- Email: [email protected]
Generation of mono-specific recombinant antibody fragments from chicken to allergenic proteins Fel d 1, Amb a 1 and whole yellow jacket venom
Abstracts S227
807
Generation of Mono-specific Recombinant Antibody Fragments From Chicken to Allergenic Proteins Fel d 1, Amb a 1 and Whole Yellow Jacket Venom
W. J. J. Finlay, E. N. Dobrovolskaia, A. Gam, J. E. Slater; Laboratory of Immunobiochemistry, CBER/FDA, Bethesda, MD. RATIONALE: We investigated the use of new methods to rapidly produce novel antibodies for the study of model allergens and complex allergen extracts. Since phage display of recombinant antibodies is a powerful method for generating useful antibodies we examined its application in antibody development against single allergens and protein mixtures. The animal model used in this study was the chicken immune system as their immunoglobulin repertoire is generated from single VH and VL genes, simplifying the antibody library construction process. METHODS: A single chicken (White Leghorn, 6 months old) was immunized with 50 µg Fel d 1, 150 µg Amb a 1 and 65 µg yellow jacket venom per dose (1st dose complete Freund’s adjuvant, subsequent doses incomplete Freund’s adjuvant). The spleen and bone marrow of the chicken were then harvested, RNA was isolated and RT-PCR was performed to generate an scFv library. Single-chain antibodies were then isolated by phage-display. The specificity of the antibodies was determined by ELISA and western blot analyses (1D and 2D). RESULTS: Mono-specific antibody fragments were generated against all target proteins and the scFv products were effectively expressed in E. coli. Purified antibody fragments exhibited highly specific recognition of their relevant proteins in ELISA. All antibodies displayed concentration-dependent absorbance curves to a dilution of 10-5 (against cognate antigen only). No cross-reactivity was observed between antigens recognized in western blots. CONCLUSIONS: This study describes the efficient generation of allergen-specific recombinant antibody fragments from chicken, to allergens from mammalian, plant and insect sources. Funding: Oak Ridge associated Laboratories
MONDAY
J ALLERGY CLIN IMMUNOL VOLUME 113, NUMBER 2
807
Generation of Mono-specific Recombinant Antibody Fragments From Chicken to Allergenic Proteins Fel d 1, Amb a 1 and Whole Yellow Jacket Venom
W. J. J. Finlay, E. N. Dobrovolskaia, A. Gam, J. E. Slater; Laboratory of Immunobiochemistry, CBER/FDA, Bethesda, MD. RATIONALE: We investigated the use of new methods to rapidly produce novel antibodies for the study of model allergens and complex allergen extracts. Since phage display of recombinant antibodies is a powerful method for generating useful antibodies we examined its application in antibody development against single allergens and protein mixtures. The animal model used in this study was the chicken immune system as their immunoglobulin repertoire is generated from single VH and VL genes, simplifying the antibody library construction process. METHODS: A single chicken (White Leghorn, 6 months old) was immunized with 50 µg Fel d 1, 150 µg Amb a 1 and 65 µg yellow jacket venom per dose (1st dose complete Freund’s adjuvant, subsequent doses incomplete Freund’s adjuvant). The spleen and bone marrow of the chicken were then harvested, RNA was isolated and RT-PCR was performed to generate an scFv library. Single-chain antibodies were then isolated by phage-display. The specificity of the antibodies was determined by ELISA and western blot analyses (1D and 2D). RESULTS: Mono-specific antibody fragments were generated against all target proteins and the scFv products were effectively expressed in E. coli. Purified antibody fragments exhibited highly specific recognition of their relevant proteins in ELISA. All antibodies displayed concentration-dependent absorbance curves to a dilution of 10-5 (against cognate antigen only). No cross-reactivity was observed between antigens recognized in western blots. CONCLUSIONS: This study describes the efficient generation of allergen-specific recombinant antibody fragments from chicken, to allergens from mammalian, plant and insect sources. Funding: Oak Ridge associated Laboratories
MONDAY
J ALLERGY CLIN IMMUNOL VOLUME 113, NUMBER 2