Application Of Multi-target Fluorescence Immunochromatographic Technique For The Detection Of Three Classes Of Antibiotics

Since Fleming’s discovery of the antibacterial effects of penicillin in1929,antibiotics had become the medical profession’s main weapon in the fight against microbial infections.They were widely used in agriculture,animal husbandry,aquaculture and the pharmaceutical industry.However,the irrational use or misuse of antibiotics had caused some residual contamination and harmfulness.Therefore,as an emerging contaminant,antibiotics pose a potential risk in terms of soil,water and food quality.Among these,sulphonamides(SAs),quinolones(FQs)and tetracyclines(TCs)were commonly used antibiotics.Overdose using of these antibiotcs was prone to residues in foods such as eggs,milk and honey,which posed a concerted threat to human health.Excess antibiotics were accumulated in the body through food and water,causing dysbiosis and allergic reactions in the human intestinal flora,leading to increased bacterial resistance and other hazards.It was also becoming increasingly common for multiple antibiotic residues to be present in food and the environment,and there was a greater potential risk if they co-exist in the water environment or in substances essential to people’s lives.However,currently commonly used detection methods did not meet the need for accurate monitoring of antibiotic residues in a relatively short period of time.Therefore,the establishment of a simultaneous rapid detection technique for multiple antibiotics was of great relevance and application.In this paper,based on the establishment of a colloidal gold immunochromatographic detection technique,a multi-target detection technique based on europium fluorescent nanoparticles was established by replacing the antibody-labelled material while increasing the number of target substances detected,which enables the simultaneous detection of three major classes of antibiotics,including sulfonamides,tetracyclines and fluoroquinolones.The results obtained were as follows.1.Based on the structure of the parent nucleus,we have synthesised artificial antigens for three classes of antibiotics.On the basis of maximum retention of antigenic reactivity,the parent nucleus structures of the various antibiotics,such as sulphonamides,tetracyclines and fluoroquinolones,were selected and designed according to their common parent nucleus structures and unique R-group,and coupled with carrier proteins such as BSA and HSA,respectively,to prepare immunogens and encapsulants.2.Studies on the broad-spectrum monoclonal antibodies specific to three antibiotic groups.The successfully prepared immunogens SAs-HSA,TCs-HSA and FQs-HSA were used to immunise Babl/c mice according to conventional animal immunisation protocols.Target mice were selected by serum potency identification and cell fusion was performed.The best cell lines were selected for the preparation of broad-spectrum monoclonal antibodies by subcloning technique and enzyme-linked immunosorbent chromatography identification,respectively.3.Study of fluorescent immunochromatographic techniques for single antibiotics.Colloidal gold immunochromatographic assays were prepared using the obtained monoclonal antibodies against SAs,TCs and FQs,respectively,while the single-weight detection limits for SAs,TCs and FQs under optimal experimental conditions were 2.4、2.4和1.2 ng/m L,respectively.4.Study of fluorescent immunochromatographic techniques for dual antibiotics.The simultaneous detections of TCs and FQs,SAs and FQs,and SAs and TCs,were achieved by increasing the number of T-lines on the basis of single-weight fluorescent immunochromatographic test strips,respectively.The limits of detection for TCs,FQs and SAs under optimal experimental conditions were 3.2、2.4和4ng/m L,respectively.5.Study of fluorescent immunochromatographic techniques for triple antibiotics.On the basis of the dual fluorescent immunochromatographic test strips,multi-target fluorescent immunochromatographic test strips were established by increasing the number of T-lines.The limits of visual detection for TCs,FQs and SAs were 3.2ng/m L,2.4 ng/m L and 4.0 ng/m L respectively,which were significantly better than those of the colloidal gold immunochromatographic method.The results for the actual samples were highly correlated with those of the HPLC with a linear correlation coefficient R~2 greater than 0.98.The accuracy and precision were also in accordance with the requirements for quantitative analysis.These results indicated that the multi-target fluorescence immunochromatographic assay developed in this paper was a promising method for the rapid screening of three types of antibiotic residues.