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Rabbit single domain antibodies specific to protein C expressed in prokaryotes
1Schweizerisches Institut für Allergie- und Asthmaforschung, Davos, Switzerland.
Abstract:
VDJ genes were amplified by the polymerase chain reaction from mRNA isolated from peripheral blood B cells of rabbits immunized with protein C. The amplified genes were cloned into a lambda phage expression vector and packaged. A library of 6 x 10(5) recombinant phages was screened with labelled protein C and 30 positive clones were found. Three of them were plaque purified and the affinity of the single domain antibodies to the antigen determined to be 10(6)-10(7) l M-1. The data indicate the feasibility of generating single domain antibody, specific to protein antigen, from rabbit.
Insights
Researchers generated single domain antibodies specific to protein C using rabbit B cells. This demonstrates a feasible method for creating targeted protein-specific antibodies from rabbits.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Rabbit peripheral blood B cells are a source of immunoglobulin genes.
- Protein C is an antigen used for immunization.
- Single domain antibodies offer therapeutic and diagnostic potential.
Purpose of the Study:
- To investigate the feasibility of generating single domain antibodies against protein C from rabbit B cells.
- To characterize the affinity of the generated antibodies.
Main Methods:
- Amplification of VDJ genes from rabbit B cell mRNA using polymerase chain reaction (PCR).
- Cloning of amplified genes into a lambda phage expression vector.
- Screening of a phage display library with labeled protein C.
- Affinity determination of purified single domain antibodies.
Main Results:
- A library of 6 x 10^5 recombinant phages was constructed.
- 30 positive clones were identified after screening with protein C.
- Three clones were plaque-purified and their antibody affinities measured.
- Affinity of the single domain antibodies ranged from 10^6 to 10^7 M^-1.
Conclusions:
- The study confirms the feasibility of generating rabbit-derived single domain antibodies specific to protein antigens.
- This method provides a viable approach for antibody discovery and development.