Human TNF‑a ELISA KitCatalog Number KHC3011 (96 tests), KHC3012 (2 × 96 tests), and KHC3011C (5 × 96 tests)Pub. No. MAN0003931 Rev.5.0 (32)CAUTION! This kit contains materials with small quantities of sodium azide and Proclin™ 300. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. In case of contact, rinse affected area with plenty of water. Proclin™ 300 is toxic, corrosive, and a skin irritant. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state, and local regulations for disposal.Note: For safety and biohazard guidelines, see the “Safety” appendix in the ELISA Technical Guide (Pub. no. MAN0006706). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.Product descriptionThe Invitrogen™ Human TNF-a ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA). This assay is designed to detect and quantify the level of human TNF-a in human serum, plasma, buffered solutions, or cell culture medium. The assay recognizes both natural and recombinant human TNF-a.Human tumor necrosis factor alpha (Hu TNF-a), also called cachectin, is a 157 AA non-glycosylated polypeptide cytokine produced by activated macrophages. Lipopolysaccharide (LPS) is a potent stimulus for TNF-a production in macrophages, and TNF-a is an important mediator of the in vivo effects of LPS. The biological activities of TNF-a may be classified as:- Immunomodulating and proinflammatory: TNF-a regulates the production of antibodies by B cells and stimulates cytotoxic T cells.- Metabolic: TNF-a strongly inhibits lipoprotein lipase and adipocyte gene expression.Contents and storageUpon receipt, store the kit at 2°C to 8°C.Materials required but not supplied•Distilled or deionized water•Microtiter plate reader with software capable of measurement at or near 450 nm•Plate washer–automated or manual (squirt bottle, manifolddispenser, or equivalent)•Calibrated adjustable precision pipettes and glass or plastic tubes for diluting solutions Before you beginIMPORTANT! Reagents are lot-specific. Do not mix or interchange different reagent lots from various kit lots.•Review the Procedural guidelines and Plate washing directions in the ELISA Technical Guide available at .•Allow reagents to reach room temperature before use. Mix toredissolve any precipitated salts.Prepare 1X Wash Buffer1.Dilute 16 mL of Wash Buffer Concentrate (25X) with 384 mL ofdeionized or distilled water. Label as 1X Wash Buffer.2.Store the concentrate and 1X Wash Buffer in the refrigerator. Usethe diluted buffer within 25 days.Sample preparation guidelines•Refer to the ELISA Technical Guide at for detailed sample preparation procedures.•Collect samples in pyrogen/endotoxin-free tubes.•Freeze samples after collection if samples will not be tested immediately. Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely and mix well (do not vortex) prior to analysis.•Avoid the use of hemolyzed or lipemic sera. If large amounts of particulate matter are present in the sample, centrifuge or filter sample prior to analysis.Pre-dilute samplesSample concentrations should be within the range of the standard curve. Because conditions may vary, each investigator should determine the optimal dilution for each application.Perform sample dilutions with Standard Diluent Buffer (serum/plasma) or with the corresponding cell culture medium (cell culture supernatant). Dilute standardsNote: Use glass or plastic tubes for diluting standards.Note: This assay has been calibrated against the International Standard preparation (87/650) for Hu TNF-a (NIBSC, Hertforshire, UK, EN6 3QG). One microgram equals 40,000 International Units.1.Reconstitute Hu TNF-a Standard to 2000 pg/mL with Standard Dilution Buffer. Refer to the standard vial label for instructions. Swirl or mixgently and allow the contents to sit for 10 minutes to ensure complete reconstitution. Label as 2000 pg/mL human TNF-a. Use the standard within 1 hour of reconstitution.2.Add 300 µL Reconstituted Standard to one tube containing 300 µL Standard Diluent Buffer and mix. Label as 1000 pg/mL human TNF-a.3.Add 300 µL Standard Diluent Buffer to each of 7 tubes labeled as follows: 500, 250, 125, 62.5, 31.2, 15.6, and 0 pg/mL human TNF-a.4.Make serial dilutions of the standard as shown in the following dilution diagram. Mix thoroughly between steps.5.Discard all remaining reconstituted and diluted standards after completing assay. Return the Standard Diluent Buffer to the refrigerator.DiluentVolumeStd5Std4Std3Std2Std1Std6Std7Std0300 μL250 pg/mL1000 pg/mL500 pg/mL15.6 pg/mL31.2 pg/mL62.5 pg/mL125 pg/mL2000 pg/mL300 μL300 μL300 μL300 μL300 μL300 μL300 μL300 μL0 pg/mLPrepare 1X Streptavidin‑HRP solutionNote: Prepare 1X Streptavidin-HRP within 15 minutes of usage.The Streptavidin-HRP (100X) is in 50% glycerol, which is viscous. To ensure accurate dilution:1.For each 8-well strip used in the assay, pipet 10 µL Streptavidin-HRP (100X) solution, wipe the pipette tip with clean absorbent paper toremove any excess solution, and dispense the solution into a tube containing 1 mL of Streptavidin-HRP Diluent. Mix thoroughly.2.Return the unused Streptavidin-HRP (100X) solution to the refrigerator.Perform ELISA (Total assay time: 4 hours)IMPORTANT! Perform a standard curve with each assay.•Allow all components to reach room temperature before use. Mix all liquid reagents prior to use.•Determine the number of 8-well strips required for the assay. Insert the strips in the frames for use. Re-bag any unused strips and frames, andstore at 2°C to 8°C for future use.a.Add 50 µL of Incubation Buffer to wells for serum or plasma samples, standards, or controls; or 50 µLof Standard Diluent Buffer to the wells for cell culture samples. Leave the wells for chromogen blanksempty.b.Add 100 µL of standards, controls, or samples (see “Pre-dilute samples” on page 2) to the appropriatewells. Leave the wells for chromogen blanks empty.c.Tap the side of the plate to mix. Cover the plate with a plate cover and incubate for 2 hours at roomtemperature.d.Thoroughly aspirate the solution and wash wells 4 times with 1X Wash Buffer.1Bind antigenCulture media +Standard DiluentBufferStandards/Serum/Plasma/Controls +Incubation Buffera.Add 100 µL Hu TNF-a Biotin Conjugate solution into each well except the chromogen blanks.2Add Biotin ConjugateAdd Streptavidin‑HRPAdd Stabilized ChromogenAdd Stop Solution1.Read the absorbance at 450 nm. Read the plate within 2 hours after adding the Stop Solution.e curve-fitting software to generate the standard curve. A four parameter algorithm provides the best standard curve fit. Optimally, thebackground absorbance may be subtracted from all data points, including standards, unknowns and controls, prior to plotting.3.Read the concentrations for unknown samples and controls from the standard curve. Multiply value(s) obtained for sample(s) by theappropriate factor to correct for the sample dilution.Note: Dilute samples producing signals greater than the upper limit of the standard curve in Standard Diluent Buffer (serum/plasma) or with the corresponding cell culture medium (cell culture supernatant) and reanalyze. Multiply the concentration by the appropriate dilution factor. Performance characteristicsStandard curve exampleThe following data were obtained for the various standards over therange of 0−1000 pg/mL human TNF-a.Inter-assay precisionSamples were assayed 18 times in multiple assays to determineprecision between assays.Intra-assay precisionSamples of known human TNF-a concentration were assayed inreplicates of 16 to determine precision within an assay.Expected valuesHuman PBMCs or whole blood were stimulated from 4 to 72 hours with lipopolysaccharide (LPS), phytohaemagglutinin (PHA), or ionomycin and phorbol myristate acetate (PMA) then evaluated for the presence of human TNF-a in this assay. Whole blood samples were pre-diluted 10-fold for the assay.Linearity of dilutionHuman serum containing 806 pg/mL of measured human TNF-a was serially diluted in Standard Diluent Buffer over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.RecoveryThe average recovery of Human TNF-a ELISA Kit added to a variety of samples is listed in the following table.SensitivityThe analytical sensitivity of ths assay is 1.7 pg/mL human TNF-a. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times. SpecificityBuffered solutions of The following panel of substances were assayed at 50 ng/mL and were found to have no cross-reactivity: human IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IFN-a, IFN-b, IFN-g, GM-CSF, OSM,MIP-1a, MIP-1b, LIF, MCP-1, G-CSF, TGF-b, RANTES; swine TNF-a; rat TNF-a; mouse TNF-a.Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at /us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at /support.Product label explanation of symbols and warningsManufacturer's address: Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, AustriaThe information in this guide is subject to change without notice.DISCLAIMERTO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified./support | /askaquestion。
人(human)肿瘤坏死因子a(TNF-a)说明书本试剂盒仅供研究使用标本：血清或者血浆一、试剂组成精密度微孔板96孔 (Microtitration Strips) 1块 2～8℃干燥保存酶标偶合液 (Conjugate ) 1瓶 12.0毫升 2～8℃冷藏保存标准品 (Standard) 5瓶各1.0毫升 2～8℃冷藏保存呈色剂A (Substrate A) 1瓶 6.0毫升 2～8℃避光冷藏保存呈色剂B (Substrate B) 1瓶 6.0毫升 2～8℃避光冷藏保存终止液 (Stopping Solution) 1瓶 6.0毫升室温保存20倍浓缩洗涤液 (Rinsing Buffer x 20) 1瓶 60.0毫升 2～8℃冷藏保存5倍浓缩样品稀释液 (Diluent x 5) 1瓶 15.0ml 2～8℃冷藏保存英文说明书，中文说明书各一份室温保存二、注意事项1. 此试剂为体外检测试剂，效期内使用，试剂应视为传染物，不同总批号的试剂不能混用。
2．使用前应将盒内各试剂取出。
室温放置至少30分钟，3．浓缩洗涤液出现结晶后，请于37℃孵育15分钟。
4．浓缩样品稀释液出现结晶后，请于37℃孵育15分钟5．若24小时内进行实验，标本可存放于2～8℃。
不需及时实验，标本-20℃保存，避免反复冻融。
6．在反复清洗微孔板，并扣干微孔中的残余液体，否则将降低精确度，造成吸光度偏离的假像。
7．加样完毕后，应注意轻微摇动微孔反应条，以便使孔中的液体充分混匀。
8．试剂盒保存于2～8℃，请勿冷冻，有效期请见盒内标示。
三、实验前准备1．使用前应将盒内各试剂取出，室温放置至少30分钟。
2．准备各种实验仪器及材料，如微量移液器，吸头，医用蒸馏水等3．浓缩洗液与医用蒸馏水1︰19倍稀释后成为应用洗涤液4．浓缩样品稀释液与医用蒸馏水1︰4倍稀释成应用样品稀释液5．用应用样品稀释液来稀释样品，按照1：100的体积比来稀释样品如10μl的样品加入到1ml的应用样品稀释液中，充分混匀待用。
TNF-T N F 根据来源和结构不同分为T N F- a 、T N F -?，其中T N F-a 与⾻质疏松关系密切。
T N F 主要由单核巨噬细胞产⽣，另外，活化的T 细胞、⾃然杀伤细胞、肥⼤细胞、软⾻细胞也能分泌这种因⼦。
单核巨噬细胞合成的TN F 是⼀个25 ku 左右的⾮糖化跨膜蛋⽩，有两种不同的受体(P55 ，P75 ) ，其分⼦量分别为55 ku 和75 ku 。
T NF 与受体结合后，信号传⼈细胞内，通过N F -xB 或活化蛋⽩(A P )．1 转录因⼦来实现其功能。
⼈的TN F ．a 基因位于第 6 对染⾊体上。
1975年E.A. Carswell等⼈发现接种卡介苗的⼩⿏注射细菌脂多糖后，⾎清中出现⼀种能使多种肿瘤发⽣出⾎性坏死的物质，将其命名为肿瘤坏死因⼦（tumor necrosis factor，TNF)。
⼋⼗年代⼈们发现其在消耗症中起了重要作⽤，⼜称恶液质素。
TNF主要由活化的巨噬细胞，NK细胞及T淋巴细胞产⽣。
1985年Shalaby把巨噬细胞产⽣的TNF命名为TNF-α，把T淋巴细胞产⽣的淋巴毒素（lymphotoxin，LT)命名为TNF-β。
虽然TNF-α与TNF-β仅有约30%的同源性，但它们却拥有共同的受体。
TNFα的⽣物学活性占TNF总活性的70 %～95 %，因此⽬前常说的TNF多指TNF-α。
1984年TNF基因的克隆开辟了临床试验的时代，是第⼀个⽤于肿瘤⽣物疗法的细胞因⼦，但因其缺少靶向性且有严重的副作⽤，⽬前仅⽤于局部治疗。
⼈类TNF-α基因于1985年成功克隆，定位于6p21.4，长约3.6 kbp，有4个外显⼦和3个内含⼦，与主要组织相容性复合体（MHC）基因紧密连锁位于HLA-B 和 HLA-C2 位点之间的 MHC3 类基因区内，由TNFA和TNFB组成，分别编码TNFα和TNFβ。
位于启动⼦区238位和308位存在单核苷酸多态性，被认为可调节TNF 的转录⽔平，与慢性⼄肝、⾃⾝免疫性疾病、胰岛素抵抗、肿瘤等多种疾病的易感性相关。
人肿瘤坏死因子α（TNF-α）酶联免疫分析（ELISA）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，细胞上清及相关液体样本中肿瘤坏死因子α（TNF-α）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肿瘤坏死因子α（TNF-α）水平。
用纯化的人肿瘤坏死因子α（TNF-α）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肿瘤坏死因子α，再与HRP标记的肿瘤坏死因子α（TNF-α）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人肿瘤坏死因子α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肿瘤坏死因子α（TNF-α）浓度。
试剂盒组成：试剂盒组成48孔配置96孔配置保存说明书1份1份封板膜2片（48）2片（96）密封袋1个1个酶标包被板1×481×962-8℃保存标准品：450pg/ml0.5ml×1瓶0.5ml×1瓶2-8℃保存标准品稀释液 1.5ml×1瓶 1.5ml×1瓶2-8℃保存酶标试剂3ml×1瓶6ml×1瓶2-8℃保存样品稀释液3ml×1瓶6ml×1瓶2-8℃保存显色剂A液3ml×1瓶6ml×1瓶2-8℃保存显色剂B液3ml×1瓶6ml×1瓶2-8℃保存终止液3ml×1瓶6ml×1瓶2-8℃保存浓缩洗涤液（20ml×20倍）×1瓶（20ml×30倍）×1瓶2-8℃保存样本处理及要求：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。
