奶制品中三聚氰胺快速检测ELISA试剂盒

Size: px

Start display at page:

Download "奶制品中三聚氰胺快速检测ELISA试剂盒"

塔黄任
6 years ago
Views:

1 Tribo TM Clenbuterol Fast Detection ELISA Kit User Guide (Catalog No. TBS21111) Enzyme-Linked Immunosorbent Assay Kit for Detection of Clenbuterol TribioScience, Inc.

2 Contents General Description 3 Intended Use 3 Safety Instructions 3 Assay Principles 3 Reagents and Materials Provided 4 Materials Required but not Provided 4 Assay Procedure 4 Calculating Results 5 Performance Data 7 Precautions 7 Storage and Expiration Date 8 Technical Assistance 8 General Limited Warranty 8 The User Guide Chinese Version

3 General Description Clenbuterol, often referred to as clen, is a potent beta-2 agonist that can stimulate beta-2 receptors in mammals, which in turn leads to fat loss by allowing the body to release and burn more stored fat. It has been used as a performance-enhancing drug by athletes and body builders (now banned by IOC), and has been illegally used for decades in the veterinary world to increase the lean yield of livestock. Clenbuterol can cause a multitude of adverse effects upon entering human body, such as dizziness, nausea, diarrhea, agitations, muscle cramps, hypertension, and even acute myocardial infarction (heart attack). Therefore, it has been banned in many countries to be used on food-producing animals. The Tribo TM Clenbuterol ELISA Kit can quickly, sensitively, and accurately determine the presence of clenbuterol in animal feed, and urine and tissue samples of animals, providing a vital tool to prevent consumption of food tainted with this toxic chemical. Intended Use The Tribo TM Clenbuterol ELISA Kit utilizes competitive ELISA for the quantitative and qualitative analysis of clenbuterol in animal feed and tissue/urine samples of farm animals. The limit of detection (LOD) of clenbuterol in ELISA Kit is 0.1ng/ml (0.1ppb). Safety Instructions To receive complete safety information on this product, contact AccuAffinity, Inc. and request Material Safety Data Sheets. Stop solution is 1N sulfuric acid. Handle with care. Assay Principle Tribo TM Clenbuterol ELISA Kit is a competitive enzyme-labeled immunoassay. Each well of the 96-well microtiter plate has been pre-coated with an anti-clenbuterol antibody. During the assay, clenbuterol standard solution or samples are added to test wells, followed by adding horse radish peroxidase (HRP)-clenbuterol conjugate, which will compete with clenbuterol in standard or sample for binding to antibody during the 30-minute incubation. After plate wash, a clear HRP substrate is added to the wells leading to a colored product only in the presence of HRP, and optical density is inversely related to clenbuterol concentrations in the samples. The accurate concentration of clenbuterol can then be determined by interpolation using the standard curve constructed in the same run

4 Reagents and Materials Provided The reagents included in the kit are sufficient for performing 96 measurements (including standards and samples). Reagents and materials in each kit include: a) 1 microtiter plate containing 12 test strips of 8 wells sealed in an aluminized pouch with dessicant. b) 6 vials each containing 0.5 ml of clenbuterol standard with 0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/ml of clenbuterol respectively. c) 1 vial containing 0.1 ml clenbuterol-hrp conjugate (100 ). d) 1 bottle containing 10 ml sample diluent (5 x). e) 1 bottle containing 10 ml assay buffer (1 x). f) 1 bottle containing 15 ml microtiter plate wash solution (20 ). g) 1 bottle containing 11 ml substrate (1 ). h) 1 bottle containing 11 ml stop solution (1 ). i) 1 microtiter plate sealers. j) 1 user guide booklet (this document). Materials required but not provided Microplate reader with 450 nm filter. Pipet capable of dispensing µl Assay Procedure Equilibrate kit components at room temperature (20-25 o C) for at least 30 min prior to running the test, and thoroughly mix all liquid components before use. Use test strips as needed on the frame, and store unused strips in the resealable bag at 2-8 o C. Number standards and samples according to positions on microtiter plate. All standards and samples need duplicate measurement for accuracy. Sample preparation: Samples need to be processed as followings before ELISA assay: For meat samples: a) Homogenize sample b) Weigh out 2.0 g ground feed c) Add 18 ml distilled water and 2 ml 1N HCl d) Homogenize and vortex for 3 min - 4 -

5 e) Centrifuge at 2000 rpm for 20 min at room temperature f) Take supernatant and adjust ph to 7-8 using 1N NaOH g) Centrifuge at 2000 rpm for 20 min at room temperature h) Use supernatant directly for ELISA assay i) Sample processed in this method has dilution factor of 10 For animal feed: 1. Weigh out 2.0 g homogenized sample 2. Add 2ml ethyl acetate and mix by vortex thoroughly 3. Centrifuge at 4000 rpm for 10 min at room temperature 4. Take 1 ml supernatant 5. Evaporate to dryness in a nitrogen evaporator 6. Add 1 ml hexane to reconstitute 7. Add 1 ml of sample diluent 8. Vortex vigorously 9. Centrifuge at 4000 rpm for 10 min at room temperature j) Discard upper layer, and transfer 50 µl bottom aqueous phase to ELISA plate k) Sample processed in this method has dilution factor of 2 For urine samples: a) Centrifuge at 4000 rpm for 5 min at room temperature b) Take supernatant and dilute 5 fold with sample diluent for ELISA assay c) Sample processed in this method has dilution factor of 5 ELISA assay: 1. Prepare Wash Solution by diluting 1 part of Wash Solution Concentrate (20x) with 19 parts of distilled water to obtain Wash Solution (1x) 2. Prepare HRP working solution by diluting 1 part of HRP conjugate (100x) with 99 parts of Assay Buffer to obtain HRP working solution (1x) 3. Add clenbuterol standard sample, or unknown sample, 20 µl/well in duplicate 4. Add HRP working solution 80 µl/well, gently shake plate by hand for 1 min, and incubate at room temperature for 30 min 5. Wash plate 4 times with wash solution (1x), 200 µl/well wash buffer each time 6. Add 100 µl/well TMB-Ultra solution, and incubate plate at room temperature for 15 min away from light 7. Add 100 µl/well stopping solution, read OD 450nm Calculating Results - 5 -

6 Two approaches can be used to obtain clenbuterol concentration results from the assay. Semi-quantitative results can be obtained with the first approach while quantitative results can be calculated with the second. Please note the negative correlation between absorbance reading (OD 450 ) and clenbuterol concentration in the sample. 1. Semi-quantitative detection of clenbuterol A simple comparison of average sample absorbance to absorption of standards will give the range of clenbuterol concentration (ng/ml or ppb) in the samples. For example, Sample 1 has an average absorbance of 0.6, and Sample 2 of 1.2, and OD 450 of standard solutions are as follows: for 0 ng/ml; for 0.1 ng/ml; for 0.3 ng/ml; for 0.9 ng/ml; for 2.7 ng/ml; for 8.1 ng/ml. It is immediately known that clenbuterol concentration of Sample 1 is between 0.9 ng/ml and 2.7 ng/ml, while Sample 2 contains ng/ml clenbuterol. 2. Quantitative calculation of clenbuterol concentration a) Calculate B/B 0 Dividing average absorbance of each standard and sample (B) by absorbance of first standard (the standard with 0 ng/ml clenbuterol concentration, B 0 ) to obtain percentage absorbance. percentage absorbance (%) = B/B 0 x 100% B average absorbance of a standard or sample B 0 average absorbance of 0 ng/ml standard b) A standard curve is obtained by graphing the percentage absorbance of standards (Y axis) versus their corresponding concentration (X axis) on semi-log graph paper (which should be a linear relationship), and sample concentration can be read from this standard curve. Alternatively, clenbuterol concentration in the samples can be calculated with regression equation correlating percentage absorbance to clenbuterol concentration. Graphing software can also be used for quick analyses of large number of samples. Performance Data Range of Standard Curve: Assay Quantitative Range: Assay Time: ng/ml ng/ml 40 min - 6 -

7 Limit of Detection (LOD): Meat Feed Urine 1 ppb 0.2 ppb 0.5 ppb Recovery: % Cross reactivity: Salbutamol 3.8% Ractopamine <0.2% Sensitivity (defined as the average of absorbance from 6 zero-standards minus 3 times of standard deviation): 0.1 ng/ml Precision: Intra-assay CV <12% Inter-assay CV <15% Precautions a) Assay kit should be stored at 2-8 o C and avoid freezing conditions; unused test strips should be sealed in reclosable bag; colorless substrate is sensitive to light so prolonged exposure to light needs to be avoided. b) Reagents should be brought to room temperature (20-25 o C) prior to use. A room temperature of lower than 20 o C or failure to equilibrate reagents or samples to room temperature could result in low OD readings for all samples. All unused portion of reagents should be put back into 2-8 o C storage immediately after use. c) Adhere to assay protocol on reaction temperature and time, and use pipet to add components whenever possible. Results are solely based on OD450 readings from plate/strip reader. d) Reagents need to be thoroughly mixed to improve reproducibility. e) During all incubation steps, avoid light and seal plate with sealer. f) If wells are dried out during plate wash steps, linearity of standard curve will be negatively affected and reproducibility will be poor. Therefore, substrate addition should be carried out immediately after tapping the plate dry (following the last wash). g) The stop solution is 1N sulfuric acid. Avoid contact with skin or clothing. Immediately clean up any spills and wash area with copious amounts of water. If contact should occur, immediately flush with copious amounts of water. h) Do not use reagents beyond expiration date. Dilution or adulteration of reagents or samples not called for in the procedure may result in adverse changes in sensitivity and OD reading. Do not substitute reagents from kits with different lot numbers

8 i) Obvious color in substrate suggests expiration and it should be discarded. When absorbance of zero-standard is lower than 0.6, the reagents may have expired. Storage and Expiration Date Storage: All components of the kit should be stored at 2-8 o C. Expiration Date: This kit expires 12 months after manufacturing date. Technical Assistance For ordering or technical assistance regarding this kit, or for additional information about TribioScience products, please info@tribioscience.com or call (650) General Limited Warranty TribioScience, Inc. warrants its manufactured products against defects in materials and workmanship when used in accordance with the applicable instructions for a period not to extend beyond a product s printed expiration date. TribioScience makes no other warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular purpose. The warranty provided herein and the data, specifications and descriptions of TribioScience products appearing in published catalogues and product literature may not be altered except by express written agreement signed by an officer of TribioScience. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and, if given, should not be relied upon. In the event of a breach of the foregoing warranty, TribioScience Inc. s sole obligation shall be to repair or replace, at its option, any product or part thereof that proves defective in materials or workmanship within the warranty period, provided the customer notifies TribioScience promptly of any such defect. The exclusive remedy provided herein shall not be deemed to have failed of its essential purpose so long as TribioScience is willing and able to repair or replace any nonconforming TribioScience product or part. TribioScience shall not be liable for consequential, incidental, special or any other indirect damages resulting from economic loss or property damage sustained by a customer from the use of its products. However, in some states the purchaser may have rights under state law in addition to those provided by this warranty. For research use only

9 肉类及饲料中克伦特罗快速检测 ELISA 试剂盒使用说明书产品编号 :TBS21111 概述克伦特罗, 又名瘦肉精, 属于 β- 兴奋剂类药物, 是一种肾上腺类神经兴奋剂当添加到动物饲料中后, 可以增加动物的瘦肉量, 同时减少饲料使用然而瘦肉精为增加瘦肉率所需用量大, 在动物体内代谢缓慢, 在肉类尤其是内脏中会有大量残留, 如果食用进入人体会直接危害人体健康, 出现晕厥心慌战栗头疼恶心呕吐肌肉振颤等中毒症状, 特别是对高血压心脏病等疾病患者危害更大, 严重的可导致死亡瘦肉精是国家明令禁止使用的饲料添加剂国家标准 GB/T 中规定了动物性食品中克伦特罗的测定方法, 适用于新鲜或冷冻的畜禽肉与内脏及其制品中克伦特罗残留的测定, 同时也适用于生物材料 ( 人或动物血液尿液 ) 中克伦特罗的测定其中的气相色谱质谱法和高效液相色谱法需要繁琐的样品前处理和复杂的仪器设备, 而酶联免疫法不仅具有简单灵敏准确等特点, 而且和其它方法相比具有同等检出限和更灵敏的线性范围, 为大多厂家和质检部门所使用用途本试剂盒用于定量定性检测肉类 ( 包括鱼虾类 ), 动物饲料样品和尿样中克伦特罗的含量试剂盒检测反应灵敏度为 0.1 μg/kg(0.1 ppb) 检测原理本试剂盒是利用免疫分析直接竞争法的原理, 在微孔板上预包被抗克伦特罗抗体检测时, 加入克伦特罗标准品或待测样品溶液及辣根过氧化物酶 (HRP) 标记的克伦特罗 ( 标记物 ), 标记物将和标准品或样品中的克伦特罗竞争性地与抗体结合洗涤后用酶底物 (TMB) 系统显色, 样本吸光值与其中所含克伦特罗量成负相关, 与标准曲线比较后即可得出样品中的瘦肉精准确含量 - 9 -

10 试剂盒组成成份每一试剂盒中的试剂足够进行 96 个测量 ( 包括标准分析孔 ) 盒中的材料如下: 1. 抗克伦特罗抗体包被微孔板 ( 可分为 12 条使用 ) 8 孔 12 条 2. 克伦特罗标准品 6 瓶, 为标准溶液 : 每瓶 0.5 ml j) 0 ng/ml; B. 0.1 ng/ml; C. 0.3 ng/ml; D. 0.9 ng/ml; E. 2.7 ng/ml; F. 8.1 ng/ml 3. 样品稀释液 (5 ) 10 ml 1 瓶 4. HRP 标记克伦特罗 (100 ) 100 μl 1 瓶 5. 分析缓冲液 10 ml 1 瓶 6. 微孔板洗涤液 (20 ) 40 ml 1 瓶 7. 显色液 (1 ) 11 ml 1 瓶 8. 终止液 (1 ) 11 ml 1 瓶 9. 封板膜 1 张 10. 自封塑料袋 1 个使用单位所需仪器微孔板酶标仪, 检测波长 450nm, 参考波长 630nm μl 200 μl 微量加样器 ;300 μl 多道加样器检测步骤 * 使用前将试剂盒置于室温 (20 25 ) 平衡 30 分钟以上, 注意每种液体试剂使用前均须摇匀 * 取出需要数量的酶标板微孔条插入框架中, 将不用的微孔条放入自封袋, 保存于 2 8 不要冷冻 * 编号 : 将样品和标准品对应微孔按序编号, 每个样品和标准品均需做二复孔样品处理方法 : 肉类 ( 包括鱼虾类 ) 样品需在 ELISA 测定前按照如下步骤进行制备 : d) 将样品匀浆 e) 称取 2 g 匀浆样品 f) 加入 18 ml 水和 2 ml 1N 盐酸, 混匀 3 分钟 g) 在室温下, 以每分钟 2000 转离心 20 分钟 h) 吸取上清, 用 1N 氢氧化钠调节 ph 至 7-8 i) 在室温下, 以每分钟 2000 转离心 20 分钟 j) 吸取上清进行 ELISA 分析 k) 经此方法处理样品稀释度为 10 倍动物饲料样品 :

11 1. 称取 2 g 磨碎样品 2. 加入 2 ml 乙酸乙酯, 充分混匀 3. 在室温下, 以每分钟 4000 转离心 10 分钟 4. 吸取 1 ml 上清液, 在氮气下吹干 5. 重溶于 1 ml 正已烷, 再加入 1 ml 样品稀释液 6. 充分混匀后, 在室温下, 以每分钟 4000 转离心 10 分钟 7. 弃去上清液, 取 50 μl 下部水相溶液进行 ELISA 分析 8. 经此方法处理样品稀释度为 2 倍尿样处理 : a) 在室温下, 以每分钟 4000 转离心 5 分钟 b) 吸取上清液, 以样品稀释液稀释 5 倍, 进行 ELISA 分析 c) 经此方法处理样品稀释度为 5 倍 ELISA 分析方法 : 配制洗涤缓冲液 : 1 份洗涤缓冲液浓缩液, 加 19 份蒸馏水配制成洗涤缓冲液配制酶标记物溶液 :1 份酶标记物浓缩液, 加 99 份分析缓冲液配制成酶标记物溶液加样 : 用加样器每孔加标准品或样本 20 μl 加酶标记物 : 用加样器每孔加 HRP 标记瘦肉精 80 μl 温育 : 轻轻振荡混匀一分钟, 用盖板膜封板后在室温 (20 25 ) 反应 30 分钟洗板 : 每孔加入微孔板洗涤液 200 μl, 倒掉后重复, 共清洗 4 次, 最后拍干显色 : 每孔加入显色液 100 μl, 轻轻振荡混匀, 室温 (20 25 ) 避光显色 10 分钟测定 : 每孔加入终止液 100 μl, 轻轻振荡混匀, 设定酶标仪于 450nm 处, 测定每孔吸光度值 (OD450 值 ) 结果判定结果判定有两种方法, 粗略判定可用第 1 种方法, 定量判定用第 2 种方法注意样本吸光值与其所含瘦肉精成负相关 l) 定性检测用样品的平均吸光度值与标准值比较即可得出其浓度范围 (ng/ml 或 ppb) 假设样品 1 的吸光度值为 0.6, 样品 2 的吸光度值为 1.2, 标准液吸光度值分别是 :0 ng/ml 为 2.300; 0.1 ng/ml 为 2.000; 0.3 ng/ml 为 1.650; 0.9 ng/ml 为 0.950; 2.7 ng/ml 为 0.400;8.1 ng/ml 为则样品 1 的浓度范围是 ng/ml; 样品 2 的浓度范围是 ng/ml m) 定量测定 a) 计算 B/B0-11 -

12 所获得的每个浓度标准溶液和样本吸光度值的平均值 (B) 除以第一个标准品 A( 零标准品 ) 的吸光度值 (B0) 再乘以 100%, 即百分吸光度值百分吸光度值 (%)=B/B0 100% B 标准溶液或样本溶液的平均吸光度值 B0 0 ng/ml 标准溶液的平均吸光度值 b) 绘制标准曲线, 计算样本中瘦肉精浓度以克伦特罗标准品浓度为 X 轴, 对应的百分吸光度值为 Y 轴, 在半对数坐标纸上绘制标准曲线图相对应每一个样品的浓度可以从标准曲线上读出也可以用回归方程法, 计算出样本溶液浓度利用计算机专业软件, 更便于大量样品的快速分析产品技术指标标准曲线范围 :0 8.1 ng/ml 试剂盒定量区间 : ng/ml 检测时间 :40 分钟样本最低检测限 : 肉类 1 ppb 饲料 0.2 ppb 尿样 0.5 ppb 回收率 % 交叉反应 : 沙丁胺醇 3.8% 莱克多巴胺 <0.2% 灵敏度 (Sensitivity, 定义为 6 个零标准品吸光度值的平均值减去 3 倍标准方差所对应的浓度 ): 0.1 ng/ml (0.1 ppb) 精密度 (Precision): 板内变差系数 (Intra-assay CV)<12% 板间变差系数 (Inter-assay CV)<15% 注意事项 1. 保存试剂盒于 2 8, 不要冷冻 ; 将不用的微孔板放进自封袋重新密封 ; 无色的显色液对光敏感, 因此要避免直接暴露在光线下

13 2. 使用之前将所有试剂温度回升至室温 (20 25 ) 室温低于 20 或试剂及样本没有回到室温可能导致所有样品的 OD 值偏低使用之后立即将所有试剂放回 2 8 冰箱 3. 严格控制反应温度和时间, 并尽量使用移液器加样 ; 结果判定以酶标仪读数为准 4. 试剂混合要均匀, 否则会出现重复性不好的现象 5. 在所有恒温孵育过程中, 避免光线照射, 用封板膜封住微孔板 6. 在洗板过程中如果出现板孔干燥的情况, 则会伴随着出现标准曲线不成线性, 重复性不好的现象所以洗板拍干后应立即进行下一步操作 7. 反应终止液为 1N 硫酸, 应避免接触皮肤和衣物 8. 不要使用超过有效日期的试剂盒, 稀释或搀杂使用会引起灵敏度 OD 值的变化不要交换使用不同批号试剂盒中的试剂 9. 显色液若有任何颜色表明变质, 应当作失效试剂处理当零标准品的吸光度值小于 0.6 时, 表示试剂可能变质存储条件和有效期贮存条件 : 在 2 8 保存试剂盒有效期 : 本试剂盒有效期为 12 个月本产品仅供研究使用

Applied Biosystems StepOne™ Real-Time PCR System Quick Reference Card for Installation

Applied Biosystems StepOne™ Real-Time PCR System Quick Reference Card for Installation Applied Biosystems StepOne Real-Time PCR System StepOne 系统安装快速参考卡本文档提供在并置布局中安装 StepOne 系统的简明指导有关完整步骤或独立安装步骤请参阅 Applied Biosystems StepOne Real-Time PCR System 安装联网和维护指南目录 1. 安装准备........................................