biāo)本勻漿充分。離心20分鐘左右（2000-30

4、00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,g|NaN3抑制辣根過氧化物酶的（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 0然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液 50 0混勻;然后從第一孔、 第二孔中各取100N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50a混勻；然后在第三孔和第四孔中先各取 50川棄掉，冉各取50M

5、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 M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必混勻后從第七、第八孔中分別取 50M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 a混勻后從第九第十孔中各取50 M棄掉。（稀釋 后各孔加樣量都為 50山濃度分別為12 mIU/L, mIU/L , mIU/L, mIU/L, mIU/L） 。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，其余各步操作相同卜待 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必然后再加待測(cè)樣品 10小

6、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7用蒸儲(chǔ)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間空白孔除外。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A50 a再加入顯色劑B50 0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必終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。11 .測(cè)定:以空白

7、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)：12 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。13 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。14 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。15 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)

8、最后乘以總稀釋倍數(shù)（x nx 5）。16 封板膜只限一次性使用，以避免交叉污染。17 底物請(qǐng)避光保存。18 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).19 所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。20 本試劑不同批號(hào)組分不得混用。21 .如與英文說明書有異，以英文說明書為準(zhǔn)。計(jì)算：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：1 .樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值為

9、以上。2 .批內(nèi)與批見應(yīng)分別小于9%和11%檢測(cè)范圍：mIU/L -15 mIU/L保存條件及有效期：1 .試劑盒保存:；2-8 Co2 .有效期:6個(gè)月Mouse InsulinDrug NamesGeneric Name:Mouse Insulin ELISA Kit.PurposeThis kit allows for the determination of Insulin concentrations in Mouse serum, tissue and other biological fluids.Principle of the assayT he kit assay Mous

10、e Insulin level in the sample, use Purified Mouse Insulin antibody to coat microtiter plate wells, make solid-phase antibody, then add Insulin to wells, Combined Insulin antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate

11、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 wavelength of 450 nm. The concentration of Mouse Insulin in the samples is then determined by comparing

12、the . of the samples to the standard curve.Materials provided with the kitSpecimen requirementscoagulation at room temperature 10-20 mins,centrifugation 20-min at the speed of2000-3000 remove supernatant, If precipitation appeared, Centrifugal again.suited EDTA, citrate or heparinized plasma as an a

13、nticoagulant,mix 10-20mins ,centrifugation 20-min at the speed of 2000-3000 remove supernatant, If precipitation appeared, Centrifugal again.sue a sterile container, centrifugation 20-min at the speed of 20003000 remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydr

14、othorax and cerebrospinal fluid Reference to it.culture supernatant-detect secretory components, collect sue a sterile container,centrifugation 20-min at the speed of 2000-3000 remove supernatant,detect the composition of cells, Dilut cell suspension with PBS Cell concentration reached 1 million / m

15、l, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20min at the speed of 2000-3000 remove supernatant, If precipitation appeared, Centrifugal again.samples- After cutting samples, check the weight,add PBS Rapidlyfrozen with liquid nitrogen, maintain

16、samples at 2-8 C aftermelting,add PBS, Homogenized by hand or Grinders, centrifugation 20min at the speed of 2000-3000remove supernatant.as soon as possible after Specimen collection,and according to the relevantliterature, and should be experiment as soon as possible after the extraction. If it can

17、' t, specimen can be kept it to preserve, Avoidrepeated freeze-thaw cycles.'t detect the sample which contain NaN3, because NaN3 inhibits HRP active. Assay procedureand add sample to Standard: set 10 Standard wells on the ELISAplates coated, add Standard 100仙 l to the first and the second we

18、l l, thenadd Standard dilution 50thepfirtstband the second well, mix; take out100 n l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50仙 l to the third and theforth well ,mix ; then take out 50仙 l from the thiodtarwelh discard,ad

19、d 50 n l to the fifth and the sixth well ,then add Standard dilution 50the fifth and the sixth well, mix ; take out 50l from the fifth and the sixthwell and add to the seventh and the eighth well, then add Standard dilution 50 仙 l to the seventh and the eighth well ,mix ; take out 50 the seventh and

20、 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 out50 n l fro m the ninth and the tenth well discard(add Sample 50仙 l to ea(well after Diluting ,(density: 12 mIU/L, mIU/L , mIU/L,mIU/L, mIU/L)sample:Set blank wells separa

21、tely (blank comparison wells don' t asample and HRP-Conjugate reagent, other each step operation is same).testing sample well. add Sample dilution 40仙 l to testing sample well, thenadd testing sample 10l (sample finatliodniluis 5-fold), add sample towells , don ' t touch the well wall as far

22、 as possible, and Gently mix.:After closing plate with Closure plate membrane ,incubate for 30 min at 37 C.liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.:Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to ever

23、y well, still for 30s then drain, repeat 5 times, dry by pat.enzyme:Add HRP- Conjugate reagent 50仙 l to each well, except blankwell.:Operation with 3.:Operation with 5.:Add Chromogen Solution A 50ul and Chromogen Solution B to eachwell, evade the light preservation for 15 min at 37Cthe reaction:Add

24、Stop Solution50仙 l to each well, Stop the reaction(theblue color change to yellow color).:take blank well as zero , Read absorbance at 450nm after Adding StopSolution and within 15min.Important noteskit takes out from the refrigeration environment should be balanced15-30 minutes inthe room temperatu

25、re, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.buffer will Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.Sample with sampler Each step, And proofread its accuracy frequently, avo

26、ids theexperimental error. add sample within 5 min, if the number of sampleis much , recommend to use Volley .the testing material content is excessively higher (The sample OD isbigger than the firststandard well ),please dilute Sample (n-fold), Please diluente andmultiplied by the dilution factor.(x n x 5).plate membrane only limits the disposable use, to avoid crosscontamination.substrate evade the light preservation.accord