Development of a Method for Pb Functionalization and Preparing Immunogenic Complex

Antibody  based  methods  have  numerous  advantages  compared  to  other  detection  methods. However, heavy metals cannot stimulate the immune antibody response; it is the main obstacle for preparation of the antibodies that used in detection methods of the metals. The production of the immunogenic Pb complexes by using cost benefit linkers of EDTA and DTPA were investigated in this  study.BSA  molecules  were  functionalized  using  EDTA  and  DTPA  linkers  in  various BSA/linker ratio, pH, incubation times and buffers. The complexes were formed after addition of the  Pb  to  the  [BSA-linker].  Different  concentrations  of  the  glutaraldehyde  were  mixed  with  the formed  complexes  and  shaken  at  room  temperature  for  12  hours.  The  prepared  conjugates  were dialyzed in phosphate buffer saline for 72 hours. Stimulation of the mice antibody responses against the  prepared  [Pb-linker-BSA]  complex  were  evaluated  using  ELISA.  In  optimized  condition,  the DTPA linker coupled Pb to each BSA molecules 2 times higher than EDTA. The optimum pH was 9.6 in EDTA binding to BSA; also, EDTA molarity was 25 times higher than Pb molarity in [PbEDTA-BSA] complexes.These conditions for DTPA conjugation to Pb and BSA are pH 7.4-9.6 and 9.6 respectively. Also, optimum molarity of DTPA was 4 times higher than Pb in [Pb-DTPABSA] complexes. Also, the higher antibody responses against Pb were stimulated by immunization with an antigenic complex that has more solubility and Pb coupled to each albumin molecules. In conclusion,  in  optimized  conditions,  DTPA more  efficient  than  EDTA  for  synthesizes  of  the immunogenic Pb complex.