重金属铅单克隆抗体的制备及ELISA检测方法的建立
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2A3>1H7>2H1>1H4,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。
The potential hazards of lead on the health of human beings and animas have drawn increasing attention. Lead is one of the most toxic heavy metals of environmental pollutants which mainly damage nervous system. It can enter the bodies through the digestive tract and the respiratory tract of human beings and animals, and then causes serious damages to the nervous system,the hematopoietic system,the digestive system, the cardiovascular system and so on.Lead pollution of environment, food and feedstuffs is the principal way that causes damages to human beings and animals.Thus, monitoring the dose of lead in environment, food, feedstuffs and packaging materials is the effective way to control the absorbing of lead and prevent the lead damage.
Immunological detection of heavy metal ions is a new, efficient and rapid detection method. Compared with traditional methods the new method has many advantages, such as high detection speed, low cost, portability, high sensitivity, excellent frequency and so on.In addition, it can be used to the rapid detection and routine testing of heavy metal ions.Immunological detection has broad application prospects in fields of clinical medicine,animal quarantine,food and feed science,drug residues and other fields.
In this study,lead ions occur chelating reaction with bifunctional chelating agent p-SCN-CHX-A"-DTPA, and get chelate compound Pb2+-SCN-CHX-A"-DTP successfully.And then the chelate compound occurs coupling reaction with carrier proteins(KLH, BSA) respectively,for getting immunogenic antigen and defined antigen. At the same time,the bifunctional chelating agent p-SCN-CHX-A"-DTPA occurs coupling reaction with carrier protein BSA,for getting another defined antigen SCN-CHX-A"-DTPA-BSA.
Immuring 6 to 8 weeks old BALB/c female mice by injecting immunogenic antigen (Pb2+-SCN-CHX-A"-DTPA-KLH, 100μg per mouse) into abdominal cavity,for inducing fusion and filtering,and then detects the antibody secreting.
Spleen cells fuse with myeloma cells (Sp2/0).Then, constructing Pb2+ monoclonal antibody secreted hybridomas through screening and cloning, and detecting the subgroups of monoclonal antibody, the concentration of antibodies in induced ascites and the affinity constants of monoclonal antibodies.
The study chooses the hybridism 2A3 to create indirect competitive ELISA detection basing on the analysis of potency and affinity of antibodies. Determining the optimum reaction conditions of reactive system,and then constructing the standard testing curve.
Results are as follows:
(1) The study has composite the immunogenic antigen ((Pb2+-SCN-CHX-A"-KLH) and defined antigens (Pb2+-SCN-CHX-A"-DTPA-BSA, SCN-CHX-A"-DTPA-BSA) successfully.
(2) When the defined antigen is 400-fold diluted (the concentration of defined antigen is 2.5μg/mL),the reactive state is optimum. The potency of serum of immune mouse is above 1:16000, it shows that the immunogenic antigen induces the mouse to create immune response successfully.The mouse secrets antibodies.
(3) Spleen cells fuse with myeloma cells (Sp2/0). Obtaining four hybridomas totally which named 1H4、1H7、2H1、2A3 respectively. All the antibodies are kind of IgM, and the concentration of antibodies in induced ascites are 3.85mg/mL,4.21mg/mL, 3.98mg/mL,4.08mg/mL respectively.The order of affinity constants of antibodies are 2A3>1H2>2H1>1H4. and all above 108mol/L,the antibodies have high affinity. The 2A3 is the best hybridism because of its optimal affinity.
(4) The optimum reaction conditions of indirect competitive ELISA are as follows: the concentration of DTPA is 0.25mg/mL,the concentration of Pb2+ monoclonal antibody is 1.02μg/mL,the optimum blocking condition is blocking for 1.5 hours with rabbit serum (10%) at 37℃,the optimum reactive conditions of second antibody is 5000-fold diluted, responding for one hour at 37℃, the best response time of substrate is 15 minutes. The study has constructed the standard testing curve of lead ions,the regression equation is Y=0.4695 X+0.0391 (R2=0.9878),and the minimum detectable concentration of lead ions is 2.072 ng/mL.
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2A3>1H7>2H1>1H4,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。
The potential hazards of lead on the health of human beings and animas have drawn increasing attention. Lead is one of the most toxic heavy metals of environmental pollutants which mainly damage nervous system. It can enter the bodies through the digestive tract and the respiratory tract of human beings and animals, and then causes serious damages to the nervous system,the hematopoietic system,the digestive system, the cardiovascular system and so on.Lead pollution of environment, food and feedstuffs is the principal way that causes damages to human beings and animals.Thus, monitoring the dose of lead in environment, food, feedstuffs and packaging materials is the effective way to control the absorbing of lead and prevent the lead damage.
Immunological detection of heavy metal ions is a new, efficient and rapid detection method. Compared with traditional methods the new method has many advantages, such as high detection speed, low cost, portability, high sensitivity, excellent frequency and so on.In addition, it can be used to the rapid detection and routine testing of heavy metal ions.Immunological detection has broad application prospects in fields of clinical medicine,animal quarantine,food and feed science,drug residues and other fields.
In this study,lead ions occur chelating reaction with bifunctional chelating agent p-SCN-CHX-A"-DTPA, and get chelate compound Pb2+-SCN-CHX-A"-DTP successfully.And then the chelate compound occurs coupling reaction with carrier proteins(KLH, BSA) respectively,for getting immunogenic antigen and defined antigen. At the same time,the bifunctional chelating agent p-SCN-CHX-A"-DTPA occurs coupling reaction with carrier protein BSA,for getting another defined antigen SCN-CHX-A"-DTPA-BSA.
Immuring 6 to 8 weeks old BALB/c female mice by injecting immunogenic antigen (Pb2+-SCN-CHX-A"-DTPA-KLH, 100μg per mouse) into abdominal cavity,for inducing fusion and filtering,and then detects the antibody secreting.
Spleen cells fuse with myeloma cells (Sp2/0).Then, constructing Pb2+ monoclonal antibody secreted hybridomas through screening and cloning, and detecting the subgroups of monoclonal antibody, the concentration of antibodies in induced ascites and the affinity constants of monoclonal antibodies.
The study chooses the hybridism 2A3 to create indirect competitive ELISA detection basing on the analysis of potency and affinity of antibodies. Determining the optimum reaction conditions of reactive system,and then constructing the standard testing curve.
Results are as follows:
(1) The study has composite the immunogenic antigen ((Pb2+-SCN-CHX-A"-KLH) and defined antigens (Pb2+-SCN-CHX-A"-DTPA-BSA, SCN-CHX-A"-DTPA-BSA) successfully.
(2) When the defined antigen is 400-fold diluted (the concentration of defined antigen is 2.5μg/mL),the reactive state is optimum. The potency of serum of immune mouse is above 1:16000, it shows that the immunogenic antigen induces the mouse to create immune response successfully.The mouse secrets antibodies.
(3) Spleen cells fuse with myeloma cells (Sp2/0). Obtaining four hybridomas totally which named 1H4、1H7、2H1、2A3 respectively. All the antibodies are kind of IgM, and the concentration of antibodies in induced ascites are 3.85mg/mL,4.21mg/mL, 3.98mg/mL,4.08mg/mL respectively.The order of affinity constants of antibodies are 2A3>1H2>2H1>1H4. and all above 108mol/L,the antibodies have high affinity. The 2A3 is the best hybridism because of its optimal affinity.
(4) The optimum reaction conditions of indirect competitive ELISA are as follows: the concentration of DTPA is 0.25mg/mL,the concentration of Pb2+ monoclonal antibody is 1.02μg/mL,the optimum blocking condition is blocking for 1.5 hours with rabbit serum (10%) at 37℃,the optimum reactive conditions of second antibody is 5000-fold diluted, responding for one hour at 37℃, the best response time of substrate is 15 minutes. The study has constructed the standard testing curve of lead ions,the regression equation is Y=0.4695 X+0.0391 (R2=0.9878),and the minimum detectable concentration of lead ions is 2.072 ng/mL.
引文
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