Ex Vivo Screening Introduction

Phage display library construction and phage library screening are important steps in phage display technology. Currently, there are many methods for phage library screening - in vivo screening, ex vivo screening, solid-phase screening, and protease substrate screening. Different phage display methods have their advantages and disadvantages. In specific experiments, the appropriate method can be selected according to the actual research needs and experimental program. Based on our rich practical experience and research platform, Creative Biolabs provides comprehensive services and technologies for ex vivo phage display.

Phage. (Creative Biolabs Authorized)

Ex Vivo Phage Display

The successful application of antibody library technology cannot be separated from antibody library screening technology. Overall screening of phage antibody libraries consists of biopanning and screening. That is, the strains carrying antibody genes that bind specifically to the antigen are first enriched from the library after several rounds of adsorption, biopanning, and amplification. Then the monoclones from the strains obtained in the previous step are selected for characterization of specificity and affinity with a view to obtaining highly specific monoclones. The whole screening process is time-consuming and laborious. In view of this, it is crucial to realize high-throughput screening of phage antibody libraries. Ex vivo screening is an effective screening method.

When the expression of the antigen is low and the immunogenicity is weak, it is difficult to achieve the purpose with the biopanning method of immobilized antigen. In addition, antigens encapsulated in solid-phase biopanning do not maintain their natural conformation and thus are not suitable for biopanning of membrane antigens or cell surface antigens. Problems caused by conformational changes can be avoided by biopanning intact cells expressing these antigens. In general, in addition to intact cells, cell membrane preparations and antigenic proteins on tissue sections maintain their natural conformation. Therefore, these materials can also be used as target materials for eluting cell surface antigens.

For adherent cells grown on cell culture plates, binding, washing, and biopanning of phage antibody libraries are similar to conventional solid phase biopanning. For sorting suspension cells, phage can be mixed with the cells to be eluted and incubated, and the phage-bound cells can be separated into the organic phase for recovery by low-speed or differential centrifugation. For labeled target cells, negatively selected cells can be added at the same time for biopanning, and the labeled target cells can be sorted out by flow cytometry (FACS) or magnetically activated cell separation (MACS). In the biopanning of phage Fab antibody library, the traditional plaque lift assay or the modified capture lift method can be used for biopanning and characterization. The biopanning method based on nitrocellulose membrane is simple and can be completed in one time without enrichment process, but it is limited by the density of clones on the plate, the resolution is 500 pfu per 100 mm dish. It is difficult to elute large-capacity antibody libraries by this method, and it is also impossible to control the rigor of the biopanning process. It has been reported in the literature that cell biopanning has been used to obtain phage antibodies that bind to cell surface receptors, but there are certain difficulties in cell biopanning, which are mainly manifested in the problems of large non-specificity, high background value, and the loss of specific ligands in the biopanning process. Due to the extremely complex composition of the cell membrane surface, with many protein, sugar, and lipid structures, the chances of non-specific phage antibodies binding to the cells are high, and the density of antigens on the membrane surface is very low, which makes antibody screening difficult and makes it hard to obtain single-chain antibodies targeting a certain antigen. Compared with solid-phase biopanning for antigen purification, cell biopanning is slower for enrichment, but rare specific phage antibodies are easily lost if too many biopanning rounds are performed. To address these problems, Sui et al. successfully screened CXCR4 antibody from a natural human antibody library by combining the Pathfinder and Step-Back methods. Nevertheless, improving the specificity and selectivity of cell biopanning is still an urgent problem to be solved.

Creative Biolabs has a wealth of knowledge and experience in antibody biopanning. We are happy to share our knowledge and experience in the fields of antibody biopanning and phage display library screening with you.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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