Abstract
Antigen Used for Immunization
Three-day-old BALB/c mice were inoculated with virus suspension by intracranial injection. At 7 days post-inoculation, the brain mass was harvested. The brain mass was grounded to suspensions in MEM media, and centrifuged at 2500 g for 15 min at 4°C. The supernatant was inactivated by ultraviolet radiation as an immunogen.
Method of Immunization
For the first immunization, equal volumes of immunogen and Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO) were hypodermically injected into mice. Equal doses of antigen in incomplete Freund's adjuvant (Sigma-Aldrich) were injected into mice on days 14 and 28 after primary immunization, respectively. The equal amount of antigen suspension mixed with saline solution was injected into mice on day 42 for the boost.
Parental Cell Line Used for Fusion
Mouse myeloma Sp2/0.
Selection and Cloning Procedure
The culture supernatants were screened using IFA for binding to viral proteins in rabies virus-infected Vero cells compared with mock-infected Vero cells.
Heavy and Light Chains of Immunoglobulin
Iso, isotype.
Specificity
MAb 1B11, 1C8, and 8H12 showed a neutralizing effect to RABV in fluorescent focus inhibition test in 96-well cell plate. Mabs against N and P protein of rabies virus were identified by immunofluorescence assay and Western blot analysis with transfected recombinant N and P plasmid Vero cells.
Specific Antigen Identified
Among the 11 MAbs, three (1B11, 1C8, and 8H12) showed a neutralizing effect to RABV, while MAb 4B7 recognized the recombinant nucleoprotein (N) of RABV expressed in Vero cells and seven MAbs (1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3) reacted specifically with phosphoprotein (P) of RABV.