A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection

Abstract

An assay based on target cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) and dual color flow cytometry for detecting antibody to MCMV is described. After optimizing conditions for this technique, kinetics of anti-MCMV IgG antibody response were tested in susceptible (BALB/c) and resistant (C57BL/6) mouse strains following primary MCMV infection. Previously published antibody kinetics were confirmed in susceptible mice, with peak IgG response seen ~8 weeks after primary infection, decreasing by 20 weeks after infection. In contrast, MCMV resistant C57BL/6 mice showed significantly lower IgG antibody responses than susceptible mice. Although several techniques have been previously described to detect murine antibody responses to MCMV, including nuclear anti-complement immunofluorescence, viral immunoblotting, complement fixation, indirect immunofluorescence, indirect hemagglutination, and enzyme-liked immunosorbent assay techniques, these techniques are all time consuming and laborious. The technique presented is a simple time efficient alternative to detect previous MCMV antibody responses in experimentally infected mice.

CD28/B7-Mediated Co-stimulation Is Critical for Early Control of Murine Cytomegalovirus Infection

“…Optimal CTL responses require professional antigen presenting cells (APC), and APC/T-cell interactions are thus critical to CTL differentiation in infected hosts (1, 19, 41). Although adoptive transfer of anti-CMV antibody is protective during acute infection (5, 22, 30, 32, 61), humoral responses to MCMV are slower to develop than cellular responses (9), and both B-cells and antibody appear dispensable during acute infection (29, 74). Thus defects in either NK or T-cell responses have significant implications for viral control, which is clinically most obvious in patients with impaired NK or T-cell immunity (10, 63, 65).…”

Increased morbidity and mortality in murine cytomegalovirus-infected mice following allogeneic bone marrow transplant is associated with reduced surface decay accelerating factor expression

“…The susceptibility of virally infected cells to complemented‐mediated cytotoxicity was determined using the lactate dehydrogenase (LDH) cytotoxicity detection kit (MK40, Takara Bio Inc., Japan), as per the manufacturer's recommended protocol and as described elsewhere, with some modification [42]. Briefly, CRL1658 cell lines (ATCC) were infected with RVG102 at multiplicity of infection (MOI) of 1 for 3 days to achieve at least a 50% infection rate [43]; some cells remained uninfected and were used as a control. About 1 × 10 4 control and infected cells were seeded per well in 96‐well culture plates in triplicate for 3–5 h at 37°C in DMEM (10% FBS and 1% P/S).…”

Allogeneic Stimulation Causes Transcriptional Reactivation of Latent Murine Cytomegalovirus

“…To confirm successful MCMV infection, mice were tested for CMV reactive antibody titers prior to skin grafting (11), and their salivary glands were tested at experiments end for MCMV DNA as previously described using PCR (12). Mice that did not develop normal antibody responses to their initial infection were not utilized.…”