Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype‐specific scFv against gliadin

Abstract

SUMMARYCoeliac disease (CD), a gastrointestinal illness characterized by intestinal malabsorption, results from gluten intolerance accompanied with immunological responses towards gliadin, an ethanol-soluble protein fraction of wheat and other cereals. The role of gliadin in eliciting immune responses in CD is still partly unclear; however, the occurrence of anti-gliadin in the sera of patients suffering from CD correlates well with clinical symptoms. In this work we report the construction of isotype-speci®c, phage-displayed scFv libraries from peripheral blood lymphocytes of a patient with CD and from a healthy control individual. V H and V L chains were ampli®ed by reverse transcription±polymerase chain reaction (RT±PCR) using a set of oligonucleotides recognizing all human variable gene families. The three scFv libraries (IgA, IgG and IgM) were selectively enriched for gliadin-binding phage. After four rounds of af®nity selection, polyclonal enrichment of gliadin-binding phage was observed in all libraries from the CD patient but in none from the healthy donor. Phagemid particles generated from single clones were demonstrated to be gliadin-speci®c, as shown by strongly positive enzymelinked immunosorbent assay (ELISA) and BiaCore signals. The V H and V L chains from samples of these monoclonal isotype-speci®c phage were sequenced to identify the most common variable regions used by the immune system to elicit antibody responses against gliadin.

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