瘦肉精克伦特罗残留检测免疫芯片技术的研究

目的：建立克伦特罗残留检测免疫芯片方法。方法：通过微阵列技术，将克伦特罗抗体与硝酸纤维素膜结合，用牛血清白蛋白封闭硝酸纤维素膜，加待测品和标样，标样中辣根过氧化酶标记的克伦特罗和待测品中的克伦特罗竞争性与克伦特罗抗体结合，借助辣根过氧化酶催化Luminol-H₂O₂化学发光反应，测定发光强度与待测品中克伦特罗浓度的相关性，并考察芯片的检测限、线性范围、回收率、信号精密度、特异性、储存稳定性。结果：本法对盐酸克伦特罗残留检测限为0.10 ng·mL^-1、定量限为0.30 ng·mL^-1，盐酸克伦特罗的质量浓度与光子信号关系成负相关性，线性范围在0.5~16.0 ng·mL^-1，其拟合方程为Y=-0.3915X³+12.5X²-140.68X+867.54，R²=0.9994，回收率为93.6%~103.3%，芯片批内与批间RSD均小于5.0%。未发现与沙丁胺醇、特布他林、莱克多巴胺、塞曼特罗、酚间羟异丙肾上腺素、异丙肾上腺素等构效关系类似物发生交叉反应。芯片贮存于2~8 ℃冰箱中，信号在12个月内基本稳定。结论：免疫芯片法检测克伦特罗残留快速、简单、准确。

Objective: To establish a method for the residue detection of clenbuterol by immunochip. Methods: Antibodies against clenbuterol were combined with cellulose nitrate membrane by microarray techniques.Cellulose nitrate membrane was blocked by bovine serum albumin.The test sample and the standard sample were added into the chip,and the standard sample was marked with horseradish peroxidase which would combine competitively with the antibodies.Then HRP catalyzed Luminol-H₂O₂ chemiluminescence reaction.Correlation between luminous intensity and the concentration of clenbuterol in the sample were measured.Limit of detection,limit of quantitation,linearity and range,precision,accuracy,specificity,storage stability were investigated. Results: The limit of detection of clenbuterol hydrochloride residues was 0.10 ng·mL^-1;the limit of quantitation was 0.30 ng·mL^-1.Correlation between clenbuterol hydrochloride concentration and its photon signal intensity was a negative correlation in the range of 0.5-16 ng·mL^-1.The equation was Y=-0.3915X +12.5X²-140.68X+867.54,R²=0.9994,the recovery was 93.6%-103.3%.RSD values were less than 5% within batch and between batches.Cross-reaction was not found with analogues of structure-activity relationship such as salbutamol,terbutaline,ractopamine,cimaterol,fenoterol,isoproterenol,etc.The chips were stable at 2-8 ℃ for 12 months. Conclusion: The method is fast,simple and accurate for detection of clenbuterol residue by immunochip.