1Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing 210029, China; 2Huadong Medical Institute of Biotechniques, Nanjing 210002, China; 3Department of Pathology, Nanjing Medical University, Nanjing 210029, China; 4Department of Pathology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China
Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.
Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F′ for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting, indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.
Results: A recombinant vector was constructed. The Fab was expressed in soluble form in E coli Top10F′. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL. The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).
Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.
Keywords: rabies; Fab engineered antibody; neutralizing antibodies