| Objective:To generate anti-idiotypic antibodies against mouse B16 melanoma and characterize responses induced by these antibodies. Methods:Monoclonal antibodies against gp75 (Ab1) and monoclonal antibodies against B16 cells(Abl) were produced by B-lymphocyte hybridoma technology. Abl were further identified by ELISA, Western Blot, immunohistochemistry and immunocytochemistry technologies. F(ab')2 fragment from Abl was prepared through pepsin cleavage. For Ab2 production, mice were immunized with F(ab')2 fragment from Abl coupled with keyhole limpet hemocyanin(KLH). Then the immune serum was identified by ELISA. Results:Six hybridoma cell lines, which could stably secrete specific mAbs against gp75, were established. They all belong to the IgGl subtype. Six hybridoma cell lines, which could stably secrete specific mAbs against B16, were established. They all belong to the IgG2a subtype. Western Blot showed that the antigen recognized by McAb D, a monoclonal antibody anginst gp75, is 75KD. The antigen recognized by McAb 4, a monoclonal antibody anginst B16 cells, is 95KD. A competitive study showed that Ab2 block the binding of Abl to Ag. Immunohistochemistry and immunocytochemistry showed that antigens recognized by Ab1 were expressed on the cell membrane and in the cytoplasma of B16. Ab2 and Ab3 were detected in the serum of mice immunized with F(ab')2 fragment from Abl. The competition experiment showed that Ag could compete with Ab2 in binding with the Ab1. Conclusions:Abl can specially recognize its corresponding antigen and trigger the immune responses. The Abl obtained could be an important tool for the further study of anti-idiotypic antibody. |
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