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Screening and Preliminary Evaluation of Anti-CEA Antibody from a Human Phage Antibody Library

Author: ChenYongFeng
Tutor: YangHePing
School: Third Military Medical University
Course: Internal Medicine
Keywords: Carcinoembryonic antigen Phage antibody Lung cancer
CLC: R73-3
Type: Master's thesis
Year: 2002
Downloads: 81
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Abstract

CEA (carcinoembryonic antigene, CEA) is a widely used clinical tumor marker, and mouse monoclonal anti-CEA monoclonal antibody has been widely used in the diagnosis of colorectal cancer, lung cancer, breast cancer recurrence monitoring, prognosis and guide therapy and imaging studies. However, the murine antibody heterogeneity severely limits the application of antibodies directed therapy and radiology imaging. To this end, a humanized antibody molecule transformation or direct preparation of human-derived monoclonal antibody increasingly important. At present, the developed human anti-CEA monoclonal antibody reported yet rarely. Phage display technology is currently the most common antibody library technology. It bypasses the technical difficulties of the hybridoma, and to simulate the mechanism of antibody diversity, human lymphocytes in the Fd and L gene fragments were cloned and amplified by RT-PCR technology, and randomly combined into the expression vector was constructed The human antibody library of a large capacity, while using phage display technology features of the unity of the genotype and phenotype of the specific molecule can be, i.e. the particular protein is expressed on the phage surface, containing the protein in the phage DNA bound corresponding to the structural gene, so that the direct use of the antigen through multiple adsorption - elution - amplification \human-derived monoclonal antibody. The present study was conducted in the Get immunoglobulin gene from a hilar lymph nodes of patients with lung cancer and constructed on the basis of human-derived phage antibody library, explore purified CEA antigen from the phage antibody library filter out the feasibility of anti-CEA monoclonal antibody. The study includes: 1. CEA purified human phage antibody library for three affinity enrichment, and two non-specific adsorption of normal human peripheral blood lymphocytes. CEA concentration gradually decreasing in the screening process, and continue to improve antibody affinity antibody library. 2. Total phagemid extract phage antibody library screening, after Nhe Ⅰ, Spe Ⅰ double digestion, electrophoresis separation and removal g Ⅲ fragment, T ₄ DNA connection ligase was transformed into XL ₁-Blue E. coli, decking obtain the expression of soluble anti-CEA monoclonal Fab fragments. 3 randomly picked the 10 monoclonal respectively expressed, the bacterial supernatants containing soluble antibody Fab fragment ELISA assay. According to the ELISA results, pick a combination of the most active monoclonal to prepare bacterial protein and 12% SDS, a team of GE protein electrophoresis and WestemBlot identification. Extracted the cloning of phage grains were sequenced using the sequencing instrument of the ABIP side SM 377% automated sequencing nuclear Pu acid sequence GenBank ten EMBL DDBJ PDB gene pool of known genes and sequencing results on the Internet homology comparison. Results showed that the measured light, the heavy chain variable region gene sequences have high homology with the sequence of human-derived immunoglobulin genes. Obtained after screening endogenous anti-CEA phage antibody library for qualitative detection of 20 cases of patients with pleural effusion, pleural CEA, and test results with clinical classic test results compared. After five screening of the phage antibody libraries, phage antibody library of anti-CEA. Extract phagemid double digestion to remove the 9111 fragment, since even after transformed into XLI Blue E. coli, the expression of soluble anti-CEA Fab fragment of monoclonal, selected randomly of 10 monoclonal for ELLISA identified, found 9 significant CEA binding activity, suggesting that the anti-CEA phage antibody libraries have high sensitivity. Pick out wherein the binding activity of the highest clones 6, preparation of the bacterial protein SDS-team GE protein electrophoresis, the results found in the 25kD at an additional protein band, the calculated value of 1/2 consistent with the theory of the antibody Fab fragment, Weste Blot also visible a lightly stained bands, that this clone expressed the anti-CEA antibody Fab fragments. Extract the cloning phagemid genome sequencing, the sequencing results on Internet, GenBank EMBL ten DDBJ PDB gene library of known genes the nuclear operators acid sequence homology comparison, results showed that the measured light and heavy chain variable region the gene sequence with the human-derived immunoglobulin gene sequence homology (91%) homology are indicated that the expression of antibody fragments human endogenous antibody Fab fragment. Will receive a human anti-CEA phage antibody library for qualitative detection of 20 cases of patients with pleural effusion, pleural CEA, and test results with clinical classic test results compared. The results showed that 10 patients with classic method to detect CEA elevated in patients with lung cancer, of its pleural effusion ELISA test OD45. Value was significantly higher than that of the positive control; the of CEA the normal ELISA test OD45. Value was significantly lower than the positive control. The test results of the results obtained in this study endogenous anti-CEA phage antibody libraries to detect pleural CEA and clinical classic Vll / correlation analysis, results were significantly correlated (Factory 0.971). In summary, this study can draw the following conclusions: 1. Research from the phage antibody library screening a human anti-CEA antibody Fab fragment, for further preparation and purification of high affinity derived anti CEA monoclonal antibodies experimental basis, suggesting that the phage antibody library screening anti-CEA monoclonal antibody is feasible. By human anti-CEA phage antibody library preliminary qualitative detection of 20 cases of CEA clinical results suggest that human anti-CEA phage antibody library and monoclonal antibodies prepared by phage display technology has potential clinical value.

Screening and Preliminary Evaluation of Anti-CEA Antibody from a Human Phage Antibody Library

Abstract

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