Ab43040

Antibody responses following HCMV infection of huBLT mice were assessed by enzyme-linked immunosorbent assay (ELISA). Costar clear polystyrene high-protein-binding enzyme immunoassay (EIA) plates (Corning) were coated in either purified HCMV virion preparations (5 ug/mL) diluted in PBS or recombinant CMV gB (0.75 ug/mL; Abcam, ab43040) diluted in a coating buffer composed of 0.015 M Na₂CO₃ and 0.035 M NaHCO₃ (Corning). After overnight incubation, plates were washed five times with 200 μl of wash buffer (PBS containing 0.25% Tween 20) then blocked for 90 min at room temperature in blocking buffer (wash buffer containing 0.89% bovine serum albumin). huBLT mouse serum was serially diluted 2-fold in blocking buffer, plated at 100 μl per well and incubated at 37 °C for 45 min. Plates were washed as above and incubated with 50 μl of secondary antibody (either anti-IgA/M/G, #609-103-130; anti-IgG, #609-1312; or anti-IgM, #609-1307, all from Rockland Inc.) for 30 min at 37 °C. Plates were washed as above prior to visualization and quantification of antibody binding by addition of chromogen OPB substrate (Invitrogen). Optical densities at 450 nm were determined using an ELISA plate reader (Synergy HTX Multi-mode Reader, BioTex). Endpoint antibody titers were calculated using log-log transformation of the linear portion of the curve.

Custom-made AD-6 (CMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYV with a GGC tag) and AD-6.4 (REFNSYKQRVKYVED with a GGC tag) were purchased from Peptide 2.0 US. Custom-made affinity-purified anti-AD-6 rabbit IgG was generated by GenScript.
A Recombinant gB fragment was purchased (Abcam; ab43040) and the vaccine gB was a kind gift of Sanofi Pasteur.
Anti-IE CMV (6F8.2, MAB8131, Millipore; 1:2000 dilution). Goat anti-mouse IgG (H + L)-AlexaFluor 568 (A11004, Invitrogen, 1:2000 dilution) and Live/Dead Aqua viability stain (Life Technologies, 1:1000 dilution). The ITC88 antibody was purchased from CreativeLabs.

Total IgG3 was measured by coating a high binding ELISA 96-well plate with an anti-human IgG mAb (Sigma-Aldrich, St Louis, MO) overnight at 4°C. Similarly, to measure IgG3 specific responses against other diseases, ELISA plates were coated with cytomegalovirus glycoprotein B (ab43040), measles virus Priorix, Schwarz strain nucleocapsid protein (ab74559) or Influenza A virus hemagglutinin H1 protein (ab69741) (Abcam, Cambridge, MA). Plates were blocked with 5% milk/ 0.05% Tween-20 in PBS for 1 hour at 37°C and plasma or isolated IgG was diluted in blocking buffer and incubated for a further hour. Following washes in 0.05% Tween 20 in PBS, IgG3 was detected with anti-human IgG3 HRP (Southern Biotech, Birmingham, AL) and following a further hour incubation TMB substrate was added and the reaction stopped with 1M H₂S0₄. To normalize for IgG levels in individuals, total IgG levels for all samples was measured as above with the exception of detecting with anti-human IgG-HRP (Sigma-Aldrich, St Louis, MO).