安定单克隆抗体的研制（一）
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摘要
目的 (1) 比较两种合成安定衍生物的方法，建立一种新的安定抗原性BSA(Bovine Serum Albumin)结合物的合成途径，为同类型小分子抗原的抗原性BSA结合物合成提供新的技术路线；(2)制备抗安定的多克隆抗体，采用ELISA法检测所合成安定抗原性BSA结合物的免疫原性和反应原性，为制备单克隆抗体提供可靠的筛选方法；(3)免疫组织化学法检测安定中毒大鼠脑组织中安定，为安定或其它小分子物质的检测提供新的方法。
方法 (1)安定抗原性BSA结合物的制备 利用有机合成方法分别采用戊二酸酐和1，3-二氯丙烷在安定的3位碳原子上连接可以与蛋白质相结合的5-羰基戊酸基或1-氯丙烷基，合成安定衍生物。薄层层析和柱层析分离合成产物，薄层色谱扫描法、傅立叶红外分光光度分析和超导核磁共振分析鉴定衍生物结构和稳定性；根据胺的制备原理将安定卤代烃衍生物与载
山西反科大学
20仍年硕士研究生毕业论文
体蛋白BSA上天冬酞胺(AsparagineN)、谷氨酞胺(Glutamine
Q)、精氨酸(ArginineR)和赖氨酸(LysineK)的游离氨基发
生亲核取代反应，制备出安定抗原性BSA结合物。紫外分光光
度法和SDS一聚丙烯酞胺凝胶电泳的方法比较安定抗原性BSA
结合物和BSA的紫外吸收峰和分子量。(2)安定多克隆抗体的
制备背部皮内多点注射安定抗原性BSA结合物和F盯end’s完
全佐剂混合物初次免疫新西兰大白兔，第21、28、35、42、49、
56天安定抗原性BSA结合物和Furend’s不完全佐剂混合物同样
方式加强免疫，第60天采血，收集血清。BSA吸收后，采用
ELISA法比较空白组、实验对照组、实验组和阳性对照组血清
的OD值，鉴定抗体的特异性;对半稀释法测定抗血清效价。(3)
脑组织中安定的免疫组织化学测定安定片粉末灌胃，复制
Wistar大鼠安定中毒模型，取大脑福尔马林固定，石蜡包埋
切片。以BSA吸收的多克隆抗血清为一抗进行SABC免疫组织
化学染色，分析。
结果(1)利用安定和戊二酸配、1，3一二氯丙烷均合成得
到安定3位5一拨基戊酸基和1一氯丙烷基取代的衍生物。傅立叶
红外和超导核磁共振分析证明其分子结构与设计一致。常温或
暴露于光线下时，戊二酸醉衍生物容易分解，卤代烃衍生物不
山西民料大学
2003年硕士研究生毕业论文
分解。安定抗原性BSA结合物的紫外吸收特征峰较BSA发生红
移、SDS一聚丙烯酞胺凝胶电泳的迁移速率与BSA差异明显。
(2)安定抗原性BSA结合物免疫新西兰大白兔，得到抗安定血
清。抗体特异性测定显示，空白组和实验对照组的OD值间无
显著性差异，与其相比，实验组和阳性对照组OD值均显著升
高;与实验组相比，阳性对照组OD值显著升高;与实验对照
组相比，实验组OD值显著升高。抗血清的效价达到1:2560。
(3)SABC染色，安定中毒大鼠大脑组织细胞膜和核膜上可见
褐色颗粒沉着，对照组未见褐色颗粒沉着。
结论采用卤代烃作为桥连剂，合成安定的卤代烃衍生
物，进一步交联于载体蛋白的技术路线适用于安定完全抗原的
合成;本实验合成的安定完全抗原可以刺激机体产生抗安定抗
体，但抗体特异性和血清效价有待于进一步提高，抗体类别有
待于进一步鉴定;本实验所建立的ELISA法为安定单克隆抗体
制备提供了可靠的筛选方法;免疫组织化学法可应用于富尔马
林固定检材内安定的定性检验。
Objective (1) To compare two synthesis of
diazepam-derivative, build a new path for the synthesis of diazepam BSA immunogenic conjugate, and provide a new technique for the synthesis of similar micro-molecular antigen. (2) To obtain polyclonal antibody specific for diazepam, detect the antibody specificity and serum titer by ELISA, and establish a screen method for monoclonal antibody study (3) To detect the diazepam in diazepam poisoned rat brain by an immunohistochemistry, and provide a new quanlitative analysis in the brain fixed with 4% formaldehyde.
Method (1) Production of diazepam BSA immunogenic conjugate Diazepam was derivatized in position 3 of the benzodiazepine ring with glutaric anhydirde and 1,3-2 chloropropane. The compound was isolated and purified by TLC and column chromatography, and assayed by TLC, Infrared Spectrophotometry and NMR spectroscopy to identify the structure and stability. The derivative (hapten) was coupled to BSA by a nucleophilic-substitution. The UV spectrometry and molecular weight of immunogenic conjugate and BSA were compared by an UV spectrophotography and a SDS-page electrophoresis to determine whether the diazepam had been coupled to BSA and the number of diazepam molecule coupled to a BSA molecule. (2) Production of
polyclonal antibody specific for diazepam The rabbits were injected at 25 subcutaneous sites near to the spine with a mixture of the immunogen and complete Freund's adjuvant on the first day. On the 21st, 28th, 35th, 42nd, 49th, and 56th days, the rabbits were injected respectively with a mixture of the immunogen and incomplete Freund's adjuvant described as former. 60th day after the first injection, the rabbit serum was obtained. The collected antisera was absorbed with BSA, OD value of antisera in four groups (Blank; Coated with BSA + Antisera absorbed with BSA; Coated with Diazepam-BSA + Antisera absorbed with BSA; Coated with Diazepam-BSA + Antisera no absorbed with BSA) was assayed by ELISA to determine the specificity. Dilute the absorbed antisera to 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, then measured their OD value to decide their titer. (3) Detection of diazepam in brain by an immunohistochemistry assay After having been poisoned with the powder of diazepam tablets, Wistar's rats were Killed , the brain was taken and fixed with 4% formaldehyde. By using antisera absorbed with BSA as the first antibody, the section of fixed brain was stained by a SABC immunohistochemistry assay.
Results (1) Using glutaric anhydirde and 1,3-2 chloropropane, two derivatives of diazepam in 3 position of the benzodiazepine ring was synthesized. TLC, Infrared Spectrophotometry and NMR spectroscopy analysis showed diazepam-glutaric-acid derivative was easy to depose, and diazepam-1, 3-2 chloropropane derivative was stable. Compared with BSA, the maximum absorbed wave-length in UV spectrometry of diazepam BSA immunogenic conjugate was shifted to longer. SDS-page electrophoresis showed there was a
significant difference between the movement speeds of BSA and diazepam BSA immunogenic conjugate. (2) After rabbits were immunized with diazepam BSA immunogenic conjugate, antisera specific for diazepam was obtained. The antibody specificity test showed: There was no significant difference between the OD values of control and test control groups; OD values of test and positive test groups were significantly higher than that of control and test control groups. The OD value of positive test group was significantly higher than that of test group. Compared with test control groups, the OD values of test group was significantly higher. The antisera titer was 1 :2560. (3) The SABC immunohistochemistry staining showed there were brown particles on cell membrane and nuclear membrane of nerve cells in brain of diazepam poisoned rats, but not in control groups.
Conclusion Using 1,3-2 chloropropane as the coupled reagent, we established a technique way for the synthesis of diazepam hapten and complete antigen. The diazepam complete antigen synthesiz
方法 (1)安定抗原性BSA结合物的制备 利用有机合成方法分别采用戊二酸酐和1，3-二氯丙烷在安定的3位碳原子上连接可以与蛋白质相结合的5-羰基戊酸基或1-氯丙烷基，合成安定衍生物。薄层层析和柱层析分离合成产物，薄层色谱扫描法、傅立叶红外分光光度分析和超导核磁共振分析鉴定衍生物结构和稳定性；根据胺的制备原理将安定卤代烃衍生物与载
山西反科大学
20仍年硕士研究生毕业论文
体蛋白BSA上天冬酞胺(AsparagineN)、谷氨酞胺(Glutamine
Q)、精氨酸(ArginineR)和赖氨酸(LysineK)的游离氨基发
生亲核取代反应，制备出安定抗原性BSA结合物。紫外分光光
度法和SDS一聚丙烯酞胺凝胶电泳的方法比较安定抗原性BSA
结合物和BSA的紫外吸收峰和分子量。(2)安定多克隆抗体的
制备背部皮内多点注射安定抗原性BSA结合物和F盯end’s完
全佐剂混合物初次免疫新西兰大白兔，第21、28、35、42、49、
56天安定抗原性BSA结合物和Furend’s不完全佐剂混合物同样
方式加强免疫，第60天采血，收集血清。BSA吸收后，采用
ELISA法比较空白组、实验对照组、实验组和阳性对照组血清
的OD值，鉴定抗体的特异性;对半稀释法测定抗血清效价。(3)
脑组织中安定的免疫组织化学测定安定片粉末灌胃，复制
Wistar大鼠安定中毒模型，取大脑福尔马林固定，石蜡包埋
切片。以BSA吸收的多克隆抗血清为一抗进行SABC免疫组织
化学染色，分析。
结果(1)利用安定和戊二酸配、1，3一二氯丙烷均合成得
到安定3位5一拨基戊酸基和1一氯丙烷基取代的衍生物。傅立叶
红外和超导核磁共振分析证明其分子结构与设计一致。常温或
暴露于光线下时，戊二酸醉衍生物容易分解，卤代烃衍生物不
山西民料大学
2003年硕士研究生毕业论文
分解。安定抗原性BSA结合物的紫外吸收特征峰较BSA发生红
移、SDS一聚丙烯酞胺凝胶电泳的迁移速率与BSA差异明显。
(2)安定抗原性BSA结合物免疫新西兰大白兔，得到抗安定血
清。抗体特异性测定显示，空白组和实验对照组的OD值间无
显著性差异，与其相比，实验组和阳性对照组OD值均显著升
高;与实验组相比，阳性对照组OD值显著升高;与实验对照
组相比，实验组OD值显著升高。抗血清的效价达到1:2560。
(3)SABC染色，安定中毒大鼠大脑组织细胞膜和核膜上可见
褐色颗粒沉着，对照组未见褐色颗粒沉着。
结论采用卤代烃作为桥连剂，合成安定的卤代烃衍生
物，进一步交联于载体蛋白的技术路线适用于安定完全抗原的
合成;本实验合成的安定完全抗原可以刺激机体产生抗安定抗
体，但抗体特异性和血清效价有待于进一步提高，抗体类别有
待于进一步鉴定;本实验所建立的ELISA法为安定单克隆抗体
制备提供了可靠的筛选方法;免疫组织化学法可应用于富尔马
林固定检材内安定的定性检验。
Objective (1) To compare two synthesis of
diazepam-derivative, build a new path for the synthesis of diazepam BSA immunogenic conjugate, and provide a new technique for the synthesis of similar micro-molecular antigen. (2) To obtain polyclonal antibody specific for diazepam, detect the antibody specificity and serum titer by ELISA, and establish a screen method for monoclonal antibody study (3) To detect the diazepam in diazepam poisoned rat brain by an immunohistochemistry, and provide a new quanlitative analysis in the brain fixed with 4% formaldehyde.
Method (1) Production of diazepam BSA immunogenic conjugate Diazepam was derivatized in position 3 of the benzodiazepine ring with glutaric anhydirde and 1,3-2 chloropropane. The compound was isolated and purified by TLC and column chromatography, and assayed by TLC, Infrared Spectrophotometry and NMR spectroscopy to identify the structure and stability. The derivative (hapten) was coupled to BSA by a nucleophilic-substitution. The UV spectrometry and molecular weight of immunogenic conjugate and BSA were compared by an UV spectrophotography and a SDS-page electrophoresis to determine whether the diazepam had been coupled to BSA and the number of diazepam molecule coupled to a BSA molecule. (2) Production of
polyclonal antibody specific for diazepam The rabbits were injected at 25 subcutaneous sites near to the spine with a mixture of the immunogen and complete Freund's adjuvant on the first day. On the 21st, 28th, 35th, 42nd, 49th, and 56th days, the rabbits were injected respectively with a mixture of the immunogen and incomplete Freund's adjuvant described as former. 60th day after the first injection, the rabbit serum was obtained. The collected antisera was absorbed with BSA, OD value of antisera in four groups (Blank; Coated with BSA + Antisera absorbed with BSA; Coated with Diazepam-BSA + Antisera absorbed with BSA; Coated with Diazepam-BSA + Antisera no absorbed with BSA) was assayed by ELISA to determine the specificity. Dilute the absorbed antisera to 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, then measured their OD value to decide their titer. (3) Detection of diazepam in brain by an immunohistochemistry assay After having been poisoned with the powder of diazepam tablets, Wistar's rats were Killed , the brain was taken and fixed with 4% formaldehyde. By using antisera absorbed with BSA as the first antibody, the section of fixed brain was stained by a SABC immunohistochemistry assay.
Results (1) Using glutaric anhydirde and 1,3-2 chloropropane, two derivatives of diazepam in 3 position of the benzodiazepine ring was synthesized. TLC, Infrared Spectrophotometry and NMR spectroscopy analysis showed diazepam-glutaric-acid derivative was easy to depose, and diazepam-1, 3-2 chloropropane derivative was stable. Compared with BSA, the maximum absorbed wave-length in UV spectrometry of diazepam BSA immunogenic conjugate was shifted to longer. SDS-page electrophoresis showed there was a
significant difference between the movement speeds of BSA and diazepam BSA immunogenic conjugate. (2) After rabbits were immunized with diazepam BSA immunogenic conjugate, antisera specific for diazepam was obtained. The antibody specificity test showed: There was no significant difference between the OD values of control and test control groups; OD values of test and positive test groups were significantly higher than that of control and test control groups. The OD value of positive test group was significantly higher than that of test group. Compared with test control groups, the OD values of test group was significantly higher. The antisera titer was 1 :2560. (3) The SABC immunohistochemistry staining showed there were brown particles on cell membrane and nuclear membrane of nerve cells in brain of diazepam poisoned rats, but not in control groups.
Conclusion Using 1,3-2 chloropropane as the coupled reagent, we established a technique way for the synthesis of diazepam hapten and complete antigen. The diazepam complete antigen synthesiz
引文
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