Cloning and expression of CMV genes.
All CMV gene fragments that encode antigens were obtained by PCR amplification with PCR primers designed to amplify specific nucleotide sequences. These gene fragments were cloned into a modified Escherichia coli
-manno-octulosonate cytidylyl transferase) epitope-embedding expression vector (G. Maine, unpublished results). Plasmids that encode recombinant proteins 4, 9, and 26 (12
) were used as template DNA to generate the CKS expression plasmids pCMV-27, pCMV-28, and pCMV-29, respectively, which express the recombinant proteins rp27, rp28, and rp29 fused to CKS, respectively. Portions of the following CMV antigenic regions were contained in three recombinant antigens: rp27 (pUL32 [pp150] and pUL44 [pp52]), rp28 (pUL83 [pp65]), and rp29 (pUL80a [pp38]). The DNA sequences of all cloned CMV genes were determined and confirmed. Bacterial clones that express the fusion proteins were grown in rich media, and the synthesis of the fusion proteins was induced as described previously (26
). After postinduction, the cells were harvested and the cell pellets were stored at −80°C until protein purification.
Purification of recombinant fusion proteins.
Insoluble fusion proteins (rp27, rp28, and rp29) were purified after lysis by a combination of detergent washes and then solubilization in 1% sodium dodecyl sulfate (26
). After solubilization, the fusion proteins were purified by Sephacryl S-300HR chromatography (Pharmacia Biotech, Piscataway, N.J.), dialyzed, and stored at −80°C until coating of microparticles.
Recombinant antigen-coated microparticles.
Purified fusion proteins were coated onto polystyrene microparticles (Polysciences, Inc., Warrington, Pa.). After coating, uncoated antigen was removed by diafiltration and the microparticles were resuspended in a microparticle diluent buffer containing Tris buffer with protein (bovine) stabilizers and antimicrobial agents. The microparticle diluent buffer also contains E. coli
CKS to competitively block the binding of anti-CKS antibodies to the solid phase. After equilibration, the microparticles were diluted to their final concentration, and two pp150 peptides, A1C2 (20mer) and F3 (43mer) (AnaSpec, Inc., San Jose, Calif.), were added. These peptides contain the identical amino acid sequence of pp150 present in the 1A (12
) and rp27 fusion proteins and are used to competitively modulate the immunoreactivity of the pp150 amino acid sequences present on the microparticles.
Purified goat anti-human IgM (μ-chain specific; Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) conjugated with alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.) (33
) was used to detect CMV IgM bound to the microparticles as described previously (28
). The instrument was calibrated with an index calibrator prepared with anti-CMV IgM (human) prepared in human serum.
The AxSYM and IMx instruments (Abbott Laboratories, Abbott Park, Ill.) are automated immunoassay analyzers that use microparticle enzyme immunoassay technology. Details of these instruments are given elsewhere (4
Human serum samples used for assay cutoff determination and preliminary performance evaluation. (i) Specimens from blood donors and pregnant women.
Specimens from random volunteer whole-blood donors (n = 300; Interstate Blood Bank, Memphis, Tenn.) and specimens randomly selected from U.S. and European populations of pregnant women (n = 199; Bartek Associates, Inc., Barrington, Ill.; University of Nantes, Nantes, France) were used to evaluate assay specificity.
(ii) Selected positive specimens.
Specimens from pregnant women and heart transplant recipients (n
= 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
) 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.
Human serum samples used for clinical evaluation.
Specimen testing at the Eastern Virginia Medical School was approved after review of the clinical protocol by the Internal Review Board (compliance no. X97-016), with signed consent from donors obtained when appropriate. Specimen testing at Dianalab did not require special approval of the Internal Review Board. Specimen testing at Abbott Laboratories, Eastern Virginia Medical School, and Dianalab with AxSYM CMV IgM assay investigational reagent lots was conducted by an Abbott Laboratories-approved clinical protocol. Frozen specimens were centrifuged prior to testing.
(i) Specimens from pregnant women.
Fresh maternal serum specimens were collected prospectively from pregnant women from Swiss (n = 599; Dianalab S.A., Geneva, Switzerland) and U.S. (n = 200; Eastern Virginia Medical School, Norfolk, Va.) populations. The average age of the women in the Swiss population was 31.1 years, with 53.1, 29.9, and 17.1% of the specimens drawn during the first, second, and third trimesters, respectively. The average age of the women in the U.S. population was 25.9 years, with 40.0 and 60.0% of the specimens drawn during the first and second trimesters, respectively. These specimens were tested with the AxSYM instrument prior to freezing and were used to evaluate assay specificity.
(ii) Selected positive specimens.
Selected frozen serum specimens (n = 39) from a Swiss population of pregnant women positive for CMV IgM antibody as determined by the IMx CMV IgM assay (Abbott Laboratories) were used to evaluate assay sensitivity. Three of these specimens overlap with the specimens listed below under Selected serial specimens.
(iii) Selected serial specimens.
A total of 17 serial specimens from three suspected CMV IgM-positive women were tested to evaluate the kinetics of appearance and disappearance of CMV-specific IgM. These women were pregnant during the evaluation. These specimens were also tested by the IMx CMV IgM and AxSYM CMV IgG assays.
(iv) Potentially cross-reactive specimens.
Potentially cross-reactive specimens, i.e., specimens known to be seropositive for a variety of specific infections and/or medical conditions, were tested to determine potential cross-reactivity in the assay. The potentially cross-reactive specimens were positive for antinuclear antibody (n = 15; Gamma Dynamics, Inc., Pompano Beach, Fla.; Boston Biomedica, Inc., West Bridgewater, Mass.), systemic lupus erythematosus (n = 16; QCP, Inc., Pompano Beach, Fla.), rheumatoid factor (RF); n = 55; (QCP, Inc.), Epstein-Barr virus (n = 26; Boston Biomedica, Inc.; BioClinical Partners, Inc., Sharon, Mass.; BioMedical Resources, Hatboro, Pa.), parvovirus B19 (n = 6), measles virus (n = 10), herpes simplex virus (n = 12), varicella-zoster virus (n = 13) (Boston Biomedica, Inc.), Hyper IgM (n = 9; Bartek Associates, Inc., Barrington, Ill.; BioClinical Partners, Inc.), and Hyper IgG (n = 8; BioClinical Partners, Inc.) or were from influenza vaccinees (n = 14; Cash Blood Bank, Pompano Beach, Fla.). These specimens were characterized by the vendor by the appropriate methodologies to verify the clinical condition or disease state. RF neutralization reagent (Abbott Manufacturing Inc., Abbott Park, Ill.) was used to neutralize RF antibodies.
(v) Precision panels.
Serum and plasma panels were prepared to evaluate the precision of the AxSYM assay. Four panel members were negative for CMV IgM, four panel members were low positive (index values, ≤1.000) for CMV IgM, and four panel members were positive for CMV IgM. The low-positive and positive serum and plasma panel members were prepared artificially by spiking CMV-negative serum or plasma with CMV IgM-positive serum.
CMV antigen detection.
The method of Wunderli et al. (34
) was used for the detection of the immediate-early antigen in human embryo fibroblasts.
Commercial CMV IgM assays and consensus interpretation.
The assay cutoff and the relative performance characteristics of the AxSYM assay were determined by testing all specimens with three commercial tests (consensus result) for the detection of CMV IgM: the Gull (Salt Lake City, Utah) CMV IgM ELISA, the Trinity Biotech/Centocor (Jamestown, N.Y., and Malvern, Pa.) CAPTIA CMV-M, and the Abbott Laboratories CMV-M EIA. The results obtained by each of the three commercial assays were interpreted according to the manufacturer's guidelines. A specimen interpretation was based upon a consensus result (two of three) of the assays. If the assays had three different results (positive, negative, and equivocal) a consensus specimen interpretation was not possible and the interpretation “none” was used. The consensus interpretation was chosen for this performance evaluation as it had been shown to agree reasonably well with the CMV IgM immunoblot assay result (16
) (data not shown). Specimens that were positive or negative by the AxSYM assay and discordant by the consensus interpretation were further resolved by CMV IgM immunoblot testing (17
Sensitivity and specificity were calculated as described by Griner et al. (6
). Agreement was calculated as follows: (TP + TN)/(TP + TN + FP + FN) × 100, where TP is the number of true-positive specimens, TN is the number of true-negative specimens, FP is the number of false-positive specimens, and FN is the number of false-negative specimens. The 95% confidence interval (CI) determined for relative sensitivity, specificity, and agreement was based on the binomial distribution by using the STATXACT-3 software (SAS Institute, Inc., Cary, N.C.) (21
). A receiver operator characteristic (ROC) analysis was used to assist the determination of the preliminary cutoff for the AxSYM assay (25
). The precision of the AxSYM assay was determined by use of National Committee for Clinical Laboratory Standards protocol EP5-T2 as a guideline (22
). The standard deviation (SD) and percent coefficient of variance (CV) were determined by a variance component analysis for a random-effects model (2
). Negative variance components were set equal to zero.