Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay.
Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.
Landini
M P
MP
Institute of Microbiology, University of Bologna, Italy.
Lazzarotto
T
T
Maine
G T
GT
Ripalti
A
A
Flanders
R
R
D000328:Adult; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000097:blood / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D004279:DNA, Viral / Q000097:blood; D005260:Female; D005814:Genes, Viral; D006801:Humans; D007124:Immunoenzyme Techniques; D007075:Immunoglobulin M / Q000097:blood*; D007231:Infant, Newborn; D016377:Organ Transplantation / Q000009:adverse effects; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013997:Time Factors
D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins