There is a continuing, intense demand for the production and selection of specific antibodies. As a cost-efficiency and time-saving approach, in vitro display provides alternative to conventional hybridomas. Creative Biolabs provides in vitro display service to meet the demands of producing and selecting antibodies.
In vitro display represents a series of emerging and innovative technologies, which can rapidly isolate and evolve the specific antibodies from large combinatorial libraries for medical applications. This technique links genotype (DNA or RNA) with phenotype (antibody fragment) to screen specific combining sites from a large library. Compared with in vivo process, in vitro display technologies have incomparable advantages. Firstly, recombinant technology has the potential to create and screen novel sequences, expanding the DNA diversity which is inaccessible by in vivo methods. In addition, since selection takes place under in vitro conditions, it does not restrict to in vivo biological constraints, with affinities as well as other desirable qualities beyond the in vivo ceiling.
Creative Biolabs has established a variety of in vitro display technologies for antibody screening, including phage display, bacterial display, yeast display, and ribosome display.
Figure 1 Diagram showing linkage of genotype and phenotype in four display technologies. scAB: single-chain antibody fragment carrying the antibody-combining site. (a) Phage display, (b) cell surface display, (c) ribosome display, and (d) mRNA display. (He and Taussig 2014)
Phage Display
Phage display is one of the most powerful and widely used in vitro display techniques for the study of protein-protein, protein-peptide, and protein-DNA interactions. Target gene encoding the interest protein is inserted into bacteriophage coat protein gene and then displayed on the phage surfaces. By displaying the interested antibodies or antibody fragments on the surface of employing phage, we can select high-affinity antibodies from millions or even billions of displayed phages. Creative Biolabs can provide the most appropriate phage display system (M13, T4 or T7) according to different properties and advantages.
Yeast Display
Yeast display is a novel technology for discovery and affinity maturation of fragment antibody, i.e. scFv and Fab, through the screening of an antibody library displayed on yeast surface. Libraries of antibody fragments are displayed on the surface of yeast for screening coupled with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques. Yeast is superior to phage for human antibody display as the eukaryotic expression environment of yeast cells is more suitable for human protein folding, modification, and translocation for display.
Bacterial Display
Bacterial display (or bacteria display or bacterial surface display) is a protein engineering technique which displays libraries containing billions of diverse molecules polypeptides (like scFv or Fab) on the surface of bacteria and, subsequently, to identify desired polypeptides using selection or high-throughput screening methods. The bacterial display is often coupled with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques which allow real-time analysis of displayed peptide properties, including target-binding affinity, specificity, and peptide stability to proteases during screening.
Ribosome Display
Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. Ribosome display couples an individual nascent protein to its corresponding mRNA through the formation of stable protein-ribosome-mRNA (PRM) complexes. In this system, the ribosome itself serves as the connector and PRMs are exposed to immobilized target molecules. From those target-binding complexes, the mRNAs are isolated, reverse transcribed and amplified as DNA for further manipulation and protein expression. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA. Ribosome display can display very large libraries, suitable for generating toxic, proteolytically sensitive and unstable proteins, and also allows amino acids modification at defined position.
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Reference
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