E-EL-H0363c

( 本试剂盒仅供体外研究使用, 不用于临床诊断!) Elabscience 人胰岛素受体抗体 (IR-Ab) 酶联免疫吸附测定试剂盒使用说明书 Human IR-Ab(Anti-Insulin Receptor) ELISA Kit 产品货号 :E-EL-H0363c 96T 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 ) Perry@elabscience.cn 电子邮箱 ( 技术 ) techsupport@elabscience.cn QQ 客服 800110755 网址 : 具体保质期请见试剂盒外包装标签 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 2017-2018 Elabscience Biotechnology Co.,Ltd. All Rights Reserved

用途 该试剂盒用于体外定量检测人 血清 血浆或其他相关生物液体中 IR-Ab 浓度 灵敏度 检测范围 特异性和重复性 灵敏度 :0.94 ng/ml 检测范围 :1.56-100 ng/ml 特异性 : 可检测样本中的人 IR-Ab, 且与其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双抗原夹心 ELISA 法 用人 IR 抗原包被于酶标板上, 实验时样品或标准品中的人 IR-Ab 会与包被抗原结合, 游离的成分被洗去 依次加入生物素化的抗原和辣根过氧化物酶标记的亲和素 生物素化的抗原与结合在包被抗原上的人 IR-Ab 结合 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 O D 值, 浓度与 OD450 值之间呈正比, 通过绘制标准曲线计算出样品中 IR-Ab 的浓度 2

试剂盒组成及保存未拆封的试剂盒可在 4 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 中文名称英文名称规格保存条件 ELISA 酶标板 ( 可拆卸 )Micro ELISA Plate(Dismountable) 8 孔 12 条 冻干标准品 Reference Standard 2 支 浓缩生物素化抗原 (100 ) Concentrated Biotinylated Detection Ag 1 支 120 μl -20, 可存放 6 个月 -20 ( 避光 ), 可存放 6 个浓缩 HRP 酶结合物 (100 ) Concentrated HRP Conjugate 1 支 120 月 μl 标准品 & 样品稀释液 Reference Standard & Sample Diluent 1 瓶 20 ml 生物素化抗原稀释液 Biotinylated Detection Ag Diluent 1 瓶 14 ml 酶结合物稀释液 HRP Conjugate Diluent 1 瓶 14 4, 可存放 6 个月 ml 浓缩洗涤液 (25 ) Concentrated Wash Buffer (25 ) 1 瓶 30 ml 底物溶液 (TMB) Substrate Reagent 1 瓶 10 ml 4 ( 避光 ) 反应终止液 Stop Solution 1 瓶 10 ml 4 封板覆膜 Plate Sealer 5 张 产品说明书 Product Description 1 份 质检报告 Certificate of Analysis 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 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, 双蒸水或去离子水 4. 吸水纸 注意事项 1. 试验中请穿着实验服并带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 2. 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 3. 请勿重复使用已稀释过的标准品 生物素化抗原工作液 酶结合物工作液 未用完的没有稀释的浓缩生物素化抗原 (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 4. 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 5. 不同批号的试剂盒组份不能混用 ( 洗涤液和反应终止液除外 ) 6. 试验中所用的 EP 管和吸头均为一次性使用, 严禁混用 3

样品收集方法 ( 具体处理方法参考官网 :http://) 1. 血清 : 全血样品于室温放置 2 小时或 4 过夜后于 1000 g 离心 20 分钟, 取上清即可检测, 收集血液的试管应为一次性的无内毒素试管 2. 血浆 : 抗凝剂推荐使用 EDTA 钠盐, 样品采集后 30 分钟内于 1000 g 离心 15 分钟, 取上清即可检测 避免使用溶血, 高血脂样品 3. 组织匀浆 : 用预冷的 PBS (0.01 M,pH=7.4) 冲洗组织, 去除残留血液, 称重后将组织剪碎 将剪碎的组织与对应体积的 PBS( 一 9 的重量体积比, 比如 1g 的组织样品对应 9 ml 的 PBS, 具体体积可根据实验需要适当调整, 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, 可以对匀浆液进行超声破碎或反复冻融 最后将匀浆液 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 ; 悬浮细 6 胞可直接离心收集 收集的细胞用冷的 PBS 洗涤 3 次 每 1 10 个细胞中加入 150-200μL PBS 重悬并通过反复冻融使细胞破碎 ( 若含量很低可减少 PBS 的体积 ) 将提取液于 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 1000 g 离心 20 分钟, 除去杂质及细胞碎片 取上清检测 样品注意事项 1. 样品收集后若在 1 周内进行检测的可保存于 4, 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 2. 试剂盒检测范围不等同于样本的浓度范围, 如果您的样品中检测物浓度高于标准品最高值, 请根据实际情况, 做适当倍数稀释 ( 建议查阅文献后先做预实验, 以确定稀释倍数 ) 3. 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 4. 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 4

检测前准备工作 : 1. 请提前 20 分钟从冰箱中取出试剂盒, 平衡至室温 读数前 15 分钟打开酶标仪预热 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 1.0mL 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, 配成 100ng/mL 的标准品工作液 然后根据需要进行倍比稀释 建议配制成以下浓度 :100,50,25,12.5,6.25,3.13,1.56,0ng/m 每管中加入 500μL 标准品 & 样品稀释液, 从 100ng/mL 的标准品工作液中吸取 500μL 到其中一支 EP 管成 50ng/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 100 50 25 12.5 6.25 3.13 1.56 0 4. 生物素化抗原工作液 : 实验前计算当次实验所需用量 ( 以 100μL/ 孔计算 ), 实际配制时应多配制 100-200μL 使用前 15 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 当日用 5. 酶结合物工作液 : 实验前计算当次实验所需用量 ( 以 100μL/ 孔计算 ), 实际配制时应多配制 100-200μL 前 15 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 当日使用 5

操作步骤 ( 第 10 页中附有简版操作概要 ) 1. 将标准品工作液依次加入到前两列孔中, 每个浓度的工作液并列加两孔, 每孔 100μL 待测样品加入到其他孔, 每孔 100μL( 若样本浓度高于检测范围, 需用标准品 & 样本稀释液稀释后取样 ) 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 90 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 加样时间控制在 10 分钟内 2. 弃去液体, 甩干, 不用洗涤 每个孔中加入生物素化抗原工作液 100μL, 混匀, 酶标板加上覆膜,37 温育 1 小时 3. 甩尽孔内液体, 每孔加洗涤液 350μL, 浸泡 1-2 分钟, 吸去或甩掉酶标板内的液体, 在厚的吸水纸上拍干 重复此洗板步骤 3 次 提示 : 此处与其他洗板步骤都可用洗板机 5. 弃去孔内液体, 甩干, 洗板 5 次, 方法同步骤 3 6. 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, 即可终止 7. 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 8. 立即用酶标仪在 450 nm 波长测量各孔的光密度 (OD 值 ) 结果判断 1. 计算每组复孔的平均 OD 值 每个标准品的平均 OD 值减去空白孔的 OD 值作为矫正值 以浓度为横坐标,O D 值为纵坐标, 在双对数坐标纸上绘出四参数逻辑函数的标准曲线 ( 作图时去掉空白组的值 ) 2. 若样品 OD 值高于标准曲线上限, 应适当稀释后重测 6

典型数据 由于实验操作条件的不同 ( 如操作者 移液技术 洗板技术和稳定条件等 ), 标准曲线的 OD 值会有所差异 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 Concentration(ng/mL) 100 50 25 12.5 6.25 3.13 1.56 0 OD 2.533 1.585 0.937 0.487 0.286 0.19 0.142 0.09 Corrected OD 2.443 1.495 0.847 0.397 0.196 0.1 0.052 -- 7

精密度板内精密度 : 低浓度样本, 中浓度样本和高浓度样本分别在 1 块板子上检测 20 次 板间精密度 : 低浓度样本, 中浓度样本和高浓度样本分别在 3 块板子上检测 20 次 Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 Mean(ng/mL) 4.79 13.99 36.95 4.98 14.13 37.46 Standard deviation 0.29 0.61 1.26 0.26 0.64 1.49 C V (%) 6.05 4.36 3.41 5.22 4.53 3.98 回收率分别往 5 个不同样本中添加已知浓度的目标蛋白, 做回收实验, 得出回收率范围和平均回收率 Sample Type Range (%) Average Recovery (%) Serum (n=5) 84-98 90 EDTA plasma (n=5) 87-102 94 线性分别往 5 个样本中添加已知浓度的目标蛋白, 做回收实验, 得出回收率范围及平均回收率 将 5 个样本分别稀释 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 Serum (n=5) EDTA plasma(n=5) Cell culture media(n=5) 1:2 1:4 1:8 1:16 Range (%) 94-106 89-102 Average (%) 100 95 0 Range (%) 87-99 81-94 Average (%) 92 86 0 Range (%) 85-99 84-97 Average (%) 92 89 0 Range (%) 87-98 83-98 Average (%) 92 90 0 8

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 标准曲线梯度差 吸液或加液不准 标准品稀释不正确 检查移液器及吸头 溶解标准品时稍微旋转瓶身, 轻轻混匀使粉末完全溶解 洗涤不完全 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 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 酶标记物失活或底物失效 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 灵敏度低 ELISA 试剂盒保存不当按说明书要求保存相关试剂 读数前未终止 OD 读数前应在每孔中加入终止液 9

操作概要 1. 在各孔中加入标准品或样品各 100 μl,37 孵育 90 分钟 2. 倒去孔内液体, 加入 100 μl 生物素化抗原工作液,37 孵育 60 分钟 3. 洗涤 3 次 4. 加入 100 μl 酶结合物工作液, 37 孵育 30 分钟 5. 洗涤 5 次 6. 加入 90 μl 底物溶液, 37 孵育 15 分钟左右 7. 加入 50 μl 终止液, 立即在 450 nm 波长处测量 OD 值 8. 结果计算 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. 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密切相关, 请务必准备充足的待测样品 10

Human IR-Ab(Anti-Insulin Receptor) ELISA Kit Synonyms: IR-Ab Catalog No : E-EL-H0363c 96T Intended use This ELISA kit applies to the in vitro quantitative determination of Human IR-Ab concentrations in serum, plasma and other biological fluids. Specification Sensitivity: 0.94 ng/ml Detection Range: 1.56-100 ng/ml Specificity: This kit recognizes Human IR-Ab in samples. No significant cross-reactivity or interference between Human IR-Ab and analogues was observed. Repeatability: Coefficient of variation is < 10%. Test principle This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Human IR-Ab. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Human IR-Ab and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human IR-Ab, biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human IR-Ab. You can calculate the concentration of Human IR-Ab in the samples by comparing the OD of the samples to the standard curve. 11

Kit components & Storage An unopened kit can be stored at 4 for 1 week. If the kit is not used within 1 week, 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 Ag (100 ) 8 wells 12 strips 2 vials 1 vial, 120 μl -20, 6 months Concentrated HRP Conjugate (100 ) 1 vial, 120 μl -20 C(shading light), 6 months Reference Standard & Sample Diluent Biotinylated Detection Ag Diluent HRP Conjugate Diluent Concentrated Wash Buffer (25 ) 1 vial, 20 ml 1 vial, 14 ml 1 vial, 14 ml 1 vial, 30 ml 4 C, 6 months Substrate Reagent 1 vial, 10 ml 4 C(shading light) Stop Solution 1 vial, 10 ml 4 C Plate Sealer 5 pieces Product Description Certificate of Analysis 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). 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 for Wash Buffer 12

Note 1. 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. 2. A freshly opened ELISA Plate may appear to have a water-like substance, which is normal and will not have any impact on the experimental results. 3. Do not reuse the diluted standard, biotinylated detection Ag working solution, concentrated HRP conjugate working solution. The unspent undiluted concentrated biotinylated detection Ag (100 ) and other stock solutions should be stored according to the storage conditions in the above table. 4. The microplate reader should have a 450(±10 nm) filter installed and a detector that can detect the wavelength. The optical density should be within 0~3.5. 5. Do not mix or use components from other lots. 6. Change pipette tips in between adding standards, in between sample additions, and in between reagent additions. Also, use separate reservoirs for each reagent. Sample collection Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4 before centrifugation for 20 min at 1000 g at 2~8. Collect the supernatant to carry out the assay. Blood collection tubes should be disposable and be nonendotoxin. Plasma: Collect plasma using EDTA-Na 2 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. Hemolysed samples are not suitable for ELISA assay! Tissue homogenates: It is recommended to get detailed references from the literature before analyzing different tissue types. For general information, hemolysed 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 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 until the cells are fully lysed. Centrifuge for 10min at 1500 g at 4. Remove the cell fragments, collect the supernatant to carry out the assay. Avoid repeated freeze-thaw cycles. 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. 13

Note for sample: 1. Samples should be assayed within 7 days when stored at 4, otherwise samples must be divided up and stored at -20 ( 1 month) or -80 ( 3 months). Avoid repeated freeze-thaw cycles. 2. 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. 3. If the sample type is not included in the manual, a preliminary experiment is suggested to verify the validity. 4. If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance. 14

Reagent preparation 1. Bring all reagents to room temperature (18~25 ) before use. Follow the Microplate reader manual for set-up and preheat it for 15 min before OD measurement. 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 100 ng/ml. Then make serial dilutions as needed. The recommended dilution gradient is as follows: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0 ng/ml. Dilution method: Take 7 EP tubes, add 500uL of Reference Standard & Sample Diluent to each tube. Pipette 500uL of the 100 ng/ml working solution to the first tube and mix up to produce a 50 ng/ml working solution. Pipette 500uL of the solution from the former tube into the latter one according to these steps. The illustration below is for reference. Note: the last tube is regarded as a blank. Don t pipette solution into it from the former tube. 100 50 25 12.5 6.25 3.13 1.56 0 4. Biotinylated Detection Ag working solution: Calculate the required amount before the experiment (100 μl/well). In preparation, slightly more than calculated should be prepared. Centrifuge the stock tube before use, dilute the 100 Concentrated Biotinylated Detection Ag to 1 working solution with Biotinylated Detection Ag Diluent. 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. Dilute the 100 Concentrated HRP Conjugate to 1 working solution with Concentrated HRP Conjugate Diluent. 15

Assay procedure (A brief assay procedure is on the 20 th page) 1. Add the Standard working solution to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100 ul for each well). Add the samples to the other wells (100 ul for each well). Cover the plate with the sealer provided in the kit. Incubate for 90 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. Remove the liquid out of each well, do not wash. Immediately add 100 μl of Biotinylated Detection Ag working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37 C. 3. Aspirate or decant the solution from each well, add 350 ul of wash buffer to each well. Soak for 1~2 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. 4. Add 100 μl of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 min at 37 C. 5. Aspirate or decant the solution from each well, repeat the wash process for five times as conducted in step 3. 6. Add 90 μl of Substrate Reagent to each well. Cover with a new plate 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 30min. 7. 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. 8. Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm. 16

Calculation of results Average the duplicate readings for each standard and samples, then subtract the average zero standard optical density. Plot a four-parameter logistic curve on log-log graph paper, with standard concentration on the x-axis and OD values on the y-axis. If the samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper 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. Concentration(ng/mL) 100 50 25 12.5 6.25 3.13 1.56 0 OD 2.533 1.585 0.937 0.487 0.286 0.19 0.142 0.09 Corrected OD 2.443 1.495 0.847 0.397 0.196 0.1 0.052-17

Precision Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human IR-Ab were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IR-Ab were tested on 3 different plates, 20 replicates in each plate. Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 Mean(ng/mL) 4.79 13.99 36.95 4.98 14.13 37.46 Standard deviation 0.29 0.61 1.26 0.26 0.64 1.49 C V (%) 6.05 4.36 3.41 5.22 4.53 3.98 Recovery The recovery of Human IR-Ab spiked at two different levels in samples throughout the range of the assay was evaluated in various matrices. Sample Type Range (%) Average Recovery (%) Serum (n=5) 84-98 90 EDTA plasma (n=5) 87-102 94 Linearity Samples were spiked with high concentrations of Human IR-Ab 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) 1:2 1:4 1:8 1:16 Range (%) 94-106 89-102 Average (%) 100 95 Range (%) 87-99 81-94 Average (%) 92 86 Range (%) 85-99 84-97 Average (%) 92 89 Range (%) 87-98 83-98 Average (%) 92 90 18

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 settings is not optimal Check pipettes. Ensure briefly spin the vial of standard and dissolve the powder thoroughly by gentle mixing. Completely aspirate wells 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. 19

Powered by TCPDF (www.tcpdf.org) 2017 年 4 月修订第七版 SUMMARY 1. Add 100 μl standard or sample to each well. Incubate for 90 min at 37 C. 2. Remove the liquid. Add 100 μl Biotinylated Detection Ag. Incubate for 1 hour at 37 C. 3. Aspirate and wash 3 times. 4. Add 100 μl HRP Conjugate. Incubate for 30 min at 37 C. 5. Aspirate and wash 5 times. 6. Add 90 μl Substrate Reagent. Incubate for 15 min at 37 C. 7. Add 50 μl Stop Solution. Read at 450 nm immediately. 8. Calculation of results. 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. The final experimental results will be closely related to the validity of products, operational skills of the operators and the experimental environments. Please make sure that sufficient samples are available. 3. To get the best results, please only use the reagents supplied by the manufacturer and strictly comply with the instructions. 4. 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. 5. 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. 6. 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 equipments, and so on. Intra-assay variance among kits from different batches might arise from the above reasons, too. Copyright 2017-2018 Elabscience Biotechnology Co.,Ltd. All Rights Reserved