磺胺类药物残留酶免疫分析的研究
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摘要
磺胺类药物是兽医临床上使用最广的一类化学合成抗菌药，主要用于细菌感染性疾病和某些原虫病的预防和治疗。但使用不合理很容易造成组织中的残留和蓄积。许多国家和组织规定了其最高残留限量(MRLs)为0.1μg/g，甚至不得检出。高效液相色谱、气-质联用等是磺胺类药物残留检测的法定方法，但成本较高，方法繁琐，不适于大量样本的测定。酶联免疫法(ELISA)检测具有特异性强、灵敏度好且适合大批样本快速测定的特点，在兽药残留检测上的应用日益广泛。关于各磺胺类药物残留免疫法检测的研究已有较多报道，但关于磺胺类药多残留(multi-residues)检测的研究才刚刚起步，而且在理论和实践中有许多问题需要解决。本研究通过化学法合成了两种具有磺胺母核结构的半抗原，制备了对多种磺胺药具有敏感性的多克隆抗体，并对多残留免疫分析的基本规律进行了探讨。另外，研制了磺胺甲基异噁唑(SMZ)抗体，建立了SMZ的ELISA检测方法，并应用于牛奶中SMZ残留的检测。
用对乙酰氨基苯磺酰氯(ASC)和对氨基苯甲酸(PABA)为反应原料，经过亲核取代、水解等化学反应，合成了保留磺胺药母核结构的半抗原化合物H1、H2，经紫外扫描、红外光谱、质谱、核磁共振以及免疫法鉴定为预期产物。用活性酯法和混合酸酐法将半抗原与载体蛋白偶联，制备了人工抗原，两种免疫原H1-HSA、H1-BSA的结合比分别为10.3：1和9.6：1。
用H1-HSA偶联物免疫兔子，制备了抗磺胺类药物抗体，抗体效价在1：10万以上。间接竞争ELISA表明，5种常用的磺胺药SO、SMM、SMD、SMZ、SC的检测限分别为0.065、0.046、0.045、0.032、0.015μg／ml，低于最高残留限量0.1μg/ml。但SD、SM_2的检测限较高为0.86、1.0μg/ml。抗体有较高的特异性，常用的4种抗生素和3种磺胺类药结构类似物与抗体无交叉反应，制备了酶标半抗原，并进行了直接竞争ELISA试验。试验结果表明磺胺药N~1端R取代基的空间位阻、电子云和电荷的分布与密度是决定抗体识别药物的重要因素。在牛奶介质中各药物的检测效果优于PBS介质中，添加SO的回收率为76.5％～107.4％，建立的间接竞争ELISA法基本能满足磺胺药多残留定性检测的要求。
用戊二醛将SMZ与载体偶联，以SMZ-BSA为免疫原制备了多克隆抗体和单克隆抗体。多克隆抗体具有较高的检测灵敏度和特异性，以其建立了测定SMZ残留的间接竞争ELISA方法。以SMZ浓度的对数为横坐标，药物对抗原抗体反应的抑制率为纵坐标，绘制了标准曲线。经计算，该方法的检测限是8.74ng／ml，定量限为71.46ng/ml，低于最高残留限量100ng／ml，抗体与其它6种磺胺药的交叉反应＜0.1％。比较了两种ELISA反应模式进行，发现抗体各作用40分钟时检测限没有降低。在牛奶介质中检测的灵敏度更高，检测限为6.14ng/ml，定量限25.4ng/ml，IC_(50)值为398ng／ml，添加回收率为71.6％～106％，RSD4.5％～10.27％，符合残留检测的要求，可用于SMZ残留测定。
Sulfonamide are synthetic bacteriostatic drug,widely used in animal husbandry and as feed additives.As a result,food derived from animals treated with sulfonamides may be contaminated with those drugs,and will influence human health, cause allergic reactions, and could result in resistance in pathogeic organisms because of the extensive use of sulfonamides.To safeguard the public heath,many countrys and organisations have established the Maximum Residues Limits(MRLS) of 0.1ug/g or even none.At present,the primary methods for detecting Sulfonamides Residues, high performance liquid chromatography(HPLC),gas chromatography(GC) and mass chromatography, are not suitable for screening a large number of samples.for they are time-consuming,costly and needing high skill.Enzyme-linked immunosorbent assy(ELISA) as a rapid,special and sensitive method has been gradually applied to veterinary drug determine. Immunoassay Screening methods for sulfonanides in foods and related materials have been reported in the literature
.But the research on the multi-residues in veterinary drug is just beginning,and there are many question eager to solve both in theory and practice.
This research synthesized two haptens H1,H2 which contain the common structure of Sulfonamide,by chemical method and developed anti-sulfonamides antibody. Anti-sulfamethoxazole antibody has been developed and the ELISA method has established for detecting SMZ ,and the method has been applied to screen SMZ residues in raw milk. The two haptens was synthesized through a series of chemical reactions of ASC and PABA.The products were successfully identified by UV scan, IR and MS .Through the EDC,DCC and MA methods,we prepared the immune antigen and coating antigen.UV-scaning identified the conjugates.The ratio of haptens to carrier protein is 10.3:1 and 9.6:lfor H1-HSA and H1-BSA.
Six rabbits were injected with the conjugates and anti-sulfonamides antibodies were developed.All of the titer of antiserum were above 1:100,000 by ELISA assay. Meanwhile,the limit of detection(LOD)of this assay was calculated to be 0.065,0.046,0.045,0.032,0.015 g/ml for SQ, SMM , SMD , SMZ , SC all of which were under the MRLS 0.1 ug/ml .However, the ELISA assay was not sensitive to SD, SM2 whose LOD are 0.86,1.0 g/ml.The antibody can be used to detect several sulfonamide drug residues .We prepared Enzyme labelled hapten and establised competitive direct ELISA.Those study showed the difference in the N1 position' s energy conformation and electrical density and property which were the determinate factors of Antibody combining with sulfonamides.
We established the immune method for detecting SMZ. SMZ was bounded to BSA or OVA using glutaraldehyde as the complete antigen, and developed monoclonal antibody and antiserum which is special and sensitive to SMZ.The competitive indirect ELISA method for screen SMZ residues was established with this
antiserum.Calibration graphes were prepared by plotting Ig drug concentration against percentage inhibition.The limit of detection of SMZ was 8.74ng/ml,and the limit of quantitative (LOQ)was 71.46ng/ml in PBS.The cross reaction with other six sulfonamides was low than 0.1% , which proved the antibody was very special.The standard curve of CiELISA was also established in raw milk condition and the curve indicated that the lower detection limit was 6.14ng/ml,the LOQ was 25.4ng/ml and the IC50 was 398ng/ml.The recovery ratio was 71.6%-106%,while SD was 4.5%-10.27%.The sensitivity and accuracy of the competitive enzyme immunoassay seemed to be satisfactory for quantitation of SMZ in milk,and fulfiled the demands of the MRLs regulation.
用对乙酰氨基苯磺酰氯(ASC)和对氨基苯甲酸(PABA)为反应原料，经过亲核取代、水解等化学反应，合成了保留磺胺药母核结构的半抗原化合物H1、H2，经紫外扫描、红外光谱、质谱、核磁共振以及免疫法鉴定为预期产物。用活性酯法和混合酸酐法将半抗原与载体蛋白偶联，制备了人工抗原，两种免疫原H1-HSA、H1-BSA的结合比分别为10.3：1和9.6：1。
用H1-HSA偶联物免疫兔子，制备了抗磺胺类药物抗体，抗体效价在1：10万以上。间接竞争ELISA表明，5种常用的磺胺药SO、SMM、SMD、SMZ、SC的检测限分别为0.065、0.046、0.045、0.032、0.015μg／ml，低于最高残留限量0.1μg/ml。但SD、SM_2的检测限较高为0.86、1.0μg/ml。抗体有较高的特异性，常用的4种抗生素和3种磺胺类药结构类似物与抗体无交叉反应，制备了酶标半抗原，并进行了直接竞争ELISA试验。试验结果表明磺胺药N~1端R取代基的空间位阻、电子云和电荷的分布与密度是决定抗体识别药物的重要因素。在牛奶介质中各药物的检测效果优于PBS介质中，添加SO的回收率为76.5％～107.4％，建立的间接竞争ELISA法基本能满足磺胺药多残留定性检测的要求。
用戊二醛将SMZ与载体偶联，以SMZ-BSA为免疫原制备了多克隆抗体和单克隆抗体。多克隆抗体具有较高的检测灵敏度和特异性，以其建立了测定SMZ残留的间接竞争ELISA方法。以SMZ浓度的对数为横坐标，药物对抗原抗体反应的抑制率为纵坐标，绘制了标准曲线。经计算，该方法的检测限是8.74ng／ml，定量限为71.46ng/ml，低于最高残留限量100ng／ml，抗体与其它6种磺胺药的交叉反应＜0.1％。比较了两种ELISA反应模式进行，发现抗体各作用40分钟时检测限没有降低。在牛奶介质中检测的灵敏度更高，检测限为6.14ng/ml，定量限25.4ng/ml，IC_(50)值为398ng／ml，添加回收率为71.6％～106％，RSD4.5％～10.27％，符合残留检测的要求，可用于SMZ残留测定。
Sulfonamide are synthetic bacteriostatic drug,widely used in animal husbandry and as feed additives.As a result,food derived from animals treated with sulfonamides may be contaminated with those drugs,and will influence human health, cause allergic reactions, and could result in resistance in pathogeic organisms because of the extensive use of sulfonamides.To safeguard the public heath,many countrys and organisations have established the Maximum Residues Limits(MRLS) of 0.1ug/g or even none.At present,the primary methods for detecting Sulfonamides Residues, high performance liquid chromatography(HPLC),gas chromatography(GC) and mass chromatography, are not suitable for screening a large number of samples.for they are time-consuming,costly and needing high skill.Enzyme-linked immunosorbent assy(ELISA) as a rapid,special and sensitive method has been gradually applied to veterinary drug determine. Immunoassay Screening methods for sulfonanides in foods and related materials have been reported in the literature
.But the research on the multi-residues in veterinary drug is just beginning,and there are many question eager to solve both in theory and practice.
This research synthesized two haptens H1,H2 which contain the common structure of Sulfonamide,by chemical method and developed anti-sulfonamides antibody. Anti-sulfamethoxazole antibody has been developed and the ELISA method has established for detecting SMZ ,and the method has been applied to screen SMZ residues in raw milk. The two haptens was synthesized through a series of chemical reactions of ASC and PABA.The products were successfully identified by UV scan, IR and MS .Through the EDC,DCC and MA methods,we prepared the immune antigen and coating antigen.UV-scaning identified the conjugates.The ratio of haptens to carrier protein is 10.3:1 and 9.6:lfor H1-HSA and H1-BSA.
Six rabbits were injected with the conjugates and anti-sulfonamides antibodies were developed.All of the titer of antiserum were above 1:100,000 by ELISA assay. Meanwhile,the limit of detection(LOD)of this assay was calculated to be 0.065,0.046,0.045,0.032,0.015 g/ml for SQ, SMM , SMD , SMZ , SC all of which were under the MRLS 0.1 ug/ml .However, the ELISA assay was not sensitive to SD, SM2 whose LOD are 0.86,1.0 g/ml.The antibody can be used to detect several sulfonamide drug residues .We prepared Enzyme labelled hapten and establised competitive direct ELISA.Those study showed the difference in the N1 position' s energy conformation and electrical density and property which were the determinate factors of Antibody combining with sulfonamides.
We established the immune method for detecting SMZ. SMZ was bounded to BSA or OVA using glutaraldehyde as the complete antigen, and developed monoclonal antibody and antiserum which is special and sensitive to SMZ.The competitive indirect ELISA method for screen SMZ residues was established with this
antiserum.Calibration graphes were prepared by plotting Ig drug concentration against percentage inhibition.The limit of detection of SMZ was 8.74ng/ml,and the limit of quantitative (LOQ)was 71.46ng/ml in PBS.The cross reaction with other six sulfonamides was low than 0.1% , which proved the antibody was very special.The standard curve of CiELISA was also established in raw milk condition and the curve indicated that the lower detection limit was 6.14ng/ml,the LOQ was 25.4ng/ml and the IC50 was 398ng/ml.The recovery ratio was 71.6%-106%,while SD was 4.5%-10.27%.The sensitivity and accuracy of the competitive enzyme immunoassay seemed to be satisfactory for quantitation of SMZ in milk,and fulfiled the demands of the MRLs regulation.
引文
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