氨苄青霉素残留ELISA检测方法的建立及检测条件优化
摘要
氨苄青霉素(Ampicillin)是目前较常用的广谱半合成青霉素，抗菌谱比青霉素G广，毒性极低，具有较强的杀菌作用。对革兰氏阴性菌和革兰氏阳性菌均有作用。在兽药临床上，氨苄青霉素多以注射方式用于治疗各种炎症、感染。如果将停药期间内的动物屠宰或挤取尚在停药期间内奶牛的牛奶，就会造成动物性食品中氨苄青霉素的残留。人们食用残留有氨苄青霉素的食品后，会诱导耐药菌株的产生，敏感的人群会产生过敏反应。发酵乳生产中残留的氨苄青霉素会抑制生产菌的生长。因此，建立针对氨苄青霉素残留的快速检测方法对控制动物性食品的卫生质量、保障消费者健康、保证工业生产等方面都具有比较重要的社会经济意义。
本研究制备了氨苄青霉素的多克隆抗体，建立了氨苄青霉素免疫检测技术。通过比较两种合成方法偶联的氨苄青霉素和BSA免疫抗原刺激机体产生抗体的亲和性和效价，比较了两种合成方法的优劣。同时，对ELISA检测条件进行了优化，建立氨苄青霉素ELISA检测技术。其主要研究结果如下：
1 通过两种方法——物理直接偶联和以戊二醛为连接物合成了氨苄青霉素和大分子蛋白质的免疫抗原和包被抗原并用紫外光谱扫描和红外吸收光谱扫描的方法对合成物质进行了鉴定。
2 将合成的氨苄青霉素免疫抗原与等量佐剂混合后多途径多次免疫兔子，第七次免疫后心脏采血，分离获得血清，并将这些血清分成3部分进行处理：一部分直接冷冻于-20℃，一部分与等量甘油混合后保藏于-20℃冰箱，另一部分血清纯化分离IgG、冷冻干燥后保存于-20℃冰箱。
3 用琼扩实验对抗血清进行分析。发现抗血清能与免疫抗原产生沉淀线，而不能与以OVA作载体蛋白的检测抗原产生沉淀线。这说明琼扩实验是一种很粗略的检测方法，能检测出血清中大量存在的载体蛋白抗体，半抗原的抗体因为量少不能在琼扩实验中产生沉淀线而被检测出来。
4 对ELISA检测中所用的HRP酶标山羊抗兔IgG的使用浓度和包被抗原类型及其浓度进行确定。实验结果表明，酶标二抗的最佳使用浓度是1600倍稀释。由于HSA和BSA有很大同源性，以其为载体蛋白的合成抗原不能作为检测抗原。同时，抗血清中存在抗桥抗体，用于检测的包被抗原和免疫抗原最好由两种合成方法含成，以消除抗桥抗体的影响。
56只兔子产生的抗血清的效价分别为：1号抗血清和2号抗血清的效价均是64×10~3，3号抗血清的效价是1×10~3，4号抗血清的效价是8×10~3，5号抗血清的效价是128×10~3，6号抗血清的效价是4×10~3。其中1、2、5号兔子产生的抗
浙江大学硕士论文
血清的效价都远远高于3、4、6号兔子产生的抗血清。而1、2、5号兔子都是以
Agl作为免疫抗原，3、4、6号兔子的免疫抗原都是Ag4。这说明不同合成方法
合成的免疫抗原有不同的免疫原性。
‘对间接ELISA检测条件进行优化，包括底物溶液的组成、包被液的选择、
包被时间和温度的选择、封闭液的选择、兔子抗血清的温育时间、HR卫酶标羊
抗兔lgG抗体的温育时间及底物加样后温育时间，并用L二(56)正交表对后6
个因素进行正交实验。正交实验证明，底物溶液的最佳配方是10ml底物缓冲液
+7 pl珑仇十200川TM[B。间接ELISA的最佳实验条件是以CBS作为包被液。加
好包被液的酶标板37℃温育2h后转入4℃冰箱温育14h包被。然后以5%甘氨酸
封闭醉标板。一抗的最佳温育时间是1 00min，二抗的最佳温育时间是120min，
加底物显色1 smin产生的阴阳性抗血清的OD月5。差值最大。包被液的类型对实验
结果的影响最大，其次是封闭液的类型和底物作用时间。由于二抗的温育时间对
实验的结果影响不是很主要，而且二抗温育40min和温育1 00min产生的实验结
果相差很少。从节约时间和成本的角度出发，在不降低实验灵敏度的前提下，二
抗的握育时间定为40min。
7用间接竞争EUSA实验对抗血清的亲和性进行测定，实验结果表明以Agl
为免疫抗原获得的抗血清的亲和性总体来说比免疫抗原为A妙的抗血清的亲和
性差。虽然Agl免疫兔子后产生的抗血清的效价比Ag4的高，但亲和性远远不
能达到犯LlsA检测抗体的要求。对于进行ELISA检测的抗血清，其亲和性的重
婆性比效价高的多。这表明以物理方法直接合成的免疫抗原比戊二醛法合成的免
痰抗原好，其免疫兔子产生的抗血清更适宜作为EUSA检侧抗体。6号抗血清是
最佳血清。
忍用氨爷青霉素的结构类似物和几种常见的抗生素与6号抗血清进行间接
竞争实脸，以测定抗血清的特异性。由于阿莫西林与氨节青霉素的结构只差一个
翔签，扰血清与阿莫西林的交叉反应率高达78%。抗血清与青霉素钾的交叉反应
率是1 .5机，与苯哇青霉素的交叉反应率是0涛%，而抗血清与硫酸链霉素、硫酸
卡那舜李和礁酸庆大霉素的交叉反应率均小子0.0125%。这说明该抗体的特异性
是好的，除了不能分辨样品中的氨节青霉素残留和阿莫西林残留，其他抗生素不
会对检翎结果产生干扰。
96号抗血清的竞争抑制实验标准曲线方程是:卜25 .046X一5.5396，检测极
限是4.1知留抽l，能达到动物性食品中氨节青霉素残留检测的要求。
Ampicillin is one of the most common semi-synthesis antibiotics. It has strong sterilizing ability and low toxicity. Ampicillin has a wider sterilizing range than penicillin G, and it's active to both G+ and G-. Ampicillin was often used to treat animals' inflammation and infection. If the animal was slaughtered or milked before ampicillin metabolized completely, there would have ampicillin residue animal food. When people eat these food, ampicillin residue remain in the food would induce anti-antibiotics bacterium. And it also would induce anaphylaxis in hypersensitive people. In industry, ampicillin residue in fermenting milk would restrain the growth of the zymogen. It's very meaningful to establish a fast detection method to detect ampicillin residue to control animal food safety, to safeguard consumers' health and to protect industry's production.
In the present study, we have produced anti-ampicillin polyclonal antibody and have established ampicillin's immunoassay method. We used two synthesis methods to synthesis immunogen. Then valued the synthesis methods by compare anti-sera produced from rabbits immunized with this two immunogen. We optimized and then established ELISA process. The main results were as following:
1. Synthesis immunogen with two methods-Physiological directly synthesis ampicillin and carrier protein conjugate; Chemical indirectly synthesis ampicillin-glutaradehyde-carrier protein conjugate. The products were scanned by UV spectrum aad infra-red spectrum to judge whether hapten was linked to the protein.
2. Synthetical antigen was mixed with same amount adjuvant and then immunized rabbits with it. After the seventh immunize, the rabbits were blooded from heart. Separated blood sera were divided into three parts for treatment: One part was directly stored at -20℃ Another part was mixed with glycerol and then stored at -20 ℃; IgG was separated from the third part serum then freeze dried and stored at -20℃.
3. Agar diffuses analysis anti-sera. We found that anti-sera reacted with immunogen but didn't react with antigen that different from immunogen only in protein carrier. This proved that agar diffuses was not a precision method in detecting an anti-hapten antibody because there was not so much antibody in the serum.
4. Determination working concentration of HRP linked goat anti-rabbitlgG antibody and working concentration of coating antigen. Working concentration of HRP linked goat anti-rabbitlgG antibody was 1600X dilution. Because HSA and BSA was very similar in Amino Acid sequence, antigen synthesized with HSA carrier protein can't be used as coating antigen. Besides, there were anti-bridge antibody in anti-sera. Coating antigen and immunogen should synthesize with different methods.
5. Working concentrations of six rabbit anti-sera were: No.1 anti-serum-64 X 103 dilution, No.2 anti-serum-64 X103 dilution, No.3 anti-serum-1 X 103 dilution, No.4 anti-serum-8 X 103 dilution, No.5 anti-serum-128 X 103 dilution, No.6 anti-serum-4X103. No.l, No.2 and No.5 anti-sera were collected from rabbits immunized with Ag1. No.3, No.4 and No.6 anti-sera were collected from rabbits immunized with Ag4. Antigen synthesized with different method has different immune ability.
6. Optimized indirect ELISA process. Our study showed that the best formula of reaction regent was 10ml buffer+7ml H2O2+200 ul TMB. The best reaction
-conditions were use CBS as coating buffer, plate should coat at 37@ for 2h then coat at 4@ for 14h, block the plate with 5% Glysine, anti-sera should incubate 100min, anti-antibody should incubate 40min.
7. Anti-sera's affinities were determined using competitive inhibitory indirect ELISA. The result showed mat anti-sera separated from rabbits immunized with Ag4(No.1, No2, No.5 anti-serum) has better affinities than anti-sera separated from rabbits immunized with Agl(No.3, No.4, N0.6 anti-serum). Though the later three anti-sera has higher working dilution than the former three ones, Physiological directly synthesis
本研究制备了氨苄青霉素的多克隆抗体，建立了氨苄青霉素免疫检测技术。通过比较两种合成方法偶联的氨苄青霉素和BSA免疫抗原刺激机体产生抗体的亲和性和效价，比较了两种合成方法的优劣。同时，对ELISA检测条件进行了优化，建立氨苄青霉素ELISA检测技术。其主要研究结果如下：
1 通过两种方法——物理直接偶联和以戊二醛为连接物合成了氨苄青霉素和大分子蛋白质的免疫抗原和包被抗原并用紫外光谱扫描和红外吸收光谱扫描的方法对合成物质进行了鉴定。
2 将合成的氨苄青霉素免疫抗原与等量佐剂混合后多途径多次免疫兔子，第七次免疫后心脏采血，分离获得血清，并将这些血清分成3部分进行处理：一部分直接冷冻于-20℃，一部分与等量甘油混合后保藏于-20℃冰箱，另一部分血清纯化分离IgG、冷冻干燥后保存于-20℃冰箱。
3 用琼扩实验对抗血清进行分析。发现抗血清能与免疫抗原产生沉淀线，而不能与以OVA作载体蛋白的检测抗原产生沉淀线。这说明琼扩实验是一种很粗略的检测方法，能检测出血清中大量存在的载体蛋白抗体，半抗原的抗体因为量少不能在琼扩实验中产生沉淀线而被检测出来。
4 对ELISA检测中所用的HRP酶标山羊抗兔IgG的使用浓度和包被抗原类型及其浓度进行确定。实验结果表明，酶标二抗的最佳使用浓度是1600倍稀释。由于HSA和BSA有很大同源性，以其为载体蛋白的合成抗原不能作为检测抗原。同时，抗血清中存在抗桥抗体，用于检测的包被抗原和免疫抗原最好由两种合成方法含成，以消除抗桥抗体的影响。
56只兔子产生的抗血清的效价分别为：1号抗血清和2号抗血清的效价均是64×10~3，3号抗血清的效价是1×10~3，4号抗血清的效价是8×10~3，5号抗血清的效价是128×10~3，6号抗血清的效价是4×10~3。其中1、2、5号兔子产生的抗
浙江大学硕士论文
血清的效价都远远高于3、4、6号兔子产生的抗血清。而1、2、5号兔子都是以
Agl作为免疫抗原，3、4、6号兔子的免疫抗原都是Ag4。这说明不同合成方法
合成的免疫抗原有不同的免疫原性。
‘对间接ELISA检测条件进行优化，包括底物溶液的组成、包被液的选择、
包被时间和温度的选择、封闭液的选择、兔子抗血清的温育时间、HR卫酶标羊
抗兔lgG抗体的温育时间及底物加样后温育时间，并用L二(56)正交表对后6
个因素进行正交实验。正交实验证明，底物溶液的最佳配方是10ml底物缓冲液
+7 pl珑仇十200川TM[B。间接ELISA的最佳实验条件是以CBS作为包被液。加
好包被液的酶标板37℃温育2h后转入4℃冰箱温育14h包被。然后以5%甘氨酸
封闭醉标板。一抗的最佳温育时间是1 00min，二抗的最佳温育时间是120min，
加底物显色1 smin产生的阴阳性抗血清的OD月5。差值最大。包被液的类型对实验
结果的影响最大，其次是封闭液的类型和底物作用时间。由于二抗的温育时间对
实验的结果影响不是很主要，而且二抗温育40min和温育1 00min产生的实验结
果相差很少。从节约时间和成本的角度出发，在不降低实验灵敏度的前提下，二
抗的握育时间定为40min。
7用间接竞争EUSA实验对抗血清的亲和性进行测定，实验结果表明以Agl
为免疫抗原获得的抗血清的亲和性总体来说比免疫抗原为A妙的抗血清的亲和
性差。虽然Agl免疫兔子后产生的抗血清的效价比Ag4的高，但亲和性远远不
能达到犯LlsA检测抗体的要求。对于进行ELISA检测的抗血清，其亲和性的重
婆性比效价高的多。这表明以物理方法直接合成的免疫抗原比戊二醛法合成的免
痰抗原好，其免疫兔子产生的抗血清更适宜作为EUSA检侧抗体。6号抗血清是
最佳血清。
忍用氨爷青霉素的结构类似物和几种常见的抗生素与6号抗血清进行间接
竞争实脸，以测定抗血清的特异性。由于阿莫西林与氨节青霉素的结构只差一个
翔签，扰血清与阿莫西林的交叉反应率高达78%。抗血清与青霉素钾的交叉反应
率是1 .5机，与苯哇青霉素的交叉反应率是0涛%，而抗血清与硫酸链霉素、硫酸
卡那舜李和礁酸庆大霉素的交叉反应率均小子0.0125%。这说明该抗体的特异性
是好的，除了不能分辨样品中的氨节青霉素残留和阿莫西林残留，其他抗生素不
会对检翎结果产生干扰。
96号抗血清的竞争抑制实验标准曲线方程是:卜25 .046X一5.5396，检测极
限是4.1知留抽l，能达到动物性食品中氨节青霉素残留检测的要求。
Ampicillin is one of the most common semi-synthesis antibiotics. It has strong sterilizing ability and low toxicity. Ampicillin has a wider sterilizing range than penicillin G, and it's active to both G+ and G-. Ampicillin was often used to treat animals' inflammation and infection. If the animal was slaughtered or milked before ampicillin metabolized completely, there would have ampicillin residue animal food. When people eat these food, ampicillin residue remain in the food would induce anti-antibiotics bacterium. And it also would induce anaphylaxis in hypersensitive people. In industry, ampicillin residue in fermenting milk would restrain the growth of the zymogen. It's very meaningful to establish a fast detection method to detect ampicillin residue to control animal food safety, to safeguard consumers' health and to protect industry's production.
In the present study, we have produced anti-ampicillin polyclonal antibody and have established ampicillin's immunoassay method. We used two synthesis methods to synthesis immunogen. Then valued the synthesis methods by compare anti-sera produced from rabbits immunized with this two immunogen. We optimized and then established ELISA process. The main results were as following:
1. Synthesis immunogen with two methods-Physiological directly synthesis ampicillin and carrier protein conjugate; Chemical indirectly synthesis ampicillin-glutaradehyde-carrier protein conjugate. The products were scanned by UV spectrum aad infra-red spectrum to judge whether hapten was linked to the protein.
2. Synthetical antigen was mixed with same amount adjuvant and then immunized rabbits with it. After the seventh immunize, the rabbits were blooded from heart. Separated blood sera were divided into three parts for treatment: One part was directly stored at -20℃ Another part was mixed with glycerol and then stored at -20 ℃; IgG was separated from the third part serum then freeze dried and stored at -20℃.
3. Agar diffuses analysis anti-sera. We found that anti-sera reacted with immunogen but didn't react with antigen that different from immunogen only in protein carrier. This proved that agar diffuses was not a precision method in detecting an anti-hapten antibody because there was not so much antibody in the serum.
4. Determination working concentration of HRP linked goat anti-rabbitlgG antibody and working concentration of coating antigen. Working concentration of HRP linked goat anti-rabbitlgG antibody was 1600X dilution. Because HSA and BSA was very similar in Amino Acid sequence, antigen synthesized with HSA carrier protein can't be used as coating antigen. Besides, there were anti-bridge antibody in anti-sera. Coating antigen and immunogen should synthesize with different methods.
5. Working concentrations of six rabbit anti-sera were: No.1 anti-serum-64 X 103 dilution, No.2 anti-serum-64 X103 dilution, No.3 anti-serum-1 X 103 dilution, No.4 anti-serum-8 X 103 dilution, No.5 anti-serum-128 X 103 dilution, No.6 anti-serum-4X103. No.l, No.2 and No.5 anti-sera were collected from rabbits immunized with Ag1. No.3, No.4 and No.6 anti-sera were collected from rabbits immunized with Ag4. Antigen synthesized with different method has different immune ability.
6. Optimized indirect ELISA process. Our study showed that the best formula of reaction regent was 10ml buffer+7ml H2O2+200 ul TMB. The best reaction
-conditions were use CBS as coating buffer, plate should coat at 37@ for 2h then coat at 4@ for 14h, block the plate with 5% Glysine, anti-sera should incubate 100min, anti-antibody should incubate 40min.
7. Anti-sera's affinities were determined using competitive inhibitory indirect ELISA. The result showed mat anti-sera separated from rabbits immunized with Ag4(No.1, No2, No.5 anti-serum) has better affinities than anti-sera separated from rabbits immunized with Agl(No.3, No.4, N0.6 anti-serum). Though the later three anti-sera has higher working dilution than the former three ones, Physiological directly synthesis
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18. Jeljaszewicz J, Mlynarczyk G, Mlynarczyk A. Antibiotic resistance in Gram-positive cocci. International Journal of Antimicrobial Agents. 2000,16:473-478
19. Therrien C, Roger C L. Molecular basis of antibiotic resistance and β-lactamase inhibition by mechanism-based inactivators:perspectives and future directions. FEMS Microbiology Reviews. 2000,24:251-262
20．李德培。抗菌促长药物耐药和残留的危害和预防，兽药与饲料添加剂，1999， 4(3)：19-20
21．刘永先，刘晓斌，任晓蓉等。1998年延安地区细菌耐药性检测，西北医学杂志，2000，5
22．杨藻晨主编。医用药理学，人民卫生出版社，1984年，第二版
23. Eva Gustavsson, Peter Bjurling, Ase Sternesj. Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity; Analytica Chimica Acta. 2002, 468:153-159
24. EEC2377/90
25．张曦，韩建众，张虹等。市售和试验猪肉内脏相关抗菌素和兴奋剂残留的检测研究，云南农业大学学报，2000，17(2)：184-187
26. Frank J. Schenck, Patrick S. Callery. Chromatographic methods of analysis of antibiotics in milk. Journal of Chromatography A. 1998, 812:99-109
27. Valérie Gaudin, Jéhanne Fontaine, Pierre Marls. Screening of penicillin residues in milk by a surface plasmon resonance-based biosensor assay: comparison of chemical and enzymatic sample pre-treatment. Analytica Chimica Acta. 2001 436:191-198
28. S J Setford, R M Van Es, Y J Blankwater, et al. Receptor binding protein amperometric assinity sensor for rapid β-lactam quantification in milk. Analytica Chimica Acta. 1999,398:13-22
29．庄无忌。各国食品和饲料农药兽药残留限量大全，北京：中国对外经济贸易出版社，1995，982-1014
30. Gianpiero Boatto, Riccardo Cerri, Amedeo Pau et al. Monitoring of benzylpenicillin in ovine milk by HPLC; Journal of Pharmaceutical and Biomedical Analysis. 1998,17:733-738.
31. Fontes E. Marques, Martins Susana, Carrapico Belmira. Determination of penicillin in milk by high pressure liquid chromatography. Toxicology Letters. 1996, 88:85
32. Lihl Stefan, Rehorek Astrid, Petz Michael. High-performance liquid chromatographic determination with electrochemical detection. Journal of Chromatography A. 1996,729(1):229-235
33. Ahrer W, Scherwenk E, Buchberger W. Determination of drug residues in water by the combination of liquid chromatography or capillary electrophoresis with electrospray mass spectrometry. Journal of Chromatography A. 2001,910(1):69-78
34. Setford S J, Van ES R M, Blankwater Y J, et al. Receptor binding protein amperometric affinity sensor for rapid β-lactam quantification in milk. Analytica Chimica Acta. 1999,398(1):13-22
35. Stefan Lihl, Astrid Rehorek, Michael Petz. High-Performance liquid chromatographic determination of penicillins by means of automated solid-phase extraction and photochemical degradation with electrochemical detection. Journal of Chromatography A. 1996,729:229-235
36. Allison J R D. Antibiotic residues in milk. Br. Vet. J. 1985,141,7-16
37. Kavanagh F. Theory and practice of microbiological assaying for antibiotics. J. Assoc. Off. Anal. Chem. 1989, 72, 6-10
38. T Hayama, Y Kido(Eds). Analytical Methods for Residual Veterinary Drugs in Food, Kindai, Tokyo, 1994, pp. 65-68
39. A Grubelnik, C Padeste, L Tiefenauer, in: Proc. 5th World Conf. Congress on Biosensors, Berlin, Germany, June 1998, p. 393)
40. GB44789. 27-34
41．何士敏，魏庆梅，李盛贤。牛奶中青霉素含量的快速测定，中国乳品工业，2000，28(3)：36-38
42. Asian-Australasian Journal of Investigative Dermatology. Application of ELISA for the penicillin antibiotic residues in live animal. 2000,115(3):589
43. Katsutani N, Shionoya H. Immunogenicity of various β-lactam antibiotic-protein conjugates and cross-reactivity of the antibodies produced in Guinea Pig. Int. Arch. Allergy Immunol. 1993,100:128-134
44. de Haan P, de Jonge A J R, Verbrugge T, et al. Three epitope-specific monoclonal antibodies against the hapten penicillin. Int. Arch. Allergy Immun. 1985,76:42-46
45. Mayorga C, Obispo T, Jimeno L, et al. Epitope'mapping of β-lactam antibiotics with the use of monoclonal antibodies. Toxicology. 1995,97:225-234
46．蔡勤仁，曾振灵，杨桂香。兽药残留免疫检测技术应用进展，动物医学进展，2002，23(2)：25-28
47．李华。奶制品中抗生素的检测，福建轻纺，2001，151(12)：5-7
48．李青。畜产品中青霉素残留的ELISA法测定，黑龙江畜牧兽医，2002，-9：6-8
49. P Cliquet, E Cox, C Van Dorpe, et al. Generation of Class-Selective Monoclonal Antibodies Against the Penicillin Group. J. Agric. Food Chem. 2001,49:3349-3355
50. H Watanabe, Atsuko Satake, Yasumasa Kido, Akio Tsuji. Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for dihydrostreptomycin in milk. Analytica Chimica Acta. 2002(472):45-53
51．黄世乐，陈祖义。氯霉素免疫抗原的合成，核农学通报，1991，12(1)：31-33
52. C Mayorga, T Obispo, L Jimeno, et al. Epitope mapping of β-lactam antibiotics with the use of monoclonal antibodies. Toxicology.
1995,97:225-234
53. Parker, C W, de Weck, A L, Kern, M and Eisen, H N. The preparation and some properties of penicillenic acid derivativers relevant to penicillin hypersensitivity. J. Exp. Med. 1961, 115:803-810
54. Levine, B B. N(α -D-Penicilloyl)amines as univalent hapten inhibitors of antibody-dependent allergic reactions to penicillin. J. Med. Pharm. Chem. 1962,5:1025-1034
55. Mozingo R and Folkers, K. The Chemistry of Penicillins, Princeton University Press. Princeton, NJ, Ch. ⅩⅧ. 1949 p. 634
56. Nagasawa, H T, Goon, D J W and De Master, E. 2, 5, 5-Trimethyl-thiazolidine-4-carboxyllic acid, a D(-)penicillamine directed pseudometabolite of ethanol, Detocication mechanism for acetaldehyde. J. Med. Chem. 1978,21:1274-1279
57. Groves D J, Morris B A. Veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas. Hybridoma. 2000, 19 (3) : 201-2014
58. Nelson P N, Reynolds G M, Waldron E E, Ward E, Giannopoulos K, Murray P G. Monoclonal antibodies. Mol Pathol. 2000,53(3):111-117
59. Deschamps R J A, Hall J C. Polyclonal and monoclonal immunoassays for picloram detection. In:Van Emon JM, Mumma RO, editors. Immunochemical methods for environmental analysis, ACS Symposium Series. Washington, DC:American Chemcial Society. 1990, 442:56-78
60. Bruce M P, Boyd V, Duch C, White J R. Dialysis-based bioreactor systems for the production of monoclonal antibodies-alternatives to ascites production in mice. J. Immunol Methods. 2002,264(1-2):59-68
61. Hudson P J, Souriau C. Recombinant antibodies for cancer diagnosis and therapy. Expert Opin Biol Ther. 2001, 1(5):845-855
62. Kang A S; Barbas C F, Janda K D, Benkovie S J, Lerner R A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci U S A. 1991,88(10):4363-4366
63. Bird R E, Hardman K D, Jacobson J W, Johnson S, Kaufman B M, Lee S M, et al. Single chain antigen binding protein. Science. 1988,242:423-426
64. Vu K B, Ghahroudi M A, Wyns L, Muyldermans S. Comparison of llama VH sequences from conventional and heavy chain antibodies. Mol Immunol. 1997,34(16-17):1121-1131
65. Smith G P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science.1985, 228:1315-1317
66．于涟主鳊。免疫学实验技术，浙江大学出版社，1992年3月第一版，p1-6
67. Remo P Vallejo, Edward R Bogus, Ralph 0 Mumma. Effects of Hapten Structure and Bridging Groups on Antisera Spexificity in Parathion Immunoassay Development. J. Agric. Food Chem. 1982, 30:572-580
68. Bertold Hock, Andrea Dankwardt, Karl Kramer, et al. Immunoehemical techniques: antibody production for pesticide analysis. A review. Analytica Chimica Acta. 1995, 311:393-405
69. Dietmar Knopp. Application of immunological methods for the determination of environmental, pollutants in human biomonitoring. A review. Analytica Chimica Acta. 1995, 311:383-392
70. Valérie Gaudin, Jéhanne Fontaine, Pierre Marls. Screening of penicillin residues in milk by a surface plasmon resonance-based biosensor assay: comparison of chemical and enzymatic sample pre-treatment. Analytica Chimica Acta. 2001 436:191-198
71. Eva Gustavsson, Peter Bjurling, Ase Sternesji. Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity. Analytica Chimica Acta. 2002, 468:153-159
72. G A Baxterm J P Ferguson, M C O'Connor and C T Elliott. Detection of Streptomycin Residues in Whole Milk Using an Optical Immunobiosensor. J. Agric Food Chem. 2001,49:3204-3207
73. Jean-Louis Marty, Delphine Garcia, Regis Rouillon. Biosensors: potential in pesticide detection. Trends in analytical chemistry. 1995, 14(7):329-333
74. Bertold Hock. Antibodies for immunosensors. A review. Analytica Chimica Acta. 1997, 347:177-186
75. A Brecht, G Gauglitz. Label free optical immunoprobes for pesticide detection. Analytica Chemica Acta. 1997, 347:219-233
76. B B Dzantiev, A V Zherdev. Electrochemical immunosensors for determination of the pesticides 2,4-dichlorophenoxyacetic and 2, 4,5-trichlorophenoxyacetic acids. Biosensors & Bioelectronics. 1996, 11(1):179-185
77. Green, M.J. (1987). New approaches in electrochemical immunoassay. In: Biosensors, fundamentals & Applications. A.P.F. Turner, I. Karube & G.S. Wilson(eds), Oxford University Press, pp. 57-65
78. Alexandra E. Botchkareva, Fabiana Fini, Sergei Eremin, et al. Development of a heterogeneous chemiluminescent flow immunoassay for DDT and related compounds. Analytica Chimica Acta. 2002, 453:43-52
79. E Mallat, C Barzen, R Abuknesha, et al. Parts per trillion level determination of isoproturon in certified and estuarine water samples with a direct optical immunosensor. Analytica Chimica Acta. 2001, 426:209-216
80．但德忠，王方强，何俊等。新型荧光光纤免疫磁珠流动分析系统研究(Ⅰ)，分析测试学报，2000，19(2)：17—20
81．但德忠，何俊、王方强等。新型荧光光纤免疫磁珠流动分析系统研究(Ⅱ)，分析测试学报，2000，19(2)：79—82
82. S A Eremin, I A Ryabova, J N Yakovleva. Development of a rapid, specific fluorescence polarization immunoassay for the herbicide chlorsulfuron. Analytica Chimica Acta. 2002, 468:229-236
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90．洪孝庄，荣康泰，韩树森，龚雄麒。军事医学科学院院刊；“桥抗体”对酶免疫分析的影响，1990，67：203-208
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8. Carman R J, Van Tassel1 R L, Wlikins T D. The normal intestinal microflora: ecology, variability and stability. J Vet Human Toxicol. 1993,35(1):11-14
9. Vollard E J, Classener H A L. Colonization resisitance. Antimicrob Agent Chem other. 1994, 38:409-414
10. Corpet D E. An Evaluation of methods to assess the effect of antimicrobial residues on the human gut flora. Vet Microb. 1993,35:199-212
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12. Carman R J, Van Tassell R L, Wlikins T D. The normal intestinal microflora: ecology, variability and stability. J Vet Human Toxicol. 1993,35(1):11-14
13．刘健华，陈杖榴。动物性食品中抗菌药残留对人体肠道菌群的影响及其研究模型与方法，动物医学进展，2003，24(2)：37-40，48
14．朱蓓蕾。动物性食品药物残留，上海科学技术出版社，1994
15. Hamscher G, Sezesny S, Abu-Qare A, Hoper H, Nau H. substances with pharmacological effects including hormonally active substances in the environment: identification of tetracyclines in soil fertilized withanimal slurry. Dtsch Tierarztl Wochenschr. 2000,107(8):332-334
16. Dijck P V, van de Voorde H. Sensitivity of environmental microorganism to antimicrobial agents. Appl Environ Microbiol. 1976,31(3):332-336
17. Strong L, Wall R, Woolford A, et al. The effect of faccally excreted ivermectin and fenbendazole on the insect colonization of cattle dung
following the oral administration of sustained rease boluses. Vet Parasitol. 1996,62(3-4):253-265
18. Jeljaszewicz J, Mlynarczyk G, Mlynarczyk A. Antibiotic resistance in Gram-positive cocci. International Journal of Antimicrobial Agents. 2000,16:473-478
19. Therrien C, Roger C L. Molecular basis of antibiotic resistance and β-lactamase inhibition by mechanism-based inactivators:perspectives and future directions. FEMS Microbiology Reviews. 2000,24:251-262
20．李德培。抗菌促长药物耐药和残留的危害和预防，兽药与饲料添加剂，1999， 4(3)：19-20
21．刘永先，刘晓斌，任晓蓉等。1998年延安地区细菌耐药性检测，西北医学杂志，2000，5
22．杨藻晨主编。医用药理学，人民卫生出版社，1984年，第二版
23. Eva Gustavsson, Peter Bjurling, Ase Sternesj. Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity; Analytica Chimica Acta. 2002, 468:153-159
24. EEC2377/90
25．张曦，韩建众，张虹等。市售和试验猪肉内脏相关抗菌素和兴奋剂残留的检测研究，云南农业大学学报，2000，17(2)：184-187
26. Frank J. Schenck, Patrick S. Callery. Chromatographic methods of analysis of antibiotics in milk. Journal of Chromatography A. 1998, 812:99-109
27. Valérie Gaudin, Jéhanne Fontaine, Pierre Marls. Screening of penicillin residues in milk by a surface plasmon resonance-based biosensor assay: comparison of chemical and enzymatic sample pre-treatment. Analytica Chimica Acta. 2001 436:191-198
28. S J Setford, R M Van Es, Y J Blankwater, et al. Receptor binding protein amperometric assinity sensor for rapid β-lactam quantification in milk. Analytica Chimica Acta. 1999,398:13-22
29．庄无忌。各国食品和饲料农药兽药残留限量大全，北京：中国对外经济贸易出版社，1995，982-1014
30. Gianpiero Boatto, Riccardo Cerri, Amedeo Pau et al. Monitoring of benzylpenicillin in ovine milk by HPLC; Journal of Pharmaceutical and Biomedical Analysis. 1998,17:733-738.
31. Fontes E. Marques, Martins Susana, Carrapico Belmira. Determination of penicillin in milk by high pressure liquid chromatography. Toxicology Letters. 1996, 88:85
32. Lihl Stefan, Rehorek Astrid, Petz Michael. High-performance liquid chromatographic determination with electrochemical detection. Journal of Chromatography A. 1996,729(1):229-235
33. Ahrer W, Scherwenk E, Buchberger W. Determination of drug residues in water by the combination of liquid chromatography or capillary electrophoresis with electrospray mass spectrometry. Journal of Chromatography A. 2001,910(1):69-78
34. Setford S J, Van ES R M, Blankwater Y J, et al. Receptor binding protein amperometric affinity sensor for rapid β-lactam quantification in milk. Analytica Chimica Acta. 1999,398(1):13-22
35. Stefan Lihl, Astrid Rehorek, Michael Petz. High-Performance liquid chromatographic determination of penicillins by means of automated solid-phase extraction and photochemical degradation with electrochemical detection. Journal of Chromatography A. 1996,729:229-235
36. Allison J R D. Antibiotic residues in milk. Br. Vet. J. 1985,141,7-16
37. Kavanagh F. Theory and practice of microbiological assaying for antibiotics. J. Assoc. Off. Anal. Chem. 1989, 72, 6-10
38. T Hayama, Y Kido(Eds). Analytical Methods for Residual Veterinary Drugs in Food, Kindai, Tokyo, 1994, pp. 65-68
39. A Grubelnik, C Padeste, L Tiefenauer, in: Proc. 5th World Conf. Congress on Biosensors, Berlin, Germany, June 1998, p. 393)
40. GB44789. 27-34
41．何士敏，魏庆梅，李盛贤。牛奶中青霉素含量的快速测定，中国乳品工业，2000，28(3)：36-38
42. Asian-Australasian Journal of Investigative Dermatology. Application of ELISA for the penicillin antibiotic residues in live animal. 2000,115(3):589
43. Katsutani N, Shionoya H. Immunogenicity of various β-lactam antibiotic-protein conjugates and cross-reactivity of the antibodies produced in Guinea Pig. Int. Arch. Allergy Immunol. 1993,100:128-134
44. de Haan P, de Jonge A J R, Verbrugge T, et al. Three epitope-specific monoclonal antibodies against the hapten penicillin. Int. Arch. Allergy Immun. 1985,76:42-46
45. Mayorga C, Obispo T, Jimeno L, et al. Epitope'mapping of β-lactam antibiotics with the use of monoclonal antibodies. Toxicology. 1995,97:225-234
46．蔡勤仁，曾振灵，杨桂香。兽药残留免疫检测技术应用进展，动物医学进展，2002，23(2)：25-28
47．李华。奶制品中抗生素的检测，福建轻纺，2001，151(12)：5-7
48．李青。畜产品中青霉素残留的ELISA法测定，黑龙江畜牧兽医，2002，-9：6-8
49. P Cliquet, E Cox, C Van Dorpe, et al. Generation of Class-Selective Monoclonal Antibodies Against the Penicillin Group. J. Agric. Food Chem. 2001,49:3349-3355
50. H Watanabe, Atsuko Satake, Yasumasa Kido, Akio Tsuji. Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for dihydrostreptomycin in milk. Analytica Chimica Acta. 2002(472):45-53
51．黄世乐，陈祖义。氯霉素免疫抗原的合成，核农学通报，1991，12(1)：31-33
52. C Mayorga, T Obispo, L Jimeno, et al. Epitope mapping of β-lactam antibiotics with the use of monoclonal antibodies. Toxicology.
1995,97:225-234
53. Parker, C W, de Weck, A L, Kern, M and Eisen, H N. The preparation and some properties of penicillenic acid derivativers relevant to penicillin hypersensitivity. J. Exp. Med. 1961, 115:803-810
54. Levine, B B. N(α -D-Penicilloyl)amines as univalent hapten inhibitors of antibody-dependent allergic reactions to penicillin. J. Med. Pharm. Chem. 1962,5:1025-1034
55. Mozingo R and Folkers, K. The Chemistry of Penicillins, Princeton University Press. Princeton, NJ, Ch. ⅩⅧ. 1949 p. 634
56. Nagasawa, H T, Goon, D J W and De Master, E. 2, 5, 5-Trimethyl-thiazolidine-4-carboxyllic acid, a D(-)penicillamine directed pseudometabolite of ethanol, Detocication mechanism for acetaldehyde. J. Med. Chem. 1978,21:1274-1279
57. Groves D J, Morris B A. Veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas. Hybridoma. 2000, 19 (3) : 201-2014
58. Nelson P N, Reynolds G M, Waldron E E, Ward E, Giannopoulos K, Murray P G. Monoclonal antibodies. Mol Pathol. 2000,53(3):111-117
59. Deschamps R J A, Hall J C. Polyclonal and monoclonal immunoassays for picloram detection. In:Van Emon JM, Mumma RO, editors. Immunochemical methods for environmental analysis, ACS Symposium Series. Washington, DC:American Chemcial Society. 1990, 442:56-78
60. Bruce M P, Boyd V, Duch C, White J R. Dialysis-based bioreactor systems for the production of monoclonal antibodies-alternatives to ascites production in mice. J. Immunol Methods. 2002,264(1-2):59-68
61. Hudson P J, Souriau C. Recombinant antibodies for cancer diagnosis and therapy. Expert Opin Biol Ther. 2001, 1(5):845-855
62. Kang A S; Barbas C F, Janda K D, Benkovie S J, Lerner R A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci U S A. 1991,88(10):4363-4366
63. Bird R E, Hardman K D, Jacobson J W, Johnson S, Kaufman B M, Lee S M, et al. Single chain antigen binding protein. Science. 1988,242:423-426
64. Vu K B, Ghahroudi M A, Wyns L, Muyldermans S. Comparison of llama VH sequences from conventional and heavy chain antibodies. Mol Immunol. 1997,34(16-17):1121-1131
65. Smith G P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science.1985, 228:1315-1317
66．于涟主鳊。免疫学实验技术，浙江大学出版社，1992年3月第一版，p1-6
67. Remo P Vallejo, Edward R Bogus, Ralph 0 Mumma. Effects of Hapten Structure and Bridging Groups on Antisera Spexificity in Parathion Immunoassay Development. J. Agric. Food Chem. 1982, 30:572-580
68. Bertold Hock, Andrea Dankwardt, Karl Kramer, et al. Immunoehemical techniques: antibody production for pesticide analysis. A review. Analytica Chimica Acta. 1995, 311:393-405
69. Dietmar Knopp. Application of immunological methods for the determination of environmental, pollutants in human biomonitoring. A review. Analytica Chimica Acta. 1995, 311:383-392
70. Valérie Gaudin, Jéhanne Fontaine, Pierre Marls. Screening of penicillin residues in milk by a surface plasmon resonance-based biosensor assay: comparison of chemical and enzymatic sample pre-treatment. Analytica Chimica Acta. 2001 436:191-198
71. Eva Gustavsson, Peter Bjurling, Ase Sternesji. Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity. Analytica Chimica Acta. 2002, 468:153-159
72. G A Baxterm J P Ferguson, M C O'Connor and C T Elliott. Detection of Streptomycin Residues in Whole Milk Using an Optical Immunobiosensor. J. Agric Food Chem. 2001,49:3204-3207
73. Jean-Louis Marty, Delphine Garcia, Regis Rouillon. Biosensors: potential in pesticide detection. Trends in analytical chemistry. 1995, 14(7):329-333
74. Bertold Hock. Antibodies for immunosensors. A review. Analytica Chimica Acta. 1997, 347:177-186
75. A Brecht, G Gauglitz. Label free optical immunoprobes for pesticide detection. Analytica Chemica Acta. 1997, 347:219-233
76. B B Dzantiev, A V Zherdev. Electrochemical immunosensors for determination of the pesticides 2,4-dichlorophenoxyacetic and 2, 4,5-trichlorophenoxyacetic acids. Biosensors & Bioelectronics. 1996, 11(1):179-185
77. Green, M.J. (1987). New approaches in electrochemical immunoassay. In: Biosensors, fundamentals & Applications. A.P.F. Turner, I. Karube & G.S. Wilson(eds), Oxford University Press, pp. 57-65
78. Alexandra E. Botchkareva, Fabiana Fini, Sergei Eremin, et al. Development of a heterogeneous chemiluminescent flow immunoassay for DDT and related compounds. Analytica Chimica Acta. 2002, 453:43-52
79. E Mallat, C Barzen, R Abuknesha, et al. Parts per trillion level determination of isoproturon in certified and estuarine water samples with a direct optical immunosensor. Analytica Chimica Acta. 2001, 426:209-216
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