N. de Vore, W. Finlay, E. Dobrovolskia, A. Gam and J. Slater
Food and Drug Administration, Rockville, MD, USA
Journal of Allergy and Clinical Immunology, 2004, 113: Issue 2, Supplement 1, Page S297
Program and Abstracts of papers presented during Scientific Sessions - AAAAI 60th Annual Meeting
Abstract
Rationale
Although many cockroach proteins bind IgE, only 5 allergenic proteins of Blattella germanica and 3 of Periplaneta americana have been identified and characterized. Phage display of scFvs (single chain fragment variable antibodies) panned against a lysate of cockroach allergens allows us to quickly identify novel cockroach allergenic proteins, and will facilitate the standardization of cockroach allergen extracts.
Methods
A 6 month old female white leghorn chicken was immunized with American cockroach extract and antibody production was verified through analysis of IgY. RNA was isolated from the spleen and bone marrow and a cDNA library was created. PCR and overlap PCR were then performed on the library to create scFvs. Phage bearing the scFvs were panned against American cockroach extract to isolate those scFvs that recognize cockroach allergens. ScFvs were used for Western blot analysis of cockroach extracts that had been separated by 1D and 2D gel electrophoresis.
Results
The scFvs recognize proteins in American cockroach extract by Western blot analysis. Nine major proteins bands migrate at 75 kD, 60 kD, 50 kD, 45 kD, 42kD, 37 kD, 31 kD, 20 kD, and 12 kD. These scFvs also recognize several proteins in German cockroach extracts, with the primary bands migrating at 300 kD, 75kD, and 12kD.
Conclusions
We have generated an scFv library that recognizes a large number of American cockroach proteins as well as several German cockroach proteins. Using this library we will identify and characterize novel German and American cockroach allergens that have similar antibody binding epitopes.
*1 Funding: Self-funded | 
