本文選題：殼聚糖微球 + 達(dá)氟沙星�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：免疫親和色譜(Immunoaffinity Chromatography,IAC),與傳統(tǒng)的樣品前處理方法相比,其操作過程簡便快速、選擇性高、能在較小的柱床體積下完成分離過程,因而被廣泛應(yīng)用于樣品前處理中,特別是目標(biāo)組分多為痕量水平以及實際樣品復(fù)雜的抗生素殘留檢測的前處理。本研究以達(dá)氟沙星(Danofloxacin,DAN)和恩諾沙星(Enrofloxacin,ENR)這兩種氟喹諾酮類抗生素作為目標(biāo)分析物,通過乳化交聯(lián)法、膜乳化法制備粒徑均一的殼聚糖微球,評價微球的粒徑及理化性質(zhì),并將其與免疫獲得的DAN、ENR多克隆抗體偶聯(lián)制備IAC柱,同時對柱子的性能和應(yīng)用前景進(jìn)行了評價。主要研究內(nèi)容和結(jié)果如下:1.采用乳化交聯(lián)法制備殼聚糖微球并對單因素優(yōu)化,確定殼聚糖分子量為600kD,氨基醛基摩爾比為2:1,殼聚糖濃度為2%,水相油相比為1/8,攪拌速率為400rpm制備殼聚糖微球,優(yōu)化的微球平均粒徑為124μm,Span值為1.08;采用膜乳化法以分子量為100kD的殼聚糖為原料制備得到的微球平均粒徑為62.9μm,Span值1.96;市售Sepharose 4B平均粒徑為86.6μm,Span值1.50;2.采用免疫原DAN-BSA免疫新西蘭白兔,純化得到DAN多克隆抗體,用icELSIA法測得純化后多克隆抗體IC50值為4.6ng/mL,與CIP、ENR、NOR和OFL的交叉反應(yīng)率均小于0.5%;純化ENR抗血清得到的ENR多克隆抗體IC50值為8.0ng/mL,與CIP、DAN、NOR和OFL的交叉反應(yīng)率均小于0.8%,兩者都有較好的親和性和特異性,達(dá)到了制備免疫親和柱的需求;3.所制備的以殼聚糖為載體DAN多克隆抗體IAC柱結(jié)合DAN的最大容量為3.9μg/mL凝膠,IAC柱的平均回收率為87.91%,IAC柱之間的變異系數(shù)(CV)為7.98%;所制備的以Sepharose 4B為載體DAN多克隆抗體IAC柱結(jié)合DAN的最大容量為2.3μg/mL凝膠,IAC柱的平均回收率為95.67%,IAC柱之間的變異系數(shù)(CV)為4.10%;所制備的以殼聚糖為載體ENR多克隆抗體IAC柱結(jié)合ENR的最大容量為4.1μg/m L凝膠,IAC柱的平均回收率為94.44%,IAC柱之間的變異系數(shù)(CV)為4.26%;所制備的以Sepharose 4B為載體ENR多克隆抗體IAC柱結(jié)合ENR的最大容量為2.5μg/mL凝膠,IAC柱的平均回收率為98.95%,IAC柱之間的變異系數(shù)(CV)為2.98%;4.將所制備的IAC柱配合HPLC用于牛奶樣品中DAN、ENR的加標(biāo)回收檢測(添加水平為25、50ng/mL),其中以殼聚糖為載體DAN多克隆抗體IAC柱的回收率為83.82%~98.76%,CV值小于4.05%;以Sepharose 4B為載體DAN多克隆抗體IAC柱的加標(biāo)回收率為90.24%~100.10%,CV值小于5.89%;將所制備的以殼聚糖為載體ENR多克隆抗體IAC柱的回收率為89.96%~99.40%,CV值小于5.02%;以Sepharose 4B為載體ENR多克隆抗體IAC柱的加標(biāo)回收率為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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O657.7;TQ460.72

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