Recognition of T cell epitopes unique to Cha o 2, the major allergen in Japanese cypress pollen, in allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen.

BACKGROUND: Pollens from species of the Cupressaceae family are one of the most important causes of respiratory allergies worldwide. Many patients with pollinosis have specific IgE to both allergens from Japanese cedar and Japanese cypress pollen. We set out to identify T cell epitopes in Cha o 2, the second major allergen of Japanese cypress pollen. METHODS: T cell lines (TCL) and T cell clones (TCC) specific to Cha o 2 were generated from allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen. T cell epitopes in Cha o 2 were identified by responses of TCL stimulated with overlapping peptides. Abilities of IL-4/IFN-gamma production by TCC were evaluated using enzyme immunoassay. RESULTS: Using TCL, 11 dominant and subdominant T cell epitopes were identified in Cha o 2. The subsets of TCC were predominantly of T helper 2-type. A T cell epitope p141-160 in Cha o 2 and corresponding peptide in Cry j 2 showed high homology. Although TCC PC.205.159 responded to stimulation with p141-160 in Cha o 2, it did not respond with corresponding peptide in Cry j 2, therefore, the T cell epitope was unique to Cha o 2. CONCLUSIONS: Eleven T cell epitopes that were identified are unique to Cha o 2. Cha o 2 is a putative aeroallergen that can potentially sensitize human T cells. We concluded that generation of T cells specific to Cha o 2 in allergic patients acts as one of the causes of continuous allergic symptoms in April.

Enzyme-linked immunosorbent assay of a linear, recombinant peptide designed for immunotherapy of Japanese cedar pollinosis.

INTRODUCTION: Cry-consensus peptide, a recombinant T-cell epitope peptide for immunotherapy of Japanese cedar pollinosis, is a linear peptide that does not have disulfide bonds because no cysteine residue exists in the molecule. We examined whether a sandwich enzyme-linked immunosorbent assay (ELISA) could be performed for linear peptides such as Cry-consensus peptide. METHODS: The 3-dimensional conformation of Cry-consensus peptide was examined by (1)H NMR analysis. Nineteen monoclonal antibodies (mAbs) that recognized various domains of Cry-consensus peptide were established to use in a sandwich ELISA. The relationship between the recognition sites of mAbs and the sensitivity of the ELISA was investigated to optimize the selection of the combination of the capture and the detection antibodies. ELISA inhibitors in serum and plasma were also studied to improve the stability and the sensitivity of determination. RESULTS: (1)H NMR analysis of Cry-consensus peptide suggested that Cry-consensus peptide molecule had no portions with rigid conformation. The sensitivity of the ELISA showed a good correlation with the distance between the respective binding sites of the capture and the detection antibodies. Human serum albumin and alpha1-acid glycoprotein strongly inhibited the binding of the capture mAb to Cry-consensus peptide in a dose-dependent manner, and heparin also inhibited the binding in the concentration at which it is used as anticoagulant. Taken together, the findings indicated that an optimized method showed good linearity and minimal variation from 0 to 1000 ng/ml of Cry-consensus peptide. DISCUSSION: These data indicate that this method is useful for monitoring Cry-consensus peptide concentrations in plasma or serum.