| Carcinoembryonic antigen (CEA) is one of tumor markers commonly used in clinics and its murine monoclonal antibodies have been widely applied in diagnosis, monitoring of recurrence, targeting therapy and image study of colorectal cancer, lung cancer and breast cancer. However, the heterogeneity of these murine monoclonal antibodies seriously restricts the application of these antibodies in antibody-targeting therapy and radioscintigraphy in vivo. Therefore, to make humanized antibodies or human antibodies is a challenge. Studies on the preparation of human anti-CEA antidobies have not been widely reported.Phage antibody library technique, a currently widely used antibody library technique, can be applied to construct large capacity of human antibody library by amplifing Fd and L gene fragments from the human lymphocytes. Based on the characteristic of the unification of genotype and phenotype of specific molecule by phage display technique, the genes of variable regions of specific human antibody can be effectively screened for the preparation of human monoclonal antibody by screening enrichment process of multiple 'adsorption-elution-amplification'. This study was conducted based on the phage antibody library constructed with immunogobulin gene successfully acquired from the local drainage lymphnodes of a patient with lung cancer to explore the feasibility of screening anti-CEA monoclonal antibody from thehuman phage antibody library. The study included:1. Three cycles of affinity enrichment of the human phage antibody library with purified CEA and 2 cycles of nonspecific absorption with human lymphocytes were performed. During the process of screening, concentrations of CEA were decreased gradually to increase the affinity of the antibodies in antibody library.2. The total phagemid of the screened phage antibody library were extracted and digested with NheI and SpeI to remove gIII fragment. Then, the phagemid DNA were electroporated into Escherichia coli XL1-Blue. The separated clones, which could express the soluble monoclonal antibody Fab fragments against CEA, were obtained.3. Ten strains that expressed soluble antibody Fab fragment were selected randomly. The supernatants of cell culture containing soluble antibody Fab fragment were evaluated by ELISA. One strain with the strongest activity was selected. The supernatant of this stain was applied to 12% SDS-PAGE and Western blot.4. Automatic gene sequencing was performed on the phagemid extracted from the strain mentioned above. The sequence was compared with database from GenBank+EMBL+DDBJ+PDB. The sequence in light and heavy chain variable regions was found to be high homology with human immunoglobulin gene sequence.5. Qualitative detection of CEA in the pleural fluid of 20 patients with hydrothorax was performed with the human anti-CEA antibody library acquired from screening and the results were compared with the typical methodcommonly applied in clinical practice.Anti-CEA antibody library was obtained after 5 cycles of screening of the human phage antibody library. The antibody library that express soluble antibodies was gained by removing gIII fragment from the phagemid. Nine of 10 monoclonal strains can bind to CEA. Clone 6 with the highest activity was selected for the preparation of protein for SDS-PAGE. A new protein band was found in a position of 25kD, which is relevant to a half of the theoretical molecular weight of antibody Fab fragment and a band with light staining could also be found by Western blot. The gene sequence in light and heavy chain variable regions was found to be high similar compared with that of human immunoglobulin gene (91% both). Qualitative detection of CEA in the pleural fluid of 20 patients with hydrothorax with our human anti-CEA phage antibody library acquired in the end was performed and the results were compared with the typical method commonly applied in clinical practice. The typical clinical examination and diagnostic results of the 20 patients with hydrotho...
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