A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) is described for the detection of immunoglobulin M and antibodies with specifity for human cytomegalovirus (CMV) early (CMV-EA) and late (CMV-LA) antigens. The emphasis is on the production of high-quality CMV antigens, CMV-EA and CMV-LA separately, and conditions for their application in the ELISA. The induction of CMV-EA and-LA in infected cell extracts was studied in detail by using human sera with defined antibody specificity for CMV-EA and CMV-LA. This resulted in the development of a simple whole cell extraction procedure that provided a high yield of CMV antigens with reproducible antigen quality. The antigens were specific for the detection of anti-CMV antibodies. The influence of autoantibodies on the determination of CMV-specific antibodies was investigated. Parallel analysis of 322 human sera by indirect immunofluorescence and ELISA showed a high correlation between both assays (r = 0.9674 for CMV-EA and 0.9362 for CMV-LA). Antibody titers determined by ELISA were equal to (for CMV-EA) or slightly higher (for CMV-LA) that those determined by immunofluorescence but significantly higher (20to 5,120-fold) than those determined by complement fixation. From 191 sera positive by ELISA (titer 240) 4 (2.1%) were negative by immunofluorescence (titer <40), and from 61 ELISA-positive sera 12 (19.6%) were negative (titer <8) when tested by complement fixation. Consequently, ELISA for CMV may prove to be more reliable for the selection of CMV-seronegative blood donors than these other methods. The use of high-quality antigens allows more economic handling of large-scale serum determinations. Possibilities for further automation are discussed.

Complement-dependent cytolytic antibodies (CyAb) to cytomegalovirus (CMV)-infected fibroblasts were detectable in acute- and convalescent-phase sera from renal allograft recipients (n = 44) and nonimmunocompromised patients (n = 14) with symptomatic CMV infection but not in sera from control donors (n = 75; P less than .001 by Wilcoxon rank sum test). Renal allograft recipients with secondary CMV infection had the highest levels of CyAb activity. Activity closely correlated with the serum antibody titer to CMV membrane antigens (r = .9106 by linear regression analysis) and was present in both the IgM and IgG fractions of human sera. IgG F(ab)2 fragments were inactive, thus implicating the classical pathway of complement activation. Maximal CMV-specific lysis was obtained with target cells expressing CMV late membrane antigens (greater than or equal to 72 hr after inoculation) irrespective of the CMV strain used. Adsorption and cold target inhibition studies indicated that the target antigens for the CyAb response are specific for the plasma membrane of CMV-infected cells and may only partly be shared by the virion envelope.

Using indirect immunofluorescence and a panel of human convalescent-phase sera, we identified cytomegalovirus (CMV) early and late membrane antigens (CMV-EMA and CMV-LMA, respectively) as separate entities on the surfaces of viable CMV-infected fibroblasts starting at 6 to 12 and 36 to 48 h postinoculation, respectively. For expression of CMV-EMA and CMV-LMA, infectious virus and active protein synthesis were required, whereas the expression of CMV-LMA, in addition, required viral DNA synthesis. Our data suggest that CMV-EMA and CMV-LMA form an individual set of CMV antigens that are different from intracellular CMV antigens and possibly (partly) different from the-viral envelope.

This paper describes an improved method for the in vitro detection of antibodies specifically directed against human cytomegalovirus (CMV)-induced membrane antigens present on the surface of CMV-infected fibroblasts (CMV-MA). Viable cells were found to be essential for specific visualization of CMV-MA staining. The addition of divalent cations (2.6 mM Ca2' and 2.2 mM Mg2+) and glucose (180 mM) to the incubation and washing buffers improved the viability and morphology of the cells and increased the cell yield at the end of the assay. Clustering of antigen-antibody complexes on the surface of viable CMV-infected cells was prevented by low-temperature incubation (0 to 4°C) rather than by the addition of agents which act on the metabolism of the cell. No interaction with the CMV-induced Fc receptor was observed at 0°C with either human sera or murine monoclonal antibodies. The specificity of the CMV-MA reaction was confirmed by using monoclonal antibodies to CMV nuclear, cytoplasmic, and membrane-associated antigens. Furthermore, a microplate modification of the membrane fluorescence test is described which is suitable for multisample screening purposes. This method can be applied to the determination of anti-CMV-MA antibody titers in human sera and to the screening of hybridoma supernatants for the presence of antibodies with specificity for CMV-MA.