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人 (Human )白细胞介素12 (1L-12/P70 )ELISA 检测试剂盒使用说明书【本试剂盒只能用于科学研究,不得用于医学诊断】 检测原理 试剂盒采用双抗体一步夹心法酶联免疫吸附试验 ( ELISA ) 。 往预 先包被 白细胞介素 12 ( IL-12/P70 ) 抗体的包被微孔中,依次加入标 本 、 标准品 、 HRP 标记的检测抗体 , 经过温育并彻底洗涤 。 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 白细胞介素 12 ( IL-12/P70 ) 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。 样品收集、处理及保存方法 1. 血清 : 使用不含热原和内毒素的试管 , 操作过程中避免任何细胞 刺激 , 收集血液后 , 3000 转离心 10 分钟将血清和红细胞迅速小心地分离。 2. 血浆 : EDTA 、 柠檬酸盐或肝素抗凝 。 3000 转离心 30 分钟取上清 。 3. 细胞上清液: 3000 转离心 10 分钟去除颗粒和聚合物。 4. 组织匀浆 : 将组织加入适量生理盐水捣碎 。 3000 转离心 10 分钟 取上清。 5. 保存 : 如果样本收集后不及时检测 , 请按一次用量分装 , 冻存于 -20 ℃ ,避免反复冻融,在室温下解冻并确保样品均匀地充分解冻 。 自备物品 1. 酶标仪( 450nm ) 2. 高精度加样器及枪头 : 0.5-10uL 、 2-20uL 、 20-200uL 、 200-1000uL 3. 37 ℃ 恒温箱 操作注意事项 \ 1. 试剂盒保存在 2-8 ℃ ,使用前室温平衡 20 分钟。从冰箱取出的 浓缩洗涤液会有结晶,这属于正常现象,水浴加热使结晶完全溶解 后再使用。 2. 实验中不用的板条应立即放回自封袋中 , 密封 ( 低温干燥 ) 保存 。 3. 浓度为 0 的 S0 号标准品即可视为阴性对照或者空白;按照说明 书操作时样本已经稀释 5 倍,最终结果乘以 5 才是样本实际浓度 。 4. 严格按照说明书中标明的时间、加液量及顺序进行温育操作。 5. 所有液体组分使用前充分摇匀。 试剂盒组成 试剂盒组成 试剂盒组成 试剂盒组成 名称96 孔配置 48 孔配置 备注 微孔酶标板 12 孔 × 8 条 12 孔 × 4 条 无 标准品 0.3mL*6 管 0.3mL*6 管 无 样本稀释液 6 mL 3 mL 无 检测抗体 -HRP 10mL 5mL 无 20 × 洗涤缓冲液 25mL 15mL 按说明书进行稀释 底物 A 6mL 3mL 无 底物 B 6mL 3mL 无 终止液 6mL 3mL 无 封板膜 2 张 2 张 无 说明书 1 份 1 份 无 自封袋 1 个 1 个 无 注:标准品( S0-S5 )浓度 依次 为: 0 、 2.5 、 5 、 10 、 20 、 40 pg/mL 试剂的准备 20 × 洗涤缓冲液的稀释 : 蒸馏水按 1 : 20 稀释 , 即 1 份的 20 × 洗涤缓 冲液加 19 份的蒸馏水。 洗板方法 洗板方法 洗板方法 洗板方法 1. 手工洗板:甩尽孔内液体,每孔加满洗涤液,静置 1min 后甩尽 孔内液体,在吸水纸上拍干,如此洗板 5 次。 2. 自动洗板机:每孔注入洗液 350 μ L ,浸泡 1min ,洗板 5 次。 操作步骤 1. 从室温平衡 20min 后的铝箔袋中取出所需板条 , 剩余板条用自封 袋密封放回 4 ℃ 。 2. 设置标准品孔和样本孔 , 标准品孔各加不同浓度的标准 品 50 μ L ; 3. 样本孔先加 待测样本 10 μ L ,再 加样本稀释液 4 0 μ L ; 空白孔不 加。 4. 除空白孔外, 标准品孔和样本孔中每孔加入辣根过氧化物酶( HRP )标记的检测抗体 100 μ L ,用封板膜封住反应孔, 37 ℃ 水浴 锅或恒温箱温育 60min 。 5. 弃去液体,吸水纸上拍干,每孔加满洗涤液,静置 1min ,甩去洗涤液,吸水纸上拍干,如此重复洗板 5 次(也可用洗板机洗板 ) 。 6. 每孔加入底物 A 、 B 各 50 μ L , 37 ℃ 避光孵育 15min 。 7. 每孔加入终止液 50 μ L , 15min 内 , 在 450nm 波长处测定各孔 的 OD 值。 结果判断 结果判断 结果判断 结果判断 绘制标准曲线:在 Excel 工作表中,以标准品浓度作横坐标,对 应 OD 值作纵坐标 , 绘制出标准品线性回归曲线 , 按曲线方程计算各样 本浓度值。 试剂盒性能 试剂盒性能 试剂盒性能 试剂盒性能 1. 准确性:标准品线性回归与预期浓度相关系数 R 值,大于等 于 0.9900 。 2. 灵敏度:最低检测浓度小于 0.1 pg/mL 。 3. 特异性:不与其它可溶性结构类似物交叉反应。 4. 重复性:板内、板间变异系数均小于 15% 。 5. 贮藏: 2-8 ℃ ,避光防潮保存。 6. 有效期: 6 个月 免责声明 1. 试剂盒仅供研究使用,不得用于临床实验或 人 体实验,否则所 产生的一切后果,由实验者承担,本公司概不负责。 2. 严格按照说明书操作,实验者违反说明书操作,后果由实验者 承担。 FOR RESEARCH USE ONLY. NOT F FOR USE IN DIAGNOSTIC PROCEDURES . . . . Human Interleukin Interleukin 12 (IL-12/P70) ELISA Kit instruction Intended use This IL-12/P70 ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of IL-12/P70 in the sample, this IL-12/P70 ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus IL-12/P70 concentration. The concentration of IL-12/P70 in the samples is then determined by comparing the O.D. of the samples to the standard curve. Sample collection and storages Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 1 0 minutes at approximately 3 000 × g. Remove serum and assay immediately or aliquot and store samples at -20 ℃ or -80 ℃ .Avoid repeated freeze-thaw cycles Plasma Plasma Plasma Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3 000 × g at 2-8 ℃ within 30 minutes of collection. Store samples at -20 ℃ or -80 ℃ . Avoid repeated freeze-thaw cycles. Cell Cell Cell Cell culture culture culture culture supernates supernates supernates supernates and and and and other other other other biological biological biological biological fluids fluids fluids fluids - - - - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 ℃ or -80 ℃ . Avoid repeated freeze-thaw cycles. Note: The samples shoule be centrifugated dequately and no hemolysis or granule was allowed. Materials required but not supplied 1. Standard microplate reader (450nm) 2. Precision pipettes and Disposable pipette tips . 3. 37 ℃ incubator P recautions 1. D o not s ub stit u t e r eage n t s fr om one k i t t o ano t he r . S t and a r d, co n j ug a t e and m i c r op l a t es a r e m a t ched f or op ti m al pe rf o r m ance. U s e on l y t he r eage n t s s upp li ed by m anu f ac t u r e r . 2. D o not r e m o v e m i c r op l a t e fr om t he st o r age bag u n t i l needed. U nu s ed stri ps s hou l d be st o r ed at 2 - 8 ° C i n t he i r pouch wit h t he de si cca n t p r o v i ded. 3 . Mix all reagents before using. R e m o v e a l l k i t r eage n t s fr om r e fri ge r a t or and a ll ow t hem t o r each r oom t e m pe r a t u r e ( 20 - 2 5 ° C ) Materials supplied Name 96 determinations 48 determinations Microelisa stripplate 12*8strips 1 2*4strips Standard 0. 3 ml *6tubes 0. 3 ml *6tubes Sample Diluent 6 .0 ml 3.0 ml HRP-Conjugate reagent 10.0 ml 5.0ml 20X W ash solution 25 ml 15ml Chromogen Solution A 6 .0 ml 3 .0ml Chromogen Solution B 6 .0 ml 3 .0ml Stop Solution 6 .0 ml 3 .0ml Closure plate membrane 2 2 User manual 1 1 Sealed bags 1 1 Note: Note: Note: Note: Standard ( S0 → S5 ) concentration was followed by: 0,2.5,5,10,20,40 pg/ml Reagent preparation 20 × wash solution:Dilute with Distilled or deionized water 1:20. A ssay procedure 1. P r ep a r e a l l r e a g e n t s be f o r e st a rti ng a ss a y p r ocedu r e. I t i s r eco mm ended t h a t a l l S t and a r ds and S a m p l es be added i n dup li c a t e t o t he Mi c r o elisa Stripp l a t e. 2. Add standard : Set Standard wells , testing sample well s. Add standard 50 μ l to3. Add Sample : Add testing s ample 1 0 μ l t hen a dd Sample Diluent 40 μ l to testing sample well ; B lank well doesn ’ t add anyting. 4. A dd 10 0 μ l of HRP-conjugate reagent to each well , c over with a n adhesive strip and i ncub a t e f or 6 0 minutes at 3 7 ° C . 5 . Aspirate each well and wash, repeating the process four times for a total of f ive washes. Wash by filling each well with Wash Solution (400 μ l ) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to goo d performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels. 6 . Add chromogen solution A 50 μ l and chromogen solution B 50 μ l to each well. Gently mix and incubate for 15 minutes at 37 ° C . Protect Protect Protect Protect from from from from light light light light . . . . 7 . Add 50 μ l Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing . 8 . R ead t he O p ti cal D en sit y ( O . D .) at 450 nm u si ng a m i c r o tit er p l a t e r eader wit h i n 15 m i nu t e s . C alculation of results 1. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm)obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis. 2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software. 3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration. 4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve. 5. The sensitivity by this assay is 0.1 pg/ml 6. Standard curveS torage : 2-8 ℃ . validity : six months. FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! ENTIRE PROCEDURE BEFORE BEGINNING! |