Preparation Of Monoclonal Antibody Against Kanamycin And Its Development Of Lateral Flow Immunoassay Strips

Kanamycin（KAN）is a kind of alkaline aminoglycoside antibiotic produced by Streptomyces kanamyceticus.It is commonly used in livestock and aquaculture.Because of its broad-spectrum antibacterial action against most Gram-negative and Gram-positive bacteria,and its advantages of low cost and convenient medication,it is widely used in the treatment of various infections caused by bacteria such as Escherichia coli,Proteus,Tuberculosis bacillus,and Staphylococcus aureus.However,excessive use of antibiotics will lead to residues in meat,eggs,dairy products and other animal-derived foods,and eventually accumulate in the human body through the internal circulatory system,resulting in a series of harm,such as ototoxicity and nephrotoxicity,and block neuromuscular joints,cause allergic reactions,and even death in severe cases.In recent years,the abuse of antibiotics has become a serious public health problem worldwide.Therefore,it is urgent to establish a fast,sensitive and reliable detection method for monitoring KAN residue.In the first chapter,the physical and chemical properties,antibacterial mechanism,toxicity mechanism,limit criteria and detection progress of target analyte KAN are systematically introduced,the types of Lateral flow immunoassay（LFIA）markers are described,and the content,purpose and significance of this study are briefly discussed.In the second chapter,Monoclonal antibody for KAN was prepared using immunology and cell fusion techniques.The whole antigen KAN-bovine serum albumin（BSA）was synthesized by glutaraldehyde method as an immunogen to immunize mice.By monitoring the serum titer of mice by Indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）,mice with high positive and inhibitory rates were selected for shock immunity.Five anti-KAN-m Ab（1D11,1C9,1E12,2E3 and 2D1）were obtained by fusion of hybridoma cells with polyethylene glycol method.Ascites were prepared by in vivo induction method and purified by n-octanoic acid-saturated ammonium sulfate method.The sensitivity of m Ab was evaluated,and the highest sensitivity m Ab 1D11 was selected for affinity and specificity evaluation,and the result was good,which was selected for follow-up experiments.In the third chapter,a rapid LFIA for the detection of KAN residues in pork and milk was established based on anti-KAN-m Ab and Colloidal gold nanospheres（Au NPs）.In this study,Au NPs and Au NPs-m Ab were synthesized by sodium citrate reduction method.Au NPs and Au NPs-m Ab were coupled by electrostatic adsorption method.Both were characterized by transmission electron microscopy,dynamic light scattering and UV-visible spectrophotometer.At the same time,the relevant parameters of the experiment were optimized.The optimized results were as follows:the optimal labeling p H of the probe was 5.5,the labeling amount of m Ab was 5μg,the concentration of KAN-BSA on the T-line was 0.6 mg/m L,the addition amount of the probe was 2.5μL,and the immune response time was 16 min.Under optimal conditions,the linear equation of Au NPs-LFIA established is y=-304.93 lg（x）+684.08（R²=0.9859）,the linear range is 2-100 ng/m L,and the Limit of detection,LOD)was 0.1124 ng/m L.The recoveries of pork and milk were 91.80%-119.50%,with coefficients of variation less than 15%.The results show that Au NPs-LFIA established in this chapter can provide technical support for the rapid detection of KAN residue.In the fourth chapter,a highly sensitive LFIA for the detection of KAN residue was established based on anti-KAN-m Ab and Fe³⁺-chelated polydopamine nanospheres（Fe@PDANs）,which have excellent absorptive capacity and strong coloritization signal.Relevant experimental conditions were optimized,including probe labeled p H,m Ab labeled amount,incubation time of m Ab and Fe@PDANs,concentration of KAN-BSA on T-line,probe supplemental amount,and immune response time.Under optimal conditions,the LOD of Fe@PDANs-LFIA was 0.0191ng/m L,2.75 times lower than that of PDANs-LFIA.Fe@PDANs-LFIA The LODs of KAN residues in pork,milk,honey and milk powder were 0.1421,0.1982,0.2901 and0.2743μg/kg,respectively,and the corresponding linear ranges were 0.5-100,0.5-50,0.5-50 and 1-20μg/kg,respectively.At the same time,the recovery experiment also proves that the established Fe@PDANs-LFIA has a good recovery rate and a low coefficient of variation,indicating that the method has a good accuracy.In the fifth chapter,the anti-KAN-m Ab synthesized in this study and Au NPs-LFIA and Fe@PDANs-LFIA established in this study were systematically summarized,and the future methods to improve the sensitivity of KAN residue detection were prospected.