| Tylosin(TYL)and tilmicosin(TIM)are both macrolides(MALs)drugs.They are special antibiotics for poultry and livestock.Because of its good intestinal absorption,rapid diffusion in the body,and high blood drug concentration,it is mainly used in clinical treatment and prevention of various respiratory tract,intestinal,reproductive tract,and motor system infections caused by Mycoplasma,Staphylococcus aureus,and Pseudobacterium.However,if such drugs are abused or unreasonably used for a long time,it is easy to cause allergic reactions,hearing loss,and even serious damage to the liver and kidney.Its residue in animal foods will pose a threat to consumer health.Therefore,the detection and supervision of these two antibiotic residues are particularly important.This study establishes a lateral flow immunochromatographic detection method for colloidal gold,latex microspheres,and time-resolved fluorescent microspheres that can simultaneously detect TYL and TIM in milk,pork,and egg samples.It aims to achieve rapid sample detection and provide technical support for the monitoring of MALs residues in animal foods.The main results obtained are as follows:(1)Establishment of colloidal gold immunochromatography method: In order to obtain colloidal gold-labeled antibody with high detection performance,the particle size of colloidal gold,the p H value of the labeling,antibody dilution,coating conditions,etc.were optimized and adjusted;A more comprehensive discussion on sample pre-treatment and sample pad technology to meet the testing needs of different samples.The optimized detection performance of the method is as follows: milk samples are directly detected without pre-treatment,and pork and egg samples can be detected after simple extraction and dilution,and the detection results can be obtained within 5 minutes.The Cut-Off values of TYL ??for milk,pork,and egg samples are: 6 ng/m L,15 ng/g,and 10 ng/g,and the Cut-Off values ??for TIM are 6 ng/m L and 8 ng/g and 4 ng/g,respectively.(2)Establishment of latex microspheres immunochromatography method: In this study,blue latex microspheres with functionalized carboxyl groups on the surface were used to optimize the technical parameters such as the p H value of carboxyl activation and antibody coupling,antibody dilution and corresponding time,etc.In order to adapt to the testing needs of the samples,the sample pad process and the sample pre-processing method are also systematically adjusted.The Cut-Off values ??of this method for residual TYL and TIM in milk,pork and egg samples are: 4 ng/m L,6 ng/g,8 ng/g and 8 ng/m L,15 ng/g,8ng/g.The sample pretreatment is consistent with the colloidal gold method,and the test result can be obtained within 5 minutes.This study is also the first to propose a lateral flow immunochromatography method for detecting TYL and TIM latex microspheres.(3)The establishment of time-resolved fluorescent microsphere immunochromatography method: time-resolved fluorescent microspheres use rare earth ions with large stoke displacement and long fluorescence half-life as a marker,and the surface is modified with functionalized carboxyl groups,which can be covalently coupled to antibodies.This study focuses on optimizing carboxyl activation efficiency,antibody labeling dosage,p H,ionic strength,and blocking time,especially for the key parameters such as surfactant and ion concentration in the sample pad treatment solution.The Cut-Off values ??of the residual TYL and TIM in milk,pork and egg samples by this method are: 2 ng/m L,3 ng/g,6 ng/g and 4 ng/m L,6 ng/g,6 ng/g.The detection costs 5 minutes.(4)Evaluation of three immunochromatographic methods: the three immunochromatographic methods established are compared in multiple dimensions in terms of sensitivity,stability,accuracy,research and development cost,and user convenience.The results show that the sensitivity of time-resolved fluorescent microsphere immunochromatography is the best,and the stability and accuracy of the three are basically the same,but the immunochromatographic methods of colloidal gold and latex microspheres are easier to operate because of the visible results.The development cost of colloidal gold immunochromatography is the lowest.Therefore,users can choose according to their needs.Compared with other literatures,which are limited to the detection of one or one type of samples,the detection samples of this method cover more common animal foods such as meat,eggs,and milk.Finally,the three established immunochromatographic methods were used to detect 40 milk samples at the same time,and confirmed by LC-MS/MS.The results show that the detection results of the three methods have a good correlation with the detection results of the LC-MS/MS method,and there are no false positive or false negative results,indicating that the method we established is reliable and accurate,and can be used to provides a variety of technical support for on-site screening of MALs in animal foods. |
