Reactivity to p52 and CM2 Recombinant Proteins in Primary Human Cytomegalovirus Infection with a Microparticle Agglutination Assay

Abstract

We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.Human cytomegalovirus (HCMV) causes morbidity in immunocompromised patients and the fetus in case of pregnancy (2,3,10,12,21). The distinction between a primary infection, reactivation, or convalescence is not easy by serological assays alone, because immunoglobulin M (IgM) antibodies can be present in sera over a long period and can reappear during reactivation (1,13,14). Besides classical IgM detection through indirect or capture assays, an alternative could be to detect antibodies against some antigenic targets. It has been shown that reactivity against the recombinant p52 (ppUL44) and CM2 (a recombinant protein of ppUL44 and pUL57) antigens is associated with primary infection (4,5,6,18,20). Reactivity against the p52 and CM2 antigens increases during primary infection. A few months after onset, reactivity against p52 sharply falls, while reactivity against CM2 can be detected for several more months in immunocompetent patients (13,20). We evaluated the CMV Multiplex Copalis assay, which is an automated qualitative test that uses coupled light scattering technology to discriminate between recent or past infection. It allows the simultaneous detection of antibody reactivity against p52, CM2, and whole-virion protein (VP). MATERIALS AND METHODSHCMV IgM serology. All sera were tested for the presence of HCMV IgM by an indirect enzyme immunoassay (EIA; Enzygnost CMV-IgM; Behring AG, Marburg, Germany). The procedures and interpretation of the results (positive, equivocal, or negative) were as recommended by the manufacturer. This included an absorption of IgG and rheumatoid factor before testing.Patients. In this evaluation 214 serum specimens obtained from 125 patients were tested. All the sera were positive or equivocal for HCMV IgM, as tested by our reference EI...

Multiplex bead array assays: Performance evaluation and comparison of sensitivity to ELISA☆

“…Few studies reported the validation of Copalis in comparison to standard ELISAs (17)(18)(19). In these studies, which were performed for the detection of CMV, syphilis, Anti-Ro/SSA and Anti-La/SSA, the assay had a sensitivity of 86, 92.2, 88.2 and 95.2% respectively using the Copalis second format; and a specificity of 81, 97.6, 93 and 97.6%.…”

Cross-Reactivity of Epstein-Barr Virus-Specific Immunoglobulin M Antibodies with Cytomegalovirus Antigens Containing Glycine Homopolymers

“…Detection of virus-specific IgM by ELISA, microagglutination, or immunoblotting is a widely used strategy to define HCMV infection as the etiology of disease in immunocompetent patients (15,17,23,26,31,32,48,49,51). In addition, screening for HCMV-specific IgM against defined viral antigens has been suggested as a method of improving the detection of maternal infection during pregnancy (8,28).…”

“…Inspection of the amino acid sequences of the HCMV proteins pp150, pp65, pUL80a, pUL44, and pUL57 revealed short glycine-rich motifs of 5 to 13 aa in the carboxy-terminal part of pUL44 and the central parts of pUL57. pUL44 and pUL57 are contained in antigen preparations widely used for the design of commercially available HCMV IgM ELISAs and agglutination assays, as well as for the design of immunoblots assays (8,23,31,32,48).…”

“…Therefore, the results of these assays may be difficult to interpret. Recombinant tests involving the dominant IgM target antigens of HCMV have been designed (26,31,32,48,49). pUL44 and pUL57 have been identified as major IgM antigens for such assays, and, consequently, most commercially available recombinant HCMV IgM assays contain fragments of one or both antigens.…”

Features of IgG-antibodies production to individual Cytomegalovirus proteins in various eye diseases (age-related macular degeneration and central serous chorioretinopathy)

“…Показано, что состав экспрессируемых вирусных белков отличается на разных стадиях CMV-инфекции (ЦМВИ). Исследование антител к индивидуальным антигенам ЦМВ позволяет уточнить фазу инфекции (первичную, хроническую, латентную), что нашло применение в клинической практике [3,14].…”