Table of Contents Introduction Description 1 Principle of the Assay 1 Limitations of the Procedure 1 General Information Materials Provided 3 Other Su

Transcription

1 This package insert must be read entirely before using this product. For proper performance, use the insert provided with each individual kit received. Catalog Number EK T EK T EK T EK T EK T For the quantitative determination of human β Nerve Growth Factor (β-ngf) concentration in cell Human β-ngf ELISA Kit culture supernates, serum and plasma. For research use only. Not for use in diagnostic procedures. 08/2016

2 Table of Contents Introduction Description 1 Principle of the Assay 1 Limitations of the Procedure 1 General Information Materials Provided 3 Other Supplies required 3 Storage 5 Precaution 7 Technical Hints 9 Assay Protocol Sample Collection and Storage 9 Reagent Preparation 11,13 Assay Procedure 15 Analysis Calculation of Results 17 Typical Data 17 Sensitivity 19 Precision 19 Recovery 21 Linearity 21 Calibration 21 Sample Values 23 Specificity 23 Assay Procedure Summary 25 目录 产品介绍 背景介绍 2 检测原理 2 试剂盒检测的局限 2 基本信息 试剂盒提供的材料 4 未提供的材料设备 4 贮存 6 注意事项 8 技术要点 10 检测步骤 样本的收集和贮存 10 试剂准备 12, 14 检测步骤 16 分析 结果计算 18 典型数据 18 灵敏度 20 精密度 20 回收率 22 稀释线性 22 校准 22 样本值 24 特异性 24 检测步骤概要 26

3 DESCRIPTION Nerve growth factor (NGF) is a neuropeptide primarily involved in the regulation of growth, proliferation, and survival of certain target neurons. NGF is initially in a 7S, 130 kda complex of 3 proteins - α-ngf, β-ngf, and γ-ngf (2:1:2 ratio) when expressed. The term "Nerve Growth Factor" usually refers to the 2.5S, 26 kda β subunit of the protein. The β subunit is the only component of the 7S NGF complex that is biologically active (i.e. acting as signaling molecules). NGF plays a critical role in the regulation of both innate and acquired immunity. Studies suggest that NGF circulates throughout the entire body via the blood plasma, and is important for the overall maintenance of homeostasis. NGF appears to promote myelin repair and could also be involved in various psychiatric disorders, such as dementia, depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimia nervosa. Dysregulation of NGF signaling has also been linked to Alzheimer's disease. Also, NGF has been shown to play a role in number cardiovascular diseases, such as coronary atherosclerosis, obesity, type 2 diabetes, and metabolic syndrome. NGF has also been shown to accelerate wound healing. PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human β-ngf has been pre-coated onto a microplate. Standard, samples and biotin-linked detect antibody specific for β-ngf are pipetted into the wells and β-ngf present is bound by the immobilized antibody and detect antibody following incubation. After washing away any unbound substances, streptavidin-hrp is added. After washing, substrate solution is added to the wells and color develops in proportion to the amount of β-ngf bound in the initial step. The color development is stopped and the intensity of the color is measured. LIMITATIONS OF THE KIT FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. 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 Assay Buffer and repeat the assay. If cell culture supernate samples require large dilution, perform an intermediate dilution in culture medium. Any variation in standard dilution, operator, pipetting technique, washing techniques, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by factors present in biological samples. Until all factors have been tested in the SunnyELISA immunoassay, the possibility of interference cannot be excluded. 1 背景介绍 神经生长因子 (NGF) 是一种神经肽, 主要参与调节某些靶神经元的生长 增殖和存活 当表达时,NGF 最初是以 7S 分子量为 130kDa 的复合物存在, 这个复合物由 α-ngf β-ngf 和 γ-ngf (2:1:2 比例 ) 三个蛋白组成 神经生长因子 通常指的是 2.5S 分子量为 26kDa 的 β 亚基蛋白 β 亚基是 7S NGF 复合物中唯一一个有生物活性的成分 ( 如作为信号分子 ) NGF 在先天性和获得性免疫中都起着重要作用 研究表明 NGF 通过血浆在机体中循环, 对整体的稳态维持很重要 NGF 可促进髓鞘的修复, 亦可参与多种精神疾病, 如老年痴呆症 抑郁症 精神分裂症 自闭症 雷特综合征 神经性厌食症, 神经性贪食症 NGF 信号失调与阿尔茨海默病有关 同样,NGF 在许多心血管疾病中发挥作用, 如冠状动脉粥样硬化 肥胖 2 型糖尿病 代谢综合征 NGF 也可促进伤口愈合 检测原理 本试剂盒采用双抗体夹心酶联免疫吸附检测技术 特异性抗人 β-ngf 单克隆抗体预包被在高亲和力的酶标板上 酶标板孔中加入标准品 待测样本和生物素化的检测抗体, 经过孵育, 样本中存在的 β-ngf 与固相抗体和检测抗体结合 洗涤去除未结合的物质后, 加入辣根过氧化物酶标记的链霉亲和素 (streptavidin-hrp) 洗涤后, 加入显色底物 TMB, 避光显色 颜色反应的深浅与样本中 β-ngf 的浓度成正比 加入终止液终止反应, 在 450 nm 波长 ( 参考波长 nm) 测定吸光度值 试剂盒检测的局限 1. 本试剂盒用于科学研究, 非诊断试剂, 不能用于临床诊断 2. 请在本试剂盒标记的有效期内使用 3. 试剂盒的试剂不能与其他批号的试剂或其他来源的试剂混合使用 4. 如果样本值高于标准曲线最高浓度, 请用检测缓冲液稀释样本, 并重新检测 如果细胞培养上清样本需要较大的稀释倍数, 请用细胞培养基进行适度稀释 5. 任何标准品稀释 操作人员 移液技术 洗涤技术 孵育温度 试剂盒保存时间的改变, 都将影响结合反应 6. 本试剂盒在设计上去除或降低了生物学样本中的一些内源性干扰因素, 并非所有可能的影响因素都已经去除 2

4 MATERIALS PROVIDED 1. β-ngf Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against human β-ngf. 2. β-ngf Standard: Recombinant human β-ngf in a buffered protein base with preservatives; lyophilized. 3. β-ngf Detect Antibody: Biotin-conjugate anti-human β-ngf detect antibody; 100 liquid. 4. Standard Diluent: In some, very rare cases, an insoluble precipitate of stabilizing protein has been seen in the Standard Diluent vial. This precipitate does not interfere in any way with the performance of the test and can thus be ignored. 5. Streptavidin-HRP: 100 liquid. 6. Assay Buffer (10 ): PBS with 0.5 % Tween-20 and 5 % BSA. 7. Substrate: TMB (tetramethyl-benzidine). 8. Stop Solution: 0.18M sulfuric acid. 9. Wash Buffer (20 ): PBS with 1 % Tween Plate Covers. 试剂盒提供的材料 组分 编号 EK11411 EK11412 EK11413 EK11414 EK11415 预包被酶标板 EK1141P 48T 96T 192T 480T 960T 标准品 EK1141S 1 vial 2 vial 4 vial 10 vial 20 vial 检测抗体 EK1141D 1 vial 1 vial 2 vial 5 vial 10 vial 标准品稀释液 EKSP80 5 ml 5 ml 10 ml 25 ml 50 ml 辣根过氧化物酶标记的链霉亲和素 E vial 1 vial 2 vial 5 vial 10 vial 10 检测缓冲液 E ml 5 ml 10 ml 25 ml 50 ml 显色底物 TMB E ml 11 ml 22 ml 55 ml 110 ml 终止液 E ml 11 ml 22 ml 55 ml 110 ml 20 洗液 E ml 50 ml 100 ml 250 ml 500 ml All reagents should be stored at 2-8. 封板膜 E OTHER SUPPLIES REQUIRED 1. Microplate reader capable of measuring absorbance at 450 nm, with correction wavelength set at 570 nm or 630 nm. 2. Pipettes and pipette tips μl to 300 μl adjustable multichannel micropipette with disposable tips. 4. Multichannel micropipette reservoir. 5. Beakers, flasks, cylinders necessary for preparation of reagents. 6. Deionized or distilled water. 7. Polypropylene test tubes for dilution. 未提供的材料设备 1. 能够检测 450 nm 吸光度的酶标仪, 参考波长 570 nm 或 630 nm 2. 移液器及枪头 μl 可调多道移液器及一次性枪头 4. 多道移液器加样槽 5. 准备试剂用的试管 离心管 量筒等 6. 蒸馏水或去离子水 7. 稀释用聚丙烯试管 3 4

5 STORAGE Store kit reagents between 2 and 8. Immediately after use remaining reagents should be returned to cold storage (2 to 8 ). Expiry of the kit and reagents is stated on labels. Expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, this reagent is not contaminated by the first handling. 贮存 试剂盒保存于 2-8, 有效期标注于标签上 只有恰当保存的试剂才是有保证的 如果试剂 盒的组分需要再次使用, 请确定上一次使用之后试剂没有污染 Unopened kit Store at 2-8. 未开封试剂盒 贮存于 2-8 请在有效期内使用 Opened/ Reconstituted Reagents Diluted Wash Buffer Diluted Assay Buffer Stop Solution Standard Diluent Substrate TMB Detect Antibody Streptavidin-HRP Standard Microplate Wells May be stored for up to 1 month at 2-8 for up to 1 month at -20 in a manual defrost freezer. Discard after use. Return unused wells to the foil pouch containing the desiccant pack, reseal along entire edge of zip-seal. May be stored for up to 1 month at 2-8. 打开的试剂盒或重组试剂 1 洗液 1 检测缓冲液终止液标准品稀释液底物 TMB 检测抗体辣根过氧化物酶标记的链霉亲和素标准品预包被酶标板 在 2-8, 大约可以贮存 1 个月 置于 -20, 大约可贮存 1 个月 使用后丢弃 未使用的板条请放回铝箔袋, 封好封口 在 2-8, 大约可贮存 1 个月 Provided this is within the expiration date of the kit. 5 6

6 PRECAUTION 1. All chemicals should be considered as potentially hazardous. 2. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. 3. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice. 4. The Stop Solution provided with this kit is an acid solution. Wear eyes, hand, face, and clothing protection when using this material. 5. Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures. 6. Do not mix or substitute reagents with those from other lots or other sources. 7. Do not use kit reagents beyond expiration date on label. 8. Do not expose kit reagents to strong light during storage or incubation. 9. Do not eat or smoke in areas where kit reagents or samples are handled. 10. Avoid contact of skin or mucous membranes with kit reagents or specimens. 11. Rubber or disposable latex gloves should be worn while handling kit reagents or specimens. 12. Avoid contact of substrate solution with oxidizing agents and metal. 13. Avoid splashing or generation of aerosols. 14. In order to avoid microbial contamination or cross- contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes. 15. Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagent. 16. Exposure to acid inactivates the conjugate. 17. Glass-distilled water or deionized water must be used for reagent preparation. 18. Substrate solution must be at room temperature prior to use. 19. Decontaminate and dispose specimens and all potentially contaminated materials as they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0 % sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite. 21. In some cases, an insoluble precipitate of stabilizing protein has been seen in the Standard Diluent. This precipitate does not interfere in any way with the performance of the test and can thus be ignored. Or remove precipitate by centrifuging at 6000 g for 5 minutes. 注意事项 1. 所有的化学试剂理应被认为具有潜在危害 2. 联科生物推荐只有经过良好实验室培训的工作人员方可操作本试剂盒 操作时请佩戴合适的防护设施, 例如白大衣 乳胶手套 安全眼镜等 3. 请避免试剂接触皮肤和眼睛 如不慎接触, 请立即用大量清水清洗 4. 试剂盒中的终止液为酸性溶液, 在使用终止液时, 请佩戴防护服, 及防护眼睛 手及面部的设施 5. 本试剂盒用于科学研究, 不能用于诊断治疗 6. 请不要使用其他批号或其他来源的试剂替代本试剂盒中的试剂 7. 请不要使用过期的试剂 8. 在试剂盒的贮存或孵育过程请避免强光照射 9. 在操作试剂盒或处理样本的区域请不要饮食 10. 不要让试剂或样本接触皮肤和粘膜 11. 在操作试剂盒或处理样本时请佩戴乳胶或一次性手套 12. 显色底物避免与氧化试剂和金属接触 13. 避免气溶胶的产生 14. 为了避免微生物的污染, 以及试剂与样本间的交叉污染, 请使用一次性枪头 15. 使用干净的容器配制试剂 16. 暴露于酸性环境会抑制结合 17. 试剂的准备必须使用蒸馏水或去离子水 18. 显色底物在使用之前必须平衡至室温 19. 样本可能含有传染性病原体, 处理样本和可能的污染材料的首选方法是 121.5, 最少 1 小时 20. 液体废弃物的处理 不含酸的液体废弃物, 加入 1.0 % 的次氯酸钠, 浸泡 30 分钟 含酸的液体废弃物, 请先中和, 再加入次氯酸钠 21. 有时标准品稀释液中可观察到蛋白沉淀, 该沉淀不影响使用, 可以忽略 或者可通过 6000 g 离心 5 分钟去除沉淀 7 8

7 TECHNICAL HINTS 1. When mixing or reconstituting protein solutions, always avoid foaming. 2. 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. 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. 4. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. 5. 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. 6. Stop Solution should be added to the plate in the same order as the Substrate Solution. 7. 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. 8. It is recommended that all samples and standards be assayed in duplicate. 9. Take care not to scratch the inner surface of the microwells. SAMPLE COLLECTION AND STORAGE Cell Culture Supernates Remove particulates by centrifugation at 300 g for 10 minutes and assay immediately or aliquot and store samples at -20. Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 10 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20. Plasma Collect plasma using EDTA, citrate or heparin as anticoagulant. Centrifuge at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20. Other biological samples might be suitable for use in the assay. Cell culture supernates, serum and plasma were tested with this assay. Note: Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples should be aliquoted and must be stored frozen at -20 to avoid loss of bioactive human β-ngf. If samples are to be run within 24 hours, they may be stored at 2 to 8. Avoid repeated freeze-thaw cycles. Prior to assay, the frozen sample should be brought to room temperature slowly and mixed gently. 技术要点 1. 重溶或者混合蛋白的时候, 始终避免气泡产生 2. 避免交叉污染, 在进行标准品加样 样本加样, 以及不同试剂加样的时候, 请更换枪头 不同的试剂, 使用不同的加样槽 3. 在应用自动洗板机的时候, 加入洗液之后, 请设置一个 30 秒的浸泡程序, 或者在不同的洗涤步骤对微孔板做 180 度的掉转, 这样可以提高分析的准确度 4. 为保证结果的精确性, 孵育时封好封板膜 5. 显色底物在添加之前应该是无色的 保持显色底物始终处于避光状态 6. 终止液的添加顺序应该与显色底物的添加顺序相同 7. 添加终止液之后, 底物的颜色应该由蓝色转变为黄色 如果底物呈现绿色, 说明终止液与显色底物没有充分混匀 8. 推荐所有的检测样本和标准品在检测中设复孔 9. 在任何情况下, 避免接触微孔板的内表面 样本采集与贮存细胞培养上清 300 g 离心 10 分钟去除沉淀物, 即刻检测, 或者分装,-20 以下贮存 血清样本分离管分离血清 在 1000 g 离心之前, 使血样凝集 30 分钟 吸取血清样本之后即刻检测, 或者分装,-20 以下贮存 血浆样本 EDTA 枸橼酸钠或肝素抗凝收集血浆样本 1000 g 离心 3 0 分钟收集样本 即刻检测, 或者分装,-20 以下贮存 本试剂盒可能适用于其它生物学样本 细胞培养上清 血清和血浆已经过验证 注意 : 检测前, 样本中可见的沉淀必须去除 不要使用严重溶血或高血脂的样本 样本应分装并贮存于 -20, 以避免人 β-ngf 活性的丢失 如果在 24 小时内检测, 样本可以存放在 2-8 避免样本的反复冻融 在检测前, 冷冻样本应该缓慢地恢复至室温, 轻柔地混匀 9 10

8 REAGENT PREPARATION Bring all reagents and samples to room temperature before use. If crystals have formed in the Buffer Concentrates, warm them gently until they have completely dissolved. Wash Buffer (1 ) Pour entire contents (50 ml) of the Wash Buffer (20 ) into a clean 1000 ml graduated cylinder. Bring to final volume of 1000 ml with glass-distilled or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2 to 25. Please note that Wash Buffer (1 ) is stable for 30 days. Assay Buffer (1 ) Pour the entire contents (5 ml) of the Assay Buffer (10 ) into a clean 100 ml graduated cylinder. Bring to final volume of 50 ml with distilled water. Mix gently to avoid foaming. Store at 2 to 8. Please note that the Assay Buffer (1 ) is stable for 30 days. Detect Antibody Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Detect Antibody solution with Assay Buffer (1 ) in a clean plastic tube as needed according to the Standards and samples. Note: Detect Antibody should be used within 30 minutes after dilution. Streptavidin-HRP Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1 ) in a clean plastic tube as needed according to the Standards and samples. Note: Streptavidin-HRP should be used within 30 minutes after dilution. Sample Dilution If your samples need to be diluted, Assay Buffer (1 ) is used for dilution of serum/plasma samples, and cell culture medium is used for dilution of culture supernates. 试剂准备检测前请将所有的试剂 样本恢复至室温 如果浓缩的试剂出现结晶,37 温浴, 直至结晶全部溶解 1 洗液吸取 20 浓缩洗液 50 ml 至 1 L 的量筒, 加蒸馏水或去离子水至 1000 ml, 轻轻混匀, 避免泡沫 转移至干净瓶内 2-25 贮存,1 洗液可稳定 30 天 1 检测缓冲液吸取 10 浓缩检测缓冲液 5 ml 至 100 ml 量筒, 加蒸馏水或去离子水至 50 ml, 轻轻混匀, 避免泡沫 2-8 贮存,1 检测缓冲液可稳定保存 30 天 检测抗体稀释前充分混匀 根据标准品和待测样本的数量, 用 1 检测缓冲液按 1:100 稀释浓缩的检测抗体 注意 : 请在 30 分钟内使用稀释后的检测抗体 辣根过氧化物酶标记的链霉亲和素稀释前充分混匀 根据标准品和待测样本的数量, 用 1 检测缓冲液按 1:100 稀释浓缩的辣根过氧化物酶标记的链霉亲和素 注意 : 请在 30 分钟内使用稀释后的辣根过氧化物酶标记的链霉亲和素 样本稀释如果样本需要稀释, 请用试剂盒提供的 1 检测缓冲液稀释血清 / 血浆样本, 用细胞培养基稀释细胞培养上清 11 12

9 Human β-ngf Standard Reconstitute Human β-ngf Standard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted standard = 2000 pg/ml). Allow the standard to reconstitute for minutes. Mix well prior to making dilutions. Use polypropylene tubes. 人 β-ngf 标准品用蒸馏水或去离子水重溶人 β-ngf 标准品, 重溶体积标注在人 β-ngf 标准品的标签上 轻柔地涡旋震荡, 确保充分混匀, 重溶后标准品的浓度为 2000 pg/ml 重溶后静置 分钟 稀释前充分混匀 请使用聚丙烯管进行标准品稀释 For serum/plasma samples, the concentrated human β-ngf standard 250 μl μl Standard Diluent serves as the high standard (1000 pg/ml). Pipette 250 μl of Standard Diluent into each tube. Use the high standard to produce a 1:1 dilution series. Mix each tube thoroughly before the next transfer. Standard Diluent serves as the zero standard (0 pg/ml). For cell culture supernates, the concentrated human β-ngf standard 250 μl μl cell culture medium serves as the high standard (1000 pg/ml). Pipette 250 μl of cell culture medium into each tube. Use the high standard to produce a 1:1 dilution series. Mix each tube thoroughly before the next transfer. Cell culture medium serves as the zero standard (0 pg/ml). 血清 / 血浆样本标准曲线的制作 : 取 250 μl 浓缩的人 β-ngf 标准品, 加入 250 μl 标准品稀释液, 作为标准曲线的最高浓度 (1000 pg/ml) 在每一个试管中加入 250 μl 标准品稀释液 使用高浓度标准品做 1:1 系列稀释 每次移液时, 请确保充分混匀 以标准品稀释液作为标准曲线的零浓度 细胞培养上清样本标准曲线的制作 : 取 250 μl 浓缩的人 β-ngf 标准品, 加入 250 μl 细胞培养基, 作为标准曲线的最高浓度 (1000 pg/ml) 在每一个试管中加入 250 μl 细胞培养基 使用高浓度标准品做 1:1 系列稀释 每次移液时, 确保充分混匀 以细胞培养基作为标准曲线的零浓度 13 14

10 ASSAY PROCEDURE Bring all reagents and samples to room temperature before use. 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 pouch containing the desiccant pack, and reseal. 3. Add 300 μl Wash Buffer (1 ) per well, and allow the Wash Buffer to sit in the wells for about 30 seconds before aspiration. Soaking is highly recommended to obtain a good test performance! Empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing. Do not allow wells to dry. 4. Add 100 μl of 2-fold diluted Standard in duplicate. Add 100 μl of Standard Diluent to Blank well in duplicate. 5. Add 50 μl of Assay Buffer (1 ) and 50 μl sample to the sample well. 6. Add 50 μl of diluted Detect Antibody to each well. Ensure reagent addition in step 4, 5 and 6 is uninterrupted and completed within 15 minutes. 7. Cover with an adhesive strip. Incubate at room temperature (18 to 25 ) for 2 hours on a microplate shaker set at 300 rpm. 8. Aspirate each well and wash, repeating the process five times for a total six washes. Wash by filling each well with 300μl Wash Buffer (1 ). 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. 9. Add 100 μl of diluted Streptavidin-HRP to each well. 10. Cover with a new adhesive strip. Incubate at room temperature (18 to 25 ) for 45 minutes on a microplate shaker set at 300 rpm. 11. Repeat aspiration/wash as in step Add 100 μl of Substrate Solution to each well. Incubate for 5-30 minutes at room temperature. Protect from light. 13. Add 100 μl of Stop Solution to each well. The color in the well should change from blue to yellow. If the color in the well is green or if the color change does not appear uniform, gently tap the plate to ensure thorough mixing. 14. Determine the optical density within 30 minutes, using microplate reader set to 450 nm. If wavelength correction is available, set to 570 nm or 630 nm. If wavelength correction is not available, subtract readings at 570 nm or 630 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate. 检测步骤检测之前请将所有的试剂 样本平衡至室温 1. 准备好所有需要的试剂及工作浓度标准品 2. 将不需要的板条拆卸下来, 放回装有干燥剂的铝箔袋, 重新封好封口 3. 加入 300 μl 1 洗液静置浸泡 30 秒 为了获得理想的实验结果浸泡是必须的 弃掉洗液之后, 在吸水纸上将微孔板拍干 洗板完成之后, 请立即使用微孔板, 不要让微孔板干燥 4. 复孔加入 100 μl 2 倍倍比稀释的标准品 空白孔复孔加入 100 μl 标准品稀释液 5. 样本孔加入 50 μl 1 检测缓冲液和 50 μl 样本 6. 每孔加入 50 μl 稀释的检测抗体 保证步骤 连续加样, 不要间断 加样过程在 15 分钟内完成 7. 使用封板膜封板 300 转 / 分钟振荡, 室温孵育 2 小时 8. 弃掉液体, 每孔加入 300 μl 洗液洗板, 洗涤 6 次 每次洗板, 在吸水纸上拍干 为获得理想的实验性能, 必须彻底移除残留液体 9. 每孔加入 100 μl 稀释的辣根过氧化物酶标记的链霉亲和素 10. 使用新的封板膜封板 300 转 / 分钟振荡, 室温孵育 45 分钟 11. 重复步骤 每孔加入 100 μl 显色底物 TMB, 避光, 室温孵育 5-30 分钟 13. 每孔加入 100 μl 终止液 颜色由蓝色变为黄色 如果颜色呈现绿色或者颜色的变化明显不均匀, 请轻轻叩击板框, 充分混匀 14. 在 30 分钟之内, 使用酶标仪进行双波长检测, 测定 450 nm 最大吸收波长和 570 nm 或 630 nm 参考波长下的 OD 值 校准后的 OD 值为 450 nm 的测定值减去 570 nm 或 630 nm 的测定值 仅使用 450 nm 测定会导致 OD 值偏高, 并且准确度降低 15 16

11 CALCULATION OF RESULTS Average the duplicate readings for each standards and sample and subtract the average zero standard optical density. Standard Concentration as horizontal axis, OD Value as the vertical axis, regressing the data, create a standard curve using computer software. The data may be linearized by plotting the log of the β-ngf concentrations versus the log of the OD and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. Note: The finally concentration of top standard is 1000 pg/ml. If instruction in this protocol have been followed samples have been diluted 1:1 (50 μl sample + 50 μl Assay Buffer), the concentration read from the standard curve must be multiplied by the dilution factor ( 2). If samples have been diluted following the instruction, the final dilution factor is 2. If sample have been diluted by other means, the concentration read from the standard curve must be multiplied by the appropriate dilution factor. TYPICAL DATA A standard curve should be generated for each set samples assayed. This standard curve is provided for demonstration only. 结果计算计算标准品和样本的平均 OD 值, 然后减去零浓度标准品的 OD 值 以标准品浓度为横坐标,OD 值为纵坐标, 用计算机软件进行回归拟合生成标准曲线 回归分析确定最佳拟合曲线 通过对浓度值和 OD 值取对数拟合, 可以对标准曲线进行线性化 此过程可能可以得到更多样本的浓度, 但数据的准确度会降低一些 注意 : 标准曲线最高浓度点的终浓度为 1000 pg/ml 如果完全按照说明书的步骤操作 (50 μl 样本 + 50 μl 检测缓冲液 ), 计算样本浓度时请乘以稀释因子 2 如果样本按照说明书进行了稀释, 最终的稀释倍数为 2 如果样本进行了其它方式的稀释, 计算样本浓度时请乘以相应的稀释倍数 典型数据每次检测, 每块酶标板都必须设立标准曲线 下面的标准曲线仅作为示例参考 pg/ml O.D. Average Corrected

12 SENSITIVITY The minimum detectable dose (MDD) of β-ngf is typically less than 0.78 pg/ml. The MDD was determined by adding two standard deviations to the mean optical density value of ten zero standard replicates and calculating the corresponding concentration. PRECISION Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in six separate assays to assess inter-assay precision 灵敏度人 β-ngf 的最低可检测浓度为 0.78 pg/ml 10 个零标准品浓度 OD 的平均值加上两倍 SD, 计算最低可检测浓度 精密度酶标板内精密度 3 个已知浓度的样本酶标板内重复测定 20 次, 评估酶标板内的精密度 酶标板间精密度 3 个已知浓度的样本酶标板间重复检测 6 次, 评估酶标板间的精密度 酶标板内精密度 酶标板间精密度 样本 平均值 (pg/ml) 标准差 变异系数 (%)

13 RECOVERY The spike recovery was evaluated by spiking 3 levels of human β-ngf into five health human serum samples. The un-spiked serum was used as blank in these experiments. The recovery ranged from 85 % to 115 % with an overall mean recovery of 98 %. LINEARITY To assess the linearity of the assay, five samples were spiked with high concentration of β-ngf in human serum and diluted with Standard Diluent to produce samples with values within the dynamic range of the assay. CALIBRATION This immunoassay is calibrated against a highly purified recombinant human β-ngf produced at MultiSciences. 回收率 5 份健康人血清加入 3 个不同浓度水平的人 β-ngf, 未加人 β-ngf 的血清作为本底, 计算回 收率 回收率的范围从 85 % 至 115 %, 平均回收率为 98 % 稀释线性 5 份健康人血清加入高浓度的人 β-ngf, 并在标准曲线的动力学范围内进行系列稀释, 评估 检测的线性 平均值 (%) 范围 (%) 1: : : : 校准 本试剂盒的标准品为联科生物校准的高纯度重组人 β-ngf 21 22

14 SAMPLE VALUES Serum/Plasma Thirty samples from apparently healthy volunteers were evaluated for the presence of β-ngf in this assay. No medical histories were available for the donors used in this study. 样本值 应用本试剂盒, 检测 30 份健康志愿者的血清样本, 志愿者的用药史不详 Sample Matrix Number of Samples Evaluated Range (pg/ml) Detectable (%) Mean of Detectable (pg/ml) 样本 类型 检测样本 数量 浓度范围 (pg/ml) 可测百分率 (%) 可测样本平均浓度 (pg/ml) Serum 30 n.d 血清 30 n.d n.d. = non-detectable. Samples measured below the sensitivity are considered to be non-detectable. Note: The sample range is non-physiological range. The sample range of healthy human will difference according to geographical, ethic, sample preparation, and testing personnel, equipment varies. The above information is only reference. SPECIFICITY This assay recognizes both natural and recombinant human β-ngf. The factors listed below were prepared at 1 ng/ml in Standard Diluent and assayed for cross-reactivity. Preparations of the following factors at 1 ng/ml in a mid-range rhβ-ngf control were assayed for interference. No significant cross-reactivity or interference was observed. n.d. = 测不到浓度值 样本的浓度值低于灵敏度被认为测不到浓度值 注意 : 此样本值范围非生理值范围 健康人样本的浓度范围因地域 种族 样本制备以及检测人员 设备的不同而有所不同 以上数据仅供参考 特异性本试剂盒识别天然和重组人 β-ngf 下述因子以 1 ng/ml 稀释于标准品稀释液中, 评估交叉反应活性 下述因子以 1 ng/ml 稀释于中等浓度的人 β-ngf 标准品中, 评估干扰影响 没有观察到明显的交叉反应和干扰影响 Human Mouse Rat 人小鼠大鼠 BDNF CNTF GDNF IFN-γ IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12 IL-17A IL-18 IL-21 IL-22 IL-23 MCP-1 NT-3 NT-4 TGF-β1 TNF-α VEGF IFN-γ IL-1β IL-2 IL-4 IL-6 IL-10 IL-17A TNF-α CNTF GDNF GDNF Rα IFN-γ IL-1β IL-2 IL-4 IL-6 IL-10 IL-17A TNF-α BDNF CNTF GDNF IFN-γ IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12 IL-17A IL-18 IL-21 IL-22 IL-23 MCP-1 NT-3 NT-4 TGF-β1 TNF-α VEGF IFN-γ IL-1β IL-2 IL-4 IL-6 IL-10 IL-17A TNF-α CNTF GDNF GDNF Rα IFN-γ IL-1β IL-2 IL-4 IL-6 IL-10 IL-17A TNF-α 23 24

15 ASSAY PROCEDURE SUMMARY 1. Prepare all reagents and standards as directed. 检测步骤概要 1. 准备所有的试剂和标准品 2. Add 100 μl 2-fold diluted Standard in duplicate. Add 100 μl Standard Diluent to Blank well in duplicate. 2. 复孔加入 100 μl 2 倍倍比稀释的标准品 空白孔复孔加入 100 μl 标准品稀释液 3. Add 50 μl Assay Buffer (1 ) and 50 μl sample to the sample well. 3. 样本孔加入 50 μl 1 检测缓冲液和 50 μl 样本 4. Add 50 μl diluted Detect Antibody to each well. Step 2, 3 and 4 should be completed within 15 minutes. 4. 每孔加入 50 μl 稀释的检测抗体 步骤 在 15 分钟内完成 5. Incubate for 2 hours at RT. 5. 室温孵育 2 小时 6. Aspirate and wash 6 times. 6. 洗涤 6 次 7. Add 100 μl diluted Streptavidin-HRP to each well. 7. 每孔加入 100 μl 稀释的辣根过氧化物酶标记的链霉亲和素 8. Incubate for 45 minutes at RT. 8. 室温孵育 45 分钟 9. Aspirate and wash 6 times. 9. 洗涤 6 次 10. Add 100 μl Substrate Solution to each well. Incubate for 5-30 minutes at RT. Protect from light. 11. Add 100 μl Stop Solution to each well. 12. Read at 450 nm within 30 minutes. Correction 570 or 630 nm. 10. 每孔加入 100 μl 显色底物, 避光, 室温孵育 5-30 分钟 11. 每孔加入 100 μl 终止液 分钟内, 在 450 nm 波长检测 OD 值, 参考波长 570 nm 或 630 nm 25 26

16 A B C D E F G H S1 S2 S3 S4 S5 S6 S7 Blank S1 S2 S3 S4 S5 S6 S7 Blank 网址 : 客服电话 : Human β-ngf ELISA Kit PLATE LAYOUT NOTE