试剂盒,人试剂盒,人刀豆素A(ConA)ELISA试剂盒使用说明书

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）。

ELISA检测试剂盒操作流程1.准备实验材料和试剂-检验样本：例如血清、尿液、细胞上清液等。

-ELISA试板：根据样本数量选择96孔或384孔的板。

- 试剂盒：包括标准品、washes缓冲液、检测抗体、底物等。

2.样本处理-对于血清等生物液体样本，可用离心将固体物质沉淀并分离下清液。

-样本需储存或运输途中应避免反复冻融。

3.实验板的板液处理-将实验板从包装中取出，将不需要的孔进行盖膜封闭。

- 根据试剂盒说明书，添加适量的板液（Coating Buffer）到每个孔中，使其充分覆盖孔内壁。

-封闭板液的孔，将实验板在2-8℃下放置静置一夜或在37℃下1-2小时孵育。

4.样本添加-倒掉盘液，用PBS或TBST洗板3次，去除残留物。

-将样本加到每个孔中，根据需要添加正样本、负样本和待测样本。

-注意每个样本的剂量和添加的体积，通过试剂盒说明书和实验设计标准控制。

5.抗体添加-清洗步骤同上，将洗板缓冲液加到孔中，重复3次。

-加入检测抗体，根据试剂盒说明书的建议将其添加到孔中。

-要确保抗体的浓度和添加的体积适宜。

6.洗涤-洗涤步骤同上，用洗涤缓冲液洗板，重复3次。

-每次洗涤时，将洗液完全加满孔，静置1分钟，然后倒出洗液。

-注意洗涤时间和洗涤次数的准确控制，以将非特异性结合物质彻底清除。

7.底物添加-清洗步骤同上，将洗液加到孔中，重复3次。

-加入底物，根据试剂盒说明书的建议将其添加到孔中。

-底物液体应完全覆盖孔底，防止反应过程中氧化。

8.反应停止-根据试剂盒说明书，将反应停止液加入到孔中，停止底物的反应。

-注意反应停止液的体积和浓度，以保证反应的准确停止。

9.色谱分析-用ELISA板读板仪读取各个孔的OD值。

-根据试剂盒说明书和实验设计的标准曲线确定样本中目标物质的含量。

-注意设置适当的控制组和标准曲线来保证测量结果的准确性。

总结：ELISA检测试剂盒的操作流程主要包括实验板处理、样本添加、抗体添加、洗涤、底物添加、反应停止和色谱分析。

简述使用elisa试剂盒的操作流程下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。

文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor.I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!ELISA试剂盒操作流程简述ELISA，全称为酶联免疫吸附测定，是一种广泛用于定量检测样本中特定蛋白质或抗原的生物分析技术。

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。

人P16ELISA试剂盒使用说明书本试剂仅供研究使用目的：本试剂盒用于测定人血清，血浆及相关液体样本中人P16的含量。

实验原理：本试剂盒应用双抗体夹心法测定标本中人P16水平。

用纯化的人P16抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入P16，再与HRP标记的P16抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。

TMB在HRP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P16呈正相关。

用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人P16浓度。

样本处理及要求：1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（2000-3000转/分）。

仔细收集上清，保存过程中如出现沉淀，应再次离心。

2. 血浆：应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂，混合10-20分钟后，离心20分钟左右（2000-3000转/分）。

仔细收集上清，保存过程中如有沉淀形成，应该再次离心。

3. 尿液：用无菌管收集，离心20分钟左右（2000-3000转/分）。

仔细收集上清，保存过程中如有沉淀形成，应再次离心。

胸腹水、脑脊液参照实行。

4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。

离心20分钟左右（2000-3000转/分）。

仔细收集上清。

检测细胞内的成份时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞浓度达到100万/ml左右。

通过反复冻融，以使细胞破坏并放出细胞内成份。

离心20分钟左右（2000-3000转/分）。

仔细收集上清。

保存过程中如有沉淀形成，应再次离心。

5. 组织标本：切割标本后，称取重量。

加入一定量的PBS，PH7.4。

用液氮迅速冷冻保存备用。

标本融化后仍然保持2-8℃的温度。

加入一定量的PBS（PH7.4），用手工或匀浆器将标本匀浆充分。

离心20分钟左右（2000-3000转/分）。

仔细收集上清。

ELISA试剂盒操作方法ELISA（Enzyme-Linked Immunosorbent Assay）是一种常用的免疫学试验技术，用于检测体内或体外的蛋白质、抗体、抗原等物质。

ELISA试剂盒是用于进行ELISA实验的一种常见装置。

下面将为您介绍ELISA试剂盒的操作方法。

1.准备工作a.预热-将试剂盒中的所有试剂预热到适当的温度，通常为室温至37°C之间。

b.样品处理-准备好待测样品，可以是血清、细胞上清液、组织提取物等。

如果样品中含有大量的蛋白质或杂质，可以进行样品处理，如稀释、蛋白质去除等。

c.标准品准备-试剂盒通常包含一系列浓度已知的标准品，用于制作标准曲线。

按照说明书的要求，用缓冲液或适当的稀释液将标准品稀释为不同浓度的工作液。

2.涂膜板的处理a.涂膜板选择-根据实验需求选择合适的涂膜板，通常有96孔、384孔等。

b.板钉定位-使用板钉或其他适当的方法将涂膜板固定在适当的工作台上。

c.预涂底物溶液加入-将试剂盒提供的预涂底物溶液加入涂膜板的孔中，注意保持各孔液面平整。

3.样品与试剂添加a.标准品和样品加入-按照实验设计的需要，将标准品和待测样品分别加入涂膜板的孔中。

b.阳性对照和阴性对照加入-根据试剂盒的要求，加入阳性对照和阴性对照。

c.洗涤液加入-在每次试剂或样品加入后，使用专用的洗涤液将孔洗涤，以清除无关物质。

4.反应与显色a.结合抗体加入-按照试剂盒说明书要求，加入适当的结合抗体，用于与待测物质结合形成复合物。

b.二抗加入-加入带有底物标记的二抗，与结合抗体结合形成复合物。

c.显色底物加入-加入含有适当底物的显色底物试剂，使底物与酶结合并发出信号。

5.反应停止与测量a.反应停止液加入-在适当的时间点，根据试剂盒的说明，加入反应停止液，停止底物的酶反应。

b.光密封膜或保护板加盖-加盖光密封膜或保护板，防止溶液蒸发或污染。

6.读取和数据处理a.读取吸光度-使用ELISA读板仪读取各孔的吸光度值。

Human kynurenine(KYN)ELISA KitCatalog Number.CSB-E13659hFor the quantitative determination of endogenic human kynurenine(KYN) concentrations in serum,urine,tissue homogenates.This package insert must be read in its entirety before using this product.If You Have ProblemsTechnical Service Contact informationPhone:86-27-87582341Fax:86-27-87196150Email:****************Web:In order to obtain higher efficiency service,please ready to supply the lot number of the kit to us(found on the outside of the box).PRINCIPLE OF THE ASSAYThis assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antigen. Standards or samples are added to the appropriate microtiter plate wells with antibody specific for KYN and Horseradish Peroxidase(HRP)conjugated goat-anti-rabbit antibody.The competitive inhibition reaction is launched between with pre-coated KYN and KYN in samples.A substrate solution is added to the wells and the color develops in opposite to the amount of KYN in the sample.The color development is stopped and the intensity of the color is measured.DETECTION RANGE7.8pmol/ml-500pmol/ml.SENSITIVITYThe minimum detectable dose of human KYN is typically less than3.9pmol/ml. The sensitivity of this assay,or Lower Limit of Detection(LLD)was defined as the lowest human KYN concentration that could be differentiated from zero.SPECIFICITYThis assay has high sensitivity and excellent specificity for detection of human KYN.No significant cross-reactivity or interference between human KYN and analogues was observed.Note:Limited by current skills and knowledge,it is impossible for us to complete the cross-reactivity detection between human KYN and all the analogues, therefore,cross reaction may still exist.PRECISIONIntra-assay Precision(Precision within an assay):CV%<8%Three samples of known concentration were tested twenty times on one plate to assess.Inter-assay Precision(Precision between assays):CV%<10%Three samples of known concentration were tested in twenty assays to assess.LIMITATIONS OF THE PROCEDURE●FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTICPROCEDURES.●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 thesamples with Sample Diluent and repeat the assay.●Any variation in Sample Diluent,operator,pipetting technique,washingtechnique,incubation time or temperature,and kit age can cause variation in binding.●This assay is designed to eliminate interference by soluble receptors,binding proteins,and other factors present in biological samples.Until all factors have been tested in the Immunoassay,the possibility of interference cannot be excluded.MATERIALS PROVIDEDReagents QuantityAssay plate1(96wells) Standard(Freeze dried)2Antibody(100x concentrate)1x60μl Antibody Diluent1x10mlHRP-conjugate(100x concentrate)1x120μlHRP-conjugate Diluent1x20ml Sample Diluent2x20mlWash Buffer(25x concentrate)1x20mlTMB Substrate1x10mlStop Solution1x10ml Adhesive Strip(For96wells)4Instruction manual1STORAGEUnopened kit Store at2-8°C.Do not use the kit beyond the expiration date.Opened kitCoated assayplateMay be stored for up to1month at2-8°C.Try to keep it in a sealed aluminum foil bag,and avoid the damp.Standard May be stored for up to1month at2-8°C.Ifdon’t make recent use,better keep it store at-20°C.AntibodyHRP-conjugateAntibodyDiluentMay be stored for up to1month at2-8°C. HRP-conjugateDiluentSample DiluentWash BufferTMB SubstrateStop Solution*Provided this is within the expiration date of the kit.OTHER SUPPLIES REQUIRED●Microplate reader capable of measuring absorbance at450nm,with thecorrection wavelength set at540nm or570nm.●An incubator which can provide stable incubation conditions up to37°C±0.5°C.●Squirt bottle,manifold dispenser,or automated microplate washer.●Absorbent paper for blotting the microtiter plate.●100ml and500ml graduated cylinders.●Deionized or distilled water.●Pipettes and pipette tips.●Test tubes for dilution.PRECAUTIONSThe Stop Solution provided with this kit is an acid solution.Wear eye,hand,face, and clothing protection when using this material.SAMPLE COLLECTION AND STORAGE●Serum Use a serum separator tube(SST)and allow samples to clot fortwo hours at room temperature or overnight at4°C before centrifugation for15minutes at1000×g.Remove serum and assay immediately or aliquot and store samples at-20°C or-80°C.Avoid repeated freeze-thaw cycles.●Urine Use a sterile container to collect urine samples.Remove anyparticulates by centrifugation for15minutes at1000xg,2-8°C and assay immediately or aliquot and store samples at-20°C or-80°C.Avoid repeated freeze-thaw cycles.Centrifuge again before assaying to remove any additional precipitates that may appear after storage.●Tissue Homogenates100mg tissue was rinsed with1X PBS,homogenized in1ml of1X PBS and stored overnight at-20°C.After two freeze-thaw cycles were performed to break the cell membranes,the homogenates were centrifuged for5minutes at5000x g,2-8°C.The supernate was removed and assayed immediately.Alternatively,aliquot and store samples at-20°C or-80°C.Centrifuge the sample again after thawing before the assay.Avoid repeated freeze-thaw cycles.Note:1.CUSABIO is only responsible for the kit itself,but not for the samplesconsumed during the assay.The user should calculate the possible amount of the samples used in the whole test.Please reserve sufficient samples in advance.2.Samples to be used within5days may be stored at2-8°C,otherwisesamples must be stored at-20°C(≤1month)or-80°C(≤2month)to avoid loss of bioactivity and contamination.3.Grossly hemolyzed samples are not suitable for use in this assay.4.If the samples are not indicated in the manual,a preliminary experiment todetermine the validity of the kit is necessary.5.Please predict the concentration before assaying.If values for these arenot within the range of the standard curve,users must determine the optimal sample dilutions for their particular experiments.6.Tissue or cell extraction samples prepared by chemical lysis buffer maycause unexpected ELISA results due to the impacts of certain chemicals.7.Owing to the possibility of mismatching between antigen from otherresource and antibody used in our kits(e.g.,antibody targets conformational epitope rather than linear epitope),some native or recombinant proteins from other manufacturers may not be recognized by our products.8.Influenced by the factors including cell viability,cell number and alsosampling time,samples from cell culture supernatant may not be detected by the kit.9.Fresh samples without long time storage are recommended for the test.Otherwise,protein degradation and denaturalization may occur in those samples and finally lead to wrong results.REAGENT PREPARATIONNote:●Kindly use graduated containers to prepare the reagent.Please don'tprepare the reagent directly in the Diluent vials provided in the kit.●Bring all reagents to room temperature(18-25°C)before use for30min.●Prepare fresh standard for each e within4hours and discardafter use.●Making serial dilution in the wells directly is not permitted.●Please carefully reconstitute Standards according to the instruction,andavoid foaming and mix gently until the crystals have completely dissolved.To minimize imprecision caused by pipetting,use small volumes and ensure that pipettors are calibrated.It is recommended to suck more than 10μl for once pipetting.●Distilled water is recommended to be used to make the preparation forreagents.Contaminated water or container for reagent preparation will influence the detection result.1.Antibody(1x)-Centrifuge the vial before opening.Antibody requires a100-fold dilution.A suggested100-fold dilution is10μl of Antibody+990μl of Antibody Diluent.2.HRP-conjugate(1x)-Centrifuge the vial before opening.HRP-conjugate requires a100-fold dilution.A suggested100-fold dilution is10μl of HRP-conjugate+990μl of HRP-conjugate Diluent.3.Wash Buffer(1x)-If crystals have formed in the concentrate,warm up toroom temperature and mix gently until the crystals have completely dissolved.Dilute20ml of Wash Buffer Concentrate(25x)into deionized or distilled water to prepare500ml of Wash Buffer(1x).4.StandardCentrifuge the standard vial at6000-10000rpm for30s before opening.Reconstitute the Standard with 1.0ml of Sample Diluent.Do not substitute other diluents.This reconstitution produces a stock solution of 500pmol/ml.Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of15minutes with gentle agitation prior to making dilutions.Pipette150μl of Sample Diluent into each tube(S0-S6).Use the stock solution to produce a2-fold dilution series(below).Mix each tube thoroughly before the next transfer.The undiluted Standard serves as the high standard(500pmol/ml).Sample Diluent serves as the zero standard (0pmol/ml).Tube S7S6S5S4S3S2S1S0pmol/ml50025012562.531.215.67.80ASSAY PROCEDUREBring all reagents and samples to room temperature before use.Centrifuge the sample again after thawing before the assay.It is recommended that all samples and standards be assayed in duplicate.1.Prepare all reagents,working standards,and samples as directed in theprevious sections.2.Refer to the Assay Layout Sheet to determine the number of wells to beused and put any remaining wells and the desiccant back into the pouch and seal the ziploc,store unused wells at4°C.3.Set a Blank well without any solution.4.Add50μl of standard and sample per well.Add50μl Antibody(1x)toeach well immediately(not to Blank well).Mix well with the pipette or shake the plate gently for60seconds.A plate layout is provided to record standards and samples assayed.5.Cover with the adhesive strip provided.Incubate for40minutes at37°C.6.Aspirate each well and wash,repeating the process two times for a total ofthree washes.Wash by filling each well with Wash Buffer(200μl)using a squirt bottle,multi-channel pipette,manifold dispenser,or autowasher, and let it stand for2minutes,complete 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.7.Add100μl HRP-conjugate(1x)to each well immediately(not to Blankwell).Cover with the adhesive strip provided.Incubate for30minutes at 37°C.8.Repeat the aspiration/wash process for five times as in step6.9.Add90μl of TMB Substrate to each well.Incubate for20minutes at37°C.Protect from light.10.Add50μl of Stop Solution to each well,gently tap the plate to ensurethorough mixing.11.Determine the optical density of each well within5minutes,using amicroplate reader set to450nm.If wavelength correction is available,set to540nm or570nm.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.Note:1.The final experimental results will be closely related to validity of theproducts,operation skills of the end users and the experimental environments.2.Samples or reagents addition:Please use the freshly prepared Standard.Please carefully add samples to wells and mix gently to avoid foaming.Do not touch the well wall as possible.For each step in the procedure,total dispensing time for addition of reagents or samples to the assay plate should not exceed10minutes.This will ensure equal elapsed time for each pipetting step,without interruption.Duplication of all standards and specimens,although not required,is recommended.To avoid cross-contamination,change pipette tips between additions of each standard level,between sample additions,and between reagent additions.Also,use separate reservoirs for each reagent.3.Incubation:To ensure accurate results,proper adhesion of plate sealersduring incubation steps is necessary.Do not allow wells to sit uncovered for extended periods between incubation steps.Once reagents have been added to the well strips,DO NOT let the strips DRY at any time during the assay.Incubation time and temperature must be observed.4.Washing:The wash procedure is plete removal of liquid at eachstep is essential to good performance.After the last wash,remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate.Insufficient washing will result in poor precision and falsely elevated absorbance reading.When using an automated plate washer,adding a30second soak period following the addition of wash buffer,and/or rotating the plate180degrees between wash steps may improve assay precision.5.Controlling of reaction time:Observe the change of color after adding TMBSubstrate(e.g.observation once every10minutes),TMB Substrate should change from colorless or light blue to gradations of blue.If the color is too deep,add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.6.TMB Substrate is easily contaminated.TMB Substrate should remaincolorless or light blue until added to the plate.Please protect it from light. 7.Stop Solution should be added to the plate in the same order as the TMBSubstrate.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 Solution has not mixed thoroughly with the TMB Substrate.ASSAY PROCEDURE SUMMARY*Please determine whether the sample needs to be diluted or the optimal dilution factor based on preliminary experiment result.CALCULATION OF RESULTSUsing the professional soft"Curve Expert"to make a standard curve is recommended,which can be downloaded from our web.Average the duplicate readings for each standard and sample and subtract the average optical density of Blank.Create a standard curve by reducing the data using computer software capable of generating 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 x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph.The data may be linearized by plotting the log of the KYN 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.人犬尿氨酸（KYN）酶联免疫试剂盒使用说明书【产品编号】CSB-E13659h【预期应用】ELISA法定量测定人血清、尿液、组织裂解液中KYN含量。

Shanghai Coon Koon Biotech Co., LtdRoom 1408，1687 Chang Yang Rd, Shanghai, China.Human Free Testosterone (F-TESTO) ELISA KitCat No:CK-bio-11536Standard Curve Range: 0.5ng/mL -12ng/mLSensitivity: 0.1ng/mLExpiration date：twelve months .Storage：2-8℃.For samples:Serum, plasma, cell culture supernatants, body fluid and tissue homogenateWhen stored at 2 -8 °C unopened reagents will retain reactivity until expiration date. Opened reagents must be stored at 2 -8 °C.OPERATION MANUALRead this manual carefully before using. The ELISA kit is based on the principle of double antibody sandwich technology.And the ELISA kits only be used for research purposes, not for medical diagnosis. Reagent preparation: Bring all reagents to room temperature before using.Intended UseFor the quantitative determination of Human Free Testosterone(F-TESTO) concentrations in serum, plasma, saliva, urine, tissue homogenate, cell culture supernates and other biological fluids.Test PrincipleThe kit was used to test the level of Human Free Testosterone(F-TESTO), based on the principle of double antibody sandwich technology enzyme linked immunosorbent assay (ELISA).Add Standard and Sample to the wells that pre-coated with objective antibody, then add HRP-Conjugate reagent to form an immune complex, incubation, by incubation and washing, removal of unbound enzyme, and then add the substrate A and B, then the solution will turn blue and finally change into yellow at the effect of acid. The color depth or light was positively correlated with the concentration of Free Testosterone (F-TESTO).Precautions1.Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.2.It is highly recommended to use the remaining reagents within 1 month before the deadline. For the expiration date, please refer to the label on the kit box. All components are stable before this expiration date.Do not use kit components beyond their expiration date.3.Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C) before use. Do not use water baths to thaw samples or reagents.e only deionized or distilled water to dilute reagents.5.Each steps add sample, should use sampler, and often proofread the accuracy to avoid the test error. Use fresh disposable pipette tips for each transfer to avoid contamination.6.Test should strict accordance with the instructions of the operation, the test results must be determined by the microplate reader.7.Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.8.Do not mix acid and sodium hypochlorite solutions.9.Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.10.All samples should be disposed of in a manner that will inactivate viruses.11.Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.12.Substrate Solution is easily contaminated. If bluish prior to use, do not use. Substrate B is sensitive to light and avoid prolonged exposure to light.MATERIALS PROVIDED WITH THE KITAll reagents provided are stored at 2-8°C. Refer to the expiration date on the label.Reagents components 96 determinations48 determinations1. Microelisa stripplate 12*8strips 12*4strips2. Standard A 0ng/mL 0ng/mL3. Standard B 0.75ng/mL 0.75ng/mL4. Standard C 1.5ng/mL 1.5ng/mL5. Standard D 3ng/mL 3ng/mL6. Standard E 6ng/mL 6ng/mL7. Standard F 12ng/mL 12ng/mL8. Sample Diluent 6.0ml 3.0ml9. HRP-Conjugate reagent 10.0ml 5.0ml10. 20X Wash solution 25ml 15ml11. Chromogen Solution A 6.0ml 3.0ml12. Chromogen Solution B 6.0ml 3.0ml13. Stop Solution 6.0ml 3.0ml14. Closure plate membrane 2 215. User manual 1 116. Sealed bags 1 1 Note: Standard (A →F) concentration was followed by: 0ng/mL ,0.75ng/mL ,1.5ng/mL , 3ng/mL ,6ng/mL ,12ng/mL .Materials required but not supplied1.37 ℃ incubator2.Microplate reader capable of measuring absorbance at 450 nm.3.Precision pipettes to deliver 2 ml to 1 ml volumes.4.100 ml and 1 liter graduated cylinders.5. Distilled water,6. Disposable test tube7. Absorbent paper8. Precision pipettes and disposable tipSpecimen Requirements1.Serum: Allow the serum to clot for 10-20 minutes at room temperature. Centrifuge (at 2000-3000 RPM) for 20 minutes. Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again.2.Blood plasma: In accordance with the requirements of sample collection, EDTA or sodium citrate should be used as anti coagulation. Add EDTA or sodium citrate and mix them for10-20 minutes. Centrifuge (at 2000-3000 RPM) for approximately 20 minutes. Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again.3.Urine: Collect by sterile tube. Centrifuge (at 2000-3000 RPM) for approximately 20 minutes. Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.4.Cell culture supernatant: Collect by sterile tubes when examining secrete components. Centrifuge (at 2000-3000 RPM) for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (PH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge (at 2000-3000 RPM) for approximately 20 minutes. Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again.5.Tissue sample: Incise sample and weigh up. Add a certain amount of PBS (PH 7.4). Freeze with liquid nitrogen immediately for later use. Thaw the sample and keep it at 2-8℃. Add a certain amount of PBS (PH 7.4) and then homogenize the sample thoroughly by hand or homogenizer. Centrifuge (at 2000-3000 RPM) for approximately 20 minutes. Collect the supernatants carefully. Aliquot and keep one for examination and freeze the others for later use.Note: 1.Samples to be used within 5 days may be stored at 2-8℃, otherwise samples must be stored at -20℃(≤1month)or -80℃(≤2months) to avoid loss of bioactivity and avoid contamination.2.Sample hemolysis will influence the result, so the samples should be centrifuged adequately and no hemolysis or granule was allowed.3.When performing the assay, bring samples to room temperature.Samples containing NaN3 can’t be tested as it inhibits the activity of Horse Radish Peroxidase (HRP).4.After collecting the sample, extraction should be immediately carried out in accordance with related documents. After extraction, experiment should be conducted immediately as well. Otherwise, keep the sample at -20℃. Avoid repeated freeze-thaw cycles.Washed plate method1.Hand-washed plate method: get rid of the liquid within the ELISA plate; in the experimental bench paved a few layers of absorbent paper, pat hard the ELISA plate several times downward; the diluted washing solution at least 0.35ml inject into the well, soaking 1-2 minutes. Repeat this process several times as needed.2. Automatic plate washing: If you have automatic washing machine, Should be skilled use, and then used in the formal experiment process.Assay procedure1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.3. Add Sample: ①Add Sample 10μl to testing sample well, then add sample diluent 40μl to testing sample well; Blank well doesn’t add anything.②Add 100μl of HRP-conjugate reagent to each well(Standard wells and testing sample wells), then cover it with seal plate membrane, gently shake and mix for 60 minutes at 37 ° C incubation.4.Preparation of washing solution: Dilute the washing concentration (20X) with distilled or deionized water for later use.5.Washing by hand: carefully remove the sealing film, drain the liquid, dried up, each well filled with washing solution, put it aside for 1 min then drain the liquid, so repeat 5 times, pat dry. (Automatic washing: Each wells inject into the wash solution 350μL, soak 1min, wash plate 5 times.)6. Color developing: firstly add 50μl chromogen solution A to each wells, then add 50μl chromogen solution B to each well as well. Shake gently to mix up. Incubate for 15 minutes at 37℃,away from light for color developing.7.Stop: Add 50μl Stop Solution to each well to stop the reaction (the blue color changes into yellow immediately at that moment). If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.8.Assay: Take blank well as zero, measure the absorbance (OD) of each well one by one under 450nm wavelength, which should be carried out within the 15 minutes after having added the stop solution.9.According to standards’ concentrations and the corresponding OD values, to calculate the linear regression equation of the standard curve. Then according to the OD value of samples, calculate the concentration of the corresponding sample.Also can use related application software.Summary of operating proceduresCalculation of results1. This standard curve is used to determine the amount in an unknown sample. Thestandard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, aresubtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.3. To determine the amount in each sample, first locate the O.D. value on the Y-axis andextend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.4. Any variation in operator, pipetting and washing technique, incubation time ortemperature, and kit age can cause variation in result. Each user should obtain their own standard curve.5. Intra-assay CV(%) is less than 7% and Inter-assay CV(%) is less than 10%.6.Standard curve : The following standard curve only for demonstration purposes, each standard curve should be generated with each assay.1.Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.2.If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor,5X.3.If specimens generate values higher than the highest standard, dilute the specimens and repeat the assay.FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDUREBEFORE BEGINNING!。

山西医科大掌硕士掌位论文白癜风患者外周血单一核细胞中Ｔｈｌ和Ｔｈ２型细胞因子的表达特点及意义（论文）专业：皮肤与性病学导师：陈丽瑛董琛姓名：尉雁［摘要］目的探讨白癜风患者中Ｔｈｌ型（ＩＬ一２，ＩＦＮ—Ｙ）和Ｔｈ一２（ＩＬ～１０）型细胞因子表达的情况。

方法采用细胞培养技术对白癜风患者和正常对照者的ＰＢＭＣ在刀豆素Ａ（ＣｏｎＡ）作用下进行体外培养；用ＥＬＩＳＡ法检测培养上清中ＩＬ一１０、ＩＦＮ—ｙ含量；采用逆转录一聚合酶链反应（ＲＴ—ＰＣＲ）法对相应培养细胞中的ＩＬ一２ｍＲＮＡ进行半定量检测。

结果（１）白癜风患者ＰＢＭＣ产生ＩＬ—ｌＯ水平低于正常对照组（Ｐ＜Ｏ．０５），ＩＦＮ—ｙ水平高于正常对照组（ＰｉＯ．０５）。

（２）白癜风患者ＰＢＭＣ表达ＩＬ一２ｍＲＮＡ相对单位高于正常对照组（Ｐ＜Ｏ．０５）。

（３）患者组和对照组ＰＢＭＣ加ＣｏｎＡ刺激孔中ＩＬ一２ｍＲＮＡ相对单位高于各自相应自然增殖孔（Ｐ＜Ｏ．０５，Ｐ＜Ｏ．０５）。

结论白癜风患者中Ｔｈｌ型细胞因子的表达增高，Ｔｈ２型细胞因子的表达下调，可能在发病机制中起一定作用。

［关键词］白癜风白细胞介素２白细胞介素１０干扰素ＩＩ型山西医科大掌司Ｅ士掌位论文ＴｈｅＣｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆｅｘｐｒｅｓｓｉｏｎｏｆＴｈｌ＆Ｔｈ２ｃｅｌｌ—ｍｅｄｉａｔｅｄｃｙｔｏｋｉｎｅｐｒｏｆｉｌｅａｎｄｉｔｓｓｉｇｎｉｆｉｃａｎｃｅｉｎｔｈｅＰｅｒｉｐｈｅｒａｌＭｏｎｏｎｕｃｌｅａｒＣｅｌｌｓｏｆｔｈｅＰａｔｉｅｎｔｓｗｉｔｈＶｉｔｉｌｉｇｏ［Ａｂｓｔｒａｃｔ］ＯｂｊｅｃｔｉｖｅＴｏｉｎｖｅｓｔｉｇａｌｅｔｈｅｅｘｐｒｅｓｓｉｏｎｏｆＴｈｌ＆Ｔｈ２ｃｅｌｌ—ｍｅｄｉａｔｅｄｃｙｔｏｋｉｎｅｐｒｏｆｉｌｅｉｎＶｉｔｉｌｉｇｏｐａｔｉｅｎｔｓ．ＭｅｔｈｏｄｓＰＢＭＣｆｒｏｍｂｏｔｈＶｉｔｉｌｉｇｏａｎｄｎｏｒｍａｌｃｏｎｔｒｏｌＳｗｅｒｅｃｕｌｔｕｒｅｄｗｉｔｈａｎｄｗｉｔｈｏｕｔＣｏｎｈ．ＣｕｌｔｕｒｅｓｕｐｅｒｎａｔａｎｔｓｗｅｒｅａｓｓａｙｅｄｆｏｒＩＬ一２．ＩＩ。