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


CONCLUSIONS
These data indicate that this method is useful for monitoring Cry-consensus peptide concentrations in plasma or serum.
Kozutsumi
Daisuke
D
Research and Development Section, Pharmaceuticals Development Department, Meiji Dairies Corporation, 540 Naruda, Odawara, Kanagawa 250-0862, Japan. daisuke_kozutsumi@meiji-milk.com
Shimizu
Kimiko
K
Morikubo
Keiko
K
Ohshiba
Yukio
Y
Yamaji
Taketo
T
Kino
Kohsuke
K
D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D032485:Cryptomeria / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000276:immunology*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis* / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy*
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C065290:Cry j I protein, Cryptomeria japonica; C065291:Cry j II protein, Cryptomeria japonica; D000939:Epitopes; D010940:Plant Proteins; D011994:Recombinant Proteins; C518796:cry-consensus peptide