Abstract
BackgroundPhage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide.ResultsHere we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv) library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA.ConclusionThese results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.
Highlights
Phage display technology is a powerful new tool for making antibodies outside the immune system, avoiding the use of experimental animals
In the past two decade, advances in phage display and antibody engineering have led to the development of phage-displayed antibody technology [10,11]
Since the method does not depend on an animal's immune system, antibodies to a wide variety of antigens, including the molecules that cannot stimulate immune system of the animals such as nonimmunogenic, "self", cell surface or toxic antigens, can be generated [16,17,18]
Summary
Phage display technology is a powerful new tool for making antibodies outside the immune system, avoiding the use of experimental animals. It was postulated that this technique would eventually replace hybridoma technology and animal immunisations Since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. In the past two decade, advances in phage display and antibody engineering have led to the development of phage-displayed antibody technology [10,11] This technology allows one to isolate antibodies directly from diverse repertoires of antibody genes, generating highaffinity binding sites without the constraint imposed by classical method for generating either polyclonal or monoclonal antibody [12,13,14,15,16]. One reason may be because of the complexity and cost of generation of the library, even though there have been some reports describing optimized protocols for the generation of efficient libraries [32,33]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
