Development Of Immunoassay For Tylosin And Tilmicosin In Animal Origin Samples

Tylosin?TYL?is a macrolide antibiotic obtained from the culture fluid of Streptomyces fradiae.Tilmicosin?TMC?is the semi-synthetic derivative of tylosin.Because of their good antibacterial properties,tylosin and tilmicosin are widely used in animal husbandry.However,the residue of these drugs can cause allergic reactions in human body and lead to antibiotic resistance to antibiotics.Therefore,strengthening the monitoring of such antibiotics in food is critical to ensuring food safety.At present,the mainly analytical methods of tylosin and tilmicosin are liquid chromatography?LC?and liquid chromatography-mass spectrometry?LC-MS?.These methods are often used as confirmatory methods because of accurate and reliable.However,these methods are unable to conduct on-site monitoring because of the requirements like high cost of equipment and professional operator.Immunoassay is a rapid and sensitive detection technology which combined antigen-antibody reaction with enzymatic reaction.Immunoassay can meet the requirement of rapid screening for agricultural products and food.Thus,it has become a research hotspot in recent years.In this paper,four haptens were designed and synthesized based on TYL and TMC.Competitive indirect enzyme immunoassay and colloidal gold immunochromatographic assay were established with the prepared monoclonal antibodies.The detailed results are as follows:?1?Four haptens were designed,synthesized and coupled with the carrier protein by active ester method.Antiserum that can specifically recognize TYL was prepared by animal-immune,and the inhibition rate of the antiserum was 89%?1 mg/mL TYL as competitors?.And then the stable and specific monoclonal antibodies were obtained by cell fusion.?2?A competitive indirect enzyme immunoassay?ciELISA?method for detecting TYL was established based on monoclonal antibody.After optimization,the limit of detection?LOD?was 0.58 ng/mL,the IC₅₀ was 4.72 ng/mL with a linear range between 1.26 ng/mL and 17.65 ng/mL.The cross-reactivity rate with TMC was 48.96%.The LOD of TMC was1.31 ng/mL,IC₅₀ was 9.64 ng/mL,and the linear detection range was 1.96⁴7.4 ng/mL.The cross-reactivity rates with other analogues were all less than 0.1%and the coefficient of variation below 15%.The average recovery of spiked milk and water samples was between 76.35%and 109.50%.The test results of the ciELISA are very compatible with that of HPLC-MS/MS.?3?The colloidal gold immunochromatographic test strip for detecting TYL was established based on the monoclonal antibody.After optimization,the IC₅₀ of the method was 18.95 ng/mL,the detection range was 1.56⁷6.92 ng/mL,and the cut-off value in buffer solution was 50 ng/mL.The cross-reactivity rate between TYL and TMC was100.69%.The IC₅₀ of TMC was 18.82 ng/mL with a detection range between 1.74 ng/mL and 203 ng/mL.The cross-reactivities with other analogues were less than 0.1%.The average recovery of spiked samples ranged from 65.96%to 126.12%.The test results of the GICA are very compatible with that of HPLC-MS/MS.