viral neutralization or vaccine efficacy), often using cell-based assays. Antibody library generation is a vital first step in all phage display projects. The method consists of building a library (left panel) of peptide or protein variantsor in the case of antibodies, an antibody gene repertoireand affinity selecting (right panel) specific antibody-phage fusions via affinity with the target of interest. This qualitative selection process requires validation using more quantitative immunoassays to assess antibody-antigen interactions such as ELISA, immunofluorescence, HTRF, complement fixation, agglutination, and/or precipitation.įollowing the characterization of antibody-antigen interactions, candidate molecules are then screened for functional activity (e.g. Begin by enriching your population of phage with high-affinity binding by exposing the library to your antigen of choice and then eluting and amplifying only those with the highest binding affinity.īacteriophage selected from the previous step are then cloned and picked in order to isolate each unique protein binder.ĭuring panning, phages displaying proteins with higher binding affinity are selected in relation to phages displaying lower affinity proteins. Panning is an iterative process for enriching phage within a population that possess high affinity binding to a target of interest compared to others. Antigen-specific clones are enriched by binding to immobilized. Various antibody-displaying phage libraries have been described, which are based on the B cell repertoire of rearranged immunoglobulin genes from spleen and bone marrow of previously immunized mice (see Sect. HIGH-THROUGHPUT, HIGH CONTENT SCREENING Phage-displayed immunoprecipitation sequencing (PhIP-seq) has enabled high-throughput profiling of human antibody repertoires. Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Using this technique, antibody genes have been cloned from multiple species or expressed directly from large man-made repertoires of antibody-encoding genes.This robust and versatile technology allows the expression of an antibody fused to a phage coat protein on the surface of a filamentous phage. PROTEIN DETECTION, QUANTITATION, ANALYSIS Antibody phage display (APD) technology has revolutionized the field of immunovirology with its application in viral disease diagnostics and antiviral therapy.NUCLEIC ACID (DNA/RNA) DETECTION & ANALYSIS.
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