Study On The Detection Method Of Cloxacillin Visualized Immunoaffinity Gel Column

In this paper,the visual immunoaffinity gel column method of cloxacillin was studied by using the principle of specific binding of antigen and antibody.A visual immunoaffinity gel column method for the detection of cloxacillin in animal-derived foods was established by optimizing various experimental indexes.Five mice were immunized with cloxacillin polyclonal antibody,and the titer of the antibodies in the five groups was determined.The results showed that the titer was 1:64000 or more.The specificity of 5 groups of cloxacillin antibodies was determined,and the inhibition curve of cloxacillin antibodies was drawn.Finally,the most sensitive cloxacillin antibody was screened out,and the regression equation was y=-0.5842x+0.9391?R²=0.9924?.The IC₅₀ value was?5.64±0.03?ng/mL,and the IC₁₅value was?1.42±0.02?ng/mL.HRP was coupled with cloxacillin to prepare cloxacillin enzyme-labeled antigen.Hydrochloric acid was used to adjust the agarose gel state to maximize its adsorption capacity.Then,cloxacillin antibody and horseradish peroxidase antibody were gel combined to prepare cloxacillin antibody and HRP antibody respectively.The visualized immunoaffinity gel column analysis method of cloxacillin was established,and it was determined that the method reached the optimal operating state under the conditions of the dilution ratio of the enzyme-labeled antigen?4000?,the HRP gel?20?,the antibody gel?10?,the sample addition time?60 s?,and the pH value of the sample diluent?5.7?.The detection limit of cloxacillin by visualized immunoaffinity gel column method was 25?g/L,and there was no cross-reaction with piperacillin,amoxicillin,ampicillin,cloxacillin,furantazone,methylsulfone and streptomycin.Pork,chicken,beef,milk,fermented milk and milk powder without cloxacillin detected by high performance liquid chromatography were selected as actual samples for the addition and recovery experiment of cloxacillin.The matrix effect could be eliminated after pretreatment and used for gel column detection.The detection limit of cloxacillin in actual animal food samples was 25?g/kg.To chlorine azole Westwood visualization immune affinity gel column are sealed in 4?and 25?for stability test,the results show that chlorine azole Westwood visualization immune affinity gel column may be retained in the 4?,3 months with good stability.The effectiveness of visualized immunoaffinity gel column method was verified by High performance liquid chromatography-mass spectrometry?HPLC-MS?.Both HPLC-MS and gel column methods were used to detect the samples,and the results were consistent with those of HPLC-MS.Therefore,the results of cloxacillin visualized immunoaffinity gel column were proved to be reliable.The successful development of the gel column opens up a new idea for the detection of multiple residues of antibiotics.This method is convenient,fast and visual,which is suitable for high-throughput screening of cloxacillin as an effective detection method,meeting the current requirements of food safety detection.