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Mol Immunol. 2014 Jun;59(2):200-7. doi: 10.1016/j.molimm.2014.02.003. Epub 2014 Mar 22.

Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.

Author information

1
National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: mueller3@niehs.nih.gov.
2
National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
3
Indoor Biotechnologies, Inc., Charlottesville, VA 22903, USA.
4
U.S. Food and Drug Administration, Bethesda, MD 20892, USA.

Abstract

Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.

KEYWORDS:

Allergen; Bla g 1; Cockroach; Epitope; Structure; scFv

PMID:
24667070
PMCID:
PMC4097036
DOI:
10.1016/j.molimm.2014.02.003
[Indexed for MEDLINE]
Free PMC Article
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