Human recombinant antibody fragments specific for a rye-grass pollen allergen: Characterization and potential applications

Claudia De Lalla, Elena Tamborini, Renato Longhi, Eleonora Tresoldi, Marco Manoni, Antonio G. Siccardi, Paolo Arosio, Alessandro Sidoli

Research output: Contribution to journalArticle

Abstract

One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with K(d) = 2.63 x 10-9 M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.

Original languageEnglish
Pages (from-to)1049-1058
Number of pages10
JournalMolecular Immunology
Volume33
Issue number13
DOIs
Publication statusPublished - Sep 1996

Fingerprint

Lolium
Immunoglobulin Fragments
Pollen
Allergens
Immunoglobulin E
Immunoglobulin Fab Fragments
Bacteriophages
Antibodies
Serum
Epitope Mapping
Periplasm
Surface Plasmon Resonance
Histamine Release
Amino Acid Substitution
DNA Sequence Analysis
Epitopes
Hypersensitivity
Clone Cells
Escherichia coli
Amino Acids

Keywords

  • Allergen
  • Anti-Lol pII
  • Human Fab
  • Phage library

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

Human recombinant antibody fragments specific for a rye-grass pollen allergen : Characterization and potential applications. / De Lalla, Claudia; Tamborini, Elena; Longhi, Renato; Tresoldi, Eleonora; Manoni, Marco; Siccardi, Antonio G.; Arosio, Paolo; Sidoli, Alessandro.

In: Molecular Immunology, Vol. 33, No. 13, 09.1996, p. 1049-1058.

Research output: Contribution to journalArticle

De Lalla, Claudia ; Tamborini, Elena ; Longhi, Renato ; Tresoldi, Eleonora ; Manoni, Marco ; Siccardi, Antonio G. ; Arosio, Paolo ; Sidoli, Alessandro. / Human recombinant antibody fragments specific for a rye-grass pollen allergen : Characterization and potential applications. In: Molecular Immunology. 1996 ; Vol. 33, No. 13. pp. 1049-1058.
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