Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
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DOI：
10.1046/j.1365-2567.2003.01728.x
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发表时间：
2003-10-01
期刊：
影响因子：
6.4
通讯作者：
Crameri, R
中科院分区：
文献类型：
--
作者：
Rhyner, C;Weichel, M;Crameri, R
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. V-H and V-L 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 V-H and V-L 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.
