Analysis of IgE reactivity to GST fusion peptides and synthetic peptides.
A, The data shown represent the results for patient 10. Immunoblots of purified GST fusion peptides Pen c 13 (A243–K274), (A243–A260) and (T261–K274); GST fusion Pen c 13 (T261–K274) mutant peptides S269A, G270A, T271A, T272A, S273A or K274A; and GST only, probed with serum from a Pen c 13-allergic patient. The protein blots were stained with Fast Green as a loading control. B, The histogram shows the quantitative densitometry of the bands. C, The critical amino acid for IgE-binding in Pen c 13 (T261–K274) was validated further using two synthetic peptides and tested for IgE-binding reactivity with pool serum from 10 allergic patients. The sequences of two synthetic peptides are listed, and the residue that was changed to alanine is underlined. Alanine substitution is shown in boldface letter.