The DNA-binding protein pUL57 of human cytomegalovirus: comparison of specific immunoglobulin M (IgM) reactivity with IgM reactivity to other major target antigens

Abstract

In this work we used PCR to amplify the DNA regions coding for two polypeptides from pUL57 of human cytomegalovirus (amino acids 540 to 601 and 1144 to 1233) and showed that both portions reacted very efficiently with immunoglobulin M in sera of acutely infected subjects. However, pUL57 is not an essential antigen for the replacement of or supplement to a cocktail of recombinant protein antigens containing portions of ppUL32,-44,-83, and-80a in immunoglobulin M serology.

Development and Clinical Evaluation of a Recombinant-Antigen-Based Cytomegalovirus Immunoglobulin M Automated Immunoassay Using the Abbott AxSYM Analyzer

“…Since detection of the humoral IgM response is improved by including both structural and nonstructural viral proteins (8,9,10,24,31), the performance of the viral antigen-based tests is directly dependent on how the virus is grown and how the viral antigens are purified. Our group and others have shown that a balanced cocktail of highly purified recombinant antigens (12,18) or peptides (5), which contain both structural and nonstructural viral antigens, can replace the virus for detection of CMV-specific IgM. In this report we describe the development of the first automated, commercially available, recombinant antigen-based CMV IgM immunoassay for the detection of CMV-specific IgM.…”

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

“…Of the sera from transplant recipients and pregnant women, we observed 15 sera that reacted with more than two viral proteins and did not show any reactivity with recombinant proteins. In order to determine whether these sera could represent true IgM-positive sera which did not react with the two recombinant proteins present in the newWB, we tested them with two other recombinant proteins that have been recently described as early gene products highly reacting with HCMVspecific IgM (UL57 [16,28]). None of the 15 sera gave a positive reaction with the two UL57 recombinant proteins (data not shown), suggesting that ppUL57 is not essential to increasing the sensitivity of the assay.…”

“…Recombinant proteins. The following Escherichia coli CMP-2-keto-3-deoxyoctulosonic acid (CMP-KDO) synthetase (CKS) recombinant proteins were used: (i) two ppUL32 regions (amino acids [aa] 595 to 614 plus 1006 to 1048) fused together which can replace the entire p150 molecule in its IgM-binding ability (21); (ii) the carboxy-terminal part of ppUL44 (aa 202 to 434), which contains highly reactive epitopes for IgM and does not contain relevant amino acid sequences cross-reacting with the homologous protein of other members of the Herpesviridae family (22); and (iii) two segments of ppUL57 (aa 540 to 601 and 1144 to 1233) previously shown to be very reactive with serum IgM (16,28). The recombinant proteins described above were obtained and characterized as previously described (12).…”

“…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.…”

Development of a New Cytomegalovirus (CMV) Immunoglobulin M (IgM) Immunoblot for Detection of CMV-Specific IgM

“…(i) Recombinant proteins. The following Escherichia coli CMP-2-keto-3-deoxyoctulosonic acid synthetase (CKS) recombinant proteins were used: (i) two ppUL32 regions (amino acids [aa] 595 to 614 and 1006 to 1048) fused together, which can replace the IgM-binding ability of the entire p150 molecule (28); (ii) the carboxy-terminal part of ppUL44 (aa 202 to 434), which contains highly reactive epitopes for IgM and does not contain relevant amino acid sequences cross-reacting with the homologous proteins of other members of the family Herpesviridae (29); (iii) two segments of ppUL57 (aa 540 to 601 and 1144 to 1233) previously shown to be very reactive with serum IgM (22,37); and (iv) a significant portion of assembly protein ppUL80a (aa 117 to 373) (13). Insoluble CKS-CMV fusion proteins were initially purified after lysis by a combination of detergent washes followed by solubilization in 8 M urea (30).…”