本文選題：頭孢氨芐 + 單克隆抗體　； 參考：《揚州大學(xué)》2017年碩士論文

【摘要】：頭孢氨芐(Cephalexin,CEX)屬于β-內(nèi)酰胺類抗生素(β-lactam antibiotics),開發(fā)距今已五十載,由于其抗菌譜廣、吸收效果好、價格低廉,在人類醫(yī)學(xué)和獸醫(yī)臨床上一直有很廣泛的應(yīng)用。但是,在細(xì)菌耐藥性日趨嚴(yán)重的今天,頭孢氨芐等抗生素殘留問題必須得到重視。目前,中國、美國、英國、歐盟等多個國家或組織都已對頭孢氨芐最大殘留限量做出標(biāo)準(zhǔn)規(guī)定。檢測分析手段的提高是應(yīng)對抗生素殘留必不可少的關(guān)鍵。檢測頭孢氨芐殘留的方法主要有微生物檢測法(包括TTC法、擴散法等)、儀器檢測法(包括UV-Vis、HPLC、LC-MS等)和免疫學(xué)檢測法(包括ELISA、FPIA、IC等)。微生物法特異性不強、靈敏度不高,而且易受到其他條件干擾,已無法滿足當(dāng)前實際工作中快速、準(zhǔn)確的檢測要求;儀器分析法靈敏度高、重復(fù)性好、特異性強,但昂貴的實驗儀器、復(fù)雜的前處理過程、操作繁瑣的方法步驟是制約該法廣泛應(yīng)用的重要因素;免疫學(xué)檢測法特異性強、靈敏度高、操作簡單、耗時少,但假陽(陰)性率高、重復(fù)性較差卻是這種方法的局限性。目前來看,將免疫學(xué)方法與儀器檢測法聯(lián)合使用是一種科學(xué)、合理、高效的檢測分析思路。將免疫學(xué)方法作為前期工作初篩方法,儀器分析法作為后期確證方法,這不僅可大規(guī)�？焖俸Y選,而且也能保證檢測結(jié)果的可靠性。本研究旨在制備出親和力高、特異性強的抗頭孢氨芐單克隆抗體,以期建立快速、簡便、準(zhǔn)確的免疫學(xué)檢測方法,為我國頭孢氨芐在動物性食品中殘留監(jiān)控提供理論和技術(shù)支持。研究包括以下四點內(nèi)容:1、頭孢氨芐人工抗原的合成與鑒定。利用偶聯(lián)劑戊二醛將頭孢氨芐與載體蛋白(BSA、OVA)偶聯(lián),分別制備免疫抗原CEX-BSA和包被抗原CEX-OVA。經(jīng)紫外分光光度法、SDS-PAGE凝膠電泳和免疫學(xué)方法,鑒定人工抗原的合成是成功的。2、抗頭孢氨芐單克隆抗體的制備與鑒定。利用單克隆抗體技術(shù),將受免小鼠B淋巴細(xì)胞與SP2/0融合,通過有限稀釋法和CiELISA篩選得到2株單克隆細(xì)胞株(2B4、5B3),并將其注入小鼠腹腔,得到腹水型單克隆抗體。經(jīng)Protein G HP親和層析柱純化后鑒定,2G4單克隆抗體效價高于1:1.28X 105,其抗體亞型為IgG2b(κ輕鏈),抗體親和力常數(shù)為K=1.51×109L/mol,抗體除了與頭孢拉定有52.55%的交叉反應(yīng)率,與頭孢噻呋、頭孢曲松、頭孢噻肟、頭孢噻吩、青霉素、鏈霉素、四環(huán)素等藥物均無明顯交叉反應(yīng),說明制備的抗體親和力高、特異性較好,為后續(xù)免疫學(xué)檢測方法的建立打下了良好的基礎(chǔ)。3、鮮奶中頭孢氨芐ELISA檢測方法的建立。本章研究中排除了鮮奶基質(zhì)干擾影響,并優(yōu)化了封閉液的成分和濃度,摸索了 2G4單克隆抗體與包被抗原CEX-OVA的最佳工作濃度,所繪制的標(biāo)準(zhǔn)抑制曲線線性關(guān)系良好R2=0.9909,在20～1000 ng/mL藥物濃度范圍里,線性方程為y=0.4674x-0.5359,IC50為 163.55 ng/mL,LOD為19.68ng/mL。通過對樣品的添加回收實驗發(fā)現(xiàn),該方法檢測靈敏度較高,樣品回收率在83.98%～102.92%之間,平均回收率為91.31%;變異系數(shù)在8.50%～14.5%之間,平均變異系數(shù)為10.75%,符合我國規(guī)定的頭孢氨芐在牛奶中最大殘留限量標(biāo)準(zhǔn),具有一定應(yīng)用價值。4、頭孢氨芐膠體金免疫層析試紙條的研制。采用檸檬酸三鈉還原法制備不同粒徑膠體金,摸索制備金標(biāo)抗體的最佳pH和最適單抗標(biāo)記量,摸索金標(biāo)墊中金標(biāo)抗體結(jié)合量,優(yōu)化NC膜中C線(羊抗鼠IgG)與T線(CEX-OVA)噴涂量,優(yōu)化了 NC膜封閉液成分,并對試紙條穩(wěn)定性、靈敏度、特異性進行鑒定。結(jié)果顯示:金標(biāo)抗體最佳pH為8.4,最佳單抗標(biāo)記量為每200 μL膠體金溶液中加入9 μL單抗;金標(biāo)墊中金標(biāo)抗體以稀釋1.5倍,30μL/cm噴量效果最佳;NC膜C線、T線的最佳噴量分別為0.8μL/cm、0.6μL/cm;封閉液選擇3%BSA的0.01 mol/L PB緩沖液比較合適;經(jīng)鑒定該試紙條檢測限為500 ng/mL,除了與頭孢拉定有輕微交叉反應(yīng),與頭孢噻呋、頭孢曲松、頭孢噻肟、頭孢噻吩、青霉素、鏈霉素、四環(huán)素反應(yīng)均呈陰性,可在4℃環(huán)境中穩(wěn)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

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S859.84

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