Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin.
Coeliac 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-specific, phage-displayed scFv libraries from peripheral blood lymphocytes of a patient with CD and from a healthy control individual. VH and VL chains were amplified 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 affinity 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-specific, as shown by strongly positive enzyme-linked immunosorbent assay (ELISA) and BiaCore signals. The VH and VL chains from samples of these monoclonal isotype-specific phage were sequenced to identify the most common variable regions used by the immune system to elicit antibody responses against gliadin.
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D002446:Celiac Disease / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005903:Gliadin / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011786:Quality Control; D020349:Surface Plasmon Resonance
D007128:Immunoglobulin Fragments; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D005903:Gliadin