莱克多巴胺(Rac)快速检测试剂盒(胶体金法)
使用说明书
一、简介
β兴奋剂是一种人和兽医作为治疗哮喘的药物。在牲畜中超剂量使用可以脂肪组织转化为肌肉组织,由于能够显著改善脂肪率和生产效率,β兴奋剂往往被滥用于畜牧养殖生产中。已经有克伦特罗或其他β兴奋剂中毒的病例报道,近来又有一些非法使用莱克多巴胺(Rac)来替代克伦特罗作为饲料添加,并存在滥用现象。
通常情况下,HPLC或GC-MS是莱克多巴胺(Rac)残留检测的确证方法,但是其前处理步骤繁琐、费用昂贵,胶体金快速检测试剂盒具有成本低廉、操作简便、不需要仪器设备等优点,已成为常规的现场筛查方法。
二、用途
定性检测动物尿液中莱克多巴胺(Rac)的残留。
三、检测原理
试剂盒采用用竞争抑制免疫层析法。在硝酸纤维素膜(NC膜)上包被检测线(T线)和对照线(C线),T线包被Rac-BSA,C线包被羊抗鼠IgG,样品孔加入样本后,在层析的过程中,样本中的莱克多巴胺(Rac)与胶体金标记的鼠抗莱克多巴胺单抗结合,抑制了胶体金标记的鼠抗莱克多巴胺单抗和NC膜T线上的Rac-BSA的结合,由此判断样本中是否含有莱克多巴胺(Rac)。
四、检测限
检测限:5ng/ml(5ppb)
五、特异性
本产品与沙丁胺醇、链霉素、四环素类、喹诺酮类等药物无交叉反应。
六、试剂盒组分
莱克多巴胺(Rac)快速检测卡 使用说明书
七、样本处理
尿液必须收集在洁净(不含任何防腐剂)的塑料尿杯或玻璃容器中。若不能及时送检,在2℃-8℃冷藏可保存4小时,长期保存需冷冻-20℃,严禁反复冻融。
八、操作步骤
(1)完整仔细阅读使用说明书。
(2)检测前尿样需回温到室温,如尿样浑浊,需3000rpm离心5min后取上清液检测。
(3)从原包装袋中取出检测试纸卡,打开后的试纸卡请在一个小时内尽快使用。
(4)将试剂卡置于干净平坦的台面上,做好样本编号标识,用吸管吸取待检样本,垂直滴加3-4滴(约60-80μL)样本于加样孔(S)内。
(5)加样后5-8min,根据示意图判定结果,其他时间结果无效。
九、结果判断
阴性(-):T线和C线都显色,表示样品中莱克多巴胺含量低于检测限。
阳性(+):T线无显色,C线显色,表示样品中莱克多巴胺含量高于检测限。
无 效:未出现C线,表明不正确的操作过程或试纸卡已变质失效。在此情况下,应再次仔细阅读说明书,并用新的试纸卡重新测试。
十、注意事项
(1)检测试纸卡在室温下一次性使用。
(2)检测时避免阳光直射。
(3)尽量不要触摸试纸卡中央的白色膜面。
十一、储存及有效期
原包装于10-30℃阴凉避光干燥处储存,切勿冷冻。打开后未使用产品的注意防潮保护,确保封口的紧密度。试剂盒在正确的储存条件下,有效期18个月。
Sample collection and storages
Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates and other biological 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 should be centrifugated adequately 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
Precautions
1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.
Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)
Materials supplied
Name |
96 determinations |
48 determinations |
Microelisa stripplate |
12*8strips |
12*4strips |
Standard |
0.3ml |
0.3ml |
Sample diluent |
6.0ml |
3.0ml |
HRP-Conjugate reagent |
10.0ml |
5.0ml |
20X Wash solution |
25ml |
15ml |
Chromogen Solution A |
6.0ml |
3.0ml |
Chromogen Solution B |
6.0ml |
3.0ml |
Stop Solution |
6.0ml |
3.0ml |
Closure plate membrane |
2 |
2 |
User manual |
1 |
1 |
Sealed bags |
1 |
1 |
Note: Standard concentration was followed by:
20、10、5、2.5、1.25、0 ng/mL.
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.
4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
5. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good 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 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
7. Add 50μl 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. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
Calculation of results
- 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.
- 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.
- 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.
- 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.
- The sensitivity by this assay is 0.1 ng/mL.
- Standard curve
Storage: 2-8℃.
validity: six months.
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!