人(Human)白细胞介素36Ra(IL-36Ra)ELISA说明书

PRODUCT INFORMATION&MANUAL Human IL-6Valukine TM ELISAVAL102For the quantitative determination of natural and recombinant human Interleukin6(IL-6)concentrationsFor research use only.Not for diagnostic or therapeutic procedures.Bio-Techne China Co.LtdP:+86(21)52380373P:8009881270F:+86(21)52381001**********************Please refer to the kit label for expiry date.Novus kits are guaranteed for3months from date of receiptVersion202209.5TABLE OF CONTENTSI.BACKGROUND (2)II.OVERVIEW (3)III.ADVANTAGES (4)IV.EXPERIMENT (7)V.KIT COMPONENTS AND STORAGE (8)VI.PREPARATION (10)VII.ASSAY PROCEDURE (12)VIII.REFERENCES (13)I.BACKGROUNDInterleukin6(IL-6)is a pleiotropicα-helical22-28kDa phosphorylated and variably glycosylated cytokine that plays important roles in the acute phase reaction, inflammation,hematopoiesis,bone metabolism,and cancer progression(1-5).Mature human IL-6is183amino acids(aa)in length and shares41%aa sequence identity with mouse and rat IL-6(6).Alternate splicing generates several isoforms with internal deletions,some of which exhibit antagonistic properties(7-10).Cells known to express IL-6include CD8+T cells,fibroblasts,synoviocytes,adipocytes,osteoblasts, megakaryocytes,endothelial cells(under the influence of endothelins),sympathetic neurons,cerebral cortex neurons,adrenal medulla chromaffin cells,retinal pigment cells,mast cells,keratinocytes,Langerhans cells,fetal and adult astrocytes,neutrophils, monocytes,eosinophils,colonic epithelial cells,B1B cells,and pancreatic islet beta cells(2,7,10-33).IL-6production is generally correlated with cell activation and is normally kept in control by glucocorticoids,catecholamines,and secondary sex steroids (2).Normal human circulating IL-6is in the1pg/mL range,with slight elevations during the menstrual cycle,modest elevations in certain cancers,and large elevations after surgery(34-38).IL-6induces signaling through a cell surface heterodimeric receptor complex composed of a ligand binding subunit(IL-6R)and a signal transducing subunit(gp130).IL-6binds to IL-6R,triggering IL-6R association with gp130and gp130dimerization(39).Gp130 is also a component of the receptors for CLC,CNTF,CT-1,IL-11,IL-27,LIF,and OSM (40).Soluble forms of IL-6R are generated by both alternative splicing and proteolytic cleavage(3).In a mechanism known as trans-signaling,complexes of soluble IL-6and IL-6R elicit responses from gp130-expressing cells that lack cell surface IL-6R(1,3). Trans-signaling enables a wider range of cell types to respond to IL-6,as the expression of gp130is ubiquitous,while that of IL-6R is predominantly restricted to hepatocytes,monocytes,and resting lymphocytes(1-3).Soluble splice forms of gp130 block trans-signaling from IL-6/IL-6R but not from other cytokines that use gp130as a co-receptor(3,41).IL-6,along with TNF-αand IL-1,drives the acute inflammatory response,is almost solely responsible for fever and the acute phase response in the liver,and is important in the transition from acute inflammation to either acquired immunity,or chronic inflammatory disease(1-4).It contributes to chronic inflammation in conditions such as obesity,insulin resistance,inflammatory bowel disease,inflammatory arthritis and sepsis when dysregulated,often involving IL-6trans-signaling(1,2).It also plays an important role in the differentiation of naive T cells to Th17inflammatory cells in the presence of TGF-β.IL-6modulates bone resorption and is a major effector of inflammatory joint destruction in rheumatoid arthritis through its promotion of Th17T cell activity(1).It contributes to atherosclerotic plaque development and destabilization(2). However,IL-6can also have anti-inflammatory effects,such as in skeletal muscle where it is secreted in response to exercise(2).It promotes hematopoiesis by being a growth factor for hematopoietic stem cells,induces B cell maturation to plasma cells and perpetuates multiple myeloma(1,42).IL-6also promotes,but probably does not initiate,other types of inflammation-associated carcinogenesis,such as colitis-associated cancer(1).II.OVERVIEWA.PRINCIPLE OF THE ASSAYThis assay employs the quantitative sandwich enzyme immunoassay technique.A monoclonal antibody specific for IL-6has been pre-coated onto a microplate.Standards and samples are pipetted into the wells and any IL-6present is bound by the immobilized antibody.After washing away any unbound substances,an enzyme-linked polyclonal antibody specific for IL-6is added to the wells.Following a wash to remove any unbound antibody-enzyme reagent,a substrate solution is added to the wells and color develops in proportion to the amount of IL-6bound in the initial step.The color development is stopped and the intensity of the color is measured.B.LIMITATIONS OF THE PROCEDURE♦FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.♦This kit is suitable for cell culture supernate,serum and plasma.♦The kit should not be used beyond the expiration date on the kit label.♦Do not mix or substitute reagents with those from other lots or sources.♦If samples generate values higher than the highest standard,dilute the samples with Diluent and repeat the assay.♦Any variation in operator,pipetting technique,washing technique,incubation time or temperature,and kit age can cause variation in binding.III.ADVANTAGESA.PRECISIONIntra-assay Precision(Precision within an assay)Three samples were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision(Precision between assays)Three samples were tested in twenty separate assays to assess inter-assay precision.Intra-assay Precision Inter-assay PrecisionSample123123Mean(pg/mL)20.677.817523.983.3177Standard Deviation 1.20 3.517.33 4.2013.325.5CV% 5.8 4.5 4.217.615.914.4B.RECOVERYThe recovery of human IL-6spiked to levels throughout the range of the assay in various matrices was evaluated.Sample Type Average%Recovery RangeCell culture media(n=4)9581-104%Serum(n=3)9380-99%Plasma(n=4)9681-109%C.SENSITIVITYThe minimum detectable dose(MDD)of IL-6is typically less than1.56pg/mL.The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.D.CALIBRATIONThis immunoassay is calibrated against highly purified E.coli-expressed recombinant human IL-6produced at R&D Systems.The NIBSC/WHO1st International Standard for IL-6(89/548),which was intended as a potency standard,was evaluated in this kit.The NIBSC/WHO standard is a CHO cell-derived recombinant human IL-6.The dose response curve of the International Standard(89/548)parallels the Valukine standard curve.To convert sample values obtained with the Valukine Human IL-6kit to approximate NIBSC89/548units,use the equation below.NIBSC(89/548)approximate value(IU/mL)=0.109×Valukine Human IL-6value (pg/mL)E.LINEARITYTo assess the linearity of the assay,samples were spiked with high concentrations of human IL-6in various matrices and diluted with Diluent1×to produce samples with values within the dynamic range of the assay.Dilution Cell culture media(n=4)Serum(n=3)Plasma(n=4)1:2Average%of Expected11210299 Range(%)105-117101-10289-106 1:4Average%of Expected111106101 Range(%)104-119102-11195-107 1:8Average%of Expected9710899 Range(%)91-105103-11693-104 1:16Average%of Expected8910998 Range(%)81-98102-11790-107 F.SAMPLE VALUESCell Culture Supernates-Human peripheral blood mononuclear cells(1×106cells/mL) were cultured in RPMI supplemented with10%fetal calf serum,50μM β-mercaptoethanol,2mM L-glutamine,100U/mL penicillin,and100μg/mL streptomycin sulfate and stimulated for3days with10μg/mL PHA.An aliquot of the cell culture supernate was removed,assayed for levels of natural IL-6,and measured6640 pg/mL.Serum-Three human serum samples were evaluated for the presence of human IL-6 in this assay.All samples measured ranged from20.5to62.5pg/mL with an average of 48.0pg/mL.Plasma-Four human plasma samples were evaluated for the presence of human IL-6 in this assay.All samples measured ranged from73.5to105pg/mL with an average of 88.6pg/mL.G.SPECIFICITYThis assay recognizes both natural and recombinant human IL-6.The following factors were prepared at50ng/mL and assayed for cross-reactivity.Preparations of the following factors at50ng/mL in a mid-range rhIL-6control were assayed for interference.No significant cross-reactivity or interference was observed.Recombinant human Recombinant mousesgp130IL-6IL-6sRIL-6sR/sgp130IV.EXPERIMENTEXAMPLE STANDARDThe standard curve is provided for demonstration only.A standard curve should be generated for each set of samples assayed.V.KIT COMPONENTS AND STORAGEA.MATERIALS PROVIDEDParts Description SizeHuman IL-6 Microplate 96well polystyrene microplate(12strips of8wells)coated with a mouse monoclonal antibodyagainst human IL-61plateHuman IL-6 Conjugate Solution of polyclonal antibody againsthuman IL-6conjugated to horseradishperoxidase1vialHuman IL-6 Standard recombinant human IL-6in a buffered proteinbase;lyophilized1vialCalibrator Diluent(5×)a5×concentrated buffered protein base1vialWash BufferConcentrate(25×)a25×concentrated solution of buffered surfactant1vial TMB Substrate TMB ELISA Substrate Solution2vials Stop Solution2N sulfuric acid1vial Plate Sealers adhesive strip3stripsB.STORAGEUnopened Kit Store at2-8°C.Do not use past kit expiration date.Opened/ Reconstituted Reagents Diluted Wash BufferMay be stored for up to1month at2-8°C.*Stop SolutionDiluent1×ConjugateTMB SubstrateStandardAliquot and store for up to1month at-20°C in a manual defrost freezer.*Avoid repeated freeze-thaw cycles. Microplate WellsReturn unused wells to the foil pouchcontaining the desiccant pack,resealalong entire edge of zip-seal.May bestored for up to1month at2-8°C.**Provided this is within the expiration date of the kit.C.OTHER SUPPLIES REQUIRED♦Microplate reader capable of measuring absorbance at450nm,with the correction wavelength set at540nm or570nm.♦Pipettes and pipette tips.♦Deionized or distilled water.♦Squirt bottle,manifold dispenser,or automated microplate washer.♦500mL graduated cylinder.D.PRECAUTIONThe Stop Solution provided with this kit is an acid solution.Wear eye,hand,face,and clothing protection when using this materialVI.PREPARATIONA.SAMPLE COLLECTION AND STORAGECell Culture Supernates-Remove particulates by centrifugation and assay immediately or aliquot and store samples at≤-20°C.Avoid repeated freeze-thaw cycles. Samples may require dilution with Calibrator Diluent1×.Serum-Use a serum separator tube(SST)and allow samples to clot for30minutes at room temperature before centrifugation for15minutes at1000x g.Remove serum and assay immediately or aliquot and store samples at≤-20°C.Avoid repeated freeze-thaw cycles.Plasma-Collect plasma using EDTA,heparin,or citrate as an anticoagulant. Centrifuge for15minutes at1000x g within30minutes of collection.Assay immediately or aliquot and store samples at≤-20°C.Avoid repeated freeze-thaw cycles.B.SAMPLE PREPARATIONSerum samples require a5-fold dilution.A suggested5-fold dilution is40μL of sample +160μL of Diluent(1×).Plasma samples require a2-fold dilution.A suggested2-fold dilution is100μL of sample+100μL of Diluent(1×).C.REAGENT PREPARATIONNote:Bring all reagents to room temperature before use.Wash Buffer-If crystals have formed in the concentrate,warm to room temperature and mix gently until the crystals have completely dissolved.Dilute20mL of Wash Buffer Concentrate(25×)into deionized or distilled water to prepare500mL of Wash Buffer. Diluent1×-Add20mL of Calibrator Diluent Concentrate5×into80mL of deionized or distilled water to prepare100mL of Diluent1×.IL-6Standard-Refer to the vial label for reconstitution volume*.This reconstitution produces a stock solution of300pg/mL.Allow the standard to sit for a minimum of15 minutes with gentle agitation prior to making dilutions.*if you have any question,please seek help from our Technical Support.Pipette667μL of Diluent1×into the100pg/mL tube.Pipette500μL of Diluent 1×into each remaining e the stock solution to produce a dilution series (below).Mix each tube thoroughly before the next transfer.The undiluted standard serves as the high standard(300pg/mL).The Diluent1×serves as the zero standard(0 pg/mL).D.TECHNICAL HINTS●When mixing or reconstituting protein solutions,always avoid foaming.●To avoid cross-contamination,change pipette tips between additions of eachstandard level,between sample additions,and between reagent additions.Also, use separate reservoirs for each reagent.●It is recommended that the samples be pipetted within15minutes.●To ensure accurate results,proper adhesion of plate sealers during incubationsteps is necessary.●TMB Substrate should remain colorless until added to the plate.Keep SubstrateSolution protected from light.Substrate Solution should change from colorless to gradations of blue.●Stop Solution should be added to the plate in the same order as the SubstrateSolution.The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution.Wells that are green in color indicate that the Stop Solutionhas not mixed thoroughly with the Substrate Solution.VII.ASSAY PROCEDURENote:Bring all reagents and samples to room temperature before use.It is recommended that all samples and standards be assayed in duplicate.1.Prepare all reagents and working standards as directed in the previous sections.2.Remove excess microplate strips from the plate frame,return them to the foil pouchcontaining the desiccant pack,and reseal.3.Add100μL of Standard,sample,or control per well.Cover with the adhesive stripprovided.Incubate for2hours at room temperature.A plate layout is provided for a record of standards and samples assayed.4.Aspirate each well and wash,repeating the process three times for a total of fourwashes.Wash by filling each well with Wash Buffer(400μL)using a squirt bottle, manifold dispenser,or plete removal of liquid at each step is essential to good performance.After the last wash,remove any remaining Wash Buffer by aspirating or decanting.Invert the plate and blot it against clean paper towels.5.Add200μL of human IL-6Conjugate to each well.Cover with a new adhesive strip.Incubate for2hours at room temperature.6.Repeat the aspiration/wash as in step4.7.Add200μL of TMB Substrate to each well.Incubate for20minutes at roomtemperature.Protect from light.8.Add50μL of Stop Solution to each well.The color in the wells should change fromblue to yellow.If the color in the wells is green or if the color change does not appear uniform,gently tap the plate to ensure thorough mixing.9.Determine the optical density of each well within10minutes,using a microplatereader set to450nm.If wavelength correction is available,set to540nm or570nm.If wavelength correction is not available,subtract readings at540nm or570nm from the readings at450nm.This subtraction will correct for optical imperfections in the plate.Readings made directly at450nm without correction may be higher and less accurate.10.CALCULATION OF RESULTS：Average the duplicate readings for each standard,control,and sample and subtract the average zero standard optical density.Createa standard curve by reducing the data using computer software capable ofgenerating a four parameter logistic(4-PL)curve-fit.As an alternative,construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.The data may be linearized by plotting the log of the IL-6 concentrations versus the log of the O.D.and the best fit line can be determined by regression analysis.This procedure will produce an adequate but less precise fit of the data.If samples have been diluted,the concentration read from the standard curve must be multiplied by the dilution factor.VIII.REFERENCES1.Naugler,W.E.and M.Karin(2008)Trends Mol.Med.14:109.2.Schuett,H.et al.(2009)Thromb.Haemost.102:215.3.Jones,S.A.(2005)J.Immunol.175:3468.4.Hodge,D.R.et al.(2005)Eur.J.Cancer41:2502.5.Rose-John,S.et al.(2006)J.Leukoc.Biol.80:227.6.Van Snick,J.et al.(1988)Eur.J.Immunol.18:193.7.Kestler,D.P.et al.(1995)Blood86:4559.8.Kestler,D.P.et al.(1999)Am.J.Hematol.61:169.9.Bihl,M.P.et al.(2002)Am.J.Respir.Cell Mol.Biol.27:48.10.Alberti,L.et al.(2005)Cancer Res.65:2.11.May,L.T.et al.(1986)A83:8957.12.Sad,S.et al.(1995)Immunity2:271.13.Cichy,J.et al.(1996)mun.227:318.14.Miyazawa,K.et al.(1998)Am.J.Pathol.152:793.15.Fried,S.K.et al.(1998)Endocrinology83:847.16.Ishimi,Y.et al.(1990)J.Immunol.145:3297.17.Jiang,S.et al.(1994)Blood84:4151.18.Xin,X.et al.(1995)Endocrinology136:132.19.Marz,P.et al.(1998)A95:3251.20.Ringheim,G.E.et al.(1995)J.Neuroimmunol.63:113.21.Gadient,R.A.et al.(1995)Neurosci.Lett.194:17.22.Kuppner,M.C.et al.(1995)Immunology84:265.23.Gagari,E.et al.(1997)Blood89:2654.24.Cumberbatch,M.et al.(1996)Immunology87:513.25.Fujisawa,H.et al.(1997)J.Interferon Cytokine Res.17:347.26.Lee,S.C.et al.(1993)J.Immunol.150:2659.fortune,L.et al.(1996)J.Neuropathol.Exp.Neurol.55:515.28.Ericson,S.G.et al.(1998)Blood91:2099.29.Melani,C.et al.(1993)Blood81:2744.cy,P.et al.(1998)Blood91:2508.31.Jung,H.C.et al.(1995)J.Clin.Invest.95:55.32.Spencer,N.F.L.and R.A.Daynes(1997)Int.Immunol.9:745.33.Campbell,I.L.et al.(1989)J.Immunol.143:1188.34.D’Auria,L.et al.(1997)Eur.Cytokine Netw.8:383.35.Yamamura,M.et al.(1998)Br.J.Haematol.100:129.36.Angstwurm,M.W.A.et al.(1997)Cytokine9:370.37.Mouawad,R.et al.(1996)Clin.Cancer Res.2:1405.38.Sakamoto,K.et al.(1994)Cytokine6:181.39.Murakami,M.et al.(1993)Science260:1808.40.Muller-Newen,G.(2003)Sci.STKE2003:PE40.41.Mitsuyama,K.et al.(2006)Clin.Exp.Immunol.143:125.42.Cerutti,A.et al.(1998)J.Immunol.160:2145.产品信息及操作手册人IL-6Valukine TM ELISA试剂盒目录号：VAL102适用于定量检测天然和重组人白介素6（IL-6）的浓度科研专用，不可用于临床诊断Bio-Techne China Co.LtdP:+86(21)52380373P:8009881270F:+86(21)52381001**********************有效期详见试剂盒包装标签Novus试剂盒确保在你收货日期3个月内有效目录I.背景 (18)II.概述 (19)III.优势 (20)IV.实验 (23)V.试剂盒组成及储存 (24)VI.实验前准备 (26)VII.操作步骤 (28)VIII.参考文献 (29)白细胞介素-6（IL-6）是一个具有α螺旋结构、22-28kDa的磷酸化和不同程度糖基化的多功能细胞因子，它在疾病急性期反应、炎症、造血、骨代谢以及癌症恶化等方面起重要作用（1-5）。

2022年修订第一版（本试剂盒仅供体外研究使用，不用于临床诊断!）产品货号：E-EL-H0149c产品规格：96T/48T/24T/96T*5Elabscience 人白介素1β(IL-1β)酶联免疫吸附测定试剂盒使用说明书Human IL-1β(Interleukin 1 Beta) ELISA Kit使用前请仔细阅读说明书。

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Copyright ©2021-2022 Elabscience Biotechnology Co.,Ltd. All Rights Reserved目录用途 (3)基本性能 (3)检测原理 (3)试剂盒组成及保存 (4)试验所需自备物品 (5)样品收集方法 (5)注意事项 (6)■ 试剂盒注意事项 (6)■ 样品注意事项 (6)样本稀释方案 (6)检测前准备工作 (7)操作步骤 (8)结果判断 (10)技术资源 (10)典型数据 (10)性能 (11)■ 精密度 (11)■ 回收率 (11)■ 线性 (11)声明 (12)Intended use (13)Character (13)Test principle (13)Kit components & Storage (14)Other supplies required (15)Sample collection (15)Note (16)■ Note for kit (16)■ Note for sample (16)Dilution Method (17)Reagent preparation (17)Assay procedure (18)Calculation of results (20)Technical resources (20)Typical data (20)Performance (21)■ Precision (21)■ Recovery (21)■ Linearity (21)Declaration (22)用途该试剂盒用于体外定量检测人 血清、血浆或其他相关生物液体中IL-1β浓度。

仅供科研使用，不得用于临床检验。

人白细胞介素6（IL-6）定量检测试剂盒（ELISA）说明书【产品名称】通用名称：人白细胞介素6（IL-6）定量检测试剂盒（ELISA）英文名称：Human Interleukin-6（IL-6）ELISA KIT【包装规格】48人份/盒，96人份/盒【预期用途】仅供科研使用，定量检测血清、血浆、细胞培养上清液中人白细胞介素6（IL-6）的浓度。

【检验原理】本试剂盒采用双抗体夹心酶联免疫吸附试验（ELISA）。

在预包被抗人白细胞介素6（IL-6）抗体（固相抗体）的微孔酶标板中，加入人白细胞介素6（IL-6）校准品和待测样本，再加入另一株HRP标记的抗人白细胞介素6（IL-6）抗体（酶标抗体），经过温育与充分洗涤，去除未结合的组分，在微孔板固相表面形成固相抗体-抗原-酶标抗体的夹心复合物。

加底物A 和B，底物在HRP催化下，产生蓝色产物，在终止液（2M 硫酸）作用下，最终转化为黄色，在酶标仪上测定吸光度（OD值），吸光度（OD值）与待测样品中人白细胞介素6（IL-6）的浓度正相关。

拟合校准品曲线，可以计算出样本中人白细胞介素6（IL-6）的浓度。

【主要组成成分】主要成分校准品浓度依次为：320、160、80、40、20、0 pg/ml。

校准品已经通过测试，结果表明HBs抗原阴性，HIV1、HIV2和HCV抗体阴性，由于不存在一种试验方法能够完全保证没有这些物质，本品必须按照具有潜在的感染性进行处理，处理过程应当遵循通用的安全措施。

需要但未提供的材料及耗材1、酶标仪2、精密移液器及一次性吸头3、蒸馏水4、洗瓶或者自动洗板机5、37℃水浴锅或恒温箱6、500ml量筒7、无粉一次性乳胶手套8、质控品（可从蓝图生物科技产品研发系统中选择）【储存条件及有效期】1、2-8℃保存，切勿冷冻，有效期6个月。

2、开封使用后，包被微孔板放入带有干燥剂的自封袋中，密闭自封袋，并将全部试剂放回2-8℃冰箱。

本试剂盒只能用于科学研究，不得用于医学诊断。

人白细胞介素8（IL-8）定量检测试剂盒（ELISA）使用说明书【试剂盒名称】人白细胞介素8（IL-8）定量检测试剂盒（ELISA）【试剂盒用途】定量检测人血清、血浆及相关液体样本中白细胞介素8（IL-8）的含量。

【检测原理】本试剂盒采用双抗体两步夹心酶联免疫吸附法（ELISA）。

将标准品、待测样本加入到预先包被人白细胞介素8（IL-8）单克隆抗体透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分，再加入酶标工作液，温育足够时间后，洗涤除去未结合的成分。

依次加入底物A、B，底物（TMB）在辣根过氧化物酶（HRP）催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样品中人白细胞介素8（IL-8）浓度呈正相关，450nm波长下测定OD值，根据标准品和样品的OD值，计算样本中人白细胞介素8（IL-8）含量。

【试剂盒组成】1酶标包被板12孔×8条7显色剂A液6mL2标准品0.3mL×6管8显色剂B液6mL320倍浓缩洗涤液25mL9终止液6mL4样本稀释液6mL10说明书1份5特殊稀释液6mL11封板膜2张6酶标试剂6mL12密封袋1个备注：标准品（1号→6号）浓度依次为：120、60、30、15、7.5、3.75pg/mL.【需要而未提供的试剂和器材】1、37℃恒温箱2、标准规格酶标仪3、精密移液器及一次性吸头4、蒸馏水5、一次性试管6、吸水纸【操作步骤】1、准备：从冰箱取出试剂盒，室温复温平衡30分钟。

2、配液：用蒸馏水将20倍浓缩洗涤液稀释成原倍的洗涤液。

3、加标准品和待测样本：取足够数量的酶标包被板，固定于框架上，分别设置标准品孔、待测样本孔和空白对照孔，记录各孔位置，在标准品孔中加入标准品50μL；待测样本孔中先加入待测样本10μL，再加样本稀释液40μL（即样本稀释5倍）；空白对照孔不加。

4、温育：37℃水浴锅或恒温箱温育30min。

碧云天生物技术/Beyotime Biotechnology 订货热线：400-168-3301或800-8283301 订货e-mail ：******************技术咨询：*****************网址：碧云天网站 微信公众号Human IL-1β ELISA Kit产品编号 产品名称包装 PI305Human IL-1β ELISA Kit96次产品简介：碧云天的Human IL-1β ELISA Kit (Human Interleukin-1β Enzyme-Linked ImmunoSorbent Assay Kit)，即人白细胞介素1β酶联免疫吸附检测试剂盒，是一种用于特异性地高灵敏地定量检测人血清、血浆、细胞或组织裂解液、或细胞培养上清液中的IL-1β的ELISA 试剂盒。

本产品检测灵敏度高，特异性强，重复性好。

多次重复检测结果表明，最小检出量为2.2pg/ml ，与人IL-1r α、IL-1sRII 、IL-1sRI 、小鼠IL-1α、小鼠IL-1β等均没有交叉反应，板内、板间变异系数均小于10%。

IL-1即白细胞介素-1(简称白介1)，其家族由IL-1α(也称IL-1F1)、IL-1β(也称IL-1F2)、IL-1受体拮抗剂(IL-1 receptor antagonist, 简称IL-1RA, 或IL-1F3)及IL-18、IL-33和IL-1F5~F10组成。

IL-1主要由巨噬细胞产生，此外几乎所有的有核细胞，如B 细胞、NK 细胞、体外培养的T 细胞、角质细胞、树突状细胞、星形细胞、成纤维细胞、中性粒细胞、内皮细胞以及平滑肌细胞均可产生IL-1。

正常情况下只有皮肤、汗液及尿液中含有一定量的IL-1，绝大多数细胞在受到外来抗原或细菌内毒素刺激后才能合成和分泌IL-1。

IL-1β在免疫和炎症反应、骨重建(bone remodeling)、发烧、碳水化合物代谢等生理、病理过程中都有重要作用。

REV20190712仅供研究，不用于临床诊断。

客服热线: 400-7060-959﹡技术支持邮箱: **************公司官网: 目录简介 ........................................................................................................................................................... - 3 -检测原理 ................................................................................................................................................... - 3 -试剂盒组分 ............................................................................................................................................... - 4 -储存条件 ................................................................................................................................................... - 5 -其他实验材料(不提供，但可协助购买) : ............................................................................................. - 5 -注意事项 ................................................................................................................................................... - 5 -样本收集处理及保存方法 ....................................................................................................................... - 6 -试剂准备 ................................................................................................................................................... - 6 -操作步骤 ................................................................................................................................................... - 8 -操作流程图 ............................................................................................................................................... - 8 -操作要点提示 ........................................................................................................................................... - 9 -结果判断 ................................................................................................................................................... - 9 -结果重复性 ............................................................................................................................................. - 10 -灵敏度 ..................................................................................................................................................... - 10 -特异性 ..................................................................................................................................................... - 10 -参考文献 ................................................................................................................................................. - 10 -该产品由北京四正柏生物科技有限公司研制。

实验步骤1. 开始ELISA 前一天，稀释包被抗体，取酶标板每孔加入100ul 包被抗体溶液，4 度过夜2. 使用前将所有试剂放置室温，强烈建议所有标准品和样品做复孔或三孔，各次检测均要求做标准曲线3. 洗涤液不少于300ul 洗板4 次，并在吸水纸上拍干板子，后续洗涤液同此4. 为了阻断非特异性结合和减少背景，每孔加200ul 样品稀释液5. 封板并在摇床上室温孵育1h6. 在上述期间，准备标准品稀释液和适当的样品稀释液（如必需）7. 标准品稀释：500 pg/ml, 250 pg/ml, 125 pg/ml,62.5 pg/ml, 31.25 pg/ml, 15.6 pg/ml, 7.8pg/ml 和0 pg/ml8. 如步骤3 洗板4 次9. 每孔加入100ul 标准品稀释液和样品到对应孔，如有需要可对样品进行稀释10. 封板并在摇床上室温孵育2h11. 如步骤3 洗板4 次12. 每孔加入100ul 已稀释的二抗，封板并在摇床上室温孵育1h13. 如步骤3 洗板4 次14. 每孔加入100ul稀释的HRP,封板并在摇床上室温孵育30min15. 如步骤3洗板5次，每次洗涤洗涤液浸泡30s至1min，有利于减少背景16. 每孔加入100UITMB显色液，并在摇床孵育15-30min，阳性孔将变为淡蓝色，此步无需封板17. 每孔加入100ul 终止液终止反应，阳性孔将由蓝色变为黄色18. 终止反应半小时内测定OD值（450nm）ELISA 实验基本原理基本原理1971 年Engvall 和Perlmann 发表了酶联免疫吸附剂测定（enzyme linked immunosorbent assay,ELISA ）用于IgG定量测定的文章，使得1966年开始用于抗原定位的酶标抗体技术发展成液体标本中微量物质的测定方法。

这一方法的基本原理是：①使抗原或抗体结合到某种固相载体表面，并保持其免疫活性。

人白介素6(IL-6)酶联免疫吸附测定试剂盒使用说明书产品编号：1910231本试剂盒仅供体外研究使用、不用于临床诊断!预期应用ELISA法定量测定人血清、血浆、细胞培养上清或其它相关生物液体中IL-6含量。

实验原理用纯化的IL-6抗体包被微孔板，制成固相载体，往微孔中依次加入标本或标准品、生物素化的IL-6抗体、HRP标记的亲和素，经过彻底洗涤后用底物(TMB)显色。

TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。

颜色的深浅和样品中的IL-6呈正相关。

用酶标仪在450nm波长下测定吸光度（值），计算样品浓度。

试剂盒组成及试剂配制1、酶标板：一块（96孔）2、标准品（冻干品）：2瓶，请临用前15分钟内配制。

每瓶以样品稀释液稀释至1ml，盖好后室温静置大约10分钟，同时反复颠倒/搓动以助溶解，其浓度为10,000 pg/ml，将其稀释为500 pg/ml后，再做系列倍比稀释（注：不要直接在板中进行倍比稀释），分别配制成500 pg/ml，250 pg/ml，125 pg/ml，62.5 pg/ml，31.2 pg/ml，15.6 pg/ml，7.8 pg/ml，样品稀释液直接作为空白孔0 pg/ml。

如配制500 pg/ml标准品：取0.5ml （不要少于0.5ml ）500 pg/ml的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中，混匀即可，其余浓度以此类推。

3、样品稀释液：1×25ml。

4、人白介素6:1×25ml。

5、HRP,100X：1×150 /瓶（1:100）。

临用前以HRP 1:100稀释（如：10 HRP / 990HRP稀释液），充分混匀，稀释前根据预先计算好的每次实验所需的总量配制（100/孔），实际配制时应多配制0.1-0.2ml。

6、HRP稀释剂：1×12ml。

7、浓洗涤液：1×50ml/瓶。

人白细胞介素1a（IL-1a）酶联免疫检测试剂盒使用说明书使用前仔细阅读本说明书。

本酶联免疫试剂盒是基于双抗体夹心技术原理，来检测人白细胞介素1a（IL-1a），只能用于研究用途，不得用于医学诊断。

用途：用于人血清、血浆及相关液体样本中白细胞介素1a（IL-1a）的测定。

工作原理本试剂盒采用的是双抗体夹心酶联免疫吸附法（ELISA）测定样品中人白细胞介素1a （IL-1a）的水平。

向预先包被了人白细胞介素1a（IL-1a）单克隆抗体的酶标孔中加入白细胞介素1a（IL-1a），温育；洗涤后，加入HRP标记过的白细胞介素1a（IL-1a）抗体。

再经过温育和洗涤，去除未结合的酶，然后加入底物A、B，产生蓝色，并在酸的作用下转化成最终的黄色。

颜色的深浅与样品中人白细胞介素1a（IL-1a）的浓度呈正相关。

试剂盒组成需要而未提供的试剂和器材1．37℃恒温箱。

2．标准规格酶标仪。

3．精密移液器及一次性吸头4．蒸馏水，5．一次性试管6．吸水纸注意事项1．从2-8℃取出的试剂盒，在开启试剂盒之前要室温平衡至少30分钟。

酶标包被板开封后如未用完，板条应装入密封袋中保存。

2．各步加样均应使用加样器，并经常校对其准确性，以避免试验误差3．建议所有标准品、样本都做双份检测。

如标本中待测物质含量过高，请先用样品稀释液稀释一定倍数（n倍）后再按说明书操作进行测定，计算时请最后乘以总稀释倍数（×n×5）。

4．严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准.5．为避免交叉污染，要避免重复使用手中的吸头和封板膜。

6．不用的其它试剂应包装好或盖好。

不同批号的试剂不要混用。

保质前使用。

7．底物B对光敏感，避免长时间暴露于光下。

洗板方法手工洗板方法：甩掉酶标板内的液体；在实验台上铺垫几层吸水纸，酶标板朝下用力拍几次；将稀释后的洗涤液至少0.35ml注入孔内，浸泡1-2分钟。

根据需要，重复此过程数次。

自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中标本要求1．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。