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Journal of Virological Methods 160 (2009) 167–171

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

erological microarray for detection of HSV-1, HSV-2, VZV,nd CMV antibodies

nne J. Jääskeläinena,b,∗, Kirsi Moilanena, Suvi Bühlerb,1, Maija Lappalainenb,lli Vapalahti a,b, Antti Vaheria,b, Heli Piiparinenb

Department of Virology, Haartman Institute, FI-00014 University of Helsinki, FinlandLaboratory Services, Helsinki University Hospital, FI-00029 Helsinki, Finland

rticle history:eceived 12 February 2009eceived in revised form 8 May 2009ccepted 18 May 2009vailable online 27 May 2009

eywords:erpesvirusntigen

a b s t r a c t

The seroprevalence of human herpesviruses is high and reactivations occur frequently. A microarray wasdesigned and tested for the detection of IgG and IgM antibodies for Puumala hantavirus (PUUV) andIgG antibodies against four herpesviruses. Initially, a microarray platform was set up using an unrelatedin-house antigen, PUUV recombinant nucleocapsid protein, to optimize the protocol for the detection ofantibodies. Detection of the four herpesviruses was set up in a microarray using the recombinant proteinsof herpes simplex virus (HSV) glycoprotein G1 and G2, varicella-zoster virus (VZV) glycoprotein E, andcytomegalovirus (CMV) pp150 phosphoprotein.

The results of the PUUV panel were in good agreement with the PUUV IgG immunofluorescent assay

iagnosisicroarray

erology

and IgM enzyme immunoassay (EIA). Seropositive and negative clinical reference panels were tested forherpesviruses by the serological microarray, and the results were compared to those of individual EIAsused for standard diagnostic purposes.

The serologic microarray for HSV, VZV and CMV antibody detection gave good specificities for IgG. How-ever, sensitivities of the assay varied depending on the herpesvirus detected. The serological microarrayshowed potential for screening purposes. The microarray based analyses were easy to perform, and HSV-1,

ibodi

HSV-2, VZV, and CMV ant

. Introduction

Severe human infections are caused by herpes simplex virusesHSVs), varicella-zoster virus (VZV), and human cytomegalovirusCMV). The first two viruses belong to the subfamily Alphaher-esvirinae and CMV belongs to the subfamily Betaherpesvirinae.he seroprevalence of human herpesviruses is high and reactiva-ions are common (Morris et al., 2002; Pebody et al., 2004; Svahnt al., 2006; Wutzler et al., 2001). The detection of herpesviruseselies on virus isolation, antigen detection, nucleic acid detectionnd also serological methods (Hanson et al., 2007; Mezzasomat al., 2002; Oladepo et al., 2000; Perkins et al., 2007; Sauerbrei

nd Wutzler, 2004; Svahn et al., 2006; Wutzler et al., 2001).fficient large-scale screening of herpesvirus antibodies in differ-nt population groups provides clinically important information.etermination of the specific immunoglobulins is of importance

∗ Corresponding author at: Department of Virology, Haartman Institute, P.O. Box1, FI-00014 University of Helsinki, Finland. Tel.: +358 50 5420743;ax: +358 9 19126491.

E-mail address: ajjaaskelainen@gmail.com (A.J. Jääskeläinen).1 Deceased.

166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2009.05.013

es could be detected on the same microarray.© 2009 Elsevier B.V. All rights reserved.

for obtaining serological proof of previous and current herpesvirusinfections.

The status of previous herpesvirus infections is importantfor immunocompromised patients, young children or preg-nant women, inter alia to determine the need for prophylacticmeasures (McKendrick et al., 2007). For immunocompromisedpatients, primary or recurrent herpesvirus infection could be fatal.Transplantation patients and organ donors are screened for her-pesviruses to ascertain their immunological status, the outcomeof which, can consequently affect clinical decisions. Primary VZVor CMV infection during pregnancy could be fatal or result insevere malformation of the fetus. Serologic tests can be used asa confirmatory test for genital herpes with atypical symptoms,in sexually transmitted disease (STD) and also prenatal clinics(Ashley-Morrow et al., 2003). Prospective studies have shown thatvirtually all patients with HSV-2 antibody have a history of gen-ital herpes (Frenkel et al., 1993; Langenberg et al., 1989; Waldand Ashley-Morrow, 2002). Pregnant women with genital HSV-

1 or HSV-2 infections are at risk of transmitting these virusesto the newborn. Studies have shown that acquiring genital her-pes toward the end of pregnancy results in a 30–50% risk ofneonatal herpes (Brown et al., 1991, 1997). Therefore, it is impor-tant to establish accurate diagnose of HSV infections in pregnant

1 irological Methods 160 (2009) 167–171

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Table 2List of commercial viral recombinant proteins.

Virus Recombinant protein Gene Immunodominant regions aa.

HSV-1 Glycoprotein G GG 84–175

TR

P

GGGG

P

68 A.J. Jääskeläinen et al. / Journal of V

omen, their partners, or STD patients with atypical symptoms.MV infection of a congenitally infected newborn (Arvaja et al.,999; Grosse et al., 2008) can also be a severe or even lethal dis-ase.

Microarray methods have been widely used in microbiology.ucleic acid-based microarrays have been used for the identifi-ation of different pathogens (Wang et al., 2002; Lovmar et al.,003; Klaassen et al., 2004; Boriskin et al., 2004; Jääskeläinen andaunula, 2006). Protein microarrays have also been developed.owever, protein microarrays are still not widely used for sero-iagnosis of viral pathogens (Mezzasoma et al., 2002; Lu et al.,005; Zhu et al., 2006; Burgess et al., 2008). Therefore, a nucleiccid-based microarray method was developed for the diagnosis ofuman herpesvirus infections (Jääskeläinen et al., 2006, 2008). A

urther aim was to study the suitability of microarray platform forerodiagnostic purposes.

. Materials and methods

Serological microarray was designed and evaluated, specificallyor the diagnosis of human herpesvirus infections. Initially, the

icroarray format for serology was tested using an antigen unre-ated to herpesviruses. Puumala hantavirus (PUUV) nucleocapsidN) recombinant protein available and evaluated thoroughly in ouraboratory (Vapalahti et al., 1996; Kallio-Kokko et al., 1998) wassed as a pilot. Both PUUV IgG and IgM antibodies were testedy microarrays. Following the observations of the initial PUUVodel format, highly purified commercial antigens for human her-

esviruses were obtained and serological microarrays were set upor the detection of HSV-1, HSV-2, VZV and CMV IgG antibodiesrom clinical specimens. Specific recombinant proteins, namely:SV-1 glycoprotein G (gG1), HSV-2 glycoprotein G (gG2), VZV gly-oprotein E (gE) and CMV phosphoprotein150 (pp150) (Ashley etl., 1998; Ashley, 2002; Hasan et al., 2002; La Rosa et al., 2005;andini et al., 1991; Wald and Ashley-Morrow, 2002), were useds antigens on the microarray. The recombinant proteins of HSV-1G1 and HSV-2 gG2 have been used in commercial EIAs to distin-uish between HSV-1 and HSV-2, and VZV gE has been found toe efficiently recognized by the human immune system. Moreover,MV pp150 is a protein of viral tegument that has been found to beighly immunogenic.

.1. Clinical specimens

Sixty-one sera were collected from patient serum panels testedor PUUV antibodies (Table 1) at Helsinki University HospitalHelsinki, Finland) during the year 2002. Of these 61 serumamples, 15 were PUUV IgG positive, 30 IgG and IgM positive,ne was IgM positive and 15 were negative for PUUV IgG and

gM.

During 2005 and 2006 the herpesvirus study material, whichncluded four batches of 119, 120, 134, and 132 serum samples wasent to Helsinki University Hospital (Finland) for the analyses ofSV-1, HSV-2, VZV, and CMV IgG antibodies, respectively.

able 1esults from PUUV IFA and EIA compared to results from microarray.

UUV EIA/IFA Microarray results

G+ G− M+

+ M− 15 13 2 0+ M+ 30 24 5 28− M+ 1 0 1 0− M− 15 0 15 0

UUV, Puumala hantavirus; G, IgG; M, IgM; E, equivocal; DSP, diagnostic specificity; DSN,* Equivocal in IgG test.

HSV-2 Glycoprotein G GG 525–578VZV Glycoprotein E ORF68 48–135CMV Phosphoprotein 150 UL32 1011–1048

2.2. Microarray design

Viral antigens of HSV-1 gG1, HSV-2 gG2, VZV gE, and CMV pp150(Table 2) purified by chromatographic methods were purchasedfrom ProsPec-Tany TechoGene Ltd. (Rehovot, Israel). ProsPec-TanyTechoGene Ltd. declare that recombinant proteins are Escherichiacoli derived and >95% pure as determined by 10% PAGE (coomassiestaining). PUUV recombinant N (PUUV-N) was produced and puri-fied as described by Vapalahti et al. (1996). The batch of PUUV-Nwas further purified by Millipore filtration. The viral antigenswere diluted in protein printing buffer solution (TeleChem Inter-national Inc., Sunnyvale, CA). The dilutions were 0.5–1, 0.25–0.50,and 0.05–0.10 �g/�l for PUUV-N. In addition, 10 and 30 �g/�l foreach of HSV-1 gG1, HSV-2 gG2, and CMV pp150. VZV gE antigenswere diluted to 0.1 and 0.3 �g/�l. All dilutions of viral antigen, neg-ative controls of rabbit myosin (Sigma–Aldrich, Helsinki, Finland),and protein printing buffer (TeleChem International Inc.) were spot-ted in duplicate on nitrocellulose-coated FAST slides (Schleicherand Schuell BioScience Inc., Keene, NH) by Biomedicum Genomics(University of Helsinki) using a microarrayer (OmniGrid®, GeneMa-chines, Huntingdon, UK). Ten-fold dilutions of human IgM and IgG(Sigma–Aldrich) were used as positive controls on the microar-ray.

2.3. Microarray reactions

In total, there were 21 separate sequence arrays on the microar-ray, which were each circled carefully using Mini Pap Pen (ZymedLaboratories Inc., South San Francisco, CA). After rinsing with PBS,the microarrays were blocked by immersion in a blocking solu-tion [2% bovine serum albumin (BSA, Sigma–Aldrich) in PBS] for90 min.

2.4. PUUV and human herpesvirus assays

PUUV IgG and IgM detection were both carried out usingmicroarray. The assay protocol for IgM detection differed from thatof IgG detection by using 1 �g/ml of Alexa 647-labeled goat anti-human IgM (Molecular Probes Europe BV, Leiden, The Netherlands)in PBS instead of 1 �g/ml Alexa 546-labeled goat anti-human IgG

(Molecular Probes Europe BV).

For human herpesviruses, IgG detection was performed usingmicroarray. IgG antibodies were detected using 1 �g/ml Alexa 546-labeled goat anti-human IgG (Molecular Probes Europe BV) in PBS.

Specificity and sensitivity %

M− E DSP DSN

15 0 100% 84.1% IgG2 1*

1 0 100% 90.6% IgM15 0

diagnostic sensitivity.

A.J. Jääskeläinen et al. / Journal of Virological Methods 160 (2009) 167–171 169

Table 3Results from herpesvirus EIAs compared to microarrays, and calculated diagnostic specificities and sensitivities.

EIA, no of samples Microarray resultsG+ G− E DSP DSN

HSV-1 G+ 62 56 5 1 80.4% 91.8% HSV-1 IgGHSV-1 G− 57 11 45 1HSV-2 G+ 24 15 9 – 95.8% 62.5% HSV-2 IgGHSV-2 G− 96 4 92 –VZV G+ 110 97 9 4 72.7% 91.5% VZV IgGVZV G− 24 6 16 2CC

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MV G+ 89 73 11MV G− 43 4 38

, IgG; E, equivocal; +, positive; −, negative; DSP, diagnostic specificity; DSN, diagno

.5. Microarray assay

Serum samples were diluted (1:200) in blocking solution, andhen 10 �l of the diluted serum samples were transferred to sep-rate arrays/wells and incubated at room temperature for 30 min.he serum dilutions were removed by rinsing followed by longerashing steps of 3 times for 3 min, in washing buffer [0.2% Tween0 (Fluka, Sigma–Aldrich Chemie GmbH, Buchs SG, Switzerland) inBS]. After this the microarray was rinsed with PBS before addingither the labeled goat anti-human IgG or the IgM in PBS. Afterncubation at room temperature for 15 min, the washing steps wereepeated then followed by a final rinsing in sterile water. Finally, theicroarray was placed in 50 ml tubes and had the moisture driven

ff by centrifuging them at 1300 × g for 3 min.

.6. EIA and immunofluorescent assays

PUUV IgG and IgM were tested by immunofluorescent assayIFA) and �-capture EIA, respectively (Kallio-Kokko et al., 1998,001).

HerpeSelect® HSV-1 IgG and HSV-2 IgG ELISA kits (Focus Diag-ostics Inc., Cypress, CA), and EIAgen HSV IgM test (Adaltis Italia.p.A., Casalecchio di Reno, Italy) were used for HSV antibody detec-ions. VIDAS CMV-IgG and -IgM Kits (bioMérieux sa, Marcy l’Etoile,rance) and EIAgen varicella-zoster IgM test (Adaltis Italia S.p.A.,)ere used for CMV and VZV antibody detection, respectively. All

IAs were performed according to the respective manufacturers’nstructions. A validated and accredited in-house sandwich-EIALaboratory Services, Helsinki University Hospital, Helsinki, Fin-and) was used for VZV IgG testing. It used VZV glycoprotein EIAntigen (Institute Virion Ltd., Rüschlikon/Zürich, Switzerland) ashe test antigen.

.7. Scanning and analyzing the microarray

Microarrays were analyzed using a ScanArray Express scanner,canArrayTM and QuantArrayTM software (PerkinElmer, Wellesley,A). Coefficient values were determined for each antigen from

epetitive results of positive and negative serum samples obtainedrom microarray (data not shown). The coefficient value for PUUV-Necombinant protein after extra Millipore filtration was 10, whereasor highly purified herpesvirus antigens it was 3 for HSV-1 gG1,SV-2 gG2, CMV pp150, and 2.3 for VZV gE. The coefficient valueas increased when the antigen contained more impurities, which

lso increased the signal intensities. This was also the case evenhen negative sera were used.

The cut-off value for each sample was calculated by multiply-

ng the mean value of intensities of the negative control spotsf the array by their respective coefficient values. Background-orrected intensities from antigen-presenting spots were comparedo the cut-off values. Intensity within +/− 10% of cut-off valueas considered an equivocal result, values over the equivocal level

5 90.5% 86.9% CMV IgG1

ensitivity.

were positive and values below equivocal were taken as nega-tive.

Diagnostic specificity (DSP) of the assay was calculated as:[TN/(TN + FP)], where TN = true negatives and FP = false positives.Diagnostic sensitivity (DSN) of the assay was calculated as:[TP/(TP + FN)], where TP = true positives and FN = false negatives.

3. Results

3.1. Protocol testing with PUUV-N recombinant protein

Sixty-two specimens tested previously by PUUV-N IgG IFA andIgM �-capture EIA were tested by the microarray method usingPUUV-N recombinant protein as the antigen (Table 2). Within thisstudy material the DSP was 100% for each of IgG and IgM. The DSNwas 84.1 and 90.6% for IgG and IgM, respectively.

3.2. Herpesvirus-specific IgG tests

The IgG antibodies were detected in these serum samples byserological herpesvirus microarray and commercial or in-house EIAkits for HSV, VZV, and CMV IgG detection (Table 3), respectively.Commercial EIA kits for HSV, VZV, and CMV IgM detection werealso used to obtain more background information.

The respective DSP and DSN were 80.4 and 91.8% for HSV-1 IgG,and 95.8 and 62.5% for HSV-2 IgG (Table 3). There were 13 HSV-1and HSV-2 IgG positive samples for the EIA method, and of these 9gave concordant results with the microarray. Three out of 4 samples,that gave discrepant results with the microarray, were only positivefor HSV-1 IgG and the one remain sample being HSV-2 IgG positive.The respective DSP and DSN were 72.7 and 91.5% for VZV IgG, and90.5 and 86.9% for CMV (Table 3).

4. Discussion

In the clinical laboratory, several standard sequential EIAs areperformed for specific herpesvirus antibody detection. Thereforethe successful combination of preferred assays on one platform andsimultaneous detection of several different herpesvirus antibodiesshould result in an efficient and fast method for screening purposes.A microarray method was chosen as being appropriate to achievethis goal and therefore the applicability of serological microarrayswas tested. Briefly, serological microarrays were designed, evalu-ated and compared against the commercial and in-house accreditedEIA methods used in clinical laboratories to assess the applicabilityof the microarray method in serology.

There is a special need to detect antibodies for several human

herpesviruses simultaneously because one herpesvirus infectioncan reactivate another or several can be reactivated simultaneously.This was also seen when we used microarrays for the detection ofDNA of eight different human herpesviruses in the plasma samplesobtained from immunocompromised patients and in the cere-

1 irolog

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70 A.J. Jääskeläinen et al. / Journal of V

rospinal fluid (CSF) samples of patients suspected of having viralncephalitis (Jääskeläinen et al., 2006, 2008). Although the detec-ion of herpesvirus DNA in the CSF is the method of choice, theerologic testing of CSF may still give specific diagnosis in prolongednfections.

In our study, different elution fractions acquired from the purifi-ation of PUUV-N recombinant protein were used in the microarrayor single virus antibody detection from the pretested serum sam-les. The results from PUUV IgG and IgM antibody testing byicroarrays gave excellent specificities and good sensitivities. We

ound that the purity of the recombinant protein had an impact onhe cut-off value. This was because the labeled anti-human anti-ody might have attached to these impurities in the absence ofuitable human antibodies against antigen with which to combine.herefore, serologically-based microarray for HSV-1, HSV-2, VZV,nd CMV IgG antibodies were set up using commercial high-purityecombinant proteins.

The specificities of the microarray method for screening forSV-1, HSV-2, VZV and CMV IgG were 80.4, 95.8, 72.7, and 90.5%,

espectively. The specificity for HSV-1 IgG was 80.4% due to 11 newositives out of 57 negative for HSV-1 IgG. Of these 11, five had HSV

gM antibodies which were detected by EIA in a clinical laboratorydata not shown). This finding may indicate that the microarrayould have detected possible IgG antibodies earlier than by the

ommercial EIA tests. If this likelihood is taken into account the DSPould rise to 88.2%. A seroprevalence for VZV of over 80% has been

alculated for Finnish children, and has been shown to increase tover 95% in adults (Aarnisalo et al., 2003; Nardone et al., 2007).ue to this high seroprevalence, it was difficult to obtain VZV IgG-egative patients samples thus only 24 were found. Therefore aouple of discriminations will impact on the results.

The sensitivities of the microarray for screening of HSV-1,SV-2, VZV and CMV IgG were 91.8, 62.5, 91.5, and 86.9%, respec-

ively. Apart from that reported for HSV-2, the sensitivity figuresan be considered good. Regarding the low sensitivity of HSV-2ata, several factors were considered. According to the manufac-urer (Prospec-Tany Technogene Ltd.), the recombinant protein wasested and found to be immunoreactive with the sera of HSV-nfected individuals. Antigens from different manufacturers mightesult in differences in sensitivity and specificity. A recombinantrotein can also lose its tertiary folding of the active form duringicroarray spotting and storage, thereby losing some of its activ-

ty and hence the ability to bind to a specific antibody. Anotherossibility could be that the glycoprotein G per se is not the best pos-ible antigen for efficient antibody detection due to the gG deficientSV-2 strains (Norberg et al., 2007).

This microarray method showed potential to be a fast screen-ng test for the detection of PUUV IgG and IgM from patients withephropathia epidemica, and positive for HSV, VZV and CMV IgG.herefore, it can be considered to provide patients’ immunologicalistory expeditiously. The time to complete simultaneous detectionf IgG antibodies against HSV-1 and -2, VZV, and CMV from only 1 �lf patient sample was approximately 3 h by the microarray. In con-lusion, this serological microarray method for HSV, VZV and CMVgG detection gave good specificities and good sensitivities with thexception of the low sensitivity for HSV-2. Several antigens shoulde tested in parallel in future in microarrays to increase sensitivitynd specificity further. New recombinant proteins should be espe-ially sought and produced for HSV-1 and HSV-2 to discriminateetween these viruses.

cknowledgements

This work received support from Helsinki University Hospi-al, Laboratory Services (Helsinki, Finland), the Orion Foundation

ical Methods 160 (2009) 167–171

(Espoo, Finland), the Instrumentarium Foundation (Helsinki, Fin-land) and Finnish National Technology Agency, Tekes. Outi Monniand Harriet Viitalahti (University of Helsinki) are thanked for spot-ting the microarrays, and Susanna Smura and Maarit Ryhänen forperforming the herpesvirus ELISA/EIA tests.

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