Analysis of a major target of the human immune response to cytomegalovirus using monoclonal antibodies

Abstract

Two murine monoclonal antibodies (C6D1 and D2B1) were found to react with a set of cytomegalovirus (CMV)-infected cell polypeptides, which comprise a major target of the human immune response to CMV. C6D1 reacted with proteins of 50 kilodaltons (KD) and 40KD molecular weight; D2B1 reacted with these two proteins plus a third of 35KD. Western blot analysis demonstrated that these protein targets also react with serum antibody from patients with acute or latent CMV infection. Immunofluorescence staining of CMV-infected diploid fibroblast cells by C6D1 and D2B1 showed that the protein targets are found in the nucleus throughout the course of viral infection. The proteins were shown to be late proteins dependent on viral DNA synthesis for their expression. Not all wild-type CMV strains tested expressed proteins that react with C6D1 and D2B1. Using an immunofluorescence stain of diploid fibroblasts infected with CMV strains from infected patients, we found that 70 of 76 (92%) wild-type strains reacted with C6D1 and 23 of 24 (96%) with D2B1. One strain was not reactive with either C6D1 or D2B1. Western blot analysis of 11 wild-type strains revealed that two isolates either lack the C6D1 and D2B1 protein targets or have forms of these proteins that migrate at different molecular weights.

Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins

“…Both LA MAbs, PV4D5 and PVSA11 (reactive with two different viral proteins and giving two distinct staining patterns), recognized all of the 135 viral strains tested, thus documenting a broad reactivity among a large number of wild isolates. Other MAbs to LA have been reported to react with most, but not all, of the wild HCMV strains tested (5).…”

Comparison of monoclonal antibodies for rapid detection of cytomegalovirus in spin-amplified plate cultures

“…At 24 and 72 h, two wells per specimen from one of the replicate plates were rinsed briefly with distilled water, fixed in methanol-acetone, and incubated for 30 min at 37°C with 0.15 ml of either UWMAb (pooled supernatants from hybridoma cultures producing CMV-specific antibodies C6D1, D2B1, F2D3, and F8D3 [7]; optimal dilution, 1:8 to 1:16) or IEMAb diluted 1:25 in phosphate-buffered saline. After being washed, cells were incubated for 30 min at 37°C with 0.15 ml of fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin G (heavy and light chains) (1:100 in phosphate-buffered saline; Organon Teknika Corp., West Chester, Pa.).…”