Generation of human monoclonal allergen-specific IgE and IgG antibodies from synthetic antibody libraries.
BACKGROUND
Allergen-specific IgE and IgG antibodies play pivotal roles in the induction and progression of allergic hypersensitivity reactions. Consequently, monoclonal human IgE and IgG4 antibodies with defined specificity for allergens should be useful in allergy research and diagnostic tests. We used combinatorial antibody libraries and subsequent recombinant production to make and assess IgE, IgG1, and IgG4 allergen-specific antibodies.


METHODS
We used phage display to select a synthetic single-chain antibody fragment (scFv) library against 3 different allergens, from bee venom, bovine milk, and apple. The scFv obtained were converted into IgG1, IgG4, and IgE antibody formats and assessed for their biochemical properties by ELISA, immunoblotting, and fluorescence-activated cell sorting.


RESULTS
Two different antibody formats for each IgG1, IgG4, and IgE antibody were produced in mammalian cells as disulfide-linked and glycosylated Ig, which were usable in allergen-specific ELISA assays and immunoblots. In addition, the recombinant IgE antibodies mediated the binding of allergens to HEK-293 cells transfected with the high-affinity IgE receptor, and this binding was blocked by corresponding IgG antibodies.


CONCLUSIONS
The use of synthetic libraries for the generation of allergen-specific recombinant IgE and IgG antibodies should have broad applications in allergological research and diagnosis.
Braren
Ingke
I
Institute of Biochemistry and Food Sciences, Division of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.
Blank
Simon
S
Seismann
Henning
H
Deckers
Susanne
S
Ollert
Markus
M
Grunwald
Thomas
T
Spillner
Edzard
E
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000918:Antibody Specificity; D001514:Bee Venoms / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D027845:Malus / Q000276:immunology; D008892:Milk / Q000276:immunology; D019151:Peptide Library; D021122:Protein Subunits / Q000378:metabolism; D017455:Receptors, IgE / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D000485:Allergens; D000911:Antibodies, Monoclonal; D001514:Bee Venoms; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D021122:Protein Subunits; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E