莫能菌素抗原及多克隆抗体研制
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摘要
莫能菌素是含羧基的多聚醚类离子载体抗生素，由于其特殊的物理化学特性及其作用机理，此类药物在生产中应用比较广泛。目前，已普遍应用于家禽、家兔等的抗球虫，成为世界上使用最早销量最大的抗球虫药物；除此以外，还作为饲料添加剂应用到养牛业和养猪业，提高牛和猪的生产性能，促进个体生长。但是不合理使用此类药物将会导致畜禽体内药物残留，对畜禽及人类均会造成危害。莫能菌素对不同动物体毒性相差较大，但总体上来说对生长及繁殖性能等都有明显的危害，对人类主要导致肌肉和心脏病变。因此，如何及时、快速有效的检测出莫能菌素残留成为国内外研究的方向。莫能菌素的检测方法中有经典比色法、薄层层析法、微生物检测法、生物自显影技术检测法、液相色谱方法及免疫检测法等多种，其中以近几年发展起来的免疫检测法较为方便快捷。多克隆抗体或单克隆抗体是免疫检测法建立的基础。本文对莫能菌素钠进行了半抗原修饰改造，制备出了莫能菌素多克隆抗体，并对其进行了抗体鉴定。此试验为建立莫能菌素免疫检测方法奠定了基础。
一、莫能菌素钠盐半抗原的修饰
生产中制备的莫能菌素以钠盐形式存在，没有直接与选用的载体蛋白质(BSA、OVA)偶联的基团，因此不能直接用于人工抗原的合成，必须加以修饰形成含羧基的物质才能偶联，合成人工抗原以便生产抗体。本试验采用三种方法(盐酸浸泡法、阳离子交换法与琥珀酸酐法)将莫能菌素钠盐改造成含羧酸基团的莫能菌素酸及莫能菌素-琥珀酸酐衍生物，并通过薄层层析与红外光谱法进行鉴定，经鉴定半抗原修饰成功。经修饰物质为多克隆抗体的制备提供基础。
二、抗莫能菌素抗体的研制
采用混合酸酐法及水溶性碳化二亚胺法，将修饰成功的半抗原莫能菌素酸(Mon)和莫能菌素-琥珀酸酐(Mon-HS)与牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)共价偶联，合成免疫抗原Mon-HS-BSA、Mon-OVA和包被抗原Mon-HS-OVA、Mon-BSA，合成的人工抗原通过双向琼脂扩散及SDS-PAGE电泳法进行鉴定，并计算出蛋白质半抗原偶联比率分别为(Mon-HS-BSA)18：1、(Mon-HS-OVA)7：1、(Mon-OVA)11：1、(Mon-BSA)20：1。用合成的免疫抗原对新西兰大白兔进行免疫，制备的抗血清经抗血清质量鉴定，结
莫能菌素抗原及多克隆杭体研制
果发现两种免疫原免疫家兔所得杭体(三免、四免、五免)均呈上升趋势，经ELISA法测
定抗体效价分别为l:12800(耳号0799)、l:6400(耳号0791)、l:25600(耳号0569);
经间接ELISA鉴定结果表明，制备的抗体均存在杭Mon杭体.
Monensin belongs to a class of compounds known as the polyether monocarboxylic acid ionophores. Because of its specific physical and chemical characteristics and the effect mechanism, this antibiotic has already applied for the production. At present, Monensin has been widely used as a feed additive to control coccidiosis in poultry and rabbits. In addition, It is used to increase the food conversion efficiency and accelerate the growth of cattle and pigs. However, using this antibiotic irrationally will lead to the remedy residue, which will damage to animals and human beings. The toxicity of Monensin is different for kinds of animals. In the mass, Monensin is harmful to growth and reproduction performance of animals , and will lead to the muscle and heart disease of the human being. Consequently, how to assay monensin rapidly and efficiently becomes the aspect of research at home and abroad. There are many methods of detecting the antibiotic such as classical colorimetric method, the TLC method, the microb
iological assay, the bioautographic method, the liquid chromatography method, the immunoassay method and so on. In these methods, Immunoassay method which has developed in these years is simple and quick. The basis of immunoassay method is the development of the polyclonal or monoclonal antibody. The present research includes the transformation of the hapten Monensin and the development of the polyclonal antibody. At the same time, we appraised the antibody. The research establishes the basis of immunoassay of Monensin. The results are shown as follows: 1. The transformation of the hapten Monensin Sodium Salt
In production Monensin is existed as Monensin sodium salt. However, Monensin sodium salt has not any group which are directly coupled with protein conjugations such as BSA,OVA. So it can not synthesize immugen directly and must be modified to the substance contained of carboxyl to produce antibody. We applied three methods including of hydrochloric acid soaking method, cation exchange method, succinate acid anhydride method for transformation to monensin acid and monensin-succinate acid anhydride
derivative. The materials of the transformation were appraised by thin layer
chromatography and Infrared spectrum. The result of appraising is approved successfully.
The materials of the transformation provide the basis for the development of polyclonal
antibody.
2. The Development of Polyclonal Antibody to Monensin
The materials (Monensin acid, Mon-HS) were coupled with protein (BSA, OVA) by the mixed acid anhydride reaction and EDC method. The conjugation included the immunogen(Mon-HS-BSA, Mon-OVA) and the coating antigen(Mon-HS-OVA, Mon-BSA). The synthesized artificial antigen were appraised by DATA and SDS-PAGE. The protein-hapten conjugate rate was 1:18(Mon-HS-BSA), 1:7(Mon-HS-OVA), l:ll(Mon-OVA),l:20(Mon-BSA) separately. High tiler polyclonal antibodies against Monensin were produced from rabbits immunized with the synthesized immunogen conjugates Mon-HS-BSA and Mon-OVA. The results show the rising trend of the antibodies to the 3rd , 4th and 5lh immunity. The final liter of antibodies were l:12800(ear No.0799), l:6400(ear No.0791) and l:25600(ear No.0569) respectively. Through the indirect competitive enzyme-linked immunosorbent assay(ELISA), there is the one to monensin among the antibodies.
一、莫能菌素钠盐半抗原的修饰
生产中制备的莫能菌素以钠盐形式存在，没有直接与选用的载体蛋白质(BSA、OVA)偶联的基团，因此不能直接用于人工抗原的合成，必须加以修饰形成含羧基的物质才能偶联，合成人工抗原以便生产抗体。本试验采用三种方法(盐酸浸泡法、阳离子交换法与琥珀酸酐法)将莫能菌素钠盐改造成含羧酸基团的莫能菌素酸及莫能菌素-琥珀酸酐衍生物，并通过薄层层析与红外光谱法进行鉴定，经鉴定半抗原修饰成功。经修饰物质为多克隆抗体的制备提供基础。
二、抗莫能菌素抗体的研制
采用混合酸酐法及水溶性碳化二亚胺法，将修饰成功的半抗原莫能菌素酸(Mon)和莫能菌素-琥珀酸酐(Mon-HS)与牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)共价偶联，合成免疫抗原Mon-HS-BSA、Mon-OVA和包被抗原Mon-HS-OVA、Mon-BSA，合成的人工抗原通过双向琼脂扩散及SDS-PAGE电泳法进行鉴定，并计算出蛋白质半抗原偶联比率分别为(Mon-HS-BSA)18：1、(Mon-HS-OVA)7：1、(Mon-OVA)11：1、(Mon-BSA)20：1。用合成的免疫抗原对新西兰大白兔进行免疫，制备的抗血清经抗血清质量鉴定，结
莫能菌素抗原及多克隆杭体研制
果发现两种免疫原免疫家兔所得杭体(三免、四免、五免)均呈上升趋势，经ELISA法测
定抗体效价分别为l:12800(耳号0799)、l:6400(耳号0791)、l:25600(耳号0569);
经间接ELISA鉴定结果表明，制备的抗体均存在杭Mon杭体.
Monensin belongs to a class of compounds known as the polyether monocarboxylic acid ionophores. Because of its specific physical and chemical characteristics and the effect mechanism, this antibiotic has already applied for the production. At present, Monensin has been widely used as a feed additive to control coccidiosis in poultry and rabbits. In addition, It is used to increase the food conversion efficiency and accelerate the growth of cattle and pigs. However, using this antibiotic irrationally will lead to the remedy residue, which will damage to animals and human beings. The toxicity of Monensin is different for kinds of animals. In the mass, Monensin is harmful to growth and reproduction performance of animals , and will lead to the muscle and heart disease of the human being. Consequently, how to assay monensin rapidly and efficiently becomes the aspect of research at home and abroad. There are many methods of detecting the antibiotic such as classical colorimetric method, the TLC method, the microb
iological assay, the bioautographic method, the liquid chromatography method, the immunoassay method and so on. In these methods, Immunoassay method which has developed in these years is simple and quick. The basis of immunoassay method is the development of the polyclonal or monoclonal antibody. The present research includes the transformation of the hapten Monensin and the development of the polyclonal antibody. At the same time, we appraised the antibody. The research establishes the basis of immunoassay of Monensin. The results are shown as follows: 1. The transformation of the hapten Monensin Sodium Salt
In production Monensin is existed as Monensin sodium salt. However, Monensin sodium salt has not any group which are directly coupled with protein conjugations such as BSA,OVA. So it can not synthesize immugen directly and must be modified to the substance contained of carboxyl to produce antibody. We applied three methods including of hydrochloric acid soaking method, cation exchange method, succinate acid anhydride method for transformation to monensin acid and monensin-succinate acid anhydride
derivative. The materials of the transformation were appraised by thin layer
chromatography and Infrared spectrum. The result of appraising is approved successfully.
The materials of the transformation provide the basis for the development of polyclonal
antibody.
2. The Development of Polyclonal Antibody to Monensin
The materials (Monensin acid, Mon-HS) were coupled with protein (BSA, OVA) by the mixed acid anhydride reaction and EDC method. The conjugation included the immunogen(Mon-HS-BSA, Mon-OVA) and the coating antigen(Mon-HS-OVA, Mon-BSA). The synthesized artificial antigen were appraised by DATA and SDS-PAGE. The protein-hapten conjugate rate was 1:18(Mon-HS-BSA), 1:7(Mon-HS-OVA), l:ll(Mon-OVA),l:20(Mon-BSA) separately. High tiler polyclonal antibodies against Monensin were produced from rabbits immunized with the synthesized immunogen conjugates Mon-HS-BSA and Mon-OVA. The results show the rising trend of the antibodies to the 3rd , 4th and 5lh immunity. The final liter of antibodies were l:12800(ear No.0799), l:6400(ear No.0791) and l:25600(ear No.0569) respectively. Through the indirect competitive enzyme-linked immunosorbent assay(ELISA), there is the one to monensin among the antibodies.
引文
1. Christopher T. Elliott, D. Glenn Kennedy et al., Methods for the detection of polyether ionophore residues in poultry[J]. Analyst, June 1998 (123): 45R-56R
2．张传津等，莫能菌素预混剂防治鸡球虫病的试验[J]．兽药与饲料添加剂，2002(6)：11-12
3. ELPotter COCooley LFRichardson et al., Effect of monensin on performance of cattle fed forage[J]. Journal of animal science, 1976 (43): 1484-1498
4. MCCalhoun LHCarrol CWLivingston et al., Effect of detary monensin on coccidial oocyst numbers feedlot performance and carcass character-rustics of lambs[J]. Journal of animal science, 1979(49): 3504-3508
5．韩兴泰．影响反刍动物瘤胃微生物蛋白合成的因素[J]．青海畜牧兽医杂志，1993，23(4)：3-6
6．崔立．反刍动物新蛋白体系的相关内容浅探[J]．饲料博览，1995(3)：14-16
7. PMgarcia-lopez et al., In vitro inhibition of microbial methane production by9, 10-anthraquinone[J], Anim. Sci. 1996(74): 2276-2284
8．秦学忠．瘤胃素作饲料添加剂[J]．国外畜牧学—草食家畜，1988(4)：30-33
9．邸怀忠．莫能菌素与拉沙里菌素对母牛群生产的效果[J]．内蒙古畜牧科学，1991(3)：26-28
10. RAMeinert and CMJyang et al., Effects of monensin on growing reproductive performance andestimated body composition in hoistein heifers[J]. Dairy. Sci., 1992(75):257-261
11. DLLalman et al., The effects of ruminnally undegradable protein propionic acid and monensin onpuberty and pregnancy in beefheifers[J], Anim. Sci., 1993(71): 1843-2852
12. Christopher JHorvath et al., Effect of monensin on selenium status and related factors
in genetically hypo-and hyperselsnemic growing swine am-j-vet-res[J]. 1992 (53):161-163
13. Fitzgerald PR and Jprotozool, 1973 (19): 332-334
14. Gwyther MJClemson university clemsonsc(硕士论文),1976
15. Bila CG, Perreira CL, Gruys E. Accidental monensin toxicosis in horses in Moza mbique[J]. J S Afr Vet Assoc, 2001, 72(3): 163-164
16. Atef M,Shalaby MA, el-Sayed MG. Influence of monensinon fertility in rats[J]. Clin Exper Pharm Physiol, 1986,13(2): 113-121
17. Rhharms, Poultry Science, 1989, 68(1): 86-88
18．俞海．莫能菌素对雏火鸡毒性试验[J]．中国兽医科技，1996，26(4)：32-33
19. Pressman, B.C., and Fahim, M., Cardiovascular toxicity of ionophores used as feed additives[J]. Adv. Exp. Med. Biol., 1983(161): 543-561
20. Caldeira C, Neves WS, Cury PM, et al. Rhabdomyolysis, acute renal failure, and death after monensin ingestion[J]. Am J Kid Dis, 2001(38): 1108-1112
21. Schweitzer D, Kimberling C, Spraker T, et al. Accidental monensin sodium intoxication of feedlot cattle[J]. J Am Vet Med Assoc, 1984(184): 1273-1276
22. Todd GC, Novilla MN, Howard LC. Comparative toxicology of monensin sodium in laboratory animals[J]. J Animal Sci, 1984(58): 1512-1517
23. Kouyou mdjian JA, Morita MP, Sato AK, et al. Fatal rhabdomyolysis after acute sodium monensin (Rumensin) toxicity: case report[J]. Arq Neuropsiquiatr, 2001(59): 596-598
24. Novilla MN .Toxic syndrome induced by ionophores[J]. Vet Hum Toxicol, 1992(34): 66-70
2．张传津等，莫能菌素预混剂防治鸡球虫病的试验[J]．兽药与饲料添加剂，2002(6)：11-12
3. ELPotter COCooley LFRichardson et al., Effect of monensin on performance of cattle fed forage[J]. Journal of animal science, 1976 (43): 1484-1498
4. MCCalhoun LHCarrol CWLivingston et al., Effect of detary monensin on coccidial oocyst numbers feedlot performance and carcass character-rustics of lambs[J]. Journal of animal science, 1979(49): 3504-3508
5．韩兴泰．影响反刍动物瘤胃微生物蛋白合成的因素[J]．青海畜牧兽医杂志，1993，23(4)：3-6
6．崔立．反刍动物新蛋白体系的相关内容浅探[J]．饲料博览，1995(3)：14-16
7. PMgarcia-lopez et al., In vitro inhibition of microbial methane production by9, 10-anthraquinone[J], Anim. Sci. 1996(74): 2276-2284
8．秦学忠．瘤胃素作饲料添加剂[J]．国外畜牧学—草食家畜，1988(4)：30-33
9．邸怀忠．莫能菌素与拉沙里菌素对母牛群生产的效果[J]．内蒙古畜牧科学，1991(3)：26-28
10. RAMeinert and CMJyang et al., Effects of monensin on growing reproductive performance andestimated body composition in hoistein heifers[J]. Dairy. Sci., 1992(75):257-261
11. DLLalman et al., The effects of ruminnally undegradable protein propionic acid and monensin onpuberty and pregnancy in beefheifers[J], Anim. Sci., 1993(71): 1843-2852
12. Christopher JHorvath et al., Effect of monensin on selenium status and related factors
in genetically hypo-and hyperselsnemic growing swine am-j-vet-res[J]. 1992 (53):161-163
13. Fitzgerald PR and Jprotozool, 1973 (19): 332-334
14. Gwyther MJClemson university clemsonsc(硕士论文),1976
15. Bila CG, Perreira CL, Gruys E. Accidental monensin toxicosis in horses in Moza mbique[J]. J S Afr Vet Assoc, 2001, 72(3): 163-164
16. Atef M,Shalaby MA, el-Sayed MG. Influence of monensinon fertility in rats[J]. Clin Exper Pharm Physiol, 1986,13(2): 113-121
17. Rhharms, Poultry Science, 1989, 68(1): 86-88
18．俞海．莫能菌素对雏火鸡毒性试验[J]．中国兽医科技，1996，26(4)：32-33
19. Pressman, B.C., and Fahim, M., Cardiovascular toxicity of ionophores used as feed additives[J]. Adv. Exp. Med. Biol., 1983(161): 543-561
20. Caldeira C, Neves WS, Cury PM, et al. Rhabdomyolysis, acute renal failure, and death after monensin ingestion[J]. Am J Kid Dis, 2001(38): 1108-1112
21. Schweitzer D, Kimberling C, Spraker T, et al. Accidental monensin sodium intoxication of feedlot cattle[J]. J Am Vet Med Assoc, 1984(184): 1273-1276
22. Todd GC, Novilla MN, Howard LC. Comparative toxicology of monensin sodium in laboratory animals[J]. J Animal Sci, 1984(58): 1512-1517
23. Kouyou mdjian JA, Morita MP, Sato AK, et al. Fatal rhabdomyolysis after acute sodium monensin (Rumensin) toxicity: case report[J]. Arq Neuropsiquiatr, 2001(59): 596-598
24. Novilla MN .Toxic syndrome induced by ionophores[J]. Vet Hum Toxicol, 1992(34): 66-70