酶联免疫吸附法检测磺胺对甲氧嘧啶残留的研究
摘要
磺胺类药物(Sulfonamides，SAs)是一类具有广谱抗菌活性的化学药物。由于它具有经济、使用方便、化学性质稳定及治疗某些疾病有特效等特点，使之在畜牧业生产上的应用十分普遍。磺胺药常作为药物和饲料添加剂用于畜禽疾病的治疗和预防，以控制某些动物疾病的发生和促进动物生长。由于大量使用或长期使用磺胺药物，使得磺胺药物在体内蓄积，造成药物在动物组织内残留。磺胺药物在动物性食品中的残留对食品卫生以及人类的健康具有潜在的危害性，因此，检测并限制肉类、乳类、蛋类等动物性食品中磺胺类药物的残留日益受到广泛关注。我国规定磺胺类药物的总残留量不得超过300ng／ml，欧盟规定磺胺类所有药剂的总残留量不得超过100μg／kg。目前检测磺胺类药物残留的化学方法有高效液相色谱法(HPLC)、薄层液相色谱法(TLC)和气相色谱法(GC)等。这些化学方法虽然非常敏感、准确，但耗时长、耗资大、人员素质要求高，不适合大量样品的筛选测定。酶联免疫吸附法(ELISA)作为一种快速、特异和灵敏度较高的免疫检测技术正逐渐应用于药物残留的检测。磺胺对甲氧嘧啶(SMD)是一类比较常用的抗菌药物和饲料添加剂，如能建立SMD残留检测的酶免疫分析方法具有非常重要的现实意义，为此，本论文开展了这方面的初步研究工作，以期探讨测定SMD残留的灵敏、快速、方便的检测方法，为科学研究与实际应用提供资料。
用戊二醛法将SMD与载体蛋白BSA偶联制备合成抗原SMD-BSA用作免疫原，同法合成包被抗原SMD-OVA，选择几种免疫程序，免疫健康家兔，筛选最佳免疫程序，获得抗血清，用双向琼脂扩散试验对抗血清进行定性测定，结果表明抗血清特异性针对SMD。用ELISA法对抗血清进行定量测定抗血清之效价，其效价为1:2560，同时，用ELISA试验对抗血清进行了鉴定，分别以OVA、SMD-OVA、BSA、SMD-BSA进行包被，结果抗血清对OVA包被孔吸光值较低，而对SMD-OVA包被孔吸光值较高，表明抗血清中的确存在抗SMD抗体，结果与琼脂扩散试验的结果一致。以上结果表明所获得的抗血清符合要求，能用于实际样品的检测。
用所制备的抗血清建立间接竞争ELISA方法。优选了ELISA的工作条件，方阵测定确定了包被抗原最佳包被浓度(50μg／mL)，抗血清最佳稀释度(1:100)，酶标抗体的最适工作稀释度(1:500)，并建立了ELISA标准工作曲线。工作曲线表明在10～2000μg／L浓度范围内呈良好的线性关系，超过2000μg／L则线性关系
扬州大学硕士学位论文
不好。该法检测底限为63pg几，低于国际规定残留限量(100抖叭g)和国内规定
残留限量(30Op叭g)的要求。
按建立的EUSA方法测定了方法的回收率，结果表明回收率较好，符合预期
效果，无基础本底的牛奶样品回收率为94.7%，含基础本底的回收率为82.6%。进
行了实际样品的测定，选取扬州鸡按药典剂量调整给鸡肌肉注射SMD，连续注射
3天，在第O一7天分别采血，按建立的ELISA方法测定血清中的SMD残留含量，
结果表明随时间延长，血清中SMD残留含量逐渐减少，第7天已低于符合规定要
求。
建立的间接竞争ELISA法的精确度以批内误差和批间误差来表示。本试验批
内误差符合要求，均小于10%，但未做批间误差，与其它结构比较类似的磺肢药
物的交叉反应率也未做。所建立的方法尚需不同实验室的操作人员以及同一实验
室的不同操作人员反复测定，以验证方法的可靠性和灵敏性。
Sulfonamides are now used widely as chemical antimicrobials and additives which are often added in animal feeds for animal growth stimulation and for prevention and treatment'of animals diseases. Owing to their characteristics of cheapness,convenience, stability, effectiveness in some animal diseases, sulfonamides are applied in stock; raising industry more and more often. But. sulfonamides will accumulate in animal tissues if they are not used properly and the residues in edible tissues will be potentially harmful to food hygiene and to human health. Therefore, their residues in the edible tissues of food-producing animals such as meat, milk and egg et al. are paid much more attention increasingly. It is stipulated in our country that the total residues of sulfanonamides should not exceed 300ng /ml while in EU the total residues should not exceed 100μg/kg. At present, the chemical methods for detecting sulfonamides residues are mainly as follows: high performance liquid chromatography (
HPLC), thin layer chr
omatography (TLC), and gas chromatography (GC) et al.. Though all these methods are very sensitive and accurate, they are not suitable for screening a large number of samples because these methods are time-taking, costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological methods is gradually applied to detecting sulfonamides residues. Sulfamethoxydiazine (SMD) is often used as an antimicrobial and an additive for domestic animals and fowls. It is necessary to establish ELISA analysis protocol for determing SMD' s residues. So, this research is launched to develop the pilot study on the establishment of this ELISA protocol with anticipation to obtain a convenient, speedful and sensitive method for the detection of the residue of SMD. This research is eager to supply some practical and useful data for further study on SMD residue analysis.
Bovine serum albumin (BSA) and ovalbumin (OVA) were used as two protein carriers respectively to couple with semiantigen SMD by glutaraldehyde method in this paper. The complete antigens SMD-BSA and SMD-OVA were thus prepared and acted as immunoantigen and coating antigen respectively in ELISA protocol. Selected a few of immunal programs to immunize four healthy experimental rabbits to observe and obtain a suitable, best program of producing antiserum. Determined the property of this antiserum with two-direction agar diffusion test and it showed that antiserum was special to SMD. And the titer of antiserum was 1 : 2560 by ELISA test. Meanwhile ,
some ELISA tests were conducted to identify the antiserum with OVA, SMD-OVA, BSA,SMD-BSA coating the microplates separately and the results indicated that the OD of the holes coated with OVA was very low while the OD of the holes coated with SMD-OVA was much high. It was clearly showed that this antiserum was sure to contain the antibody of SMD and this result was up to the need of request of practical application.
Indirect competitive ELISA (icELISA) was established with this antiserum. The suitable working condition of icELISA was obtained by several grid tests which were used to calculate the suitable concentration and dilution of the coating antigen, enzyme labelled IgG and antiserum relatively. The most appropriate concentration and dilution of them was 50μg/mL, 1 : 500 and 1 : 100 correspondingly. The standard curve of icELISA was also established and the curve indicated that the lowest detection limit was 63μg/L which was under the demanded detection limit of 100μg/kg (EU) and 300 μg/kg (domestic). The curve had a favorable linearity relation within the concentration range of 10-2000μg/L. However the curve had a rather unfavorable linearity relation at the concentration exceeding over 2000 μg/L.
The recovery ratio was favorable to accord with anticipation. One was 94.7% with no background and the other was 82.6% with added background beforehand. On the basis of the established protocol, two hens 20 months old were taken as practical test samples. One h
用戊二醛法将SMD与载体蛋白BSA偶联制备合成抗原SMD-BSA用作免疫原，同法合成包被抗原SMD-OVA，选择几种免疫程序，免疫健康家兔，筛选最佳免疫程序，获得抗血清，用双向琼脂扩散试验对抗血清进行定性测定，结果表明抗血清特异性针对SMD。用ELISA法对抗血清进行定量测定抗血清之效价，其效价为1:2560，同时，用ELISA试验对抗血清进行了鉴定，分别以OVA、SMD-OVA、BSA、SMD-BSA进行包被，结果抗血清对OVA包被孔吸光值较低，而对SMD-OVA包被孔吸光值较高，表明抗血清中的确存在抗SMD抗体，结果与琼脂扩散试验的结果一致。以上结果表明所获得的抗血清符合要求，能用于实际样品的检测。
用所制备的抗血清建立间接竞争ELISA方法。优选了ELISA的工作条件，方阵测定确定了包被抗原最佳包被浓度(50μg／mL)，抗血清最佳稀释度(1:100)，酶标抗体的最适工作稀释度(1:500)，并建立了ELISA标准工作曲线。工作曲线表明在10～2000μg／L浓度范围内呈良好的线性关系，超过2000μg／L则线性关系
扬州大学硕士学位论文
不好。该法检测底限为63pg几，低于国际规定残留限量(100抖叭g)和国内规定
残留限量(30Op叭g)的要求。
按建立的EUSA方法测定了方法的回收率，结果表明回收率较好，符合预期
效果，无基础本底的牛奶样品回收率为94.7%，含基础本底的回收率为82.6%。进
行了实际样品的测定，选取扬州鸡按药典剂量调整给鸡肌肉注射SMD，连续注射
3天，在第O一7天分别采血，按建立的ELISA方法测定血清中的SMD残留含量，
结果表明随时间延长，血清中SMD残留含量逐渐减少，第7天已低于符合规定要
求。
建立的间接竞争ELISA法的精确度以批内误差和批间误差来表示。本试验批
内误差符合要求，均小于10%，但未做批间误差，与其它结构比较类似的磺肢药
物的交叉反应率也未做。所建立的方法尚需不同实验室的操作人员以及同一实验
室的不同操作人员反复测定，以验证方法的可靠性和灵敏性。
Sulfonamides are now used widely as chemical antimicrobials and additives which are often added in animal feeds for animal growth stimulation and for prevention and treatment'of animals diseases. Owing to their characteristics of cheapness,convenience, stability, effectiveness in some animal diseases, sulfonamides are applied in stock; raising industry more and more often. But. sulfonamides will accumulate in animal tissues if they are not used properly and the residues in edible tissues will be potentially harmful to food hygiene and to human health. Therefore, their residues in the edible tissues of food-producing animals such as meat, milk and egg et al. are paid much more attention increasingly. It is stipulated in our country that the total residues of sulfanonamides should not exceed 300ng /ml while in EU the total residues should not exceed 100μg/kg. At present, the chemical methods for detecting sulfonamides residues are mainly as follows: high performance liquid chromatography (
HPLC), thin layer chr
omatography (TLC), and gas chromatography (GC) et al.. Though all these methods are very sensitive and accurate, they are not suitable for screening a large number of samples because these methods are time-taking, costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological methods is gradually applied to detecting sulfonamides residues. Sulfamethoxydiazine (SMD) is often used as an antimicrobial and an additive for domestic animals and fowls. It is necessary to establish ELISA analysis protocol for determing SMD' s residues. So, this research is launched to develop the pilot study on the establishment of this ELISA protocol with anticipation to obtain a convenient, speedful and sensitive method for the detection of the residue of SMD. This research is eager to supply some practical and useful data for further study on SMD residue analysis.
Bovine serum albumin (BSA) and ovalbumin (OVA) were used as two protein carriers respectively to couple with semiantigen SMD by glutaraldehyde method in this paper. The complete antigens SMD-BSA and SMD-OVA were thus prepared and acted as immunoantigen and coating antigen respectively in ELISA protocol. Selected a few of immunal programs to immunize four healthy experimental rabbits to observe and obtain a suitable, best program of producing antiserum. Determined the property of this antiserum with two-direction agar diffusion test and it showed that antiserum was special to SMD. And the titer of antiserum was 1 : 2560 by ELISA test. Meanwhile ,
some ELISA tests were conducted to identify the antiserum with OVA, SMD-OVA, BSA,SMD-BSA coating the microplates separately and the results indicated that the OD of the holes coated with OVA was very low while the OD of the holes coated with SMD-OVA was much high. It was clearly showed that this antiserum was sure to contain the antibody of SMD and this result was up to the need of request of practical application.
Indirect competitive ELISA (icELISA) was established with this antiserum. The suitable working condition of icELISA was obtained by several grid tests which were used to calculate the suitable concentration and dilution of the coating antigen, enzyme labelled IgG and antiserum relatively. The most appropriate concentration and dilution of them was 50μg/mL, 1 : 500 and 1 : 100 correspondingly. The standard curve of icELISA was also established and the curve indicated that the lowest detection limit was 63μg/L which was under the demanded detection limit of 100μg/kg (EU) and 300 μg/kg (domestic). The curve had a favorable linearity relation within the concentration range of 10-2000μg/L. However the curve had a rather unfavorable linearity relation at the concentration exceeding over 2000 μg/L.
The recovery ratio was favorable to accord with anticipation. One was 94.7% with no background and the other was 82.6% with added background beforehand. On the basis of the established protocol, two hens 20 months old were taken as practical test samples. One h
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