人粒細(xì)胞巨噬細(xì)胞集落刺激因子(GM-CSF)酶聯(lián)免疫分析試劑盒使用說明書解讀

1、人粒細(xì)胞巨噬細(xì)胞集落刺激因子（GM-CSF）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)定大鼠血清，血漿及相關(guān)液體樣本中粒細(xì)胞巨噬細(xì)胞集落刺激因子（ GM-CSF）的含量。實(shí)驗(yàn)原理：本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人粒細(xì)胞巨噬細(xì)胞集落刺激因子（ GM-CSF）水平。用純化的人粒細(xì)胞巨噬細(xì)胞集落刺激因子（ GM-CSF）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粒細(xì)胞巨噬細(xì)胞集落刺激因子（ GM-CSF），再與 HRP標(biāo)記的粒細(xì)胞巨噬細(xì)胞集落刺激因子（ GM-CSF）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。TM

2、B 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。顏色的深淺和樣品的粒細(xì)胞巨噬細(xì)胞集落刺激因子 （ GM-CSF）呈正相關(guān)。用酶標(biāo)儀在 450nm 波長下測(cè)定吸光度（ OD 值），通過標(biāo)準(zhǔn)曲線計(jì)算樣品中人粒細(xì)胞巨噬細(xì)胞集落刺激因子（ GM-CSF）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(gè)1個(gè)酶標(biāo)包被板 1 × 48 1 ×96 2-8 保存標(biāo)準(zhǔn)品： 45ng/L 0.5ml × 1 瓶 0.5ml × 1 瓶 2-8 保存標(biāo)準(zhǔn)品稀釋液 1.5ml ×

3、1 瓶 1.5ml ×1 瓶 2-8 保存酶標(biāo)試劑 3 ml ×1 瓶 6 ml ×1 瓶 2-8 保存樣品稀釋液 3 ml ×1 瓶 6 ml × 1 瓶 2-8 保存顯色劑 A 液 3 ml × 1 瓶 6 ml ×1 瓶 2-8 保存顯色劑 B 液 3 ml × 1 瓶 6 ml ×1 瓶 2-8 保存終止液 3ml ×1 瓶 6ml × 1 瓶 2-8 保存濃縮洗滌液 （20ml× 20 倍）× 1 瓶 （20ml×30 倍）× 1 瓶

4、2-8 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)/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（ 200

5、0-3000 轉(zhuǎn) /20 分鐘左右分）。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2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

6、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 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. 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 l ，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 50 l 棄掉，再各取 50 l 分別加到第五、第六孔中，再

7、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 l 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 30ng/L ，20ng/L ，10 ng/L ，5ng/L ， 2.5 ng/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）、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 1

8、0l （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 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，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 l ，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 ，輕輕蕩混勻， 37 避光顯色15 分鐘 .10. 終止：每孔加終止液 50 l

9、，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)：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 3 大于標(biāo)準(zhǔn)品孔第

10、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)（× n×5）。5 封板膜只限一次性使用，以避免交叉污染。6 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：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

11、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 0.990 以上。2. 批內(nèi)與批見應(yīng)分別小于 9%和 11%檢測(cè)范圍：2ng/L -40ng/L保存條件及有效期：1. 試劑盒保存：； 2-8 。2有效期： 6 個(gè)月如果次技術(shù)資料， 請(qǐng)加我扣扣 835041457 或 mobile call 一五一二一零七二二五三Rat interleukin 1 FOR RESEARCH USE ONLYDrug NamesGeneric Name：Rat interleukin1(GM-CSF) ELISA Kit.P

12、urposeThis kit allows for the determination of GM-CSF concentrations in Rat serum, blood plasma,and other biological fluids.Principle of the assayThe kit assay Rat GM-CSF level in the sample, use Purified Rat GM-CSF antibody to coatmicrotiter plate wells, make solid-phase antibody, then add GM-CSF t

13、o wells, Combined GM-CSFantibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, afterwashing Completely, Add TMBsubstrate solution,TMB substrate becomesblue color At HRPenzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and thecolor ch

14、ange is measured spectrophotometrically at a wavelength of 450 nm. Theconcentration of GM-CSF in the samples is then determined by comparing the O.D. of the samplesto the standard curve.5Materials provided with the kitMaterials provided withthe kit48determinations 96 determinations StorageUser manua

15、l 1 1Closure plate membrane 2 2Sealed bags 1 1Microelisa stripplate 1 1 2-8Standard ：45ng/L 0.5ml×1 bottle 0.5ml× 1 bottle 2-8Standard diluent 1.5ml×1 bottle 1.5ml× 1 bottle 2-8HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8Sample diluent 3ml ×1 bottle 6ml 

16、5; 1 bottle 2-8 Chromogen Solution A 3ml×1 bottle 6ml× 1 bottle 2-8Chromogen Solution B 3ml×1 bottle 6ml× 1 bottle 2-8Stop Solution 3ml × 1 bottle 6ml ×1 bottle 2-8 wash solution（ 20ml× 20 fold ） × 1bottle（ 20ml× 30 fold ） × 1bottle2-8 Specimen requi

17、rements1. serum- coagulation at room temperature 10-20 mins， centrifugation20-min at the speed ofprecipitation appeared, Centrifugal again.3.remove supernatant, If precipitation appeared, Centrifugal again. The Operation ofHydrothorax and cerebrospinal fluid Reference to it.4. cell culture supernata

18、nt-detect secretory components, collect sue a sterile container,composition of cells, Dilut cell suspension with PBS（ PH7.2-7.4 ）, Cellconcentrationreached 1 million / ml, repeated freeze-thaw cycles, damage cells and release ofintracellularcomponents, centrifugation20-min at the speed of 2000-3000s

19、upernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add PBS （ PH7.2-7.4 ）, Rapidlyfrozen with liquid nitrogen, maintain samples at 2-8 after melting,add PBS（ PH7.4）,6remove supernatant.6. extract as soon as possible after Specimen colle

20、ction,and according to the relevantliterature, and should be experiment as soon as possible after theextraction. If it cant,specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Cant detect the sample which contain NaN3, because NaN3inhibits HRP active.Assay procedure1.Dilute

21、 and add sample to Standard: set 10 Standard wells on the ELISA plates coated, addStandard 100 l to the first and the second well, then add Standard dilution 50 l to the first andthe second well, mix; take out 100l form the first and the second wellthen add it to the thirdand the forth well separate

22、ly. then add Standard dilution 50 l to thethird and the forthwell ,mix ; then takeout 50 lfrom thethird and theforthwell discard,add50 l to the fifth andthesixth well ,thenadd Standard dilution50l to thefifthand the sixthwell, mix ; take out 50 lfrom the fifth and the sixth well and add to the seven

23、th and the eighth well, then add Standarddilution 50l to the seventh and the eighth well ,mix ; take out 50 l from the seventh and theeighthwelland add to the ninthand the tenthwell,add Standard dilution50l to the ninth andthe tenth well, mix , take out 50l from the ninth and the tenth welldiscard(a

24、dd Sample 50l toeach well after Diluting ,(density: 30ng/L,20ng/L ,10 ng/L,5ng/L,2.5 ng/L)2.add sample ： Set blank wells separately (blank comparison wells don t add sample andHRP-Conjugate reagent,othereach step operationissame). testingsamplewell. add Sampledilution 40 l to testing sample well, th

25、en add testing sample 10l(sample final dilution is5-fold), add sample towells ,dont touch the wellwall as faras possible,and Gently mix.3.Incubate:After closing plate with Closure platemembrane,incubate for30 min at 37 .4.Configurateliquid:30-fold(or20-fold)wash solution diluted30-fold(or 20-fold) w

26、ith distilledwater and reserve.5.washing ： Uncover Closure platemembrane, discardLiquid,dry by swing,add washing bufferto 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 3.78.washing

27、 ： Operation with 5.9.color ：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight preservation for 15 min at 3710.Stop the reaction： Add Stop Solution50 l to each well, Stop thereaction(the blue colorchange to yellow color).11.assay ：take blankwell as zero ,Read absorb

28、ance at450nmafterAddingStop Solution andwithin 15min.important notes1. The kit takes out from the refrigerationenvironmentshould be balanced15-30 minutes inthe room temperature,ELISA platescoatedif has not use up afteropened,the plate shouldbe stored in Sealed bag.2. washing buffer will Crystallizat

29、ion separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental error. addsample within 5mins, ifthe number ofsample ismuch ,recommend to use Volley .4. if th

30、e testing material content is excessively higher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilutionfactor. （× n× 5）.5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. Th

31、e substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take themicrotiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective materialprocess.9. Do not mix reagents with those from other lots.8C