Mouse anti-nuclear Antibody IgG (anti-ana IgG) ELISA Kit Catalog No. CSB-E12912m (96 tests) This immunoassay kit allows for the in vitro semi-quantitative determination of mouse anti-ana IgG concentrations in 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
PRINCIPLE OF THE ASSAY The microtiter plate provided in this kit has been pre-coated with purified nuclear antigen. Samples are then added to the appropriate microtiter plate wells and incubated. Then add Horseradish Peroxidase (HRP)-conjugated anti-mouse IgG to each well and incubate. Finally, a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. 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. Calculate the valence of anti-ana IgG in the samples. SPECIFICITY This assay recognizes mouse anti-ana IgG. No significant cross-reactivity or interference was observed. SENSITIVITY The minimum detectable dose of mouse anti-ana IgG is typically less than 1 ng/ml. MATERIALS PROVIDED Reagent Quantity Assay plate 1 Sample Diluent 1 x 20 ml HRP-anti-mouse IgG Diluent 1 x 10 ml HRP-anti-mouse IgG 1 x 120µl (1:100) Wash Buffer 1 x 20 ml (25 concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml Negative Control 1 x 800 µl 2
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. 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 up 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. Sample Dilute 2µl sample using 198µl Sample Diluent (1:100), respectively. 3. HRP-anti-mouse IgG Dilute to the working concentration using HRP-anti-mouse IgG 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. 3
SAMPLE COLLECTION AND STORAGE Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 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 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. ASSAY PROCEDURE Bring all reagents and samples to room temperature before use. It is recommended that all samples, and controls be assayed in duplicate. 1. Add 100µl of Negative Control. Add 100µl of Sample per well. Cover with the adhesive strip. Incubate for 30 minutes at 37 C. 2. Aspirate each well and wash, repeating the process for a total of five 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. 3. Add 100µl of HRP-anti-mouse IgG working solution to each well. Incubate for 30 minutes at 37 C. HRP-anti-mouse IgG working solution may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform. 4. Wash plate five times as before. 4
5. Add 90µl of TMB Substrate to each well. Incubate for 10 minutes at 37 C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 6. Add 50µl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7. Determine the optical density of each well within 15 minutes, using a microplate reader set to 450 nm. CALCULATION OF RESULTS For calculation the valence of mouse anti-ana IgG antibody, compare the sample well with control. The well appears significant blue color is determined as Positive. The well not contains significant blue is determined as Negative. While OD sample / OD negative 2.1: Positive While OD sample / OD negative <2.1: Negative 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. Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. 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. 5
TECHNICAL HINTS When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips 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. 6
小鼠抗核抗体 (ANA)IgG 酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用 产品编号 产品编号 :CSB-E12912m 最低检测限 最低检测限 :1 ng/ml 特异性 : 本试剂盒可同时检测小鼠 anti-ana IgG, 且与其他相关抗体无交 叉反应 有效期 有效期 :6 个月 预期应用 预期应用 :ELISA 法半定量测定小鼠血清 血浆或其它相关生物液体中 anti-ana IgG 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 本试剂盒是用 ELISA 间接法检测的, 用纯化的核抗原包被酶标板, 制成固相载体 向微孔中先加入待测样品进行反应, 然后再加入辣根过氧化物酶标记抗小鼠 IgG 进行反应, 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 anti-ana IgG 呈正相关 用酶标仪在 450nm 波长下测定吸光度 (OD 值 ), 评估样本中抗体的含量 7
试剂盒组成及试剂配制 1. 酶联板 (Assay plate ): 一块 (96 孔 ) 2. 样本稀释液 (Sample Diluent): 1 20ml/ 瓶 3. 辣根过氧化物酶标记抗小鼠 IgG 稀释液 (HRP-anti-mouse IgG Diluent): 1 10ml/ 瓶 4. 辣根过氧化物酶标记抗小鼠 IgG(HRP-anti-mouse IgG): 1 120µl/ 瓶 (1:100) 5. 底物溶液 (TMB Substrate): 1 10ml/ 瓶 6. 浓洗涤液 (Wash Buffer): 1 20ml/ 瓶, 使用时每瓶用蒸馏水稀释 25 倍 7. 终止液 (Stop Solution): 1 10ml/ 瓶 (2N H 2 SO4) 8. 阴性质控品 (Negative Control): ):): 1 800ul/ 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 小时或室温过夜后于 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 8
标本的稀释原则 : 试验前用样本缓冲液按 1:100 的比例稀释样本 所以使用 2ul 的样本 +198ul 样本稀释液加入聚苯乙烯试管中 混合均匀 阴性质控品已经准备好不需要稀释 辣根过氧化物酶标记抗小鼠 IgG 的稀释原则 : 临用前以辣根过氧化物酶标记抗小鼠 IgG 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抗小鼠 IgG 加 990µl 辣根过氧化物酶标记抗小鼠 IgG 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 如样品浓度过高时, 用样品稀释液进行稀释, 以使样品符合试剂盒的检测范围 1. 加样 : 分别设阴性孔 待测样品孔 阴性孔加阴性质控品 100µl, 余孔加待测样品 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 30 分钟 2. 甩干, 洗板 5 次, 每次浸泡 1-2 分钟,200µl/ 每孔, 甩干 3. 每孔加辣根过氧化物酶标记抗小鼠 IgG 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 ),37,30 分钟 4. 弃去孔内液体, 甩干, 洗板 5 次, 每次浸泡 1-2 分钟,350µl/ 每孔, 甩干 5. 依序每孔加底物溶液 90µl,37 避光显色 10min 6. 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 7. 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 9
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 辣根过氧化物酶标记抗小鼠 IgG 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辣根过氧化物酶标记抗小鼠 IgG 工作液 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 1. 目测 : 在白色背景下观察各孔显色情况, 有明显变蓝者为阳性, 无色或蓝色不明显者为阴性 2. 酶标仪检测 : 每孔加终止液 50ul, 酶标仪 450nm, 空白孔调零 测定各孔 OD 值 临界值 = 阴性对照 x2.1 阳性大于等于临界值阴性小于临界值 10
注意事项 1. 当混合蛋白溶液时应尽量轻缓, 避免起泡 2. 洗涤过程非常重要, 不充分的洗涤易造成假阳性 3. 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 4. 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 5. 在配制检测溶液工作液时, 请以相应的稀释液配制, 不能混淆 6. 底物请避光保存 7. 不要用其它生产厂家的试剂替换试剂盒中的试剂 11
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