Detection of anti-HCMVpp65 antibody by ELISA and immunoblot assay in immunized BALB/c mice. The IgG against HCMVpp65 or HeLa extract from pp651-167 (n = 11), pp65336-439 (n = 17), SA-C3d (n = 5) or PBS (n = 2) immunized mice. (a) ELISA assays for anti-HCMV reactivity against purified HCMV virions. Sera were 500X diluted and positivity was defined by mean + 3 s.e.m. of SA-C3d-immunized sera. O.D.450 > 0.50 was considered to be positive. (b) Immunoblot analysis on anti-HCMV reactivity against purified HCMV virions. Sera were 500X diluted. Top panel: pp651-167 (n = 5), pp65336-439 (n = 5) and PBS (n = 2). Lane 1 to 2 and 3 to 4, PBS-immunized sera at 8 and 12 weeks, lane 5 to 9 and 15 to 19, pp65 to 1-167-immunized sera, lane 10 to 14 and 20 to 24, pp65336-439-immunized sera, lane 25, N: 1,000X diluted healthy control serum, lane 26, P: 1,000X diluted SLE patient's serum. Bottom panel: SA-C3d (n = 5), pp651-167 (n = 6) and pp65336-439 (n = 12). Lane 1 to 5, SA-C3d-immunized sera, lane 6, N: 1,000X diluted healthy control serum, lane 7 to 8, P: 1,000X diluted SLE patients' sera, lane 9 to 14, pp651-167-immunized sera, lane 15 to 26, pp65336-439-immunized sera. (c) ELISA assays for anti-HeLa reactivity against total HeLa lysate. Sera were 500X diluted. O.D.450 > 0.48 was considered to be positive. (d) Immunoblot analysis with mouse sera at eight weeks post-immunization against HCMV and total HeLa lysate. Top panel: purified HCMV virion blot. Bottom panel: total HeLa lysate blot. Lane1 to 4, C3d-immunized sera, lane 5 to 8, pp651-167-immunized sera, lane 9 to 12, pp65167-336-immunized sera, lane 13 to 16, pp65336-439-immunized sera. Molecular mass markers (kD) are shown on the left. MW: molecular weight. w: weeks of post-immunization. Graphs depict mean ± s.e.m. values. Unpaired Student t test was performed. Results with a P-value of < 0.05 were considered to be significant. These results are representative of triplicated experiments.
Hsieh et al. Arthritis Research & Therapy 2011 13:R162 doi:10.1186/ar3481