Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens

Abstract

DNA fragments from eight different reading frames of human cytomegalovirus (HCMV) were generated by PCR and subsequently cloned and expressed in Escherichia coli in fusion with glutathione S-transferase. The recombinant viral antigens were evaluated in immunoblot analyses. The most reactive antigens were purified and further evaluated in ELISAs. For this, sera from healthy blood donors and immunocompetent individuals with acute HCMV infection, and follow-up sera from transplant recipients with acute primary HCMV infection were used. The results of our experiments indicate that only three particular recombinant polypeptides from two viral proteins are necessary for serodiagnosis. While a fragment covering amino acids (aa) 495 to 691 of ppl50 (150/1) was the most suitable antigen for the identification of infected individuals in general, immunoglobulin M antibodies against the C-terminal parts of ppl50 (aa 862 to 1048; 150/7) and p52 (aa 297 to 433; 52/3) proved to be excellent serological markers to monitor acute HCMV infection. The selected recombinant antigens enable the improvement of serodiagnosis of HCMV-related diseases, especially during the early stages of infection.

An antigen fragment encompassing the AD2 domains of glycoprotein B from two different strains is sufficient for differentiation of primary vs. recurrent human cytomegalovirus infection by ELISA

“…Puri®ed recombinant proteins or synthetic peptides were coated on 96-well polystyrene microtiter plates according to a previously established procedure [Vornhagen et al, 1994]. For coating, antigens were diluted in 0.01 M carbonate buffer (pH 9.5) to ®nal concentrations of 0.5 to 1.0 mg/ml.…”

“…Primary HCMV infection can, theoretically, be identi®ed by IgG antibody seroconversion. Sensitive and speci®c enzyme immunosorbent assays (ELISA) based on recombinant antigens have been developed to detect IgG antibodies directed to HCMV [Vornhagen et al, 1994]. However, recent serum specimens are only infrequently available and, consequently, IgG seroconversion can usually not be documented.…”

A novel Western blot test containing both viral and recombinant proteins for anticytomegalovirus immunoglobulin M detection

“…Serum IgM to HCMV can be detected by a variety of different procedures, but its detection has been hampered by several technical problems causing interassay variability. The most widely used procedure for IgM detection is enzyme immunoassay (EIA) (18,(24)(25)(26)(27). Many different EIAs for HCMV IgM are available commercially, and poor agreement has been found among the results obtained with different EIA kits (13).…”

“…Recombinant antigens containing significant portions of ppUL32 (rp150), ppUL44 (rp52), and pUL57 have been obtained by us and by other researchers and evaluated serologically (12,16,27,28). The aim of the present work was the development, optimization, and preliminary clinical evaluation of an ideal serological test for detection of HCMV-specific IgM.…”

The DNA-binding protein pUL57 of human cytomegalovirus is a major target antigen for the immunoglobulin M antibody response during acute infection

“…Crude preparations of extracellular virus particles have been used as antigens for IgM enzyme-linked immunosorbent assay (ELISA); thus, the performance of such assays has been limited (9). Recently, the viral proteins pp150 (UL32) and p52 (UL44) have been identified as major target antigens for the IgG-and IgM-specific antibody responses (8,9,11,17). However, some antigen fragments revealed IgM-specific reactivities with sera from healthy blood donors without signs of acute infection (17).…”