牛白細(xì)胞介素2IL-2酶聯(lián)免疫分析ELISA

1、牛白細(xì)胞介素 2（IL-2 ）酶聯(lián)免疫分析（ ELISA ）試劑盒使用說明書本試劑盒僅供研究使用。使用目的： 本試劑盒用于測(cè)定牛血清、血漿及相關(guān)液體樣本中白細(xì)胞介素2（ IL-2 ）的含量。實(shí)驗(yàn)原理本試劑盒應(yīng)用間接法測(cè)定標(biāo)本中牛白細(xì)胞介素2（IL-2 ）水平。用純化的牛白細(xì)胞介素2（IL-2 ）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已知濃度的白細(xì) 胞介素 2（IL-2 ）標(biāo)準(zhǔn)品和未知濃度的白細(xì)胞介素2（ IL-2 ）待檢樣品，溫育后，加入生物素標(biāo)記的抗 IgG 抗體，再與鏈霉親和素 -HRP 結(jié)合，形成免疫復(fù)合物，經(jīng)過徹底洗滌后加底 物 TMB 顯色。 TMB 在 HRP 酶

2、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。 顏色的深淺和樣品中的白細(xì)胞介素 2（IL-2 ）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)算樣品中牛白細(xì)胞介素2（ IL-2 ）濃度。試劑盒組成120 倍濃縮洗滌液20ml× 1 瓶8標(biāo)準(zhǔn)品 S1（ 240ng/L ）0.5ml × 1瓶2鏈霉親和素 -HRP6ml× 1 瓶標(biāo)準(zhǔn)品 S2（ 160ng/L）0.5ml × 1瓶3酶標(biāo)包被板12孔×8 條標(biāo)準(zhǔn)品 S3（ 80ng/L ）0.5ml × 1瓶4生物素標(biāo)記的抗 -IgG 抗體6ml&#

3、215; 1 瓶標(biāo)準(zhǔn)品 S4（ 40ng/L ）0.5ml × 1瓶5顯色劑 A 液6ml× 1 瓶標(biāo)準(zhǔn)品 S5（ 20ng/L）0.5ml × 1瓶6顯色劑 B 液6ml× 1/瓶9說明書1份7終止液6ml× 1 瓶10封板膜2張標(biāo)本要求1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。 2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)凍融操作步驟1. 根據(jù)待測(cè)樣品數(shù)量加上標(biāo)準(zhǔn)品的數(shù)量決定所需的板條數(shù)。 每個(gè)標(biāo)準(zhǔn)品和空白孔建議做復(fù)

4、 孔。每個(gè)樣品根據(jù)自己的數(shù)量來定，能使用復(fù)孔的盡量做復(fù)孔。、標(biāo)準(zhǔn)品孔、待測(cè)樣2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品，其余各步操作相同）品孔。然后在標(biāo)準(zhǔn)品孔中加入標(biāo)準(zhǔn)品50l，在樣本反應(yīng)孔中加入50 微升樣本，蓋上封板膜，輕輕振蕩混勻， 37溫育 45 分鐘。3. 配液：將 30 倍濃縮洗滌液用蒸餾水 30 倍稀釋后備用30 秒后棄去，如此4. 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(fù) 4 次，拍干。5. 加生物素標(biāo)記的抗 -IgG 抗體：每孔加入生物素標(biāo)記的抗 -IgG 抗體 50l 。37溫育 30 分 鐘6. 洗滌：操作同 4。7. 加鏈霉親和素 -HRP ：每

5、孔加入 50l 的鏈酶親和素 -HRP，輕輕振蕩混勻， 37溫育 30分鐘。8. 洗滌：操作同 4。9. 顯色：每孔先加入顯色劑 A 50l，再加入顯色劑 B 50l，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(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。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

6、， 將樣品的 OD 值待入方程式， 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。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 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)先將樣本稀釋一定倍數(shù)（ n 倍）后再測(cè)定，計(jì)算時(shí)請(qǐng)最后乘以稀釋倍數(shù)（×5×n）。5

7、 封板膜只限一次性使用，以避免交叉污染。 6本試劑不同批號(hào)組分不得混用。顯色劑B 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. 如與英文說明書有異，以英文說明書為準(zhǔn)。線形范圍：15ng/L -300 ng/L規(guī)格：96 人份 /盒 保存條件及有效期 1試劑盒保存： ； 2-8。 2有效期： 6 個(gè)月Bovine IL-2FOR RESEARCH USE ONLYPackage size: 96 determinations.Drug NamesGeneric Nam：e Bovine IL-2 ELISA Kit

8、.PurposeThis kit allows for the determination of IL-2 concentrations in Bovine serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay Bovine IL-2 level in the sam，pluese Purified Bovine IL-2 antibody to coat microtiter plate wells, make solid-phase antibody, Samples whi

9、ch including standards of known IL-2 concentrations and unknowns are pipetted into coated microtiter wells, after Incubating, add Biotinylateda nti-IgG,andC ombined Streptavidin-HRPb, ecome antibody - antigen- enzyme-antibodcyo mplex,a fter washingC ompletely,Add TMB substrates olution, TMB substrat

10、e becomesb lue color At HRP enzyme-catalyzedA, nd at the effecto f acid the color finally become yellow, The intensity of this coloured product is directly proportional to the concentration of AFP present in the samples. measure the optical densit (OD) at 450 nm with microtiter plate reader, calcula

11、te Bovine IL-2 concentration by standard curv.Materials provided with the kit1wash solution20ml×1bottle81 Standard(24n0g/L )0.5ml ×1 bottle2Streptavidin-HRP6ml ×1 bottle2 Standard(610ng/L )0.5ml ×1 bottle3Microelisa stripplate12well 8×strips3 Standard(8n0g/L )0.5ml ×1 b

12、ottle4Biotinylated anti-IgG6ml ×1 bottle4 Standard(4n0g/L )0.5ml ×1 bottle5Chromogen Solution A6ml ×1 bottle5 Standard(02ng/L)0.5ml ×1 bottle6Chromogen Solution B6ml ×1 bottle9User manual17Stop Solution6ml ×1 bottle10Closure plate membrane2Specimen requirements1. Can

13、9; t detect the sample which coNnataNin3 , because NaN3 inhibits HRP a.c tive2. extract as soon as possible after Specimen collection, Extracted according to therelevant literature, and should be experiment as soon as possible after the extraction. If it can not be tested immediately,s pecimenc an b

14、e kept in -20 to preserve,A void repeated freeze-thaw cycles.Assay procedure1. Determine the number of microwell strips required to test the desired number of samples,. Each sample, standard and blank should be assayed in duplicate.2. add sample： Set blank wells separaltye (blank comparison wells do

15、n' t add sample andELISA reagent, other each step operation is same).0 A dd L5 of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate, and Gently mix. Incubate for 45 min at 373. Configuratel iquid: 20 times of wash solution diluted 20 times with distilled wa

16、ter and reserve.4. washing：remove Liquid, dry by swing, add washing buffer to every well, still for 30 second then remove, repeat 4 times.5. add Biotinylated anti-IgG: Add diluted Biotinylated anti-IgG 50ul to all wells, Incubate for 30 min at 376. washing： Operation with 4.7. add streptavidin-HRP :

17、 Add streptavidin-HRP 50ul to all wells, Gently mix Incubate for 15 min at 378. washing： Operation with 4.9. color： Add Chromogen Solution A 50ul and Chromogen Solution B to each well,Incubate for 15 min at 3710. Stop the reactio：n Add Stop Solutio5n0 tlo each well, Stop the reaction(the blue color

18、change to yellow color Immediately).11. assay： take blank well as zero , measure the optical densit (OD) at 450 nm after AddingStop Solution and within 15min.CalculateTake the standardd ensity as the horizontal,t he OD value for the vertical ,draw the standard curve on graph paper, Find out the corr

19、esponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculatet he sample density, mu

20、ltipliedb y the dilution multiple, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature then use, ELISA plates coated if has not use up after opened, the plate should be stored in Seale

21、d bag.2. washing buffer will Crystallizations eparation,it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with samplerE ach step, And proofreadi ts accuracyf requently,a voids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. Please make specification curve when you assay, had better make duplicate well, if in the sample the testing material content is excessively high (The sample OD is