Isolation of Human scFv Antibodies Specific for House Dust Mite Antigens from an Asthma Patient by Using a Phage Display Library

ABSTRACT

BACKGROUND: In order to characterize human antibodies with specificity for mite allergens at the molecular level, a scFv phage display library was constructed using peripheral blood mononuclear lymphocytes from an asthma patient allergic to mite as Ig gene sources. METHODS: Immunoglobulin VH and V gene fragments were obtained by polymerase chain reaction, and randomly combined in pCANTAB-5E vector. The resulting human scFv phage display library had 3 X 10(4) independent clones, and biopanning was performed with house dust mite extracts. RESULTS: Four scFv clones specific for house dust mite extract were isolated. Immunoblot assay showed that our clones reacted to 25 kDa and 50~60 kDa proteins with unknown identity in mite extracts. Sequence analysis indicated that two clones (b7 and c15) are identical, and all clones belong to human VH3 subgroup. On the other hand, light chain usage was different in that two clones (a2 and b7/c15) belonging to V kappa 4 subgroup, but a4 used V kappa 1 light chain gene. CONCLUSION: Our approach should facilitate provision of useful information on the antibody responses against allergens at the molecular level in humans.