人IFN-γ Elisa試劑盒

1、人IFN-定量分析酶聯(lián)免疫檢測試劑盒本試劑盒僅供科研使用。用于體外定量檢測人血清、血漿或細(xì)胞培養(yǎng)上清液中的IFN-濃度。使用前請仔細(xì)閱讀說明書并檢查試劑組分是否完整,如有疑問請與北京博凌科為生物科技有限公司聯(lián)系，我們將提供力所能及的幫助。如您有其它需求，請登錄北京博凌科為生物科技有限公司網(wǎng)站或致電本公司。IFN-簡介：被稱為II 型干擾素的IFN-，是由143個殘基構(gòu)成的，有20和25kDa亞型的糖蛋白，以頭尾相連的同型糖蛋白的形式存在。IFN-由T淋巴細(xì)胞和NK細(xì)胞產(chǎn)生，用以抗病毒和干擾增殖，此外，IFN-還有與免疫調(diào)節(jié)相關(guān)的幾種功能包括調(diào)節(jié)細(xì)胞的增殖和程序性死亡，刺激或抑制不同基因的表達(dá)。

2、IFN-能通過誘導(dǎo)產(chǎn)生吲哚胺2，3-雙加氧酶來抗弓形蟲和衣原體的感染。IFN-是效應(yīng)強烈的單核吞噬細(xì)胞增強劑，IFN-是通過增強單核吞噬細(xì)胞的的Mac-1的表達(dá)來達(dá)到增強胞飲作用和吞噬作用的。IFN-通過增加巨噬細(xì)胞的氧自由基和TNF-的釋放來達(dá)到增強殺腫瘤細(xì)胞的作用。IFN-選擇性地提高包括因LPS 刺激的B 細(xì)胞的IgG2a 和因2型T 細(xì)胞呈遞抗原而引起的B 細(xì)胞的IgG3的釋放量。有報道稱IFN-能引起細(xì)胞的自分泌IFN-作用的增強。檢測原理:本試劑盒采用雙抗體夾心ELISA法檢測樣本中IFN-的濃度。IFN-捕獲抗體已預(yù)包被于酶標(biāo)板上，當(dāng)加入標(biāo)本或參考品時，其中的IFN-會與捕獲抗體

3、結(jié)合，其它游離的成分通過洗滌的過程被除去。當(dāng)加入生物素化的抗人IFN-抗體后，抗人IFN-抗體與IFN-接合，形成夾心的免疫復(fù)合物，其它游離的成分通過洗滌的過程被除去。隨后加入辣根過氧化物酶標(biāo)記的親合素。生物素與親合素特異性結(jié)合，親合素連接的酶就會與夾心的免疫復(fù)合物連接起來；其它游離的成分通過洗滌的過程被除去。最后加入顯色劑，若樣本中存在IFN-將會形成免疫復(fù)合物，辣根過氧化物酶會催化無色的顯色劑氧化成藍(lán)色物質(zhì)，在加入終止液后呈黃色。通過酶標(biāo)儀檢測，讀其450nm處的OD值，IFN-濃度與OD450值之間呈正比，通過參考品繪制標(biāo)準(zhǔn)曲線，對照未知樣本中OD值，即可算出標(biāo)本中IFN-濃度。人定量分

4、析酶聯(lián)免疫檢測試劑盒組成:組分規(guī)格預(yù)包被板12條或6條樣本分析緩沖液1瓶5ml/3ml 標(biāo)準(zhǔn)品稀釋液10ml/5ml標(biāo)準(zhǔn)品4/2(凍干生物素化抗體1瓶10ml/5mlHRP連接的酶結(jié)合物1瓶10ml/5ml濃縮洗滌液20×30ml/瓶TMB底物1瓶10ml/5ml 終止液1瓶5ml/3ml 封板膠紙3/2張說明書1份標(biāo)本收集:1.標(biāo)本的收集請按下列流程進(jìn)行操作：A.細(xì)胞上清標(biāo)本離心去除懸浮物后即可；B.血清標(biāo)本應(yīng)是自然凝固后，取上清，避免在冰箱中凝固血液；C.血漿標(biāo)本，推薦用EDTA的方法收集若待測樣本不能及時檢測；D.標(biāo)本收集后請分裝，凍存于20，避免反復(fù)凍融。2.血清標(biāo)本不應(yīng)添加

5、任何防腐劑或抗凝劑；3.標(biāo)本應(yīng)清澈透明，檢測前樣本中如有懸浮物應(yīng)通過離心去除；4.請勿使用溶血，高血脂或污染的標(biāo)本檢測，否則結(jié)果將不準(zhǔn)確。注：正常人血清或血漿樣本請用標(biāo)本緩沖液做倍比稀釋后再檢測。注意事項:1.試劑盒請保存在28。2.濃縮洗滌液因在低溫下可能有結(jié)晶，請水浴加熱使結(jié)晶完全溶解后再配制工作液。3.若標(biāo)準(zhǔn)品復(fù)溶后，請在三天內(nèi)用完。4.底物請勿接觸氧化劑和金屬。5.加樣時，請及時更換槍頭，避免交叉污染。6.嚴(yán)禁混用不同批號的試劑盒組份。7.充分混勻?qū)ΡＷC反應(yīng)結(jié)果的準(zhǔn)性很重要，在加液后請輕輕叩擊邊緣以保證混勻。8.室溫反應(yīng)，請嚴(yán)格控制在2528。9.洗滌過程是至關(guān)重要的，洗滌不充分會使精

6、確度下降并導(dǎo)致結(jié)果誤差較大。10.試驗中標(biāo)準(zhǔn)品和樣本檢測時建議作雙復(fù)孔。11.加樣過程中避免氣泡的產(chǎn)生。12.血清和血漿標(biāo)本的檢測時，檢測抗體的孵育時間應(yīng)適當(dāng)延長。檢測前準(zhǔn)備工作:1.試劑盒自冰箱中取出后應(yīng)置室溫平衡20分鐘；每次檢測后剩余試劑請及時于28保存。2.將濃縮洗滌液用雙蒸水或去離子水稀釋(1份加19份水。3. 標(biāo)準(zhǔn)品:加入標(biāo)準(zhǔn)品稀釋液0.25ml至凍干標(biāo)準(zhǔn)品瓶中使IFN-終濃度達(dá)到1000pg/ml，靜置15分鐘后輕輕混懸待徹底溶解，用標(biāo)準(zhǔn)品稀釋液倍比梯度稀釋后依次加入檢測孔中。（標(biāo)準(zhǔn)曲線取七個點，最高濃度為1000pg/ml,標(biāo)準(zhǔn)品稀釋液直接加入作為0濃度.） 洗滌方法:自動洗板

7、機或人工洗板：每孔洗滌液為300ul，注入與吸出間隔15-30秒。洗板5次。最后一次洗板完成后將板倒扣著在厚吸水紙上用力拍干。實驗過程需自備的材料：1.不同規(guī)格的加樣槍及相應(yīng)的槍頭；2.酶標(biāo)儀；3自動洗板機；4去離子水或雙蒸水；操作步驟:1.通過計算并確定一次性實驗所需的板條數(shù)，取出所需板條放置在框架內(nèi)，暫時用不到板條請放回鋁箔袋密封，保存于4。2.建議設(shè)置本底較正孔，即空白孔,設(shè)置方法為該孔只加TMB 顯色液和中止液。每次實驗均需做標(biāo)準(zhǔn)品對照并畫出標(biāo)準(zhǔn)曲線。3.分別將標(biāo)本或不同濃度標(biāo)準(zhǔn)品(100ul/孔加入相應(yīng)孔中，用封板膠紙封住反應(yīng)孔，室溫孵育120分鐘。對于血清或血漿標(biāo)本，請加入50ul

8、樣本分析緩沖液后加50ul標(biāo)本,如稀釋量大，請將樣本與樣本分析緩沖液等量加入，不足部分用標(biāo)準(zhǔn)品稀釋液補充至100ul。4.洗板5次，且最后一次置厚吸水紙上拍干。5.加入生物素化抗體工作液(100ul/孔。用封板膠紙封住反應(yīng)孔，室溫孵育60分鐘。6.洗板5次，且最后一次置厚吸水紙上拍干。7.加入酶結(jié)合物工作液(100ul/孔。用封板膠紙封住反應(yīng)孔，避光室溫孵育20分鐘。8.洗板5次，且最后一次置厚吸水紙上拍干。9.加入顯色劑TMB100ul/孔，避光室溫孵育20分鐘。10.加入終止液50ul/孔,混勻后即刻測量OD450值。結(jié)果判斷:1.復(fù)孔的值在20%的差異范圍內(nèi)結(jié)果才有效，復(fù)孔的值平均后可作

9、為測量值。2.每個標(biāo)準(zhǔn)品或標(biāo)本的OD值應(yīng)減去本底校正孔的OD值。3.手工繪制標(biāo)準(zhǔn)曲線。以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，OD值作縱坐標(biāo)，以平滑線連接各標(biāo)準(zhǔn)品的坐標(biāo)點。通過標(biāo)本的OD值可在標(biāo)準(zhǔn)曲線上查出其濃度。4.若標(biāo)本OD 值高于標(biāo)準(zhǔn)曲線上限，應(yīng)適當(dāng)稀釋后重測，計算濃度時應(yīng)乘以稀釋倍數(shù)。典型數(shù)值和參考曲線濃度pg/ml典型OD 值1典型OD 值2OD 平均值人IFN-參考標(biāo)準(zhǔn)曲線 靈敏度，特異性和重復(fù)性:1.靈敏度：多次重復(fù)結(jié)果表明，最小檢出量為1.8pg/ml。2.特異性：與小鼠IFN-, 大鼠IFN-, 馬IFN-, 狗IFN-, 貓IFN-等沒有交叉反應(yīng)。3.重復(fù)性：板內(nèi)，板間變異系數(shù)均<10

10、%.參考文獻(xiàn):1、Naylor,S.L., Sakaguchi, A.Y., Shows, T.B., et al.(1983J.Exp.Med. 157:1020-10272、Wheelock,E. F.(1965Science 146:3103、Billiav,A.(1996Adv. Immunol . 62:61ELISA Kit for the Quantitative Analysis of Human IFN-The human IFN-ELISA (enzyme-linkedimmunosorbent assay kit is used for detection of huma

11、n IFN-in cell culture supernatants,human serum and plasma. THE ELISA KIT IS FOR RESEARCH USE ONLY . Please read this instruction manual carefully and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call us for other aim.

12、IntroductionIFN-,also called Type II interferon, is a 143amino acid residue glycoprotein with MW of 20or 25kDa and exists as a head-to-tail homodimeric protein. Produced by T lymphocytes and natural killer (NKcells, IFN-induces an anti-viral or anti-proliferative state. In addition, IFN-has several

13、properties related to immunoregulation including cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes. IFN-can induces the expression of indoleamine 2,3-dioxygenase, which is believed against Toxoplasma and Chlamydia . IFN- is a potent activator of mononu

14、clearphagocytes, IFN-stimulates the ed the expression of Mac-1, augments endocytosis and phagocytosis by monocytes , and activates macrophages to kill tumor cells by releasing reactive oxygen intermediates and TNF-.IFN- selectively enhances both IgG2a secretion by LPS-stimulated B cells and IgG3secr

15、etion in T cell independent type 2antigen-mediated B cell activation. It has also been reported to induce its own expression.Principles of the TestThe kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human IFN-.An anti-human IFN-monoclonal antibody has been absorbed onto t

16、he wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human IFN-in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human IFN-biotin-conjugated antibody were added and binds t

17、o human IFN-captured by the first antibody, which formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a wash step. After this, subtrate solution would be added and catalyzed by the HRP, and a coloured prod

18、uct is formed. The intensity of the colored product is used to calculate in proportion to the amount of human IFN-in the original specimen . Materials provided with the kits:reagent96/48TestKit Assay Buffer5ml/3ml IFN-Antibody-Coated Wells12strips/6strips Standard Diluent10ml IFN-Standard4/2vial(sDe

19、tetion Antibody10ml/5mlStreptavidin-HRP10ml/5mlWash Buffer Concentrate 20×30ml TMB10ml/5ml Stop Solution5ml/3ml Plate Covers3/2Complete Instruction Manual 1Specimen Collection1.Collecting specimen as following:A.The particulate of the cell culture supernatants should be removed before use.B.Ser

20、um was obtained from clot at room temperature.C.Please collect plasma with EDTA.D.Assay immediately or store samples at 20. Avoid free-thaw cycles.2Antiseptic and anticoagulant should not appear in Serum samples.3.Any particulate should be removed from samples before use.4. Do not use grossly hemoly

21、zed or lipemic samples.Note:Srongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.Precautions for use:1.Please storage the Kit at 28。2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.3. Ple

22、ase discard the dissolved standard after 3days for use.4.Avoid contact of substrate solution with oxidizing agents and metal.5.Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.6. Do not mix or substitute reagents with those from other lot

23、s or other sources.7. To ensure the adequate mixure of added reagents, please tap gently the plate after the wells were filled with liquid.8. Incubation temperature should be 2528.9. Wash step was crucial for whole assay process.10. Duplicate wells of the same sample were recommended in assay proces

24、s.11. Avoid the foam while pour the liquid into wells.12.For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90minutes.Reagent Preparation1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refriger

25、atory again as soon as possible.2.Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.3. Add 0. 25ml standard diluent to bottle wait15minutes for complete dissolution. And in turn add the half concentration diluent by standard diluent .Wash step:Automated microplate

26、washer or operating by pipette:Each well should be pour into 300ul wash buffer and soak 15or 30seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer byaspirating.Invert the plate and blot it against clean paper towels.Materials Required But No

27、t Provided1. pipettes and pipette tips2. Microwell strip reader capable of reading at 450nm (540nm as3. automated microplate washer4.Glass-distilled or deionized waterAssay procedure1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.2.Blank well we

28、re recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient density of standard for standard curve.3.Add 100ul of standard or sample.Cover with the Plate Covers provided.Incubate for 2hours at room temperature If assay the serum sample,you sho

29、uld add 50l assay buffer with 50l sample into the wells,if the protein concentration is higher than the range of the Kit, add the same quantitys assay buffer with the sample, the deficiency should be complemented with sample diluent to 100l per well. 4.Five times wash process were repeated. 5.Add 10

30、0ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature. 6.Five times wash process were repeated. 7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature. 8.Five times wash process w

31、ere repeated. 9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature. 10.Add 50ul of stop solution to each well, determine the optical density of each well within 10 minutes. Calculation of Results 1.Duplicates should be within 20 per cent of the mean. Average absorbance values f

32、or each set of duplicate samples were used as detection results. 2.The blank absorbance values of subtract should be deducted. 3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD valure (on the Y axis vs. concentration (on the X axis. The sample concentration was obtained based on its OD value founding in the standard concentration curve. 4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again. Typical Data and Standard Curve co