Generation Of A Naïve Human Scfv Antibody Library For The Production Of Human Monoclonal Antibodies By Using Phage Display Technology
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Date
2015-09
Authors
Lim, Bee Nar
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Abstract
Naive antibody libraries are able to overcome the limitation of traditional hybridoma method in producing monoclonal antibodies. Phage display biopanning is a more cost effective and less time consuming technology for antibody production. Construction of a naive antibody library with a large diversity is crucial to ensure successful enrichment of antibodies against various target antigens. A naïve scFv antibody library was constructed from 90 donors of different ethnic groups (Malay, Chinese and Indian) with an equal gender distribution. All possible antibody V-genes were amplified by conventional PCR method and cloned into a phagemid vector. Two different approaches were used to generate the antibody library, namely PCR assembly and two step cloning. Both antibody libraries constructed had an estimated size of 2x109. However, the PCR assembly method showed an insert rate of 67 % only while two step cloning reached 80 %. The antibody library was used to screen for monoclonal antibodies against three different types of antigens, ubiquitin, hemolysin E and HIV matrix protein p17 (MAp17). Selection of monoclonal antibody was carried out using two different techniques, semi-automated and conventional plate biopanning. Human monoclonal scFv antibody was successfully obtained for all the antigens. V-gene usage of heavy chain and light chain of all the antibody clones were studied. Heavy chain of antibody against ubiquitin and hemolysin E were made up from IGHV3 whereas the heavy chain for MAp17 was from IGHV1. Meanwhile the light chain for the antigen was from IGKV1, IGLV2 and IGLV1 respectively. The antibodies enriched were also evaluated in soluble form for binding activities. In addition, a novel method to increase antibody diversity was introduced utilizing lambda exonuclease to create single-stranded DNA (ssDNA) as template for mutagenesis. Two types of randomization were demonstrated using this method, chain shuffling and CDR3 random mutagenesis. The best condition for ssDNA generation was incubating 1 μg of DNA with 10 U lambda exonuclease at 37 oC for 30 mins. In conclusion, the diverse naïve scFv library generated was able to generate soluble monoclonal scFv antibodies against the target antigens.
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Medicine