Construction of Antibody Library and Production of Polyclonal Antibodies Specific to Human Protein C

Abstract

Background: Thromboembolism can occur at any age, from infancy to adulthood. The genetic risk factors for thromboembolism in the Thai population are protein C and protein S mutations. The diagnosis depends on the activity level of proteins in the plasma. Management of acute thrombotic events in severe protein C deficiency typically requires replacement with protein C in the form of fresh frozen plasma or protein C concentrate. At present, the measurement of protein C and protein S activities level is limited due to the cost and availability of the tests. Therefore, it can take a lengthy time to determine protein C levels, resulting in a delay in treatment, especially in severe cases. Objective: To create an antibody library on filamentous bacteriophage for generating antigen specific monoclonal antibodies and to produce polyclonal antibodies specific to protein C. Results: Recombinant protein C was constructed and produced from human embryonic kidney 293 cells. Protein C was used as an antigen to immunize mice and rabbits. The antibody titers after final boosting immunization reached up to 50,000 in mice and 10,000 in rabbits. The phage antibody library with a capacity of approximately 3×10⁵ CFU was obtained from mice lymphocytes. Polyclonal antibodies were purified from rabbit sera using affinity chromatography and achieved a yield of 2 mg/mL rabbit sera. The purified polyclonal antibodies were able to detect protein C by immunoblotting and ELISA. Conclusion: The present study demonstrated that polyclonal antibodies could be produced from immunized rabbits and the antibody library obtained from immunized mice is beneficial for further isolation of monoclonal antibodies against protein C. Keywords: Protein C; Recombinant protein; Antibody library; Monoclonal antibody; Polyclonal antibody