摘要
四环素类(Tetracyclines,TCs)抗生素是由链霉菌产生的一类广谱抗生素,常用于各种动物传染性疾病的治疗以及动物生长促进剂。动物性食品中TC滥用及超标残留给人类健康带来了严重威胁,因此,迫切需要研究能对其进行快速准确的检测方法。本课题合成了TC完全抗原,制备了TC特异性抗体,以此为基础,建立了快速、准确、灵敏的TC残留阻抗型免疫电化学传感检测方法。
合成了TC完全免疫原和包被原。对TC苯环上4位和7位碳原子衍生化后,通过重氮化法分别合成了TC免疫原和包被原。同时对载体蛋白BSA进行了羧基化,减少BSA自身的偶联,提高所合成抗原的纯度及偶联比。经过HPLC-MASS、凝胶色谱、红外图谱、紫外图谱鉴定,免疫原和包被原均合成成功,且由羧基化的BSA制备的免疫原(TC-aBSA)比未经羧基化的BSA制备的免疫原(TC-BSA)偶联比高,分别为11:1和7:1,包被原的偶联比为5:1。
制备了TC特异性抗体并通过间接竞争ELISA方法对抗体进行了表征。用制备的TC免疫原TC-BSA和TC-aBSA各免疫两只新西兰大白兔,其中三只产生了针对TC的抗血清,经硫酸铵法初步纯化后,效价分别为14.4万、25.6万、32万左右。取效价为32万的抗血清建立间接竞争ELISA法,测得OD450值与TC标准品浓度在40～120 ng/kg范围内呈线性关系,线性回归方程为:Y=-0.0064X+1.1308,R2=0.9937,TC的半数抑制率IC50为97μg/kg,与土霉素(OTC)、金霉素(CTC)的交叉反应率分别为100%和85%。
建立了基于阻抗技术和纳米材料的电化学免疫传感检测方法。以制备的TC抗体为识别元件,通过L-半胱氨酸(L-Cys)和纳米金(Nano-Au)自组装技术设计传感器界面固定抗体,通过电化学交流阻抗技术,建立了快速、准确、灵敏的TC免疫电化学传感检测方法。实验得出工作电极修饰的最佳条件如下:L-Cys自组装浓度为0.5%,pH值为4.5,时间为2 h;自组装Nano-Au粒径为20 nm,pH 6.0;抗体包被浓度为100μg/mL,pH 7.0,时间为1 h;1% BSA封闭时间为40min;优化后阻抗测定初始电压为0.2 V。本方法室温下检测时间为15 min,对TC的响应线性范围为5～85μg/kg,最低检测限1μg/kg。鸡肉中样品加标回收率平均为79.37%,工作电极保存一个月以内性能稳定,偏差在5%以内。
Tetracyclines (TCs) are a group of broad-spectrum antibiotics produced by Streptomyces and commonly used for medical purposes as well as animal growth promotion agent. At present more and more serious abusing of TCs resulted serious question of residue tetracycline exceeded the standard which posed a serious threat to human health. Therefore, there is an urgent need to develop a fast and accurate method to detection of them. This thesis through the preparation of specific antibodies to tetracyclines, a new method of immunosensor which based on electrochemical impedance was developed.
In my study two different methods was used to derive the tetracycline, and synthesized a completely immune antigen and coating antigen, respectively. Meanwhile, the carrier protein BSA was undergone carboxylation, which reduced self-polymerization of BSA and improved the purity of the conjugation. After identification by HPLC-MASS, gel chromatography, infrared spectra, UV map, the synthesis of complete immune antigen and coating antigen were successful, and the coupling ratio of artificial antigen(TC-aBSA) synthesized with carboxylic BSA was higher than with BSA without carboxylation(TC-BSA), 11:1 and 7:1, respectively, the coupling ratio of coating antigen was 5:1.
Two synthesized artifical antigen TC-aBSA and TC-BSA were used to immune two New Zealand rabbits, respectively. Three of them produced antiserum for TC and after purification by the methody of SAS, the titers of the three antiserum was 144000, 256000 and 300000, respectively. Indirect competitive enzyme-linked immunosorbent assay method was developed based on the highest titer serum which found that OD450 value is linear with the concentration of TC between 40～120 ng/kg(R2=0.9937), IC50 value was 97μg/mL, cross reactivities with two similar chemical structure compounds OTC, CTC were 100% and 85%.
Using the prepared antibody as sensitive material, through self-assembling of L-Cys and nano-Au particles, antibody was immobilized onto the working gold electrode. The optimum processing conditions were as following: The self-assembling concentration of L-Cys was 0.5%, pH value was 4.5 and time was 2 h; The nano-Au particles diameter used for self-assembling was 20 nm, pH value was 6.0; The concentration of antibody was 100μg/mL, pH value was 7.0, time was 1 h; Detetion time was 15 min; Initial voltage was 0.2 V. The immunosensor has a good linear relationship with the concentration of TC between 5～85μg/kg(R2=0.9908), LOD was 1μg/kg, recovering rate from chicken was 79.37%; The working electrodes was stable within one month.
引文
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