Development Of Rabbit IgG And Chicken IgY-based Enzyme-linked Immunosorbent Assay(ELISA) For The Detection Of Mebendazole

Mebendazole(MBZ)is a broad-spectrum benzimidazole insect repellent.It is widely used in livestock and aquaculture,and it is easily be residual in animal-derived foods.The current methods for detecting MBZ have disadvantages.such as expensive equipment,high cost,and not suitable for rapid detection of a large number of samples.Therefore,the development of simple,rapid,sensitive,and low-cost detection methods for MBZ residues is of great significance for ensuring the safety of animal-derived foods.In this study,the active ester method was used to construct the MBZ artificial antigen.Artificial antigen was used to immunize New Zealand big-eared rabbits and laying hens to prepare MBZ-specific IgG and IgY.Then the indirect competitive ELISA detection method based on IgG and IgY was established.The results are as follows:The active lipid method was used to couple MBZ(MBZ-3C)containing three carbon arms with porcine thyroglobulin(Thy)and bovine fetal bovine serum albumin(BSA)to prepare immunogens and coatings.After immuning New Zealand big-eared white rabbits and laying hens,we collected and separated serum regularly.Meanwhile we extracted IgY antibodies by polyethylene glycol precipitation.from collected eggs.The IC50 of IgG obtained from rabbit Ⅰ,rabbit Ⅱ and rabbit Ⅲ before immunization to detect MBZ was 9.68 ng m L-1,13.13 ng m L-1and 9.06 ng m L-1,respectively.The IgG sensitivity of rabbit Ⅲ was higher.The IC50 of MBZ for IgY in layer Ⅰ and layer Ⅱ were 5.41 ng m L-1 and 7.14 ng m L-1,respectively.By optimizing the working conditions of the indirect competitive ELISA,a rapid detection method based on IgG and IgY was established.The result of the IgG-based ELISA detection method was that the IC50 was 7.41 ng m L-1 and the minimum detection limit was 0.27 ng m L-1,The linear detection range is0.92 ng m L-1-54.49 ng m L-1.The IgY-based ELISA detection method has an IC50 of 3.65 ng m L-1,a minimum detection limit of 0.25 ng m L-1,and a linear detection range of 0.63-25.2 ng m L-1.Cross-examination of the MBZ indirect competition ELISA method based on IgG and IgY showed that the obtained two antibodies have very good specificity for MBZ,only with amino mebendazole(CR(IgG)=90.5%,CR(IgY)=70.7%)There is a certain cross-reaction.The recovery test showed that the animal muscle tissue sample can eliminate the interference of the IgG and IgY-based MBZ indirect competition ELISA method by the sample matrix at a 10-fold dilution.Based on the indirect competitive ELISA detection method of IgG,the recovery rate of MBZ in animal muscle is between 83.15% and 110.65%,and the coefficient of variation is between 5.35% and 14.27%.Based on the detection method of IgY,MBZ in animal muscle tissue.The recoveries were between 89.63% and 110.51%,and the coefficient of variation was between 4.32% and 10.85%.The above results show that both IgG and IgY-based ELISA can be used for the analysis of MBZ residues in animal-derived foods.