| Fluoroquinolones(FQs)consist of two six-membered heterocyclic compound,carboxyl groups at C-3,F atoms at C-6,a piperazine compounds at C-7,and a substituent group at C-1.Due to its broad antibacterial spectrum and high efficiency,the quinolones that achieve their bactericidal action by inhibiting the replication of bacterial DNA are widely used in the field of animal and human medicine to deal with some diseases.Fluoroquinolones(FQs)are widely used in animal foods due to their remarkable effects which leads to drug abuse and residue,as a result,the drug eventually endangers human health through the food chain.Ingestion of large amounts of fluoroquinolones in a short period can cause acute poisoning,and long-term intake of excessive quinolones can lead to increased bacterial resistance and create "super bacteria."Therefore,the detection and control of fluoroquinolone residues is particularly important.In this study,a chemiluminescent enzyme immunoassay technique and a lanthanide fluorescent microsphere lateral flow dipstick technique were established to detect fluoroquinolone residues by preparing a broad-spectrum specific monoclonal antibody against fluoroquinolone.Test Ⅰ Preparation of broad-spectrum monoclonal antibodies to fluoroquinolones(FQs)In order to obtain a broad-spectrum monoclonal antibody against fluoroquinolone,norfloxacin(NOR),which is closer to the parent structure of the fluoroquinolone,was selected as the hapten for complete antigen preparation.The mice were immunized with two complete antigens,one is NOR-1,which is prepared by modifying the hapten NOR in the drug structure;the other is NOR-BSA,which is synthesized directly from the hapten by the carbodiimide method.At the same time,using NOR-1-OVA and indirect ELISA,the fusion cells were positively screened according to sensitivity and broad spectrum specificity.The titer and sensitivity of serum were determined to screen mice with higher drug sensitivity and cross-reaction rate of fluoroquinolone,which were used to prepare anti-FQs monoclonal antibodies by hybridoma technology.The NOR-1-OVA was used as a coating to screen hybridoma cells according to the sensitivity of the cell supernatant using ic-ELISA method,and the screened cells are used to prepare monoclonal antibodies by intracorporeal induction of ascites.The results showed that the mice which was immuned complete antigen NOR-1-BSA had higher sensitivity and cross-reactivity rate.The sensitivity(IC50)of the anti-FQs monoclonal antibody to elven fluoroquinolones was below 11 ng/ml and the cross-reactivity rate was 16.2%～104.2%,at the same time,the monoclonal antibodies are not specific for other non-quinolones..In this study,monoclonal antibodies prepared by drug modification was conformed the requirements of broad spectrum,which was used to establish chemiluminescence of fluoroquinolone(FQs)residues and lanthanide fluorescent microsphere test strip technology.Text Ⅱ Development of direct detection of elven fluoroquinolones by direct competitive chemiluminescence enzyme immunoassayTo detect fluoroquinolone in food-borne animal tissues,a direct competitive chemiluminescent enzyme immunoassay was established.A complete antigen prepared by modification of norfloxacin(NOR)was used to immunize mice to prepare a monoclonal antibody having broad spectrum specificity for elven fluoroquinolones.The optimal dilution of the coating and the enzyme-labeled antibody was determined by a two-factor crossover experiment.The high-throughput detection of elven drugs in fluoroquinolones was completed by optimizing the conditions of coating,the type of blocking solution,and the optimal reaction time and evaluate this method in the sensitivity,crossover rate and precision.The results show that the optimal reaction conditions are that the optimal NOR-1-OVA coating antigen amount in the coatings was 0.3 μg/ml,the dilution ratio of enzyme-labeled antibodies was 1:8000,coated overnight at 4℃,the blocking solution is 1%BSA,and the competitive reaction time was 1 hour.The half-inhibition(IC50)of the elven detectable fluoroquinolones was from 1.44～9.62 ng/ml,the cross-reactivity rate was from 15.0%～109.1%,and the cross-reaction rate with other non-quinolones was less than 0.01%.The detection limit of this method was from 0.05 to 0.27 ng/mL,the average recovery rate was from 73.3%to 103.0%and the coefficient of variation was from 1.9%to 11.8%.Therefore,this method with the high sensitivity and good atopic can be reliably used as a conventional method to detect fluoroquinolone residues in animal-derived foods.Test Ⅲ Lanthanide-labeled immunochromatographic strips for the rapid detection of fluoroquinolones(FQs)In this study,a lanthanide fluorescent lateral flow dipstick for the detection of fluoroquinolone residues in food-borne animal tissues was prepared.In this experiment,the lateral flow dipstick consists of labeled fluorescent microspheres which were prepared by monoclonal antibodies against fluoroquinolone(FQs)and lanthanide fluorescent microspheres,a detection lines prepared from complete antigen(NOR-1-OVA),and a control line prepared by the goat anti-mouse secondary antibody.The buffer,reaction time,coating concentration of T-line and dosage of microsphere were optimized during the detection process,and the specificity,sensitivity and precision of the method were further evaluated.The results showed that the test strip had the highest detection sensitivity when the T-line coated antigen NOR-1-OVA 1 mg/mL,the microsphere dose was 0.4 μL,and the reaction time was 15 min.The established method for the detection of elven drugs in fluoroquinolones(FQs)has an sensitivity(IC50)of 2.02～13.44 ng/ml,a detection limit was less than 1 ng/mL,and the average intra-assay and inter-assay coefficient of difference was less than 10%,in the meantime,sample recovery rate is between 75.0%and 102.0%.The results showed that the immunochromatographic test strip established in this experiment has high sensitivity and specificity,simple process,and it can be applied to preliminary rapid screening of elven FQs drug residues in animal food. |
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