粒径均一壳聚糖微球免疫亲和柱的制备及其应用

发布时间：2018-05-11 00:37

本文选题：壳聚糖微球 + 达氟沙星　；参考：《江苏大学》2017年硕士论文

【摘要】：免疫亲和色谱(Immunoaffinity Chromatography,IAC),与传统的样品前处理方法相比,其操作过程简便快速、选择性高、能在较小的柱床体积下完成分离过程,因而被广泛应用于样品前处理中,特别是目标组分多为痕量水平以及实际样品复杂的抗生素残留检测的前处理。本研究以达氟沙星(Danofloxacin,DAN)和恩诺沙星(Enrofloxacin,ENR)这两种氟喹诺酮类抗生素作为目标分析物,通过乳化交联法、膜乳化法制备粒径均一的壳聚糖微球,评价微球的粒径及理化性质,并将其与免疫获得的DAN、ENR多克隆抗体偶联制备IAC柱,同时对柱子的性能和应用前景进行了评价。主要研究内容和结果如下:1.采用乳化交联法制备壳聚糖微球并对单因素优化,确定壳聚糖分子量为600kD,氨基醛基摩尔比为2:1,壳聚糖浓度为2%,水相油相比为1/8,搅拌速率为400rpm制备壳聚糖微球,优化的微球平均粒径为124μm,Span值为1.08;采用膜乳化法以分子量为100kD的壳聚糖为原料制备得到的微球平均粒径为62.9μm,Span值1.96;市售Sepharose 4B平均粒径为86.6μm,Span值1.50;2.采用免疫原DAN-BSA免疫新西兰白兔,纯化得到DAN多克隆抗体,用icELSIA法测得纯化后多克隆抗体IC50值为4.6ng/mL,与CIP、ENR、NOR和OFL的交叉反应率均小于0.5%;纯化ENR抗血清得到的ENR多克隆抗体IC50值为8.0ng/mL,与CIP、DAN、NOR和OFL的交叉反应率均小于0.8%,两者都有较好的亲和性和特异性,达到了制备免疫亲和柱的需求;3.所制备的以壳聚糖为载体DAN多克隆抗体IAC柱结合DAN的最大容量为3.9μg/mL凝胶,IAC柱的平均回收率为87.91%,IAC柱之间的变异系数(CV)为7.98%;所制备的以Sepharose 4B为载体DAN多克隆抗体IAC柱结合DAN的最大容量为2.3μg/mL凝胶,IAC柱的平均回收率为95.67%,IAC柱之间的变异系数(CV)为4.10%;所制备的以壳聚糖为载体ENR多克隆抗体IAC柱结合ENR的最大容量为4.1μg/m L凝胶,IAC柱的平均回收率为94.44%,IAC柱之间的变异系数(CV)为4.26%;所制备的以Sepharose 4B为载体ENR多克隆抗体IAC柱结合ENR的最大容量为2.5μg/mL凝胶,IAC柱的平均回收率为98.95%,IAC柱之间的变异系数(CV)为2.98%;4.将所制备的IAC柱配合HPLC用于牛奶样品中DAN、ENR的加标回收检测(添加水平为25、50ng/mL),其中以壳聚糖为载体DAN多克隆抗体IAC柱的回收率为83.82%~98.76%,CV值小于4.05%;以Sepharose 4B为载体DAN多克隆抗体IAC柱的加标回收率为90.24%~100.10%,CV值小于5.89%;将所制备的以壳聚糖为载体ENR多克隆抗体IAC柱的回收率为89.96%~99.40%,CV值小于5.02%;以Sepharose 4B为载体ENR多克隆抗体IAC柱的加标回收率为96.52%~99.84%,CV值小于1.93%。
[Abstract]:Compared with the traditional sample pretreatment method, Immunoaffinities ChromatographyCon (IAC) has the advantages of simple and rapid operation, high selectivity, and can complete the separation process in a small column bed volume, so it has been widely used in sample pretreatment. In particular, the target components are preprocessed for the detection of antibiotic residues at trace levels and in actual samples. In this study, two fluoroquinolones (Danofloxacinine DAN) and enrofloxacinine (ENR) were used as target analyzers. Chitosan microspheres with uniform particle size were prepared by emulsification and crosslinking method and membrane emulsification method, and the size and physicochemical properties of the microspheres were evaluated. The IAC column was prepared by coupling it with the immunized Dannr polyclonal antibody, and the performance and application prospect of the column were evaluated. The main contents and results are as follows: 1. Chitosan microspheres were prepared by emulsification and crosslinking. The molecular weight of chitosan was 600kD, the molar ratio of amino to aldehyde was 2: 1, the concentration of chitosan was 2 / 1, the ratio of water to oil was 1 / 8, and the stirring rate was 400rpm. The average particle size of the optimized microspheres is 124 渭 m Span = 1.08, the average diameter of the microspheres prepared by membrane emulsification method is 62.9 渭 m Span value from chitosan with molecular weight of 100kD, and the average particle size of marketable Sepharose 4B is 86.6 渭 m Span value 1.50m2. New Zealand white rabbits were immunized with immunogen DAN-BSA and DAN polyclonal antibodies were purified. The IC50 value of purified polyclonal antibody was 4.6 ng / mL, the cross reaction rate with CIPENRNOR and OFL was less than 0.5 by icELSIA method, the IC50 value of ENR polyclonal antibody obtained from purified ENR antiserum was 8.0 ng / mL, and the cross reaction rate with ENR DANNOR and OFL was less than 0.8%, both of which had good results. Affinity and specificity, The requirement of preparing immune affinity column was reached. The maximum capacity of the DAN polyclonal antibody column combined with DAN was 3.9 渭 g/mL gel column, the average recovery was 87.91% and the coefficient of variation was 7.98. The DAN polyclonal antibody IAC column with Sepharose 4B as carrier was prepared. The maximum capacity of DAN was 2.3 渭 g/mL gel column, the average recovery rate was 95.6767 g/mL column, the coefficient of variation between columns was 4.10, and the maximum capacity of ENR polyclonal antibody column combined with ENR was 4.1 渭 g / mL IAC column with chitosan as carrier, and the average capacity of ENR column was 4.1 渭 g / mL. The recovery was 94.440.The coefficient of variation (CV) was 4.26.The maximum capacity of ENR polyclonal antibody IAC column combined with ENR was 2.5 渭 g/mL gel column. The average recovery rate was 98.95%. The coefficient of variation was 2.98%. The prepared IAC column combined with HPLC was used for the detection of DANN ENR in milk samples by standard addition and recovery (addition level was 255ng / mL), in which the recovery rate of DAN polyclonal antibody IAC column with chitosan as carrier was 83.82% and 98.76% CV was less than 4.05; Sepharose 4B was used as carrier for DAN polyclonal antibody. The recoveries of the IAC column were 90.24 and 100.100.100.10, the CV of the ENR polyclonal antibody IAC column with chitosan as carrier was 89.96 and 99.400.40C < 5.02, and the CV of ENR polyclonal antibody IAC with Sepharose 4B as carrier was 96.52 ~ 99.84% and < 1.93% respectively.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.7;TQ460.72

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