Abstract
Antigen Used for Immunization
Virus-like particle (VLP) of foot-and-mouth disease virus serotype O assembled by VP1, VP2, VP3, and VP4 that expressed in Escherichia coli.
Method of Immunization
VLP of FMDV serotype O (100 μg/mouse) with complete Freund's adjuvant (50% v/v) through hypodermic injection. This procedure took place three times at a 14-day interval.
Parental Cell Line Used for Fusion
Mouse myeloma Sp2/0.
Selection and Cloning Procedure
The supernatant of the fusion cells was tested by LPBE Kit for detecting antibodies of foot-and-mouth disease virus type O (Lanzhou, Lanzhou Veterinary Research Institute, China) for the verification of Mab production.
Heavy and Light Chains of Immunoglobulin
Specificity
The specificity of selected Mabs was analyzed by indirect immunofluorescent assay in the 293T cells with transient expression of VP1, VP2, or VP3. In brief, the pCAGGS plasmid harboring the genes of VP1, VP2, or VP3 was constructed and confirmed by sequencing. The 293T cells were seeded in six-well plates (3.0 × 105 per well) and incubated for 24 hours, and then the cells were transfected with three recombinant pCAGGS plasmids using Lipofectamine 2000 (Qiagen, Valencia, CA) according to the manufacturer's instructions. At 48 hours post-transfection, the cells were fixed with 80% acetone (4°C, 30 minutes) and washed twice with phosphate buffered saline (PBS) (0.01 M, pH 7.2). After that, the fixed cells were incubated with the Mabs produced in this study (dilution of 1:500) at 37°C for 1 hour and washed twice with PBS, then the cells were incubated at 37°C for 1 hour with fluorescein isothiocyanate-conjugated goat antimouse IgG (Sigma-Aldrich, St. Louis, MO) at the dilution of 1:400. After washing with PBS, images were captured by a fluorescence microscope (ECLIPSE Ts2, Nikon, Japan). Mock-infected cells were used as controls. 6G6 reacted with VP2 and 7D2 reacted with VP1.
Specific Antigen Identified
MAb 6G6 recognized the recombinant VP2 protein expressed in 293T cells, whereas MAb 7D2 recognized the recombinant VP1 protein expressed in 293T cells.