Fig. 3

Identification and characterization of mAbs against EMCV. Note: A. Identification of 45G3 in Vero cells infected with EMCV and Sf9 cells infected with Ac-P12A-3C recombinant baculoviruses via IFA; bar: 50 μm. B. Identification of 45G3 in Vero cells infected with EMCV and Sf9 cells infected with Ac-P12A-3C recombinant baculoviruses via Western blotting. In Figures a and b, Sf9 cells were infected with Ac-P12A-3C (lane 1) and wild-type Acanthamoeba castellanii virus (WtAc, lane 2), respectively. Western blot analysis was conducted using a custom-made anti-VP1 monoclonal antibody (figure a) and a 45G3 antibody (figure b) to detect the presence of the VP1 protein. A distinct band at approximately 35 kDa, indicative of VP1 expression, was observed in both figures and is marked with a red arrow. Similarly, in Figures c and d, BHK cells were infected with EMCV (lane 1) or served as uninfected controls (lane 2). Western blot analysis was performed using the same custom-made anti-VP1 monoclonal antibody (figure c) and the 45G3 antibody (figure d). A band corresponding to the expected size of VP1 (35 kDa) was detected in both infected cell lysates, as indicated by the red arrow. These findings confirm the specific and successful expression of the VP1 protein in virus-infected cells. C. Immunoelectron microscopy (IEM) detection. In figures a, b, and c, the primary antibody used for immunostaining was 45G3, which was applied at a dilution of 1:100 to ensure optimal binding specificity and sensitivity. Conversely, in figure d, a mouse IgG antibody served as the primary antibody used as a control. D. 3D binding epitopes predicted by AlphaFold. Green: VP1, pink: H1, and purple: K1. The red regions indicate the antibody variable regions, with the amino acids at the interaction sites marked