CSB-E04577h

Human Interferon γ (IFN-γ) ELISA Kit Catalog No. CSB-E04577h (96T) This immunoassay kit allows for the in vitro quantitative determination of human IFN-γ concentrations in cell culture supernates, serum, plasma and other biological fluids. Expiration date six months from the date of manufacture FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. CUSABIO BIOTECH CO., Ltd. http://www.cusabio.com/ http://www.cusabio.cn/ E-mail: cusabio@cusabio.com cusabio@cusabio.cn 1

INTRODUCTION Interferon-gamma (IFN-γ) is a dimerized soluble cytokine that is the only member of the type II class of interferons. This interferon was originally called macrophage-activating factor. The IFN-γ monomer consists of a core of six α-helices and an extended unfolded sequence in the C-terminal region. This is shown in the structural models below. The α-helices in the core of the structure are numbered 1 to 6. Full length IFN-γ is 143 amino acids in length, the models are 126 amino acids in length. Affinity for the glycosaminoglycan heparan sulfate resides solely within the deleted sequence of 17 amino acids. In contrast to interferon-α and interferon-β which can be expressed by all cells, IFN-γ is secreted by Th1 cells, Tc cells, dendritic cells and NK cells. Also known as immune interferon, IFN-γ is the only Type II interferon. It is serologically distinct from Type I interferons and it is acid-labile, while the type I variants are acid-stable. IFN-γ has antiviral, immunoregulatory, and anti-tumour properties. It alters transcription in up to 30 genes producing a variety of physiological and cellular responses. Amongst the effects are: Increase antigen presentation of macrophages. Activate and increase lysosome activity in macrophages. Suppress Th2 cell activity. Cause normal cells to increase expression of class I MHC molecules. Promotes adhesion and binding required for leukocyte migration Promotes NK cell activity. Activation by IFN-γ is achieved by its interaction with a heterodimeric receptor consisting of 2

IFNGR1 & IFNGR2 (interferon gamma receptors). IFN-γ binding to the receptor activates the JAK-STAT pathway. In addition, IFN-γ activates APCs and promotes Th1 differentiation by upregulating the transcription factor T-bet. IFN-γ is the hallmark cytokine of Th1 cells (whereas Th2 cells produce IL-4 and Th17 cells produce IL-17). NK cells and CD8+ cytotoxic T cells also produce IFN-γ. IFN-γ suppresses osteoclast formation by rapidly degrading the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway, which otherwise stimulates the production of NFκB. PRINCIPLE OF THE ASSAY The microtiter plate provided in this kit has been pre-coated with an antibody specific to IFN-γ. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IFN-γ and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain IFN-γ, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of IFN-γ in the samples is then determined by comparing the O.D. of the samples to the standard curve. 3

DETECTION RANGE 0.78 ng/ml-50 ng/ml. The standard curve concentrations used for the ELISA s were 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml, 1.56 ng/ml, 0.78 ng/ml. SPECIFICITY This assay recognizes recombinant and natural human IFN-γ. No significant cross-reactivity or interference was observed. SENSITIVITY The minimum detectable dose of human IFN-γ is typically less than 0.195 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. MATERIALS PROVIDED Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120µl HRP-avidin 1 x 120µl Wash Buffer 1 x 20 ml (25 concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml 4

STORAGE 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. REAGENT PREPARATION Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 50 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (50 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). 5

3. Biotin-antibody Dilute to the working concentration specified on the vial label using Biotin-antibody Diluent(1:100), respectively. 4. HRP-avidin Dilute to the working concentration specified on the vial label using HRP-avidin Diluent(1:100), respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. OTHER SUPPLIES REQUIRED Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. SAMPLE COLLECTION AND STORAGE Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 C. Avoid repeated freeze-thaw cycles. Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 C. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay. 6

ASSAY PROCEDURE Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. 1. Add 100µl of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 C. 2. Remove the liquid of each well, don t wash. 3. Add 100µl of Biotin-antibody working solution to each well. Incubate for 1 hour at 37 C. Biotin-antibody working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform. 4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (200µl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. 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. 5. Add 100µl of HRP-avidin working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37 C. 6. Repeat the aspiration and wash three times as step 4. 7. Add 90µl of TMB Substrate to each well. Incubate for 30 minutes at 37 C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 8. Add 50µl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 9. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. 7

CALCULATION OF RESULTS Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. 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 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 IFN-γ 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. LIMITATIONS OF THE PROCEDURE 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. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. 8

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 Quantikine Immunoassay, the possibility of interference cannot be excluded. TECHNICAL HINTS When mixing or reconstituting protein solutions, always avoid foaming. 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. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution 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 Substrate Solution. 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 Substrate Solution. 9

人 γ 干扰素 (IFN-γ) 酶联免疫分析试剂盒说明书本试剂盒仅供研究使用 产品编号 产品编号 :CSB-E04577h 检测范围 检测范围 :0.78 ng/ml - 50 ng/ml 最低检测限 最低检测限 :0.195 ng/ml 特异性 : 本试剂盒可同时检测天然或重组的人 IFN-γ, 且与其他相关蛋白无 交叉反应 有效期 有效期 :6 个月 预期应用 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 IFN-γ 含量 说明 : 1. 试剂盒保存 :-20 ( 较长时间不用时 );2-8 ( 频繁使用时 ) 2. 浓洗涤液低温保存会有盐析出, 稀释时可在水浴中加温助溶 3. 中 英文说明书可能会有不一致之处, 请以英文说明书为准 4. 刚开启的酶联板孔中可能会含有少许水样物质, 此为正常现象, 不会对实验结果造成任何影响 实验原理 : 用纯化的抗体包被微孔板, 制成固相载体, 往包被抗 IFN-γ 抗体的微孔中依次加入标本或标准品 生物素化的抗 IFN-γ 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 IFN-γ 呈正相关 用酶标仪在 450nm 波长下测定吸光度 (OD 值 ), 计算样品浓度 10

试剂盒组成及试剂配制 : 1. 酶联板 (Assay plate ): 一块 (96 孔 ) 2. 标准品 (Standard): 2 瓶 ( 冻干品 ) 3. 样品稀释液 (Sample Diluent): 1 20ml/ 瓶 4. 生物素标记抗体稀释液 (Biotin-antibody Diluent): 1 10ml/ 瓶 5. 辣根过氧化物酶标记亲和素稀释液 (HRP-avidin Diluent): 1 10ml/ 瓶 6. 生物素标记抗体 (Biotin-antibody): 1 120µl/ 瓶 (1:100) 7. 辣根过氧化物酶标记亲和素 (HRP-avidin): 1 120µl/ 瓶 (1:100) 8. 底物溶液 (TMB Substrate): 1 10ml/ 瓶 9. 浓洗涤液 (Wash Buffer):1 20ml/ 瓶, 使用时每瓶用蒸馏水稀释 25 倍 10. 终止液 (Stop Solution): 1 10ml/ 瓶 需要而未提供的试剂和器材 : 1. 标准规格酶标仪 2. 高速离心机 3. 电热恒温培养箱 4. 干净的试管和 Eppendof 管 5. 系列可调节移液器及吸头, 一次检测样品较多时, 最好用多通道移液器 6. 蒸馏水, 容量瓶等 标本的采集及保存 : 1. 血清 : 全血标本请于室温放置 2 小时或 4 过夜后于 1000 x g 离心 20 分钟, 取上清即可检测, 或将标本放于 -20 或 -80 保存, 但应避免反复冻融 2. 血浆 : 可用 EDTA 或肝素作为抗凝剂, 标本采集后 30 分钟内于 2-8 C 1000 x g 离心 15 分钟, 或将标本放于 -20 或 -80 保存, 但应避免反复冻融 3. 细胞培养物上清或其它生物标本 :1000 x g 离心 20 分钟, 取上清即可检测, 或将标本放于 -20 或 -80 保存, 但应避免反复冻融 注 : 标本溶血会影响最后检测结果, 因此溶血标本不宜进行此项检测 11

标本的稀释原则 : 首先通过文献检索的方式了解待测样本的大致含量, 确定适当的稀释倍数 只有稀释至标准曲线的范围内, 检测的结果才是准确的 稀释的过程中, 应做好详细的记录 最后计算浓度时, 稀释了 N 倍, 标本的浓度应再乘以 N 本产品不推荐稀释样本, 如果个别样本 OD 值过高, 可以稀释后再检测 标准品的稀释原则 : 2 瓶, 每瓶临用前以样品稀释液稀释至 1ml, 盖好后静置 10 分钟以上, 然后反复颠倒 / 搓动以助溶解, 其浓度为 50 ng/ml, 做系列倍比稀释后, 分别稀释 50 ng/ml,25 ng/ml,12.5 ng/ml,6.25 ng/ml,3.12 ng/ml,1.56 ng/ml,0.78 ng/ml, 样品稀释液直接作为标准浓度 0 ng/ml, 临用前 15 分钟内配制 如配制 25 ng/ml 标准品 : 取 0.5ml( 不要少于 0.5ml)50 ng/ml 的上述标准品加入含 0.5ml 样品稀释液的 Eppendorf 管中, 混匀即可, 其余浓度以此类推 生物素标记抗体的稀释原则 : 临用前以生物素标记抗体稀释液稀释, 稀释前根据预先计算好的每次实验所需的总量配制 ( 每孔 100µl), 实际配制时应多配制 0.1-0.2ml 如 10µl 生物素标记抗体加 990µl 生物素标记抗体稀释液的比例配制, 轻轻混匀, 在使用前一小时内配制 辣根过氧化物酶标记亲和素的稀释原则 : 临用前以辣根过氧化物酶标记亲和素稀释液稀释, 稀释前根据预先计算好的每次实验所需的总量配制 ( 每孔 100µl), 实际配制时应多配制 0.1-0.2ml 如 10µl 辣根过氧化物酶标记亲和素加 990µl 辣根过氧化物酶标记亲和素稀释液的比例配制, 轻轻混匀, 在使用前一小时内配制 操作步骤 : 实验开始前, 请提前配置好所有试剂, 试剂或样品稀释时, 均需混匀, 混匀时尽量避免起泡 每次检测都应该做标准曲线 如样品浓度过高时, 用样品 12

稀释液进行稀释, 以使样品符合试剂盒的检测范围 1. 加样 : 分别设空白孔 标准孔 待测样品孔 空白孔加样品稀释液 100µl, 余孔分别加标准品或待测样品 100µl, 注意不要有气泡, 加样将样品加于酶标板孔底部, 尽量不触及孔壁, 轻轻晃动混匀, 酶标板加上盖或覆膜, 37 反应 120 分钟 为保证实验结果有效性, 每次实验请使用新的标准品溶液 2. 弃去液体, 甩干, 不用洗涤 每孔加生物素标记抗体工作液 100µl( 取 1µl 生物素标记抗体加 99µl 生物素标记抗体稀释液的比例配制, 轻轻混匀, 在使用前一小时内配制 ),37,60 分钟 3. 温育 60 分钟后, 弃去孔内液体, 甩干, 洗板 3 次, 每次浸泡 1-2 分钟, 200µl/ 每孔, 甩干 4. 每孔加辣根过氧化物酶标记亲和素工作液 ( 同生物素标记抗体工作液 ) 100µl,37,60 分钟 5. 温育 60 分钟后, 弃去孔内液体, 甩干, 洗板 5 次, 每次浸泡 1-2 分钟, 200µl/ 每孔, 甩干 6. 依序每孔加底物溶液 90µl,37 避光显色 (30 分钟内, 此时肉眼可见标准品的前 3-4 孔有明显的梯度蓝色, 后 3-4 孔梯度不明显, 即可终止 ) 7. 依序每孔加终止溶液 50µl, 终止反应 ( 此时蓝色立转黄色 ) 终止液的加入顺序应尽量与底物液的加入顺序相同 为了保证实验结果的准确性, 底物反应时间到后应尽快加入终止液 8. 用酶联仪在 450nm 波长依序测量各孔的光密度 (OD 值 ) 在加终止液后 15 分钟以内进行检测 实验备注 1. 用户在初次使用试剂盒时, 应将各种试剂管离心数分钟, 以便试剂集中到管底 2. 每次实验留一孔作为空白调零孔, 该孔不加任何试剂, 只是最后加底物溶液及 2N H 2 SO4 测量时先用此孔调 OD 值至零 3. 为防止样品蒸发, 试验时将反应板放于铺有湿布的密闭盒内, 酶标板加上盖或覆膜 4. 未使用完的酶标板或者试剂, 请于 2-8 保存 标准品 生物素标记抗体工作液 辣根过氧化物酶标记亲和素工作液请依据所需的量配置使用 请勿重复使用已稀释过的标准品 生物素标记抗体工作液 或辣根过氧化物酶标记亲和素工作液 13

5. 建议检测样品时均设双孔测定, 以保证检测结果的准确性 洗板方法 : 手工洗板方法 : 吸去 ( 不可触及板壁 ) 或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 ; 将推荐的洗涤缓冲液至少 0.3ml 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 计算 : 以标准物的浓度为横坐标 ( 对数坐标 ),OD 值为纵坐标 ( 普通坐标 ), 在半对数坐标纸上绘出标准曲线, 根据样品的 OD 值由标准曲线查出相应的浓度 ; 再乘以稀释倍数 ; 或用标准物的浓度与 OD 值计算出标准曲线的直线回归方程式, 将样品的 OD 值代入方程式, 计算出样品浓度, 再乘以稀释倍数, 即为样品的实际浓度 注意事项 : 1. 本操作说明适用于 48T 试剂盒, 48T 试剂盒中酶联板中酶联板 标准品标准品 生物素标记抗体及辣根过氧化物酶标记亲和素减半 2. 当混合蛋白溶液时应尽量轻缓, 避免起泡 3. 洗涤过程非常重要, 不充分的洗涤易造成假阳性 4. 一次加样时间最好控制在 5 分钟内, 如标本数量多, 推荐使用排枪加样 5. 请每次测定的同时做标准曲线, 最好做复孔 6. 如标本中待测物质含量过高, 请先稀释后再测定, 计算时请最后乘以稀释倍数 7. 在配制标准品 检测溶液工作液时, 请以相应的稀释液配制, 不能混淆 8. 底物请避光保存 9. 不要用其它生产厂家的试剂替换试剂盒中的试剂 14

Notes 15

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