Abstract
Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.
Highlights
Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure
To overcome the drawback associated with the need to transform bacteria, there have been developed several modifications of the method, that are based on selection in cell-free conditions, in particular, a ribosome display
This method is based on a coupled cell-free transcription and translation system, where complexes consisting of transcripts, ribosomes, and growing protein chains corresponding to the repertoire of antigen-binding regions of antibodies are subjected to affinity selection on the immobilized antigen
Summary
Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. One of the drawbacks of the method is the requirement to clone the repertoire of fragments encoding the antigen-binding fragments into a special phagemid and to obtain a pool of transformed cells to produce libraries of phage particles This stage is considered to be the bottleneck of the method, limiting the diversity of the resulting libraries. It can be assumed that by combining the methods of ribosome and phage display it will be possible to overcome the weak spots of both methods and to increase the overall efficiency of selection In this case, it would be possible to preliminarily select variants in a cell-free system, achieving enrichment with high-affinity variants, and clone the resulting repertoire of fragments into a phagemid and carry out the final selection using phage display
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