Development Of Immunoassay For Florfenicol?florfenicol Amine And Thiamphenicol

As an analogue of broad-spectrum antibiotics,florfenicol?FF?and thiamphenicol?TAP?are widely used in the prevention and treatment of bacterial infectious diseases.However,it was reported that florfenicol and thiamphenicol can cause drug-resistant strains easily,and both show toxicity to human and animal,including hematological toxicity,embryo toxicity and immunotoxicity,which threatens the health of consumers.Therefore,it is very important to strengthen the monitoring of florfenicol and thiamphenicol,and it is necessary to control the residues in animal derived food of these two drugs.For now,the main detection methods of thiamphenicol,florfenicol and its metabolite,florfenicol amine,are instrument methods,including liquid chromatography?LC?,high performance liquid chromatography-mass spectrometry?HPLC-MS/MS?.Although these technologies are accurate and relable,but they also have shortcomings,such as tedious operation,high cost,proficient experts and low throughout.As a rapid,sensitive,and high throughout analytical method,immunoassay can be used conjunction with the instrument techniques to meet the fast screening needs of large quantities of samples.In this study,a series of haptens were designed and synthesized to the preparation of high-affinity polyclonal antibodies and monoclonal antibodies.After the screening of antibodies-coating antigens,three indirect competitive enzyme-linked immunosorbent assays?ELISA?for different detection performance were established.The following are the main contents:?1?Based on the structure of florfenicol and thiamphenicol,several haptens were designed and synthesized?FF,FFA,FFD,FFM,FFAC,TAPN?.Antiserum was obtained by animal immunity after coupled to carrier protein,it was identified that two rat antiserum which immunized by FFD-BSA and FFD-KLH show specificity against the targets and exhibit a high affinity of 1:128000.The inhibition rates of the antiserums were 80%and74%at 1?g/mL florfenicol,respectively.The rabbit antiserum and mouse monoclonal antibody were prepared by the two immune antigens?FFD-BSA and FFD-KLH?.After screening of antibody-coating antigen,the combination of different detection results were obtained.?2?Based on rabbit pAb-FFD-KLH1 and coating antigen FFM-OVA,an ciELISA for the simultaneous detection of florfenicol and thiamphenicol was established.The ciELISA showed an IC₅₀ value of 1.32 ng/mL for florfenicol and 3.21 ng/mL for thiamphenicol,respectively.The linear ranges were ranged from 0.31 ng/mL to 5.61 ng/mL with a limit of detection of 0.12 ng/mL for florfenicol,and 0.6 ng/mL to 17.01 ng/mL with a limit of detection of 0.24 ng/mL for thiamphenicol,respectively.There is no cross reaction with other analogues.The kit stored at 37?can be kept for 6 days,at 4?for more than half a year.The average recoveries of florfenicol and thiamphenicol of spiked samples ranged from 77%to 116%with a coefficient of variation of less than 16%.Good correlations between the results of ciELISA and HPLC-MS/MS were obtained.Therefore,this ciELISA provided a valid detection method for florfenicol and thiamphenicol in real samples.?3?Based on rabbit pAb-FFD-KLH2 and coating antigen FF-OVA,an ciELISA for the simultaneous detection of florfenicol and florfenicol amine were established.The ciELISA showed an IC₅₀ of 8.94 ng/mL for florfenicol and 10.96 ng/mL for florfenicol amine,respectively.The linear ranges were ranged from 2.32 ng/mL to 34.46 ng/mL with a limit of detection of 1.06 ng/mL for florfenicol,and 3.05 ng/mL to 39.45 ng/mL with a limit of detection of 1.45 ng/mL for florfenicol amine,respectively.There is no cross reaction with other analogues.The kit stored at 37?can be kept for 6 days,at 4?for more than half a year.The average recoveries of florfenicol and florfenicol amine of spiked samples ranged from 64.8%to 96.4%with a coefficient of variation of less than 15%.Good correlations between the results of ciELISA and HPLC-MS/MS were obtained.Therefore,this ciELISA provided a valid detection method for florfenicol and florfenicol amine in real samples.?4?Based on murine mAb-FFD-BSA and coating antigen FFD-OVA,an ciELISA for the detection of florfenicol was established.The ciELISA showed an IC₅₀ value of 9.48ng/mL,the linear ranges were ranged from 1.75 ng/mL to 51.36 ng/mL with a limit of detection of 0.64 ng/mL,there is no cross reaction with the other analogues.The kit stored at 37?can hold for 6 days,at 4?for more than half a year.The average recoveries of florfenicol of spiked samples ranged from 80%to 120%with a coefficient of variation of less than 15%.Good correlations between the results of ciELISA and HPLC-MS/MS were obtained.Therefore,this ciELISA provided a valid detection method for florfenicol in real samples.