磺胺甲噁唑单抗的制备及初步应用
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摘要
磺胺甲噁唑(Sulfamethoxazole, SMZ),在兽医临床上主要用于呼吸道和泌尿道感染。其与抗菌增效剂甲氧苄氨嘧啶(TMP)联合应用药物复方新诺明,具有抗菌谱广、疗效确切、性质稳定、使用方便、价格低廉、国内能大量生产等诸多优点,使得它成为目前兽医临床上磺胺类药物(Sulfonamides, SAs)中应用最广泛的抗菌药物之一。与此同时,滥用、滥添加于动物和动物饲料中的事件屡见不鲜,最终导致其在动物性产品中残留过高,这不仅对动物本身有很大的毒副作用,也对人类健康产生极大的损害!许多国家和组织规定了磺胺类药物的最高残留限量(Maximum Residues Limits,MRL),韩国、美国、欧共体(EEC)、以及在国际食品法典委员会(CAC)下设立的食品中兽药残留法典委员会(CCRVDF)等都规定了磺胺类总量和或具体单个磺胺类药物的含量不得超过0.1mg/kg,日本更是对个别磺胺类药物规定不得检出。因此,开发和建立高效、准确、低廉的磺胺类药检测方法具有极其重要的现实意义。
目前,关于磺胺类药物的检测方法主要有传统方法,包括微生物学测定法、滴定法、分光光度法等;色谱技术,包括气相色谱(GC)、高效液相色谱(HPLC)、气/液-质联用(GC/LC-MS)、毛细管电泳法(CE)、超临界流体色谱技术(SFC)等;免疫学分析法,包括酶联免疫吸附法(ELISA)、荧光免疫测定法(FIA)、胶体金免疫测定法(CGIA)、免疫传感器(IB)等。传统方法灵敏度不高,特异性不强,易被其它物质干扰,且分析时间较长;色谱技术虽然灵敏度高,但检测仪器化程度高,设备昂贵,操作技术要求高,前处理要求高;免疫学方法特异性强、灵敏度高、前处理简单、操作简便、易推广使用,其中酶联免疫吸附法同时可检测多个样品,特异性强、灵敏度高、速度快,是一个准确性、安全性高、实用性强的检测技术。
本研究运用化学合成方法制备SMZ完全抗原,采用单克隆抗体技术制备灵敏度高、特异性强、效价高的单抗,建立了ELISA测定方法,并初步应用于猪肉、鸡蛋、蜂蜜和牛奶中SMZ药物的检测。
由于磺胺甲噁唑分子量很小,仅为253.28,为半抗原。本研究利用磺胺甲噁唑具有的伯氨基结构,通过重氮法将其与载体蛋白HSA和BSA偶联,制备的两种人工抗原偶联比分别为:23∶1和21∶1。
用SMZ-BSA免疫7周龄BALB/c雌性小鼠后,融合前血清效价已达1:400,000以上。利用单克隆抗体技术和现代细胞融合技术,经过筛选,获得两株单克隆抗体杂交瘤细胞株3G11和2H10,稳定性实验表明,这两株单抗细胞株可稳定地分泌抗SMZ单克隆抗体。
通过建立间接竞争ELISA方法,测定腹水最佳工作浓度在1∶1×106以上。用3G11制备的腹水作出的SMZ药物抑制标准曲线,其线性方程为:y =0.206x -0.0317(R2=0.9885),LOD为3.29ng/ml,LOQ为23.06ng/ml。通过对同类药物以及两种非磺胺类药物的交叉反应实验表明该SMZ单抗的特异性良好,对其他药物的抑制率均小于10%。
另外,本研究对应用磺胺甲噁唑较多的动物性食品进行了添加回收试验。对样品处理方法进行了优化。测得在牛奶中药物添加回收率为98.9%～129.1%,在蛋黄、蛋清以及全蛋中的添加回收率分别为78.7%～102.3%、71.9%～85.3%和79.3%～93.6%,在猪肉中的添加回收率为89.7%～116.2%,在蜂蜜中的添加回收率为74%～111.7%。试验表明本实验所制备的磺胺甲噁唑单克隆抗体可用于对于动物性食品中磺胺甲噁唑残留的检测,为磺胺甲噁唑残留酶联免疫快速检测试剂盒或免疫胶体金检测试纸条的开发提供了优质的抗体。
Sulfamethoxazole(SMZ), is mainly used for respiratory and urinary tract infection in the veterinary clinic. Cotrimoxazole(TMP-SMZ),which is SMZ combined with Antibacterial synergist trimethoprim (TMP) , has wide antibiotic spectrum, formal curative effect, stable property, easy to use, inexpensive and can be domesticly mass-produced etc. advantages, making it become one of the most widely used Sulfonamides antibiotics in veterinary clinic. At the same time, abuse, overcharging add in the animal and animal feed become common occurrence events, which leading to too high residual in animal products. It not only has great poison side effects for the animals themselves, but also has tremendous damage on the human health . Many countries and organizations have prescription for the maximum residue limit (MRL) of Sulfonamides. South Korea, the United States, the European Community (EEC), Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) of Codex Alimentarius Commission (CAC), etc. have the prescription presents total Sulfonamides and/or specific individual Sulfonamides must be no more than 0.1 mg/kg MRL, and even Japan ordains that certain individual Sulfonamide should not be detected. Therefore, it has important practical significance to develop efficient, accurate and inexpensive Sulfonamides inspection methods.
At present, the main methods used for Sulfonamides detection are the traditional methods, including microbiology method, titration, spectrophotometry etc. ; chromatography technology, including gas chromatography (GC), high-performance liquid chromatography (HPLC), gas / liquid chromatography-mass spectrometry (GC / LC-MS), capillary electrophoresis (CE), supercritical fluid chromatography (SFC); immunological analysis, including the ELISA, Fluoro-immunoassay (FIA), colloidal gold immunization method (CGIA), immune biosensor (IB), and so on. Traditional methods are low sensitive and specific, easy to be interfered with other substances, and long time needed; although the chromatography has high sensitivity, but it also needs high-level detection equipment, expensive equipment, high technical requirements of operation, and preprocessing is complicated; Immunological methods are high specific, high sensitive, simple preprocessing, easy to operate and to promote the use. And the ELISA can detect a number of samples simultaneously, with high specificity, high sensitivity, high speed. It is an accurate, safe and practical detection technology.
In this research, the chemical synthesis methods were used for preparation of SMZ complete antigens. Monoclonal antibodies with high sensitivity, specificity and valence were prepared through monoclonal antibody technology. The ELISA detection method was founded, and was preliminary applied in the detection of SMZ in pork, eggs, honey and milk.
As SMZ molecular weight is very low, only 253.28, is a semi-antigen. Diazo method was used to make the SMZ combined with the carrier protein HSA and BSA by aryl-primary amino group structure of SMZ. Coupling ratio of the two artificial antigen is 23:1 and 21:1 respectively.
SMZ-BSA was used to immunize 7-week BALB/ c female mouse. The serum titer had reached more than 1:400000 before fusion. Through the monoclonal antibody technology and modern cell fusion technique, two monoclonal hybridoma cell line called 3G11 and 2H10 were obtained. Stability experiments showed that these two cell lines could secrete monoclonal antibody against SMZ stably.
Through the foundation of indirect competition ELISA method, the best work concentration of ascites was got, which is 1:1×106 above. SMZ standard curve was prepared by 3G11 ascites, with the linear equation: y = 0.206x -0.0317 (R2 = 0.9885), LOD 3.29 ng/ml, LOQ 23.06 ng/ml. The cross reaction experiment of congeneric Sulfonamides drugs and two non-Sulfonamides drugs showed that the SMZ monoclonal antibody has good specificity, with less than 10 percent inhibition rate anti other drugs.
In addition, additive reclamation test had been done for animal foods which with relatively more SMZ used. The preprocessing approach of samples was optimized. The measured drug additive reclamation test rates were: milk 98.9% to129.1%, egg yolk 78.7% to 102.3%, egg white 71.9% to 85.3%, the whole eggs79.3% to 93.6%, pork 89.7% to 116.2%, honey 74% to 111.7%. Experiment results showed that the SMZ monoclonal antibody prepared by this research can be used for animal food SMZ residue detection. It provided a high quality antibody for the development of SMZ residual ELISA rapid detection kit or immune colloidal gold detection paper.
目前,关于磺胺类药物的检测方法主要有传统方法,包括微生物学测定法、滴定法、分光光度法等;色谱技术,包括气相色谱(GC)、高效液相色谱(HPLC)、气/液-质联用(GC/LC-MS)、毛细管电泳法(CE)、超临界流体色谱技术(SFC)等;免疫学分析法,包括酶联免疫吸附法(ELISA)、荧光免疫测定法(FIA)、胶体金免疫测定法(CGIA)、免疫传感器(IB)等。传统方法灵敏度不高,特异性不强,易被其它物质干扰,且分析时间较长;色谱技术虽然灵敏度高,但检测仪器化程度高,设备昂贵,操作技术要求高,前处理要求高;免疫学方法特异性强、灵敏度高、前处理简单、操作简便、易推广使用,其中酶联免疫吸附法同时可检测多个样品,特异性强、灵敏度高、速度快,是一个准确性、安全性高、实用性强的检测技术。
本研究运用化学合成方法制备SMZ完全抗原,采用单克隆抗体技术制备灵敏度高、特异性强、效价高的单抗,建立了ELISA测定方法,并初步应用于猪肉、鸡蛋、蜂蜜和牛奶中SMZ药物的检测。
由于磺胺甲噁唑分子量很小,仅为253.28,为半抗原。本研究利用磺胺甲噁唑具有的伯氨基结构,通过重氮法将其与载体蛋白HSA和BSA偶联,制备的两种人工抗原偶联比分别为:23∶1和21∶1。
用SMZ-BSA免疫7周龄BALB/c雌性小鼠后,融合前血清效价已达1:400,000以上。利用单克隆抗体技术和现代细胞融合技术,经过筛选,获得两株单克隆抗体杂交瘤细胞株3G11和2H10,稳定性实验表明,这两株单抗细胞株可稳定地分泌抗SMZ单克隆抗体。
通过建立间接竞争ELISA方法,测定腹水最佳工作浓度在1∶1×106以上。用3G11制备的腹水作出的SMZ药物抑制标准曲线,其线性方程为:y =0.206x -0.0317(R2=0.9885),LOD为3.29ng/ml,LOQ为23.06ng/ml。通过对同类药物以及两种非磺胺类药物的交叉反应实验表明该SMZ单抗的特异性良好,对其他药物的抑制率均小于10%。
另外,本研究对应用磺胺甲噁唑较多的动物性食品进行了添加回收试验。对样品处理方法进行了优化。测得在牛奶中药物添加回收率为98.9%～129.1%,在蛋黄、蛋清以及全蛋中的添加回收率分别为78.7%～102.3%、71.9%～85.3%和79.3%～93.6%,在猪肉中的添加回收率为89.7%～116.2%,在蜂蜜中的添加回收率为74%～111.7%。试验表明本实验所制备的磺胺甲噁唑单克隆抗体可用于对于动物性食品中磺胺甲噁唑残留的检测,为磺胺甲噁唑残留酶联免疫快速检测试剂盒或免疫胶体金检测试纸条的开发提供了优质的抗体。
Sulfamethoxazole(SMZ), is mainly used for respiratory and urinary tract infection in the veterinary clinic. Cotrimoxazole(TMP-SMZ),which is SMZ combined with Antibacterial synergist trimethoprim (TMP) , has wide antibiotic spectrum, formal curative effect, stable property, easy to use, inexpensive and can be domesticly mass-produced etc. advantages, making it become one of the most widely used Sulfonamides antibiotics in veterinary clinic. At the same time, abuse, overcharging add in the animal and animal feed become common occurrence events, which leading to too high residual in animal products. It not only has great poison side effects for the animals themselves, but also has tremendous damage on the human health . Many countries and organizations have prescription for the maximum residue limit (MRL) of Sulfonamides. South Korea, the United States, the European Community (EEC), Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) of Codex Alimentarius Commission (CAC), etc. have the prescription presents total Sulfonamides and/or specific individual Sulfonamides must be no more than 0.1 mg/kg MRL, and even Japan ordains that certain individual Sulfonamide should not be detected. Therefore, it has important practical significance to develop efficient, accurate and inexpensive Sulfonamides inspection methods.
At present, the main methods used for Sulfonamides detection are the traditional methods, including microbiology method, titration, spectrophotometry etc. ; chromatography technology, including gas chromatography (GC), high-performance liquid chromatography (HPLC), gas / liquid chromatography-mass spectrometry (GC / LC-MS), capillary electrophoresis (CE), supercritical fluid chromatography (SFC); immunological analysis, including the ELISA, Fluoro-immunoassay (FIA), colloidal gold immunization method (CGIA), immune biosensor (IB), and so on. Traditional methods are low sensitive and specific, easy to be interfered with other substances, and long time needed; although the chromatography has high sensitivity, but it also needs high-level detection equipment, expensive equipment, high technical requirements of operation, and preprocessing is complicated; Immunological methods are high specific, high sensitive, simple preprocessing, easy to operate and to promote the use. And the ELISA can detect a number of samples simultaneously, with high specificity, high sensitivity, high speed. It is an accurate, safe and practical detection technology.
In this research, the chemical synthesis methods were used for preparation of SMZ complete antigens. Monoclonal antibodies with high sensitivity, specificity and valence were prepared through monoclonal antibody technology. The ELISA detection method was founded, and was preliminary applied in the detection of SMZ in pork, eggs, honey and milk.
As SMZ molecular weight is very low, only 253.28, is a semi-antigen. Diazo method was used to make the SMZ combined with the carrier protein HSA and BSA by aryl-primary amino group structure of SMZ. Coupling ratio of the two artificial antigen is 23:1 and 21:1 respectively.
SMZ-BSA was used to immunize 7-week BALB/ c female mouse. The serum titer had reached more than 1:400000 before fusion. Through the monoclonal antibody technology and modern cell fusion technique, two monoclonal hybridoma cell line called 3G11 and 2H10 were obtained. Stability experiments showed that these two cell lines could secrete monoclonal antibody against SMZ stably.
Through the foundation of indirect competition ELISA method, the best work concentration of ascites was got, which is 1:1×106 above. SMZ standard curve was prepared by 3G11 ascites, with the linear equation: y = 0.206x -0.0317 (R2 = 0.9885), LOD 3.29 ng/ml, LOQ 23.06 ng/ml. The cross reaction experiment of congeneric Sulfonamides drugs and two non-Sulfonamides drugs showed that the SMZ monoclonal antibody has good specificity, with less than 10 percent inhibition rate anti other drugs.
In addition, additive reclamation test had been done for animal foods which with relatively more SMZ used. The preprocessing approach of samples was optimized. The measured drug additive reclamation test rates were: milk 98.9% to129.1%, egg yolk 78.7% to 102.3%, egg white 71.9% to 85.3%, the whole eggs79.3% to 93.6%, pork 89.7% to 116.2%, honey 74% to 111.7%. Experiment results showed that the SMZ monoclonal antibody prepared by this research can be used for animal food SMZ residue detection. It provided a high quality antibody for the development of SMZ residual ELISA rapid detection kit or immune colloidal gold detection paper.
引文
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