重金属铅单克隆抗体的制备及ELISA检测方法的建立
发布时间：2019-06-09 17:19
【摘要】： 铅对人和动物健康的潜在危害,已日益引起人们的关注。铅是环境污染物中毒性很大并且以神经毒性为主的一种重金属元素,其可通过消化道和呼吸道进入人和动物体内,对神经、造血、消化、心血管等系统和肾脏造成损害。环境铅污染和食品、饲料铅污染是造成人和动物铅损伤的主要途径,因此,监测环境,食品、饲料原料和食品、饲料包装材料及食品、饲料中铅的含量,是控制人和动物铅摄入量,预防和减少铅对人和动物危害的重要措施。 重金属离子的免疫学检测方法是一种新型、高效、快速的检测方法。与传统检测方法相比该方法具有检测速度快、费用低廉、简单易携、灵敏度高和选择性的特点。且可以用于重金属离子的快速检测和常规检测,在临床医学、动物检疫、食品饲料科学、药物残留等领域都有着很广泛的应用前景。 本研究采用重金属铅离子与双功能螯合剂ρ-SCN-CHX-A"-DTPA进行螯合反应,制得Pb2+-SCN-CHX-A"-DTPA螯合物,再将Pb2+-SCN-CHX-A"-DTPA螯合物分别与载体蛋白KLH、BSA偶联,制备免疫抗原和针对Pb2+单克隆抗体的检测抗原。再使用双功能螯合剂p-SCN-CHX-A"-DTPA与载体蛋白BSA偶联,制备检测螯合剂的检测抗原。 选取6～8周龄BALB/c雌性小鼠4只,腹腔注射合成的免疫抗原Pb2+-SCN-CHX-A"-DTPA-KLH,100μg/只,诱导小鼠机体产生对免疫抗原的免疫应答,检测其所分泌的抗体。 取免疫小鼠脾细胞与骨髓瘤细胞(Sp2/0)进行融合、筛选及克隆,获得分泌抗Pb2+单抗的杂交瘤细胞株,并对细胞株所分泌的抗Pb2+单抗的亚类、细胞株诱生的腹水单抗的浓度及单抗亲和常数进行测定。 在对单抗的效价、亲和力等进行分析的基础上,选择单抗2A3用于建立间接竞争ELISA检测方法,确定反应系统最佳工作条件,并建立标准检测曲线。 结果： (1)成功制备了的免疫抗原pb2+-SCN-CHX-A"-DTPA-KLH和检测抗原pb2+-SCN-CHX-A"-DTPA-BSA以及针对螯合剂的检测抗原SCN-CHX-A"-DTPA-B SA。(2)检测抗原作1：400稀释(即检测抗原浓度为2.5μg/mL)时反应效果最佳,免疫小鼠血清效价均达1：16000以上。结果表明合成的免疫抗原Pb2+-SCN-CHX-A"-DTPA-KLH成功诱导小鼠机体产生了对免疫抗原的免疫应答,并分泌针对免疫抗原的特异性抗体。 (3)免疫小鼠脾细胞与骨髓瘤细胞(Sp2/0)进行融合、筛选及克隆,共得四株抗Pb2+杂交瘤细胞株,分别命名为IH4、1H7、2H1、2A3。四株抗Pb2+杂交瘤细胞所分泌的抗体均为IgM亚类。四株杂交瘤细胞所诱生的小鼠腹水抗体浓度分别为3.85mg/mL,4.21mg/mL,3.98mg/mL,4.08mg/mL,单抗亲和常数2A31H72H11H4,且均在108mol/L以上,单抗具有较高的亲和力。由于2A3单抗亲和常数最大,因此抗Pb2+的杂交瘤细胞株2A3最佳。 (4)建立的间接竞争ELISA最佳工作条件为：螯合剂DTPA的最佳工作浓度为4 mM。抗原Pb2+-SCN-CHX-A"-DTPA-BSA (1mg/mL)最佳工作浓度为0.25mg/mL;亢铅单抗最佳工作浓度为1.02μg/mL;最佳封闭条件为10%兔血清37℃封闭1.5h；二抗最佳工作条件为5000倍稀释(0.16μg/mL),37℃作用1h；底物最佳作用时间为15min。在间接竞争ELISA最佳工作条件下,建立了铅离子标准检测曲线,其回归方程为Y=0.4695X+0.0391,相关系数R2=0.9878,铅离子最低检出浓度为2.072ng/mL。
[Abstract]:The potential harm of lead to human and animal health has attracted more and more attention. Lead is a heavy metal element which is toxic in environmental pollutants and is mainly neurotoxic, which can enter human and animal body through digestive tract and respiratory tract, and can cause damage to nervous, hemopoietic, digestive, cardiovascular system and kidney. lead pollution in the environment, food, feed and lead pollution is the main way to lead to human and animal lead damage, therefore, the content of lead in the monitoring environment, the food, the feed raw materials and the food, the feed packaging material and the food and the feed is the lead intake of the control person and the animal, Important measures to prevent and reduce lead to human and animal hazards. The immunological detection of heavy metal ions is a new, efficient and rapid test. Compared with the traditional detection method, the method has the advantages of high detection speed, low cost, simple and easy carrying, high sensitivity and selectivity, and can be used for rapid detection and routine detection of heavy metal ions, and has wide application in the fields of clinical medicine, animal quarantine, food feed science, drug residue and the like The preparation method of Pb2 +-SCN-CHX-A-DTPA conjugate was carried out by using the heavy metal lead ion and the double-functional iron-binding agent to prepare the Pb2 +-SCN-CHX-A "-DTPA conjugate. The Pb2 +-SCN-CHX-A-DTPA conjugate was then coupled with the carrier protein KLH and BSA respectively to prepare the immune antigen and the detection antigen for the Pb2 + monoclonal antibody. The immune antigen Pb2 +-SCN-CHX-A "-DTPA-KLH,100. m A. the antibody secreted by the cell strain is detected, and the spleen cells of the immunized mouse are fused, screened and cloned in the myeloma cell (Sp2/0) to obtain the hybridoma cell strain which secretes the anti-Pb2 + monoclonal antibody, and the subclass of the anti-Pb2 + monoclonal antibody secreted by the cell line and the ascites sheet induced by the cell strain The concentration of the antibody and the affinity constant of the monoclonal antibody were determined. On the basis of the analysis of the titer and the affinity of the monoclonal antibody, the monoclonal antibody 2A3 was selected to establish the indirect competitive ELISA detection method and the reverse antibody was determined. The system shall be Results: (1) The successfully prepared immune antigen pb2 +-SCN-CHX-A "-DTPA-KLH and test antigen pb2 +-SCN-CHX-A"-DTPA-BSA and the detection antigen SCN-CHX-A "-DTPA-B SA. (2) When the antigen is diluted 1:400 (i.e., the concentration of the test antigen is 2.5. mu.g/ mL), the effect of the reaction is the best, and the serum titer of the immunized mice is 1:16000 or more. The result shows that the synthesized immune antigen Pb2 +-SCN-CHX-A"-DTPA-KLH for the binding agent successfully induced the production of the mouse body (3) The spleen cells of the immunized mice and the myeloma cells (Sp2/0) were fused, screened and cloned to obtain four strains. Pb2 + hybridoma cell strain, named IH4, 1H7, 2H, respectively 1, 2A3. The anti-Pb2 + hybridoma cells of the four hybridoma cells were all IgM subclasses. The concentration of the mouse ascites antibody induced by the four hybridoma cells was 3.85 mg/ mL, 4.21 mg/ mL, 3.98 mg/ mL, 4.08 mg/ mL, and the monoclonal antibody affinity constant of 2 A31H7 2H11H4, and all above 108 mol/ L, and the monoclonal antibody has a high affinity. The affinity constant of the 2A3 monoclonal antibody is the largest, so the anti-Pb2 + hybridoma cell line 2A3 is the best. The optimal working conditions of indirect competitive ELISA were as follows: the optimal working concentration of the mixture of DTPA was 4 mM, the best working concentration of the antigen Pb2 +-SCN-CHX-A "-DTPA-BSA (1 mg/ mL) was 0.25 mg/ mL, the best working concentration of the high-activity lead was 1.02. m The optimum working time of the substrate was 15 min. Under the optimum working condition of indirect competitive ELISA, the standard detection curve of lead ion was established. The regression equation was Y = 0.469X + 0.0391.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：S854.4;R392
本文编号：2495737
[Abstract]:The potential harm of lead to human and animal health has attracted more and more attention. Lead is a heavy metal element which is toxic in environmental pollutants and is mainly neurotoxic, which can enter human and animal body through digestive tract and respiratory tract, and can cause damage to nervous, hemopoietic, digestive, cardiovascular system and kidney. lead pollution in the environment, food, feed and lead pollution is the main way to lead to human and animal lead damage, therefore, the content of lead in the monitoring environment, the food, the feed raw materials and the food, the feed packaging material and the food and the feed is the lead intake of the control person and the animal, Important measures to prevent and reduce lead to human and animal hazards. The immunological detection of heavy metal ions is a new, efficient and rapid test. Compared with the traditional detection method, the method has the advantages of high detection speed, low cost, simple and easy carrying, high sensitivity and selectivity, and can be used for rapid detection and routine detection of heavy metal ions, and has wide application in the fields of clinical medicine, animal quarantine, food feed science, drug residue and the like The preparation method of Pb2 +-SCN-CHX-A-DTPA conjugate was carried out by using the heavy metal lead ion and the double-functional iron-binding agent to prepare the Pb2 +-SCN-CHX-A "-DTPA conjugate. The Pb2 +-SCN-CHX-A-DTPA conjugate was then coupled with the carrier protein KLH and BSA respectively to prepare the immune antigen and the detection antigen for the Pb2 + monoclonal antibody. The immune antigen Pb2 +-SCN-CHX-A "-DTPA-KLH,100. m A. the antibody secreted by the cell strain is detected, and the spleen cells of the immunized mouse are fused, screened and cloned in the myeloma cell (Sp2/0) to obtain the hybridoma cell strain which secretes the anti-Pb2 + monoclonal antibody, and the subclass of the anti-Pb2 + monoclonal antibody secreted by the cell line and the ascites sheet induced by the cell strain The concentration of the antibody and the affinity constant of the monoclonal antibody were determined. On the basis of the analysis of the titer and the affinity of the monoclonal antibody, the monoclonal antibody 2A3 was selected to establish the indirect competitive ELISA detection method and the reverse antibody was determined. The system shall be Results: (1) The successfully prepared immune antigen pb2 +-SCN-CHX-A "-DTPA-KLH and test antigen pb2 +-SCN-CHX-A"-DTPA-BSA and the detection antigen SCN-CHX-A "-DTPA-B SA. (2) When the antigen is diluted 1:400 (i.e., the concentration of the test antigen is 2.5. mu.g/ mL), the effect of the reaction is the best, and the serum titer of the immunized mice is 1:16000 or more. The result shows that the synthesized immune antigen Pb2 +-SCN-CHX-A"-DTPA-KLH for the binding agent successfully induced the production of the mouse body (3) The spleen cells of the immunized mice and the myeloma cells (Sp2/0) were fused, screened and cloned to obtain four strains. Pb2 + hybridoma cell strain, named IH4, 1H7, 2H, respectively 1, 2A3. The anti-Pb2 + hybridoma cells of the four hybridoma cells were all IgM subclasses. The concentration of the mouse ascites antibody induced by the four hybridoma cells was 3.85 mg/ mL, 4.21 mg/ mL, 3.98 mg/ mL, 4.08 mg/ mL, and the monoclonal antibody affinity constant of 2 A31H7 2H11H4, and all above 108 mol/ L, and the monoclonal antibody has a high affinity. The affinity constant of the 2A3 monoclonal antibody is the largest, so the anti-Pb2 + hybridoma cell line 2A3 is the best. The optimal working conditions of indirect competitive ELISA were as follows: the optimal working concentration of the mixture of DTPA was 4 mM, the best working concentration of the antigen Pb2 +-SCN-CHX-A "-DTPA-BSA (1 mg/ mL) was 0.25 mg/ mL, the best working concentration of the high-activity lead was 1.02. m The optimum working time of the substrate was 15 min. Under the optimum working condition of indirect competitive ELISA, the standard detection curve of lead ion was established. The regression equation was Y = 0.469X + 0.0391.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：S854.4;R392
【引证文献】
相关硕士学位论文 前2条
1 吴兵;斑点叉尾洶病毒单克隆抗体及血清免疫球蛋白多克隆抗体的制备及初步应用[D];华中农业大学;2011年
2 马燕玲;抗ω-芋螺毒素MⅦA单克隆抗体的制备与鉴定[D];福建农林大学;2013年
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