The aim of this study was to set up an easy screening tool for herpesvirus antibodies. A successful combination of preferred immunological assays on one platform and simultaneous detection of several herpesvirus antibodies is the goal. A microarray method was considered appropriate for achieving this goal and so a serological microarray was designed (V). Briefly, a microarray was designed, evaluated and compared to the commercial and in-house accredited EIAs used in daily diagnostics by HUSLAB (Helsinki, Finland).
The PUUV-N recombinant protein was first immobilized on a microarray for single virus antibody detection from previously tested serum samples. The results from PUUV IgG and IgM antibody testing gave excellent specificities and sensitivities. The specificities were 100% for both PUUV IgG and IgM, and the sensitivities were 84.1% and 90.6% for PUUV IgG and IgM, respectively.
The high level of purity of recombinant proteins was found to be essential for accurate screening. Therefore, a serological microarray for HSV-1, HSV-2, VZV, and CMV antibodies was set up using commercial high-purity recombinant proteins and tested with patient samples. The HSV G glycoproteins gG1 for HSV-1 and gG2 for HSV-2, which have been used in serological assays to distinguish between HSV-1 and HSV-2, were used as antigens for HSVs in microarrays [Ashley 2002, Ashley et al. 1998, Wald, Ashley-Morrow 2002]. VZV gE, which has been found to be efficiently recognized by the immune system, and CMV pp150, a protein of viral tegument that has been found to be highly immunogenic, were immobilized on the microarray [Fields BN, Knipe DM & Howley PM 1996; La Rosa et al. 2005, Landini et al. 1991].
The specificities for HSV-1, HSV-2, VZV and CMV IgG were 75.9%, 94.7%, 82.6%, 81.4%, respectively (Table 5). The specificity of HSV-1 IgG was below 80% because the microarray method detected 14 new positives for HSV-1 IgG out of 58 negative with EIA. Of these 14, five had HSV IgM antibodies as determined by EIA, which may indicate that microarray detected possible IgG antibodies earlier than commercial EIA. The sensitivities for HSV-1, HSV-2, VZV and CMV IgG were 93.3%, 65.2%, 76.1%, and 83.0%, respectively (Table 5). These sensitivities, except for HSV-2, could be considered to be good.
TABLE 5. Results from herpesvirus-specific EIAs compared to microarrays, and calculated diagnostic specificities and sensitivities.
Herpesvirus EIA, no
of samples MICROARRAY Results G+ G- E DSP DSN
HSV-1 G+ 49 44 4 1 75.9% 93.3% HSV-1 IgG
HSV-2 G+ 10 5 5 -
HSV-1, and -
2 G+ 13 13 of which 9* double and 3 HSV-1 pos,
1 HSV-2 pos - - 94.7% 65.2% HSV-2 IgG HSV-1 G- 58 14 44 - HSV-2 G- 95 5 90 - M+ M- E DSP DSN HSV M+ 15 4 11 - 95.9% 26.7% HSV IgM HSV M- 98 4 94 - HSV M E 1 1 G+ G- E DSP DSN VZV G+ 110 89 18 3 82.6% 76.1% VZV IgG VZV G- 23 4 19 - M+ M- E DSP DSN VZV M+ 58 46 6 6 43.5% 88.5% VZV IgM VZV M- 69 39 30 - VZV M E 6 2 2 2 G+ G- E DSP DSN CMV G+ 91 73 15 3 81.4% 83.0% CMV IgG CMV G- 43 8 35 - M+ M- E DSP DSN CMV M+ 14 12 2 - 99.1% 85.7% CMV IgM CMV M- 118 1 113 4 CMV M E 2 2 -
G, IgG; M, IgM, E, equivocal; +, positive; -, negative; DSP, diagnostic specificity; DSN, diagnostic sensitivity.
*Nine samples had dual antibodies against HSV-1 and -2.
Good specificities for HSV and CMV IgM (95.9% and 99.1%, respectively) were achieved but the specificity for VZV IgM was only 43.5% (Table 5). Sixty-nine samples were VZV-IgM negative by immunoassay but 39 of these samples were positive according to the microarray. However, 31 out of the 39 samples had high levels of VZV-IgG antibodies, and it could be speculated that the false positives might be due to IgG adsorbing before IgM in the microarray. Sensitivities of the microarray method for HSV, VZV and CMV IgM varied being 26.7%, 88.5%, and 85.7%, respectively (Table 5).
The EIA for HSV IgM detection used in HUSLAB (Helsinki, Finland) does not differentiate the HSV-1 and HSV-2 IgM antibodies. With the microarray, HSV-1 and HSV-2 antibodies were both detected, and differentiation was achieved when IgG antibodies for both were present in the sample. The
sensitivity of the HSV-2 IgG was only 65.2% with the microarray. The lower sensitivity of HSV IgM could be due to a failure to properly detect that the sample was positive for HSV-2 IgM. According to the manufacturer (Prospec- Tany Technogene Ltd.), the recombinant protein was immunoreactive with sera of HSV-infected individuals. However, the recombinant protein can be denaturated during microarray spotting and storage, thereby losing some of its activity and ability to bind specific antibody. One possibility could also be that glycoprotein G is not the best possible antigen for efficient antibody detection due to the gG deficient HSV-2 strains [Nordberg et al. 2007]. At the moment, the commercial kits contain other antigens such as UV-light inactivated HSV-1 and HSV-2 particles, which are used in HSV IgM tests (Adaltis Italia S.p.A).
The sensitivities and specificities of our assay were promising, but some optimization is still needed to achieve a reliable serological tool for diagnosis. However, this microarray method might be used as a fast screening test for detection of IgG antibodies to HSV-1, VZV and CMV, in order to provide an immunological history. The herpesvirus-specific EIAs are often quantitative assays and used to monitor antibody levels of sample. Accurate EIAs are commercially available, but when used as a screening tool they could be laborious, expensive and sample-consuming. Some specific EIAs even need 100 l of sample, e.g. the VIDAS-system (bioMérieux sa) for CMV antibody detection. The microarray method uses sample efficiently, and only 1 l is needed for simultaneous detection of different antibodies. Microarrays cannot not yet be considered a highly reliable quantitative method, although some published studies of viral antibody detection have been carried out [Lu et al. 2005; Mezzasoma et al. 2002]. However, as a screening tool, the microarray could be a superior method. After screening, some of the results can be confirmed by specific EIA to reveal quantitative antibody levels for diagnosis of acute infections. This could potentially reduce the amount of expensive EIAs used in daily diagnostics.