Reliable methods for the detection of cytomegalovirus (CMV) strain-specific serological responses are lacking. We describe a simple and reliable enzyme-linked immunosorbent assay method developed to detect antibodies against the polymorphic epitopes within the two envelope glycoproteins of CMV, glycoproteins H and B. This assay is useful for the detection of serologic responses to CMV strains and the identification of CMV reinfections.Cytomegalovirus (CMV) is an important pathogen in immunocompromised hosts and a frequent cause of congenital infection. CMV isolated from clinical samples exhibits extensive genetic variation (7,12,13), and CMV reinfections have been demonstrated to occur in seropositive individuals. However, it is thought that these reinfections have little untoward consequences with respect to congenital infections. Recent studies documenting higher rates of congenital CMV infection in populations with nearly universal seroreactivity to CMV suggest that infection with new or different virus strains could be responsible for the intrauterine transmission of CMV in immune mothers (5,17,18). The frequency and consequences of infection with multiple CMV strains are unclear because of the lack of reliable methods for the accurate identification of CMV strain-specific antibody responses. By utilizing the defined heterogeneity within the antibody binding epitopes on envelope glycoprotein H (gH) and gB of the AD169 and Towne strains of CMV, an enzyme-linked immunosorbent assay (ELISA) method was developed to distinguish serological responses against infection with different CMV strains.Serum samples from 96 CMV-seropositive women participating in an ongoing study and 51 seronegative individuals were tested for anti-CMV strain-specific antibodies. Informed consent was obtained from the study participants, and the study was conducted in accordance with the guidelines of the Institutional Review Board for Human Use of the University of Alabama at Birmingham.Purified recombinant antigens based on polymorphic antibody binding sites defined on gH (antigen gpUL75) and gB (antigen gpUL55) were used as antigens (Fig. 1). The gH antigens were constructed as -galactosidase fusion proteins containing the coding region for amino acids (aa) 15 to 142 of gpUL75 from the AD169 strain (the AP86 antigen) and aa 14 to 42 of the Towne strain (the TO86 antigen) of CMV (16).The recombinant peptides were expressed in Escherichia coli and were purified as described previously (8). gB antigens were prepared as six-His-tag-labeled peptides by cloning the coding region (aa 1 to 116) from strains AD169 (the AD55 antigen) and Towne (the TO55 antigen) (9) into expression vector pET21a (EMD, Gibbstown, NJ) by using the HindIII and BamHI endonuclease restriction sites. The peptides were expressed in E. coli Rosetta cells and were purified by using Talon Superflow metal affinity columns (Clonetech, Mountain View, CA). A positive control antigen was constructed by cloning the antigen domain 1 (AD-1) region of the gene coding gB, which ha...
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