Preparation Of Monoclonal Antibody Against Lead And Establishment Of ELISA Method

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.