头孢氨苄单克隆抗体的研制及免疫学检测方法的初步建立

发布时间：2018-04-26 00:02

本文选题：头孢氨苄 + 单克隆抗体　；参考：《扬州大学》2017年硕士论文

【摘要】：头孢氨苄(Cephalexin,CEX)属于β-内酰胺类抗生素(β-lactam antibiotics),开发距今已五十载,由于其抗菌谱广、吸收效果好、价格低廉,在人类医学和兽医临床上一直有很广泛的应用。但是,在细菌耐药性日趋严重的今天,头孢氨苄等抗生素残留问题必须得到重视。目前,中国、美国、英国、欧盟等多个国家或组织都已对头孢氨苄最大残留限量做出标准规定。检测分析手段的提高是应对抗生素残留必不可少的关键。检测头孢氨苄残留的方法主要有微生物检测法(包括TTC法、扩散法等)、仪器检测法(包括UV-Vis、HPLC、LC-MS等)和免疫学检测法(包括ELISA、FPIA、IC等)。微生物法特异性不强、灵敏度不高,而且易受到其他条件干扰,已无法满足当前实际工作中快速、准确的检测要求;仪器分析法灵敏度高、重复性好、特异性强,但昂贵的实验仪器、复杂的前处理过程、操作繁琐的方法步骤是制约该法广泛应用的重要因素;免疫学检测法特异性强、灵敏度高、操作简单、耗时少,但假阳(阴)性率高、重复性较差却是这种方法的局限性。目前来看,将免疫学方法与仪器检测法联合使用是一种科学、合理、高效的检测分析思路。将免疫学方法作为前期工作初筛方法,仪器分析法作为后期确证方法,这不仅可大规模快速筛选,而且也能保证检测结果的可靠性。本研究旨在制备出亲和力高、特异性强的抗头孢氨苄单克隆抗体,以期建立快速、简便、准确的免疫学检测方法,为我国头孢氨苄在动物性食品中残留监控提供理论和技术支持。研究包括以下四点内容:1、头孢氨苄人工抗原的合成与鉴定。利用偶联剂戊二醛将头孢氨苄与载体蛋白(BSA、OVA)偶联,分别制备免疫抗原CEX-BSA和包被抗原CEX-OVA。经紫外分光光度法、SDS-PAGE凝胶电泳和免疫学方法,鉴定人工抗原的合成是成功的。2、抗头孢氨苄单克隆抗体的制备与鉴定。利用单克隆抗体技术,将受免小鼠B淋巴细胞与SP2/0融合,通过有限稀释法和CiELISA筛选得到2株单克隆细胞株(2B4、5B3),并将其注入小鼠腹腔,得到腹水型单克隆抗体。经Protein G HP亲和层析柱纯化后鉴定,2G4单克隆抗体效价高于1:1.28X 105,其抗体亚型为IgG2b(κ轻链),抗体亲和力常数为K=1.51×109L/mol,抗体除了与头孢拉定有52.55%的交叉反应率,与头孢噻呋、头孢曲松、头孢噻肟、头孢噻吩、青霉素、链霉素、四环素等药物均无明显交叉反应,说明制备的抗体亲和力高、特异性较好,为后续免疫学检测方法的建立打下了良好的基础。3、鲜奶中头孢氨苄ELISA检测方法的建立。本章研究中排除了鲜奶基质干扰影响,并优化了封闭液的成分和浓度,摸索了 2G4单克隆抗体与包被抗原CEX-OVA的最佳工作浓度,所绘制的标准抑制曲线线性关系良好R2=0.9909,在20～1000 ng/mL药物浓度范围里,线性方程为y=0.4674x-0.5359,IC50为 163.55 ng/mL,LOD为19.68ng/mL。通过对样品的添加回收实验发现,该方法检测灵敏度较高,样品回收率在83.98%～102.92%之间,平均回收率为91.31%;变异系数在8.50%～14.5%之间,平均变异系数为10.75%,符合我国规定的头孢氨苄在牛奶中最大残留限量标准,具有一定应用价值。4、头孢氨苄胶体金免疫层析试纸条的研制。采用柠檬酸三钠还原法制备不同粒径胶体金,摸索制备金标抗体的最佳pH和最适单抗标记量,摸索金标垫中金标抗体结合量,优化NC膜中C线(羊抗鼠IgG)与T线(CEX-OVA)喷涂量,优化了 NC膜封闭液成分,并对试纸条稳定性、灵敏度、特异性进行鉴定。结果显示:金标抗体最佳pH为8.4,最佳单抗标记量为每200 μL胶体金溶液中加入9 μL单抗;金标垫中金标抗体以稀释1.5倍,30μL/cm喷量效果最佳;NC膜C线、T线的最佳喷量分别为0.8μL/cm、0.6μL/cm;封闭液选择3%BSA的0.01 mol/L PB缓冲液比较合适;经鉴定该试纸条检测限为500 ng/mL,除了与头孢拉定有轻微交叉反应,与头孢噻呋、头孢曲松、头孢噻肟、头孢噻吩、青霉素、链霉素、四环素反应均呈阴性,可在4℃环境中稳定保持180 d。
[Abstract]:Cephalexin (CEX) is a beta lactam antibiotic (beta -lactam antibiotics). It has been developed for fifty years. Because of its wide antimicrobial spectrum, good absorption effect and low price, it has been widely used in the human medical and veterinary clinic. However, the antibiotic residues in cephalosporin are asked today. At present, many countries and organizations such as China, the United States, the United Kingdom, the EU and other countries have made standard regulations for the maximum residue limit of cefampicin. The improvement of detection and analysis means is essential to the residue of antibiotics. The main methods for detecting cefampicin residues are microbial detection (including TTC, diffusion, etc.). Instrument detection methods (including UV-Vis, HPLC, LC-MS, etc.) and immunological detection methods (including ELISA, FPIA, IC, etc.). Microbiological methods are not specific, sensitive, and easy to be disturbed by other conditions. It is unable to meet the rapid and accurate detection requirements in the current practical work, and the instrumental analysis method is highly sensitive, repeatable and highly specific, but is expensive but expensive. Experimental apparatus, complicated pretreatment process and complicated method and procedure are important factors restricting the wide application of the method. Immunological detection method is highly specific, sensitive, simple and time-consuming, but the false positive rate is high, and the repeatability is poor. At present, the immunology method is combined with the instrument detection method. Combined use is a scientific, rational and efficient method of detection and analysis. Immunology is used as a preliminary screening method for early work, and instrumental analysis is used as a later confirmation method. This method can not only be used for large-scale rapid screening, but also can ensure the reliability of the detection results. This study aims to prepare a high affinity and specific anti cephalosporin single gram. In order to establish a rapid, simple and accurate method of immunological detection to provide theoretical and technical support for the residue monitoring of cephaloprim in animal foods. The following four points are included: 1, synthesis and identification of the artificial antigen of cephalopaxin, coupled with the coupling agent amyl two aldehyde and BSA, OVA. Do not prepare immune antigen CEX-BSA and envelope antigen CEX-OVA. via ultraviolet spectrophotometry, SDS-PAGE gel electrophoresis and immunological methods. The synthesis of artificial antigen is a successful.2, the preparation and identification of anti cephalexin monoclonal antibody. Using monoclonal antibody technique, the B lymphocyte of mice is fused with SP2/0 by the finite dilution method. 2 monoclonal cell lines (2B4,5B3) were screened by CiELISA and injected into the abdominal cavity of mice to obtain the ascites monoclonal antibody. The monoclonal antibody was purified by Protein G HP affinity chromatography column. The antibody titer of 2G4 was higher than that of 1:1.28X 105. The antibody subtype was IgG2b (kappa light chain), the antibody affinity constant was K=1.51 x 109L/mol, and the antibody was associated with cephalosporin. There was no obvious cross reaction with ceftif, ceftriaxone, cefotaxime, cefotaxime, cefotaxime, cefotaxime, penicillin, streptomycin, and tetracycline, which showed that the prepared antibody had high affinity and good specificity, which provided a good foundation.3 for the establishment of subsequent immunological detection methods, and ELISA in fresh milk was used to detect cefamaxine in fresh milk. In this chapter, the effect of fresh milk matrix interference was excluded, the composition and concentration of the closed liquid were optimized, and the optimum working concentration of 2G4 monoclonal antibody and envelope antigen CEX-OVA was explored. The linear relation of the standard inhibition curve was good R2=0.9909, and the linear equation was y=0.46 in the range of 20~1000 ng/mL drug concentration. 74x-0.5359, IC50 is 163.55 ng/mL, and LOD is 19.68ng/mL. through the experiment of adding the sample. It is found that the method has high sensitivity, the recovery rate is from 83.98% to 102.92%, the average recovery is 91.31%, the coefficient of variation is 8.50% to 14.5%, and the average coefficient of variation is 10.75%, which is in line with our country's prescribed cephalin in milk. The maximum residue limit standard has a certain application value.4, the preparation of the colloidal gold immunochromatography strip of cefamoxin. Using the three sodium citrate reduction method to prepare the different particle size colloid gold, explore the best pH and the optimum monoclonal antibody for preparing the gold standard antibody, explore the binding amount of the gold mark antibody in the gold mark pad, and optimize the C line in the NC membrane (Sheep anti rat IgG) and T Line (CEX-OVA) spraying, optimized the composition of the NC membrane sealing solution, and identified the stability, sensitivity and specificity of the paper strip. The results showed that the best pH of the gold standard antibody was 8.4, the best monoclonal antibody was added to 9 mu L mAb per 200 L colloid gold solution; the gold mark antibody in the gold pad was diluted by 1.5 times, and the effect of 30 u L/cm was the best; NC membrane C line, T The optimum spray amount of the line was 0.8 L/cm and 0.6 mu L/cm, and the 0.01 mol/L PB buffer solution of the closed solution was suitable for selecting 0.01 mol/L PB, and the test strip detection limit was 500 ng/mL, except for the slight cross reaction with cefadine, and the reaction was negative with ceftif, ceftriaxone, cefotaxime, penicillin, streptomycin, tetracycline. Keep 180 D. stable at 4 centigrade

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.84

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