Tetherin-Specific Monoclonal Antibodies from H. pylori NAP Immunization

Tetherin-specific mouse hybridomas were generated following vaccination of mice with Helicobacter pylori neutrophil-activating protein chimera (NAP). NAP chimera proteins significantly enhance immunogenicity of antigens and methods of protein expression, mouse immunization, and hybridoma isolation have been previously described (48 (link)– (link)50 (link)). NAP-tetherin was cloned into the bacterial expression plasmid pET28a+ with His6 and MBP N-terminal tags for purification, separated by a TEV cleavage site. BL21(DE3) E. coli were transformed with the pET28 NAP-tetherin and His-tagged protein was purified with a NiNTA kit (Qiagen). Six- to 8-week-old BALB/c mice were intraperitoneal injected with 50 μg of purified NAP-tetherin with Freund’s adjuvant and boosted with 25 μg additional antigen by intravenous injection. Spleen cells were collected and fused with myeloma line Sp2/0-Ag14 (ATCC) using polyethylene glycol 4000. Hybridomas were screened for tetherin specific reactivity by antigen-mediated ELISA and immunoblotting. Two tetherin specific MAbs were identified, 34F11 and 35H2. Hybridoma supernatant was directly used as primary antibodies for experiments (1:20 to 1:50 dilution).