Mouse anti p24 monoclonal antibody

293T cells were transfected with pNL4-3.Luc.R-E- plasmids using TransIT-LT1 Transfection Reagent (Clontech) according to the manufacturer's instructions. It should be noted that cells were transfected in the absence of the VSV-G protein to prevent infection of cells with newly produced virions. Equal volume of culture supernatants (900 μl) was layered onto 500 μl of 20% sucrose in phosphate-buffered saline and centrifuged at 20,000 × g for 2 hours at 4°C. Pelleted virions were resuspended in 1 × NuPAGE LDS sample buffer containing 2% β-mercaptoethanol. Lysed virions were subjected to Western blot. The p24 CA protein was probed with mouse anti-p24 monoclonal antibody (Abcam) followed by HRP-conjugated goat anti-mouse IgG antibody (KPL). To check transfection efficiency, 293T cells were lysed with cell culture lysis reagent (Promega) and used to measure luciferase activity with a luciferase assay kit (Promega) on a luminometer.

To determine the p24 Gag expression levels in the rMpg or rBCG strain, we conducted western blot and enzyme-linked immunosorbent assay (ELISA) analyses as previously described²⁴ . The expression of p24 in the rMpg-p24 or rBCG-p24 strain was determined using a mouse anti-p24 monoclonal antibody (Abcam, Cambridge, USA; 1:1,000 dilution). As an internal control, mycobacterial HSP65 (Abcam, 1:1,000 dilution) was used. To assess the stable expression of p24, the p24 expression level in the rMpg-p24 strain after various passages (after 1, 4, 6, 8, 10 and 12 passages) was also determined. The passage process was conducted from plate to plate (7H10 agar plate with or without kanamycin), and the colonies from each passage were cultured in 7H9 broth medium for 2 weeks prior to performing each experiment. Additionally, the same amount of protein was used to detect the p24 levels using a p24 ELISA kit (in triplicate wells) (ABL, Rockville, USA) as suggested by the manufacturer³⁶ . All the groups were analyzed in two independent experiments.