Research On Fluorescence Immunoassay Technology For Veterinary Drug Residues Based On FRET

Objective Based on the characteristics of the specific binding between antigen and antibody,the rapid detection method of diethylstilbestrol and estradiol residues in milk is established by using quantum dot labeling technology and resonance energy transfer technology respectively,which is a benchmark for the national veterinary drug residue monitoring system and veterinary drug safety evaluation system Provide perfect technical support.Methods(1)Establishment of fluorescence immunoassay method.The estrogen antigen coated on the microplate competes with the estrogen small molecules in the system to bind the estrogen antibody.After removing the complex formed by the small molecule and the antibody,the second antibody is labeled with quantum dots to capture the antibody adsorbed in the microplate,and the fluorescent signal is directly detected and converted into the quantitative detection result of the target.Using orthogonal design to optimize the experimental conditions of four factors and five levels,the fluorescence immunoassay of diethylstilbestrol and estradiol in milk was established respectively for the detection and evaluation of actual samples.(2)Research on the method of resonance energy transfer based on quantum dots.Label the estradiol antibody and the specific sequence single-stranded deoxyribonucleic acid on the surface of the quantum dot;another specific-sequence single-stranded deoxyribonucleic acid is coupled to the estradiol antigen to form a complementary pairing with the single-stranded deoxyribonucleic acid of the modified quencher Stabilize the complex.When functionalized quantum dots coexist with the complex,the antibody specifically recognizes and captures the antigen,the surface of the quantum dot and the nucleic acid binding region of the quencher end complementarily pair to form a stable structure,and resonance energy transfer occurs between the two,and the quantum dot is quenched.When estradiol is present,it competes with the antigen to separate part of the antigen and antibody,the quencher binding region is not hybridized,the complex probe is released,and the fluorescence signal changes,which can be used for quantitative analysis of estradiol.(3)The methodological comparison of fluorescence immunoassay,liquid chromatography-mass spectrometry and enzyme-linked immunosorbent assay results is carried out,the stability of the method is evaluated,and it is applied to batch sample detection for investigation.Results The optimal conditions of the experiment were optimized through orthogonal design,and the standard curve of the fluorescence immunoassay for diethylstilbestrol was established.The linear range is 0.418-195.065 ng/m L,the linear equation is y=18.995-6.434x,and the goodness of linear fit is R~2=0.997,the lowest detection limit is 0.109 ng/m L;the standard curve of fluorescence immunoassay for estradiol has been established,the linear range is between 0.472-66.597 ng/m L,the linear equation is y=7.858-3.197x,the linear goodness of fit is R~2=0.987,the lowest detection limit is 0.236 ng/m L.A standard curve for detecting estradiol based on resonance energy transfer was established.The linear range is between3.125-100.000ng/m L,the linear equation is y=406.96x+1350.12,the goodness of linear fit R~2=0.993,and the minimum detection limit is 0.500ng/m L.Fluorescence immunoassay methodological comparison results are consistent,and the recovery rate of diethylstilbestrol is between 95.83%-107.80%,which is statistically different from the liquid chromatography-mass spectrometry tandem method(P=0.011<0.05),and the enzyme-linked immunosorbent assay The difference was not statistically significant(P=0.253>0.05).The recovery rate of estradiol was between 101.21%and110.04%,which was statistically different from the liquid chromatography-mass spectrometry tandem method(P=0.002<0.05),and the difference from the enzyme-linked immunosorbent assay was statistically significant(P=0.031<0.05).Conclusions Two methods for the detection of estrogen in milk have been successfully established.The detection method based on resonance energy transfer can quickly complete the detection within 1 hour,which is economical and convenient;the results of the method comparison suggest that the detection result of the fluorescence immunoassay method is compared with that of liquid chromatography.-The mass spectrometry tandem method and the enzyme-linked immunosorbent method have the same results,which can be used for batch detection of actual milk samples.