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

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.

Fig. 1

Crystal structure of scFv and analysis. Panel A shows a diagram of the two polypeptide chains of the scFv molecules (molecules 1 and 2), with colors corresponding to the ribbon diagram shown in panel B. The distances between the visible termini are indicated in angstroms. The abbreviations are LC (derived from light chain), HC (derived from heavy chain). Panel C shows an electrostatic rendering of the surface of the scFv. The dashed black ellipse indicates the antibody-combining site.

Fig. 2

Representative ELISA data. The four curves shown correspond to Wild type (blue), E79A (red), T184A (green), and D76A (purple). The lines are the best least-squares fit to a sigmoidal function (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 3

ELISA results mapped to the structure of Bla g 1. Residues on the structure of Bla g 1 are color coded according to the results of the ELISA experiments listed in Table 1. Magenta indicates residues with a significant difference in ΔΔG compared to WT, yellow indicates no significant change, and green indicates the mutation was created but failed to express (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 4

Two possible orientations of Bla g 1 binding to scFv. The lowest energy structure of the two major clusters from the docking results are displayed: panel A represents cluster 1 and panel B represents cluster 2. Bla g 1 helices are rendered as colored cylinders and the scFv is rendered with a solid, gray surface and oriented the same in panels A and B. Bla g 1 subunit α is colored magenta and subunit β is colored tan. Cluster 2 was judged more likely based on the residue interactions in the epitope.

Fig. 5

Simulation of occluded regions of Bla g 1 by the scFv. The scFv is rendered as a ribbon diagram with a semitransparent surface and colored gray. Bla g 1 is rendered as a ribbon diagram and a semi transparent surface. The surface is colored as scFv epitope residues (red), likely occluded (orange), likely accessible (yellow), and accessible (green) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 6

Binding of Bla g 1 and Per a 1 with anti-Bla g 1 scFv. Beads coupled with anti Bla g 1 and anti Bla g 3 scFv were mixed and analyzed for binding with Bla g 1 (A) and Bla g 3 (B) in E6Cg and their homologs (Per a 1 and Per a 3) in American cockroach extracts (ACr). The binding is presented as 4 Parametric logistic sigmoidal curve generated using GraphPad Prism 5 by least squares fit method.