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Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Published on: September 25, 2011
Expression of a prototypic anti-colorectal cancer polyclonal antibody library in mammalian cells
Liyan Chen1, Meredith A Liebman, Sanda Teodorescu-Frumosu
1Department of Pathology and Laboratory Medicine, Boston University, School of Medicine, Boston, MA 02118, USA.
We describe the production of a prototypic polyclonal antibody library (PCAL), a standardized mixture of full-length IgG polyclonal antibodies for which the genes are available. The PCAL was generated by mass transfer of heavy and light chain variable region gene pairs, selected for binding to human colorectal cancer cells, from a Fab phage display vector to a mammalian IgG expression vector. Following transfection of the IgG vector library into Sp2/0 myeloma cells, clones were characterized for IgG expression and binding to the colorectal cancer cells by ELISA, and for diversity by DNA fingerprinting, nucleotide sequencing, and immunoblot analysis. The results showed that 76-84% of the library clones produce IgG and of those 72-79% bind antigen. Furthermore, preliminary analysis showed clonal diversity at both the DNA and antigen-binding levels. When depleted of reactivity to normal tissue, polyclonal antibody libraries to cancer cells may be efficacious for cancer therapy.
We describe the production of a prototypic polyclonal antibody library (PCAL), a standardized mixture of full-length IgG polyclonal antibodies for which the genes are available. The PCAL was generated by mass transfer of heavy and light chain variable region gene pairs, selected for binding to human colorectal cancer cells, from a Fab phage display vector to a mammalian IgG expression vector. Following transfection of the IgG vector library into Sp2/0 myeloma cells, clones were characterized for IgG expression and binding to the colorectal cancer cells by ELISA, and for diversity by DNA fingerprinting, nucleotide sequencing, and immunoblot analysis. The results showed that 76-84% of the library clones produce IgG and of those 72-79% bind antigen. Furthermore, preliminary analysis showed clonal diversity at both the DNA and antigen-binding levels. When depleted of reactivity to normal tissue, polyclonal antibody libraries to cancer cells may be efficacious for cancer therapy.
