Production of hybridoma cells and testing of monoclonal antibodies directed against interleukin-12

Abstract

Interleukin-12 is a cytokine that plays an important role is the regulation of the cell mediated immune responses. It is produced by macrophages and antigen presenting cells and causes the proliferation of natural killer cells and enhances their lytic ability. In order to further understand the effect of IL-12 on NK cells, a monoclonal antibody (a monogenous preparation of antibody molecules all exhibiting the same antigenic specificity) could be used to remove IL-12 activity. To prepare a monoclonal antibody against IL-12, a mouse was injected with interleukin-12 antigen. An enzyme-linked immunosorbant assay was used to measure antibody production. A hybridoma cell line was produced by the fusion of splenic B cells (antibody-producing cells) and P3X murine myeloma (cancer) cells. Fourteen variable hybridoma cultures E, F, G, H, I, J, K, L, M, N, O, P, Q, R, were the result of this fusion. After confirming that all fourteen cultures were secreting antibodies against interleukin-12, one culture (G) was selected and subcloned to create the monoclonal antibodies H3, H4, H9, G6, G7, F7, F8, E9, D9. Subcloning involved isolation of a cell that would continuously divide to create a clone of genetically identical cells. Further confirmation of continual antibody secretion was done followed by a second subcloning of F8 and D9. The project was ended when the second subclone of F8 and D9 no longer secreted antibodies against interleukin-12. the remainder of the project will include a repeat cloning of the hydridomas producing monoclonal antibodies to obtain a twice cloned monoclonal antibody against interleukin-12. Further work will be needed to determine the isotype and specificity of the monoclonal antibodies produced as part of this study.