| Subject | carcinoembryonic antigen; chimeric protein; enhanced green fluorescent protein; monoclonal antibody; single chain fragment variable antibody; animal experiment; binding affinity; cancer cell; enzyme linked immunosorbent assay; Escherichia coli; gene expression; human cell; in vitro study; mouse; polymerase chain reaction; Amino Acid Sequence; Base Sequence; Carcinoembryonic Antigen; Cloning, Molecular; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Green Fluorescent Proteins; Hybridomas; Immunotherapy; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Sequence Analysis, DNA; Single-Chain Antibodies; Spectrometry, Fluorescence; Tumor Markers, Biological; Escherichia coli |
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| Abstract | According to World Health Organization (WHO), cancer is a leading cause of death worldwide, accounting for 7.4 million deaths (around 13% of all deaths) in 2004. Monoclonal/recombinant antibodies, which specifically target clinical biomarkers of disease, have increasingly been applied as powerful tools in cancer imaging and therapy, a fact that is highlighted by some nine FDA-approved monoclonal antibodies (MAbs) or their immunoconjugates (as of December 2008) for use in cancer treatment. In this study, five monoclonal antibodies (MAbs) were generated and characterized against carcinoembryonic antigen (CEA), which is widely used clinically as both a blood and tissue tumor marker of epithelial malignancy. Variable domains (VH and VL) of one the stable MAbs with highest affinity were PCR-amplified and assembled as single-chain antibody fragment (scFv). Following the cloning and expression of scFv antibody fragments in Escherichia coli, the functional binding and specificity of the recombinant antibody were confirmed by ELISA. To develop a direct in vitro detection of CEA-positive cancer cells, scFv DNA was genetically fused to enhanced green fluorescent protein (EGFP) gene and expressed in bacteria. The chimeric fluorescent protein is able to specifically detect CEA-positive cell lines; no cross-reactivity was observed with a negative control cell line. This strategy will likely allow the establishment of a rapid, single-step detection assay of CEA, which is considered to be one of the best predictors of malignancy among all other tumor markers. © Copyright 2011, Mary Ann Liebert, Inc. |
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