E-EL-M0372c

Elabscience ( 本试剂盒仅供体外研究使用, 不用于临床诊断!) 产品货号 :E-EL-M0372c 产品规格 :96T/48T/24T/96T*5 Elabscience 小鼠 β- 骨胶原交联 (β-ctx) 酶联免疫吸附测定试剂盒使用说明书 Mouse β-ctx(beta Crosslaps) ELISA Kit 使用前请仔细阅读说明书 如果有任何问题, 请通过以下方式联系我们 : 销售部电话 027-65022280,027-87854967 技术部电话 027-87526315 电子邮箱 ( 销售 ) 电子邮箱 ( 技术 ) 网址 : Perry@elabscience.cn techsupport@elabscience.cn 具体保质期请见试剂盒外包装标签 请在保质期内使用试剂盒 联系时请提供产品批号 ( 见试剂盒标签 ), 以便我们更高效地为您服务 Copyright 2020-2021 Elabscience Biotechnology Co.,Ltd. All Rights Reserved

目录 用途 3 基本性能 3 检测原理 3 试剂盒组成及保存 4 试验所需自备物品 5 样品收集方法 5 注意事项 6 试剂盒注意事项 6 样品注意事项 6 样本稀释方案 6 检测前准备工作 7 操作步骤 8 结果判断 10 典型数据 10 性能 11 精密度 11 回收率 11 线性 11 问题分析 12 声明 13 Intended use 14 Character 14 Test principle 14 Kit components & Storage 15 Other supplies required 16 Sample collection 16 Note 17 Note for kit 17 Note for sample 17 Dilution Method 18 Reagent preparation 18 Assay procedure 19 Calculation of results 21 Typical data 21 Performance 22 Precision 22 Recovery 22 Linearity 22 Troubleshooting 23 Declaration 24 2

用途 该试剂盒用于体外定量检测小鼠 血清 血浆或其他相关生物液体中 β-ctx 浓度 基本性能性能灵敏度 46.88 pg/ml 检测范围 78.13-5000 pg/ml 特异性 可检测样本中的小鼠 β-ctx, 且与其它类似物无明显交叉反应 重复性板内, 板间变异系数均 < 10% 检测原理 本试剂盒采用竞争 ELISA 法 用抗原包被于酶标板上, 实验时样品或标准品中的与包被的竞争生物素标记的抗单抗上的结合位点, 游离的成分被洗去 加入辣根过氧化物酶标记的亲和素, 生物素与亲和素特异性结合而形成免疫复合物, 游离的成分被洗去 加入显色底物 (TMB),TMB 在辣根过氧化物酶的催化下呈现蓝色, 加终止液后变成黄色 用酶标仪在 450nm 波长处测 OD 值, 浓度与 OD450 值之间呈反比, 通过绘制标准曲线计算出样品中的浓度 3

试剂盒组成及保存 未拆封的试剂盒可在 2-8 保存一周 ; 如果一周以后才使用试剂盒, 请拆开试剂盒按照下表中的条件分别保存各组分 中文名称规格开封后保存条件 ELISA 酶标板 Micro ELISA Plate 冻干标准品 Reference Standard 浓缩生物素化抗体 (100 ) Concentrated Biotinylated Detection Ab (100 ) 浓缩 HRP 酶结合物 (100 ) Concentrated HRP Conjugate(100 ) 标准品 & 样品稀释液 Reference Standard & Sample Diluent 生物素化抗体稀释液 Biotinylated Detection Ab Diluent 酶结合物稀释液 HRP Conjugate Diluent 浓缩洗涤液 (25 ) Concentrated Wash Buffer (25 ) 底物溶液 (TMB) Substrate Reagent 反应终止液 Stop Solution 封板覆膜 Plate Sealer 产品说明书 Manual 质检报告 Certificate of Analysis 96T:8 孔 12 条 48T:8 孔 6 条 24T:8 孔 3 条 -20, 可存放 6 个月 96T*5:5 块 96T 酶标板 96T:2 支 48T/24T:1 支 96T*5:10 支 96T:1 支 120 μl 48T/24T:1 支 60 μl 96T*5:5 支 120 μl 96T:1 支 120 μl -20 ( 避光 ), 可存放 6 个月 48T/24T:1 支 60 μl 96T*5:5 支 120 μl 96T/48T/24T:1 瓶 20 ml 96T*5:5 瓶 20 ml 96T/48T/24T:1 瓶 2-8, 14 ml可存放 6 个月 96T*5:5 瓶 14 ml 96T/48T/24T:1 瓶 14 ml 96T*5:5 瓶 14 ml 96T/48T/24T:1 瓶 30 ml 96T*5:5 瓶 30 ml 96T/48T/24T:1 瓶 2-8 ( 10 ml避光 ) 96T*5:5 瓶 10 ml 96T/48T/24T:1 瓶 2-8 10 ml 96T*5:5 瓶 10 ml 96T/48T/24T:5 张 96T*5:25 张 1 份 1 份 说明 : 所有试剂瓶盖须旋紧以防止蒸发和微生物的污染 试剂体积以实际发货版说明书为准 相关试剂在分装时会比标签上标明的体积稍多一些, 请在使用时量取而非直接倒出 4

试验所需自备物品 1. 酶标仪 (450nm 波长滤光片 ) 2. 高精度移液器,EP 管及一次性吸头 :0.5-10μL, 2-20μL, 20-200μL, 200-1000 3.37 恒温箱 4. 双蒸水或去离子水 5. 吸水纸 6. 加样槽 样品收集方法 ( 具体处理方法参考官网 :http:///list-detail-241.html) 1. 血清 : 全血样品于室温放置 1 小时或 2-8 过夜后于 2-8,1000 g 离心 20 分钟, 取上清即可检测 2. 血浆 : 抗凝剂推荐使用 EDTA-Na2, 样品采集后 30 分钟内于 2-8,1000 g 离心 15 分钟, 取上清即可 3. 组织匀浆 : 用预冷的 PBS (0.01M, ph=7.4) 冲洗组织, 去除残留血液, 称重后将组织剪碎 将剪碎的对应体积的 PBS( 一般按 1:9 的重量体积比, 比如 1 g 的组织样品对应 9 ml 的 PBS, 具体体积可根据实验需当调整, 并做好记录 推荐在 PBS 中加入蛋白酶抑制剂 ) 加入玻璃匀浆器中, 在冰上充分研磨 为了进一步裂解组织细胞, 可以对匀浆液进行反复冻融或超声破碎 最后将匀浆液于 2-8,5000 g 离心 5-10 分钟, 取上清检测 4. 细胞提取液 : 贴壁细胞用冷的 PBS 轻轻清洗, 然后用胰蛋白酶消化,1000 g 离心 5 分钟后收集细胞 ; 悬浮 6 细胞可直接离心收集 收集的细胞用冷的 PBS 洗涤 3 次 每个细胞中加入 10 150-200 μl PBS 重悬 ( 推荐在 P 中加入蛋白酶抑制剂 ; 若含量很低可减少 PBS 的体积 ) 并通过反复冻融或超声使细胞破碎 将提取液于 2-8,1500 g 离心 10 分钟, 取上清检测 5. 细胞培养上清或其他生物体液 : 收集液体后于 2-8,1000 g 离心 20 分钟, 除去杂质及细胞碎片 取上清检测 5

注意事项 试剂盒注意事项 1) 本试剂盒仅供体外研究使用, 不用于临床诊断 2) 试验中请穿着实验服并戴乳胶手套做好防护工作 特别是检测血液或者其他体液样品时, 请按国家生物试验室安全防护条例执行 3) 刚开启的酶标板孔中可能会有少许水样物质, 此为正常现象, 不会对实验结果造成任何影响 暂时不用的板条应拆卸后放入备用铝箔袋, 按照上述表格中保存条件存放 4) 请勿重复使用已稀释过的标准品 生物素化抗体工作液 酶结合物工作液 未用完的浓缩生物素化抗体 (100 ) 浓缩 HRP 酶结合物 (100 ) 酶标板及其他原液按照上述表格中保存条件存放 5) 检测使用的酶标仪需要安装能检测 450±10nm 波长的滤光片, 光密度范围在 0-3.5 之间 建议使用时提前 15 分钟预热 6) 请勿使用其他批号或其他来源的试剂混合或替代本试剂盒中的试剂 7) 试验中所用的 EP 管和吸头均为一次性使用, 严禁混用 8) 请勿使用过期的试剂 样品注意事项 1) 收集血液的试管应为一次性无内毒素试管 避免使用溶血, 高血脂样品 2) 样品收集后若在 1 周内进行检测可保存于 2-8, 若不能及时检测, 请按一次使用量分装, 冻存于 -20 (1 个月内检测 ), 或 -80 (3 个月内检测 ), 避免反复冻融 在检测前, 冷冻过的样本应缓慢地融化并离心除去冻融过程产生的沉淀物 室温混匀后使用 3) 试剂盒检测范围不等同于样本中待测物的浓度范围, 建议实验前通过相关文献预估样本中待测物的浓度并通过预实验确定样本的实际浓度情况 如果样品中待测物浓度过高或过低, 请对样本做适当的稀释或者浓缩 4) 若所检样本不在说明书所列样本之中, 建议做预实验验证其检测有效性 5) 若使用化学裂解液制备组织匀浆或细胞提取液, 由于引入某些化学物质会导致 ELISA 测值出现偏差 6) 某些重组蛋白可能与试剂盒中捕获或检测抗体不匹配而出现不能检测的情况 样本稀释方案 请提前预估样本的浓度范围, 如果您的检测样本需要稀释, 参考稀释方案如下 : 稀释 100 倍一步稀释 取 : 5 μl 样本到 495 μl 标准品 & 样本稀释液内, 做 100 倍稀释 ; 稀释 1000 倍两步稀释 取 : 5μL 样本到 95μL 标准品 & 样本稀释液内, 做 20 倍稀释, 再取 5μL 20 倍稀释样本到 245μL 标准品 & 样本稀释液内, 做 50 倍稀释, 总共稀释 1000 倍 ; 稀释 100000 三步稀释 取倍 : 5μL 样本到 195μL 标准品 & 样本稀释液内, 做 40 倍稀释, 再取 5μL 40 倍稀释样本到 245μL 标准品 & 样本稀释液内, 做 50 倍稀释, 最后取 5μL 2000 倍稀释样本到 245μL 标准品 & 样本液内, 做 50 倍稀释, 总共稀释 100000 倍 ; 每步稀释时取液量不少于 3μL, 稀释倍数不超过 100 倍 每步稀释都需混合均匀, 避免起泡 6

检测前准备工作 : 1. 提前 20 分钟从冰箱中取出试剂盒, 平衡至室温 (18-25 ) 如果试剂盒需分多次使用, 请仅取出本次实验所需的酶标板条和试剂, 剩余板条和试剂需按照指定条件保存 2. 洗涤液 : 将浓缩洗涤液用双蒸水稀释 (1:24) 提示: 从冰箱中取出的浓缩洗涤液可能有结晶, 属于正常现象, 可用 40 水浴微加热使结晶完全溶解后再配制洗涤液 当日使用 3. 标准品工作液将标准品于 10000 g 离心 1 分钟, 加入标准品 & 样品稀释液 1 ml 至冻干标准品中, 旋紧管盖, 静置 10 分钟, 上下颠倒数次, 待其充分溶解后, 轻轻混匀, 避免起泡, 配成 5000pg/mL 的标准品工作液 ( 或加入 1 ml 标准品 & 样品稀释液后, 静置 1-2 分钟, 用低速涡旋仪充分混匀 可通过低速离心去除涡旋过程中产生的气泡 ) 然后根据需要进行倍比稀释 建议配制成以下浓度:5000,2500,1250,625,312.5, 倍比稀释方法: 取 7 支 EP 管, 每管中加入 500μL 标准品 & 样品稀释液, 从 5000pg/mL 的标准品工作液 500μL 到第一支 EP 管中混匀配成 2500pg/mL 的标准品工作液, 按此步骤往后依次吸取混匀 如下图 提示 : 最后一管直接作为空白孔, 不需要再从倒数第二管中吸取液体 5000 2500 1250 625 312.5156.2578.13 0 4. 生物素化抗体工作液 : 实验前计算当次实验所需用量 ( 以 50 μl/ 孔计算 ), 实际配制时应多配制 100-200 使用前 15 分钟, 将浓缩生物素化抗体于 800 g 离心 1 分钟, 以生物素化抗体稀释液将 100 浓缩生物素化抗体稀释成 1 工作浓度 ( 例如 :10 μl 浓缩液 +990 μl 稀释液 ) 5. 实验前计算当次实验所需用量 ( 以 100 μl/ 孔计算 ), 实际配制时应多配制 100-200 μl 使用前 15 分缩 HRP 酶结合物于 800 g 离心 1 分钟, 以酶结合物稀释液将 100 浓缩 HRP 酶结合物稀释成 1 工作浓度 ( 例 :10 μl 浓缩液 +990 μl 稀释液 ) 7

操作步骤 1. 分别设定标准孔 空白孔和样本孔 标准孔加入 50μL 倍比稀释的标准品, 空白孔加入 50μL 标准品 & 样本稀释液, 其余孔加入 50μL 待测样本 ( 建议所有的待检样本和标准品在检测中设立复孔 ) 立即每孔加入配好的生物素化抗体工作液 50μL 给酶标板覆膜,37 孵育 45 分钟 提示 : 加样时将样品加于酶标板底部, 尽量不触及孔壁, 轻轻晃动混匀, 避免产生气泡 加样时间宜控制在 10 分钟内 2. 甩尽孔内液体, 在洁净的吸水纸上拍干 每孔加洗涤液 350 μl, 浸泡 1 分钟, 吸去或甩掉酶标板内的液体, 拍干 重复此洗板步骤 3 次 提示 : 此处与其他洗板步骤都可使用洗板机 ( 参考北京拓普 DEM-3 型洗板机参数设置 :2 点吸, 每孔加入洗涤液 350μL, 振板 5 秒, 吸液 0.5 秒 ) 洗板完成后请立即进行下步操作, 不要让微孔板干燥 3. 每孔加酶结合物工作液 100μL, 酶标板加上覆膜,37 温育 30 分钟 4. 甩尽孔内液体, 洗板 5 次, 方法同步骤 2 5. 每孔加底物溶液 (TMB)90μL, 酶标板加上覆膜,37 避光孵育 15 分钟左右 提示 : 根据实际显色情况酌情缩短或延长, 但不可超过 30 分钟 当标准孔出现明显梯度时 ( 前 4 个显色孔出现明显蓝色梯度 ), 即可终止 提前 15 分钟打开酶标仪预热 6. 每孔加终止液 50μL, 终止反应 提示 : 终止液的加入顺序应尽量与底物溶液的加入顺序相同 7. 立即用酶标仪在 450nm 波长测量各孔的光密度 (OD 值 ) 8

操作一览表 9

结果判断 1. 计算标准品和样本复孔的平均 OD 值, 以浓度为横坐标,OD 值为纵坐标, 在双对数坐标轴上拟合四参数逻辑函数的标准曲线, 操作详情请参阅 Elabscience 官网 / 微信公众号内技术文章 2. 若样品 OD 值低于标准曲线下限, 应适当稀释后重测并在计算样本浓度时乘以相应的稀释倍数 典型数据 以下数据和曲线仅供参考, 实验者需根据自己的实验建立标准曲线 pg/ml OD 标曲 5000 0.415 2500 0.562 1250 0.796 625 1.112 312.5 1.456 156.25 1.752 78.13 1.96 0 2.231 10

性能 精密度 板内精密度 : 低, 中, 高浓度样本分别在 1 块板子上检测 20 次 板间精密度 : 低, 中, 高浓度样本分别在 3 块板子上检测 20 次 批内变异系数 批间变异系数 样本 1 2 3 1 2 3 数量 20 20 20 20 20 20 平均值 (pg/ml) 264.20768.901936.80 252.10764.901935.40 标准差 16.1034.6093.0014.60 32.90 71.60 变异系数 (%) 6.09 4.50 4.80 5.79 4.30 3.70 回收率 分别往不同样本中添加已知浓度的小鼠 β-ctx, 做回收实验, 得出回收率范围和平均回收率 样本类型 回收率范围 (%) 平均回收率 (%) 血清 (n=8) 88-103 94 血浆 (EDTA)(n=8) 91-104 98 细胞培养基 (n=8) 85-96 91 线性 将添加有小鼠 β-ctx 的样本分别稀释 2 倍,4 倍,8 倍,16 倍做回收实验, 得出回收率范围及平均回收率 血清 (n=5) 血浆 (EDTA) (n=5) 细胞培养基 (n=5) 1:2 1:4 1:8 1:16 回收率范围 (%)95-110 88-100 89-104 平均回收率 (%)102 95 97 回收率范围 (%)85-96 87-100 100-116 平均回收率 (%)91 94 106 回收率范围 (%)85-101 85-98 94-107 平均回收率 (%)92 92 101 回收率范围 (%)82-95 92-107 96-110 平均回收率 (%)89 99 103 11

问题分析若实验效果不好, 请及时对显色结果拍照, 保存实验数据, 保留所用板条及未使用试剂, 然后联系我公司技术支持为您解决问题 同时您也可以参考以下资料 : 问题描述可能原因相应对策 吸液或加液不准 检查移液器及吸头 标准曲线梯度差 标准品稀释不正确 洗涤不完全 溶解标准品时稍微旋转瓶身, 轻轻混匀使粉末完全溶解 保证洗涤时间和洗涤次数及每孔的加液量 孵育时间太短 保证充足的孵育时间 显色很弱或无色 实验温度不正确试剂体积不够或漏加稀释不正确酶标记物失活或底物失效 使用推荐的实验温度 检查吸液及加液过程, 保证所有试剂按顺序足量添加 混合酶结合物和底物, 通过迅速显色来检查判断 读数数值低 酶标仪设置不正确 在酶标仪上检查波长及滤光片设置 提前打开酶标仪预热 变异系数大加液不正确检查加液情况 检测抗体的工作浓度过高 使用推荐的稀释倍数 背景值高 酶标板洗涤不完全 保证每步清洗完全 ; 如果用自动洗板机, 请检查所有的出口 是否有堵塞 ; 是否使用试剂盒配备的洗涤液 洗液有污染 配制新鲜的洗液 ELISA 试剂盒保存不当按说明书要求保存相关试剂 灵敏度低 读数前未终止 OD 读数前应在每孔中加入终止液 12

声明 1. 限于现有条件及科学技术水平, 尚不能对所有原料进行全面的鉴定分析, 本产品可能存在一定的质量技术风险 2. 本试剂盒在研发过程中去除 / 降低了生物学样本中的一些内源性干扰因素, 并非所有可能影响的因素均已去除 3. 最终的实验结果与试剂的有效性 实验者的相关操作以及当时的实验环境等因素密切相关, 本公司只对试剂盒本身负责, 不对因使用试剂盒所造成的样本消耗负责, 请使用者使用前充分考虑到样本可能的使用量, 预留充足的样本 4. 为了达到好的实验结果, 请只使用本公司试剂盒内提供的试剂, 不要混用其他制造商的产品, 严格按照说明书操作 5. 由于操作过程中试剂制备以及酶标仪参数设置不正确, 可能导致结果异常, 实验前请仔细阅读说明书并调整好仪器 6. 即使是相同人员操作也可能在两次独立实验中得到不同的结果, 为保证结果的重现性, 需要控制实验过程中每一步的操作 7. 试剂盒发货前会经过严格的质检, 然而, 因为运输条件 实验设备差异等等因素影响, 用户检测结果可能跟出厂数据不一致 不同批次间试剂盒间的差异也可能来自上述原因 8. 本试剂盒未与其他厂家同类试剂盒或不同方法检测同一目的物的产品进行对比, 所以不排除检测结果不一致的情况 9. 试剂盒仅供研究使用, 如将其用于临床诊断或任何其他用途, 我公司将不对因此产生的问题负责, 亦不承担任何法律责任 13

Mouse β-ctx(beta Crosslaps) ELISA Kit Catalog No : E-EL-M0372c Size:96T/48T/24T/96T*5 Intended use This ELISA kit applies to the in vitro quantitative determination of Mouse β-ctx concentrations in serum, plasma and other biological fluids. Character Item Sensitivity Detection Range Specificity 46.88 pg/ml 78.13-5000 pg/ml This kit recognizes Mouse β-ctx in samples. No significant cross-reactivity or interference between Mouse β-ctx and analogues was observed Repeatability Coefficient of variation is < 10% Test principle This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Mouse β-ctx. During the reaction, Mouse β-ctx in samples or Standard competes with a fixed amount of Mouse β- CTx on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Mouse β-ctx. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450±2 nm. The concentration of Mouse β-ctx in the samples is then determined by comparing the OD of the samples to the standard curve. 14

Kit components & Storage An unopened kit can be stored at 2-8 for 1 month. If the kit is not supposed to be used within 1 month, store the items separately according to the following conditions once the kit is received. Item Specifications Storage Micro ELISA Plate (Dismountable) Reference Standard Concentrated Biotinylated Detection Ab (100 ) Concentrated HRP Conjugate (100 ) 96T: 8 wells 12 strips 48T: 8 wells 6 strips 24T: 8 wells 3 strips 96T*5: 5 plates, 96T 96T: 2 vials 48T/24T: 1 vial 96T*5: 10 vials 96T: 1 vial, 120 μl 48T/24T: 1 vial, 60 μl 96T*5: 5 vials, 120 μl 96T: 1 vial, 120 μl 48T/24T: 1 vial, 60 μl 96T*5: 5 vials, 120 μl -20, 6 months -20 C(shading light), 6 months Reference Standard & Sample Diluent 96T/48T/24T: 1 vial, 20 ml 96T*5: 5 vials, 20 ml Biotinylated Detection Ab Diluent 96T/48T/24T: 1 vial, 14 ml 96T*5: 5 vials, 14 ml 2-8 C, 6 months HRP Conjugate Diluent 96T/48T/24T: 1 vial, 14 ml 96T*5: 5 vials, 14 ml Concentrated Wash Buffer (25 ) 96T/48T/24T: 1 vial, 30 ml 96T*5: 5 vials, 30 ml Substrate Reagent 96T/48T/24T: 1 vial, 10 ml 96T*5: 5 vials, 10 ml Stop Solution 96T/48T/24T: 1 vial, 10 ml 96T*5: 5 vials, 10 ml 2-8 C(shading light) 2-8 C Plate Sealer Product Description Certificate of Analysis 96T/48T/24T: 5 pieces 96T*5: 25 pieces 1 copy 1 copy Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution. The volume of reagents in partial shipments is a little more than the volume marked on the label, please use accurate measuring equipment instead of directly pouring into the vial(s). 15

Other supplies required Microplate reader with 450 nm wavelength filter High-precision transfer pipette, EP tubes and disposable pipette tips Incubator capable of maintaining 37 Deionized or distilled water Absorbent paper Loading slot Sample collection (More detailed information please view our website: http:///list detail-241.html) Serum: Allow samples to clot for 1 hour at room temperature or overnight at 2-8 before centrifugation for 20 min at 1000 g at 2-8. Collect the supernatant to carry out the assay. Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15 min at 1000 g at 2-8 within 30 min of collection. Collect the supernatant to carry out the assay. Tissue homogenates: It is recommended to get detailed references from the literature before analyzing different tissue types. For general information, hemolyzed blood may affect the results, so the tissues should be minced into small pieces and rinsed in ice-cold PBS (0.01M, ph=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (tissue weight (g): PBS (ml) volume=1:9) with a glass homogenizer on ice. To further break down the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5-10 min at 5000 g at 2-8 to get the supernatant. Cell lysates: For adherent cells, gently wash the cells with moderate amount of pre-cooled PBS and dissociate the cells using trypsin. Collect the cell suspension into a centrifuge tube and centrifuge for 5 min at 1000 g. Discard the medium and wash the cells 3 times with pre-cooled PBS. For each 1 10 6 cells, add 150-250 μl of pre-cooled PBS to keep the cells suspended. Repeat the freeze-thaw process several times or use an ultrasonic cell disrupter until the cells are fully lysed. Centrifuge for 10 min at 1500 g at 2-8. Remove the cell fragments, collect the supernatant to carry out the assay. Cell culture supernatant or other biological fluids: Centrifuge samples for 20 min at 1000 g at 2-8. Collect the supernatant to carry out the assay. 16

Note Note for kit 1) For research use only. Not for use in diagnostic procedures. 2) Please wear lab coats, eye protection and latex gloves for protection. Please perform the experiment following the national security protocols of biological laboratories, especially when detecting blood samples or other bodily fluids. 3) A freshly opened ELISA plate may appear a water-like substance, which is normal and will not have any impact on the experimental results. Return the unused wells to the foil pouch and store according to the conditions suggested in the above table. 4) Do not reuse the reconstituted standard, biotinylated detection Ab working solution, concentrated HRP conjugate working solution. The unspent undiluted concentrated biotinylated detection Ab (100 ) and other stock solutions should be stored according to the storage conditions in the above table. 5) The microplate reader should be able to be installed with a filter that can detect the wave length at 450±10 nm. The optical density should be within 0-3.5. Follow the Instructions of the Microplate Reader for set-up and preheat it for 15 min before OD measurement. 6) Do not mix or substitute reagents with those from other lots or sources. 7) Change pipette tips in between adding of each standard level, between sample adding and between reagent adding. Also, use separate reservoirs for each reagent. 8) The kit should not be used beyond the expiration date on the kit label. Note for sample: 1) Tubes for blood collection should be disposable and be non-endotoxin. Samples with high hemolysis or much lipid are not suitable for ELISA assay. 2) Samples should be assayed within 7 days when stored at 2-8, otherwise samples must be divided up and stored at -20 ( 1 month) or -80 ( 3 months). Avoid repeated freeze-thaw cycles. Prior to assay, the frozen samples should be slowly thawed and centrifuged to remove precipitates. 3) Please predict the concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. 4) If the sample type is not included in the manual, a preliminary experiment is suggested to verify the validity. 5) If a lysis buffer is used to prepare tissue homogenates or cell lysates, there is a possibility of causing a deviation due to the introduced chemical substance. 6) Some recombinant protein may not be detected due to a mismatching with the coated antibody or detection antibody. 17

Dilution Method Please predict the concentration range of the sample in advance. If your test sample needs dilution, please refer to the dilution method as follows: For 100 fold dilution: One-step dilution. Add 5 μl sample to 495 μl sample diluent to yield 100 fold dilution. For 1000 fold dilution:two-step dilution. Add 5 μl sample to 95 μl sample diluent to yield 20 fold dilution, then add 5 μl 20 fold diluted sample to 245 μl sample diluent, after this, the neat sample has been diluted at 1000 fold successfully. For 100000 fold dilution:three-step dilution. Add 5 μl sample to 195 μl sample diluent to yield 40 fold dilution, then add 5 μl 40 fold diluted sample to 245 μl sample diluent to yield 50 fold dilution, and finally add 5 μl 2000 fold diluted sample to 245 μl sample diluent, after this, the neat sample has been diluted at 100000 fold successfully. Reagent preparation 1. Bring all reagents to room temperature (18-25 ) before use. If the kit will not be used up in one assay, please only take out the necessary strips and reagents for present experiment, and store the remaining strips and reagents at required condition. 2. Wash Buffer: Dilute 30 ml of Concentrated Wash Buffer with 720 ml of deionized or distilled water to prepare 750 ml of Wash Buffer. Note: if crystals have formed in the concentrate, warm it in a 40 water bath and mix it gently until the crystals have completely dissolved. 3. Standard working solution: Centrifuge the standard at 10,000 g for 1 min. Add 1.0 ml of Reference Standard &Sample Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a working solution of 5000 pg/ml(or add 1 ml of Reference Standard &Sample Diluent, let it stand for 1-2 min and then mix it thoroughly with a vortex meter of low speed. Bubbles generated during vortex could be removed by centrifuging at a relatively low speed). Then make serial dilutions as needed. The recommended dilution gradient is as follows: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 0 pg/ml. Dilution method: Take 7 EP tubes, add 500uL of Reference Standard & Sample Diluent to each tube. Pipette 500uL of the 5000 pg/ml working solution to the first tube and mix up to produce a 2500 pg/ml working solution. Pipette 500uL of the solution from the former tube into the latter one according to this step. The illustration below is for reference. 5000 2500 1250 625 312.5 156.25 78.13 0 4. Biotinylated Detection Ab working solution: Calculate the required amount before the experiment (50 μl/well). In preparation, slightly more than calculated should be prepared. Centrifuge the Concentrated Biotinylated Detection Ab at 800 g for 1 min, then dilute the 100 Concentrated Biotinylated Detection Ab to 1 working solution with Biotinylated Detection Ab Diluent(Concentrated Biotinylated Detection Ab: Biotinylated Detection Ab Diluent= 1: 99). 5. Concentrated HRP Conjugate working solution: Calculate the required amount before the experiment (100 μl/well). In preparation, slightly more than calculated should be prepared. Centrifuge the Concentrated HRP Conjugate at 800 g for 1 min, then dilute the 100 Concentrated HRP Conjugate to 1 working solution with HRP Conjugate Diluent(Concentrated HRP Conjugate: HRP Conjugate Diluent= 1: 99). 18

Assay procedure 1. Determine wells for diluted standard, blank and sample. Add 50 μl each dilution of standard, blank and sample into the appropriate wells (It is recommended that all samples and standards be assayed in duplicate). Immediately add 50 μl of Biotinylated Detection Ab working solution to each well. Cover the plate with the sealer provided in the kit. Incubate for 45 min at 37. Note: solutions should be added to the bottom of the micro ELISA plate well, avoid touching the inside wall and causing foaming as much as possible. 2. Decant the solution from each well, add 350 μl of wash buffer to each well. Soak for 1 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times. Note: a microplate washer can be used in this step and other wash steps. Make the tested strips in use immediately after the wash step. Do not allow wells to be dry. 3. Add 100 μl of HRP Conjugate working solution to each well. Cover the plate with a new sealer. Incubate for 30 min at 37 C. 4. Decant the solution from each well, repeat the wash process for 5 times as conducted in step 2. 5. Add 90 μl of Substrate Reagent to each well. Cover the plate with a new sealer. Incubate for about 15 min at 37 C. Protect the plate from light. Note: the reaction time can be shortened or extended according to the actual color change, but not more than 30 min. Preheat the Microplate Reader for about 15 min before OD measurement. 6. Add 50 μl of Stop Solution to each well. Note: adding the stop solution should be done in the same order as the substrate solution. 7. Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm. 19

Assay Procedure Summary 20

Calculation of results Average the duplicate readings for each standard and samples. Plot a four parameter logistic curve on log-log axis, with standard concentration on the x-axis and OD values on the y-axis. If the OD of the sample under the lowest limit of the standard curve, you should re-test it with an appropriate dilution. The actual concentration is the calculated concentration multiplied by the dilution factor. Typical data As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only. pg/ml OD Standard Curve 5000 0.415 2500 0.562 1250 0.796 625 1.112 312.5 1.456 156.25 1.752 78.13 1.96 0 2.231 21

Performance Precision Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse β-ctx were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse β-ctx were tested on 3 different plates, 20 replicates in each plate, respectively. Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 Mean 264.20 768.90 1936.80 252.10 764.90 1935.40 Standard deviation 16.10 34.60 93.00 14.60 32.90 71.60 C V (%) 6.09 4.50 4.80 5.79 4.30 3.70 Recovery The recovery of Mouse β-ctx spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices. Sample Type Range (%) Average Recovery (%) Serum (n=8) 88-103 94 EDTA plasma (n=8) 91-104 98 Cell culture media (n=8) 85-96 91 Linearity Samples were spiked with high concentrations of Mouse β-ctx and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay. Serum (n=5) EDTA plasma(n=5) Cell culture media(n=5) 1:2 1:4 1:8 1:16 Range (%) 95-110 88-100 89-104 Average (%) 102 95 97 Range (%) 85-96 87-100 100-116 Average (%) 91 94 106 Range (%) 85-101 85-98 94-107 Average (%) 92 92 101 Range (%) 82-95 92-107 96-110 Average (%) 89 99 103 22

Troubleshooting Problem Causes Solutions Poor standard curve Low signal Deep color but low value Inaccurate pipetting Improper standard dilution Wells are not completely aspirated Insufficient incubation time Incorrect assay temperature Inadequate reagent volumes Improper dilution HRP conjugate inactive or TMB failure Plate reader setting is not optimal Check pipettes. Ensure briefly spin the vial of standard and dissolve the powder thoroughly by gentle mixing. Completely aspirate wells in between steps. Ensure sufficient incubation time. Use recommended incubation temperature. Bring substrate to room temperature before use. Check pipettes and ensure correct preparation. Mix HRP conjugate and TMB, rapid coloring. Verify the wavelength and filter setting on the Microplate reader. Open the Microplate Reader ahead to preheat. Large CV Inaccurate pipetting Check pipettes. High background Low sensitivity Concentration of target protein is too high Plate is insufficiently washed Contaminated wash buffer Improper storage of the ELISA kit Stop solution is not added Use recommended dilution factor. Review the manual for proper wash. If using a plate washer, check that all ports are unobstructed. Prepare fresh wash buffer. All the reagents should be stored according to the instructions. Stop solution should be added to each well before measurement. 23

Powered by TCPDF (www.tcpdf.org) Declaration 1. Limited by current conditions and scientific technology, we can't conduct comprehensive identification and analysis on all the raw material provided. So there might be some qualitative and technical risks for users using the kit. 2. This assay is designed to eliminate interference by factors present in biological samples. Until all factors have been tested in the ELISA immunoassay, the possibility of interference cannot be excluded. 3. The final experimental results will be closely related to the validity of products, operational skills of the operators, the experimental environments and so on. We are only responsible for the kit itself, but not for the samples consumed during the assay. The users should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance. 4. To get the best results, please only use the reagents supplied by the manufacturer and strictly comply with the instructions. 5. Incorrect results may occur because of incorrect operations during the reagents preparation and loading, as well as incorrect parameter settings of the Micro-plate reader. Please read the instructions carefully and adjust the instrument prior to the experiment. 6. Even the same operator might get different results in two separate experiments. In order to get reproducible results, the operation of every step in the assay should be controlled. 7. Every kit has strictly passed QC test. However, results from end users might be inconsistent with our data due to some variables such as transportation conditions, different lab equipment, and so on. Intra-assay variance among kits from different batches might arise from the above reasons too. 8. Kits from different manufacturers or other methods for testing the same analyte could bring out inconsistent results, since we haven t compared our products with those from other manufacturers. 9. The kit is designed for research use only, we will not be responsible for any issues if the kit is applied in clinical diagnosis or any other related procedures. 24