IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins.
Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (p52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (pp150, aa 994-1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELISA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection.
Vornhagen
R
R
Biotest AG, Research Department, Dreieich, Germany.
Hinderer
W
W
Sonneborn
H H
HH
Bein
G
G
Matter
L
L
The
T H
TH
Enders
G
G
Jahn
G
G
Plachter
B
B
D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000276:immunology / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000175:diagnosis / Q000276:immunology / Q000821:virology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology*; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; C412912:CM2 fusion protein, Cytomegalovirus; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D016056:Immunodominant Epitopes; D007075:Immunoglobulin M; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C056367:pp150 protein, Cytomegalovirus