Isolation and molecular characterization of single-chain Fv antibodies raised against pollen allergens from Japanese cedar (Cryptomeria japonica D. Don).
We report here the isolation and molecular characterization of single-chain Fv (scFv) antibodies raised against two major allergens, Cryj1 and Cryj2, in the pollen of Cryptomeria japonica by the phage display method. Recombinant phages that produced scFv antibodies that bound to Cryj1 or Cryj2 were isolated by selection with immobilized antigens in microtiter plates. After selection of six Cryj1- and four Cryj2-specific scFv antibodies with strong binding activity, we performed pairwise interaction analysis of them by surface plasmon resonance. The analysis revealed that the scFv antibodies against Cryj1 bound to only four non-overlapping epitopes, with dissociation constants that ranged from 4.84x10(-9) M to 1.62x10(-7) M. By contrast, four Cryj2-specific scFv antibodies inhibited each other's binding to Cryj2, with dissociation constants from 1.11x10(-7) M to 4.21x10(-7) M. Our results indicate that recombinant technology provides a time-saving method for the production of antibodies against pollen allergens.
Yamada
Shinya
S
Numazu Technical Support Center, Industry Institute of Shizuoka Prefecture, Numazu, Shizuoka, Japan.
Ohta
Toshiya
T
Iizuka
Chikayo
C
Ozawa
Kazumichi
K
Katayama
Yoshihiro
Y
Kajita
Shinya
S
D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant / Q000276:immunology*; D032485:Cryptomeria / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D011058:Pollen / Q000276:immunology*; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D012995:Solubility; D020349:Surface Plasmon Resonance
D052179:Antigens, Plant; D000939:Epitopes; D019151:Peptide Library; D057127:Single-Chain Antibodies