Back to Search
Start Over
Trimolecular complex formation of IgE, Fc epsilon RI, and a recombinant nonanaphylactic single-chain antibody fragment with high affinity for IgE.
- Source :
-
Journal of immunology (Baltimore, Md. : 1950) [J Immunol] 2009 Apr 15; Vol. 182 (8), pp. 4817-29. - Publication Year :
- 2009
-
Abstract
- IgE is a central molecule in allergic disease. We have isolated cDNAs coding for the heavy and light chains of a murine mAb specific to human IgE and expressed a recombinant single-chain variable fragment (ScFv) derived thereof in Escherichia coli. The purified recombinant ScFv has a molecular mass of 28 kDa as measured by mass spectrometry and shows a beta-sheet fold as determined by circular dichroism. In biosensor-based studies it was demonstrated that the ScFv rapidly and stably binds to human IgE with an affinity of K(D) of 1.52 x 10(-10) M, which is almost as high as the affinity of IgE for FcepsilonRI, and that the ScFv is able to recognize FcepsilonRI-bound IgE and to prevent IgE binding to FcepsilonRI. The ScFv reacts specifically with IgE but not with other isotypes, allows the measurement of allergen-specific IgE in serum samples, and specifically targets cells that contain FcepsilonRI- or FcepsilonRII-bound IgE or that secrete IgE. Using negative-stain electron microscopy we demonstrated the formation of bimolecular complexes consisting of two ScFv molecules and one IgE and trimolecular complexes consisting of IgE, FcepsilonRI, and ScFv in which only one ScFv is able to bind to IgE. Accordingly, we found that the ScFv does not cross-link basophil-bound IgE and hence does not induce histamine release or activation of basophils as demonstrated by FACS analysis of CD203c expression and by histamine release experiments. In vivo skin testing confirmed the lack of allergenic activity of the ScFv. The recombinant ScFv may represent a universal tool for the IgE-targeted treatment of allergies.
- Subjects :
- Allergens immunology
Amino Acid Sequence
Anaphylaxis genetics
Anaphylaxis metabolism
Animals
Base Sequence
Basophils immunology
Circular Dichroism
Humans
Immunoglobulin E genetics
Immunoglobulin E metabolism
Immunoglobulin Fab Fragments genetics
Immunoglobulin Fab Fragments metabolism
Immunoglobulin Fragments
Mice
Mice, Inbred BALB C
Molecular Sequence Data
Protein Binding genetics
Protein Binding immunology
Recombinant Proteins biosynthesis
Recombinant Proteins genetics
Recombinant Proteins metabolism
Anaphylaxis immunology
Immunoglobulin E biosynthesis
Immunoglobulin Fab Fragments biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1550-6606
- Volume :
- 182
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Journal of immunology (Baltimore, Md. : 1950)
- Publication Type :
- Academic Journal
- Accession number :
- 19342660
- Full Text :
- https://doi.org/10.4049/jimmunol.0800726