頭孢曲松單克隆抗體的制備及ELISA方法的初步建立
發(fā)布時(shí)間：2019-05-15 17:42
【摘要】：頭孢曲松(Ceftraxone,CTRX)是第三代頭孢類抗生素,其抗菌譜廣,抑菌能力強(qiáng),常被用于動(dòng)物感染性疾病的治療。頭孢曲松的濫用,易導(dǎo)致人與動(dòng)物多種系統(tǒng)的疾病,以過敏反應(yīng)和結(jié)石癥最為嚴(yán)重。頭孢曲松為人用抗生素,目前已被農(nóng)業(yè)部列為禁止用于動(dòng)物飼養(yǎng)過程的人用藥品。目前檢測(cè)頭孢類抗生素殘留可采用理化檢測(cè)法、微生物檢測(cè)法和免疫學(xué)檢測(cè)法。理化檢測(cè)法所需儀器設(shè)備價(jià)格昂貴、專業(yè)技術(shù)性強(qiáng)及前處理過程繁瑣,以上因素決定了此法并不適用于基層大量樣品的檢測(cè)。微生物檢測(cè)法具有細(xì)菌培養(yǎng)耗時(shí)較長(zhǎng)、特異性不高、準(zhǔn)確度低等缺點(diǎn)。免疫學(xué)檢測(cè)法,以酶聯(lián)免疫吸附法(ELISA)為主,基于抗原抗體的特異性結(jié)合,其特異性強(qiáng)、靈敏度高、操作方法簡(jiǎn)單,適用于大量樣品的篩選。1.CTRX完全抗原的合成及鑒定。本研究采用戊二醛法合成兩種完全抗原。完全抗原經(jīng)紫外光譜掃描及SDS-PAGE凝膠電泳分析鑒定,可以初步判定完全抗原偶聯(lián)成功。完全抗原經(jīng)免疫小鼠、細(xì)胞融合及ELISA測(cè)定,最終證明兩種完全抗原制備是成功的。利用BCA法測(cè)得CTRX-BSA和CTRX-OVA蛋白濃度分別為5.5 mg/mL和5.0 mg/mL。2.CTRX單抗制備。采用常規(guī)免疫方案免疫BALB/c小鼠,免疫原使用人工抗原CTRX-BSA,ELISA檢測(cè)包被原使用CTRX-OVA。在小鼠五免后第7d尾靜脈采血,檢測(cè)血清抗體效價(jià)及其藥物抑制情況,血清效價(jià)達(dá)1:16000以上。通過細(xì)胞融合技術(shù)、ELISA篩選方法和有限稀釋亞克隆法獲得兩株可持續(xù)分泌針對(duì)CTRX單克隆抗體的雜交瘤細(xì)胞株,分別命名為3E7和7A7。通過小鼠體內(nèi)誘生法制備腹水,共18 mL,BCA法測(cè)其蛋白濃度分別為20.93 mg/mL 和 21.47 mg/mL;效價(jià)分別為 32000 和 128000。采用 HiTrap Protein G HP 純化柱純化1mL 7A7腹水,得到純化后的7A7腹水約5 mL,純化后的蛋白濃度為1.1 mg/mL,蛋白回收率為5.1%。SDS-PAGE凝膠電泳結(jié)果顯示,腹水純化效果良好。交叉實(shí)驗(yàn)結(jié)果表明,該CTRX單抗與頭孢氨芐、頭孢噻呋、頭孢拉定、頭孢噻肟、頭孢噻吩幾乎沒有交叉反應(yīng),且對(duì)CTRX的抑制率為100%。3.ELISA檢測(cè)方法的建立。利用純化后的7A7腹水建立檢測(cè)CTRX殘留的CiELISA方法。包被原最佳稀釋濃度1:3000,純化后7A7抗體的工作濃度1:16000。CTRX濃度在1.72～2000 ng/mL時(shí),標(biāo)準(zhǔn)曲線線性關(guān)系良好。其線性方程為y=0.2541x-0.0184(R2=0.9875),IC50為108.68 ng/L,LOD 為 1.72 ng/mL,LOQ 為 13.12 ng/mL。4.豬肉中CTRX添加回收實(shí)驗(yàn)使用豬肉進(jìn)行添加回收實(shí)驗(yàn),豬肉樣品經(jīng)過一系列前處理后用于實(shí)驗(yàn)測(cè)定。實(shí)驗(yàn)結(jié)果顯示樣品處理液稀釋8倍以上時(shí),基本可以消除樣品基質(zhì)的干擾。在20～1000 ng/mL藥物濃度范圍內(nèi),線性關(guān)系良好,線性方程為y=0.4466x-0.4738,相關(guān)系數(shù)R2=0.9869,IC50 為 150.52 ng/mL,LOD 為 16.38 ng/mL,LOQ 為 44.53 ng/mL。用 50、400、800 ng/mL的CTRX標(biāo)準(zhǔn)品溶液做添加回收實(shí)驗(yàn),經(jīng)檢測(cè)豬肉樣品回收率在81%～98.7%之間。
[Abstract]:Ceftriaxone (Ceftraxone,CTRX) is the third generation of cephalosporin antibiotics. Ceftriaxone (Ceftriaxone) is often used in the treatment of animal infectious diseases because of its wide antibacterial spectrum and strong bacteriostatic ability. The abuse of ceftriaxone is easy to lead to many diseases of human and animal systems, especially allergic reaction and stone formation. Ceftriaxone, a human antibiotic, has been listed by the Ministry of Agriculture as a human drug banned from animal feeding. At present, physicochemical detection, microbial detection and immunological detection can be used to detect cephalosporin antibiotic residues. The instrument and equipment needed for physical and chemical detection are expensive, professional and technical, and the pretreatment process is tedious. The above factors determine that this method is not suitable for the detection of a large number of samples at the base level. Microbial detection has many disadvantages, such as long time consuming, low specificity and low accuracy. Immunological detection, mainly enzyme-linked immunosorbent assay (ELISA), is based on the specific binding of antigen and antibody, which has strong specificity, high sensitivity and simple operation method, and is suitable for screening a large number of samples. 1. Synthesis and identification of CTRX complete antigen. In this study, two complete antigens were synthesized by glutaraldehyde method. The complete antigen was identified by UV spectrum scanning and SDS-PAGE gel electrophoresis analysis, and the complete antigen coupling success could be preliminarily determined. The complete antigen was successfully prepared by immunizing mice, cell fusion and ELISA assay. The concentrations of CTRX-BSA and CTRX-OVA proteins were determined by BCA method to be 5.5 mg/mL and 5.0 mg/mL.2.CTRX monoclonal antibodies, respectively. BALB/c mice were immunized with routine immunization regimen, and the immunogen was detected by artificial antigen CTRX-BSA,ELISA and coated with CTRX-OVA.. On the 7th day after the fifth immunization, the blood samples were collected from the tail vein of mice. The serum antibody titer and its drug inhibition were detected. The serum titer was more than 1 鈮，
本文編號(hào)：2477675
[Abstract]:Ceftriaxone (Ceftraxone,CTRX) is the third generation of cephalosporin antibiotics. Ceftriaxone (Ceftriaxone) is often used in the treatment of animal infectious diseases because of its wide antibacterial spectrum and strong bacteriostatic ability. The abuse of ceftriaxone is easy to lead to many diseases of human and animal systems, especially allergic reaction and stone formation. Ceftriaxone, a human antibiotic, has been listed by the Ministry of Agriculture as a human drug banned from animal feeding. At present, physicochemical detection, microbial detection and immunological detection can be used to detect cephalosporin antibiotic residues. The instrument and equipment needed for physical and chemical detection are expensive, professional and technical, and the pretreatment process is tedious. The above factors determine that this method is not suitable for the detection of a large number of samples at the base level. Microbial detection has many disadvantages, such as long time consuming, low specificity and low accuracy. Immunological detection, mainly enzyme-linked immunosorbent assay (ELISA), is based on the specific binding of antigen and antibody, which has strong specificity, high sensitivity and simple operation method, and is suitable for screening a large number of samples. 1. Synthesis and identification of CTRX complete antigen. In this study, two complete antigens were synthesized by glutaraldehyde method. The complete antigen was identified by UV spectrum scanning and SDS-PAGE gel electrophoresis analysis, and the complete antigen coupling success could be preliminarily determined. The complete antigen was successfully prepared by immunizing mice, cell fusion and ELISA assay. The concentrations of CTRX-BSA and CTRX-OVA proteins were determined by BCA method to be 5.5 mg/mL and 5.0 mg/mL.2.CTRX monoclonal antibodies, respectively. BALB/c mice were immunized with routine immunization regimen, and the immunogen was detected by artificial antigen CTRX-BSA,ELISA and coated with CTRX-OVA.. On the 7th day after the fifth immunization, the blood samples were collected from the tail vein of mice. The serum antibody titer and its drug inhibition were detected. The serum titer was more than 1 鈮，
本文編號(hào)：2477675
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