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

Lazzarotto, Tiziana; Maine, Gregory T.; Monte, Paola Dal; Ripalti, Alessandro; Landini, Maria Paola

doi:10.1128/jcm.35.2.393-397.1997

Cited by 24 publications

(12 citation statements)

References 28 publications

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“…A CMV IgM serological reference standard was needed to define the "truth" for a specimen with respect to the presence of CMV-specific IgM apart from virus detection. During development of the recombinant antigen-based test, we also devel-oped two versions of a CMV IgM immunoblot assay which can be considered a reference test for CMV IgM serology (13,15,16,17). The CMV IgM immunoblot assay (16,17) was used as a benchmark for development of this recombinant antigenbased CMV IgM test on the AxSYM and IMx immunoassay analyzers.…”

Section: Discussionmentioning

confidence: 99%

“…(ii) Selected positive specimens. Specimens from pregnant women and heart transplant recipients (n ϭ 73) positive for CMV IgM antibody as determined with the Enzygnost anti-HCMV/IgM kit (Behring AG, Marburg, Germany) or by the CMV IgM immunoblot assay (16,17) were used to evaluate assay sensitivity. Due to the low natural prevalence of CMV IgM antibody in the general population, it was necessary to run selected positive specimens.…”

Section: Methodsmentioning

confidence: 99%

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

Maine¹,

Stricker²,

Schüler³

et al. 2000

J Clin Microbiol

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A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Maine¹,

Stricker²,

Schüler³

et al. 2000

J Clin Microbiol

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show abstract

“…In this report, we describe the development of a new CMV IgM immunoblot test for detection of CMV-specific IgM which contains structural viral proteins purified from viral particles and recombinant structural and nonstructural proteins obtained by molecular biology. The reason we developed the newWB test (19), composed of an upper portion consisting of viral proteins resulting from electrophoretic separation of whole viral particles and a lower portion containing recombinant proteins, was to improve the specificity of the WB assay. Although this assay proved to be much better than the conventional WB, it is difficult to interpret correctly because of the presence of some minor viral bands and the lack of band uniformity.…”

Section: Discussionmentioning

confidence: 99%

“…These proteins are not present in the conventional WB, which contains only viral structural proteins. Therefore, we have developed an improved version of the WB assay (newWB) (19). Viral structural polypeptides separated by polyacrylamide gel electrophoresis (PAGE) were subjected to WB, along with three recombinant proteins contain-ing significant portions of ppUL32(p150), ppUL44(p52), and ppUL57(p130), as well as two additional control proteins (the carrier CKS protein as a negative control and human chain as a positive control).…”

mentioning

confidence: 99%

“…Second, the viral reactive bands on the newWB are not uniform in size due to the various amounts of posttranslational modification of the different viral proteins that occur. For example, the vp150 reactive band is very sharp and thin, whereas the vp65 reactive band is very broad (19). These differences make it difficult to determine the cutoff intensity of the bands accurately.…”

mentioning

confidence: 99%

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

et al. 1998

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We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a μ-specific conjugate. The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals. The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100%. We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history. Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests. Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring.

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Use of recombinant glycoprotein antigens gB and gH for diagnosis of primary human cytomegalovirus infection during pregnancy

Eggers¹,

Radsak

Enders³

et al. 2001

J. Med. Virol.

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The major risk factor for intrauterine transmission of human cytomegalovirus (HCMV) is a primary infection during pregnancy. The neutralizing antibody response appeared after an average of 13 weeks after seroconversion and therefore the absence of neutralizing titers in HCMV IgG positive pregnant women is a reliable marker for primary infection. Determination of neutralizing antibody, however, is time-consuming and labor-intensive. For this reason an immunoblot assay for detection of neutralizing antibodies was developed based on the use of recombinant antigens representing neutralizing epitopes of glycoproteins (gp) gB (gpUL55) and gH (gpUL75) of HCMV. In this study, 93.6% of sera of pregnant women with prior infection recognized the gp-specific epitopes corresponding to a nonresponder rate of 6.4% relative to the neutralizing antibody. In primary infection the gp-response in general coincided with the appearance of neutralizing antibody. Intriguingly, lack of HCMV gB-specific antibodies was correlated with a lower risk of intrauterine fetal infection (P < 0.05).

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

Cited by 24 publications

References 28 publications

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

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

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

Use of recombinant glycoprotein antigens gB and gH for diagnosis of primary human cytomegalovirus infection during pregnancy

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