Preparation And Application Of Immunoaffinity Column With Size-uniformed Chitosan Microspheres As Support Matrix

Immunoaffinity chromatography(IAC),compared with other pre-tretment methods,it has advantages of time saving,high specificity and completing the separation process at a small bed volume,is therefore widely used in the pretreatment of samples,especially used for screening and quantification of antibiotic residues with low level of the target analyte and the complexity of the real sample matrices.In this study,emulsion chemical crosslinking and membrane emulsification methods were used to prepare size-uniformed chitosan microspheres and size distribution and physicochemical properties of the microspheres were also characterized.Subsequently,chitosan microsphere-based IAC column were prepared by covalently coupling the microspheres with polyclonal antibodies against danofloxaein(DAN)and enrofloxain(ENR)and their performance and potential application were also evaluated.The main contents and results are as follows:1.Emulsion chemical crosslinking method was used to prepare chitosan microspheres with the following parameters,chitosan molecular weight 600 kD,2:1 of molar ratio between amino group and aldehyde group,2% of chitosan concentration,1:8 of water to oil volume ration,400 rpm stirring speed,and the mean particle size of the obtained microsparticle was 124?m with a Span value of 1.08.The mean particle size of microsphere prepared with 100 kD chitosan by membrane emulsification was 62.9?m with a Span value of 1.96.The average particle size of commercially obtained Sepharose 4B is 86.6?m with a Span value of 1.50.2.The New Zealand white rabbits were immunized by the immunogen DAN-BSA to generate polyclonal antibody.The IC50 value of the obtained DAN polyclonal antibody was 4.6 ng/m L,and the cross-reactivities to its analogues(CIP?ENR?NOR and OFL)were less than 0.5%.The IC50 value of the ENR polyclonal antibody was 8.0 ng/mL,and the cross-reactivities to its analogues(CIP?DAN?NOR and OFL)were less than 1%.3.The capacity of the chitosan-based DAN IAC column was about 3.9?g DAN/m L adsorbent.The average recovery was 87.91% with the coefficient of variation(CV)of 7.98% among the columns.The capacity of the Sepharose 4B-based DAN IAC column was about 2.3?g DAN/mL adsorbent.The average recovery was 95.67% with the coefficient of variation(CV)of 4.10% among the columns.The capacity of the chitosan-based ENR IAC column was about 4.1?g ENR/m L adsorbent.The average recovery was 94.44% with the coefficient of variation(CV)of 4.26% among the columns.The capacity of the Sepharose 4B-based ENR IAC column was about 2.5?g ENR/mL adsorbent.The average recovery was 98.95% with the coefficient of variation(CV)of 2.98% among the columns.4.IAC-based clean up combined with HPLC method was applied in the detection in milk samples with DAN and ENR spiking level of 25 and 50 ng/m L,respectively.The chitosan-based IAC column exhibited a extraction recovery of DAN ranged from 83.82% to 98.76% with CV less than 4.05%.The Sepharose 4B-based IAC column exhibited a extraction recovery of DAN ranged from 90.24% to 100.10% with CV less than 5.89%.The chitosan-based IAC column exhibited a extraction recovery of ENR ranged from 89.96% to 99.40% with CV less than 5.02%.The Sepharose 4B-based IAC column exhibited a extraction recovery of ENR ranged from 96.52% to 99.62% with CV less than 1.93%.