Preparation Of Monoclonal Antibody Against Azithromycin And Initial Establishment Of Immunoassay For Azithromycin

Azithromycin（AZM）is the earliest 15-membered ring macrolide antibiotic developed in the late 1980s,which has the advantages of widely antibacterial spectrum,long half-life,stability in acidic conditions,and low side effects.AZM has been widely used in clinical patients infected with o various bacterial,mycoplasma and chlamydia.On the other hand,as AZM plays a certain role in the growth,antibacterial and anticancer of foodborne animals,it is also widely used in veterinary clinic.Although AZM has been widely used in the past few decades,AZM residues in animal food may cause harm to food safety and human health.In 2005,AZM was banned as a veterinary drug for livestock and poultry in China.Although it is banned by the state,many livestock and poultry farmers still use AZM illegally due to its good antibacterial effect and convenient use and low price.Therefore,in order to ensure the safety of animal-derived food,it is necessary to establish a sensitive,specific,convenient and accurate AZM detection method.In this study,the immunogen AZM-BSA and the coating antigen AZM-OVA were synthesized by coupling AZM to the bovine serum albumin（BSA）and ovalbumin（OVA）with the activated ester method.Hybridoma cell line 50D10 was obtained by fusing mouse myeloma cells Sp2/0 with splenocytes from the BALB/c mice immunized with AZM-BSA.The cell line was then injected into BALB/c mice to producing ascites.The ascites was collected and purified by octanoic acid-ammonium sulfate method.The of purified ascites titer was 1:4.0×10⁵,the heavy chain subtype was Ig G1,and the light chain wasκ.Good antibody specificity was demonstrated by the cross-reactivity rates of the m Ab with erythromycin,roxithromycin,doramectin,clarithromycin,tilmicosin and tylosin were less than 0.1%.Establishing AZM indirect competitive ELISA was detected by using titration of ELISA test,the best dilution concentration of AZM-OVA was 0.1 mg/L and the best working concentration of ascites was 1:4.0×10⁵.After optimizing the experimental conditions,the coating buffer was Na₂CO₃-Na HCO₃,the coating condition was 4℃for 16h,the optimal blocking time was 90 min,the secondary antibody dilution concentration is1:5000,the competitive reaction temperature was 37℃and time was 60 min.When the linear range was 0.5～8μg/L,the standard curve of AZM to antibody-antigen reaction was prepared with the linear equation y=-0.6699x+0.5828（R²=0.9905）,IC₅₀=1.33μg/L,LOD=0.287μg/L.The recovery rates of AZM added into milk ranged from 79.6%to104.1%,the coefficients of variation between 16.4%and 27.3%.In summary,AZM m Ab with high sensitivity and specificity has been prepared in this experiment research.In addition,an indirect competitive ELISA method for AZM detection in food residues was established on this basis.In order to conduct the rapid screening of a large number of samples on site and provide a more efficient means for food safety testing.