Alphaherpesvirus antigen quantitation to optimize the diagnosis of herpes B virus infection

Journal of Virological Methods 103 (2002) 15–25

Alphaherpesvirus antigen quantitation to optimize thediagnosis of herpes B virus infection

David Katz *, Wei Shi, Peter W. Krug, Julia K. HilliardDepartment of Biology, Viral Immunology Center, Georgia State Uni�ersity, 50 Decatur Street, Atlanta, GA 30303, USA

Received 5 June 2001; received in revised form 11 September 2001; accepted 11 September 2001

Abstract

Standardized, quantified virus antigen stocks are essential for dependable quality control of diagnostic assays. Fivesimple, rapid and economical direct enzyme linked immunoassays (dELISA) were developed to standardize andoptimize antigen from five major cross-reacting alphaherpesviruses: herpes B virus, herpesvirus papio 2, langurmonkey herpesvirus, herpes simplex virus-1 and herpes simplex virus-2. Each dELISA relied on pools of convalescentsera from rhesus monkeys, baboons, langurs and humans. Conjugates were prepared from purified IgG preparations,fractionated from the same sera and then labeled with peroxidase. Serum coated microplates could be stored at−70 °C for at least 1 year before use. The duration of the test was �2.5 h if plates were prepared at an earlier time.Virus antigen titers could be determined from titration curves or from single dilutions using a standard curve. Thesensitivity of detection was �8×105 PFU/ml. This sensitivity sufficed for the determination of viral antigen mass inlive or detergent treated virus stocks that usually contain at least 1×108 PFU/ml. The assays were valuable forquality assurance of diagnostic serological assays for herpes B virus and other alphaherpesviruses. © 2002 Publishedby Elsevier Science B.V.

Keywords: Alphaherpesviruses; Herpes B virus; Quantitative enzyme immunoassay; dELISA

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1. Introduction

Herpes B virus (BV) (Cercopithecine herpes�irus1), whose natural hosts are macaques (Macacaspp.), is the only known monkey herpesvirus toinfect and cause fatal disease in man. Otherviruses, closely related serologically to this group,are herpesvirus papio 2 (HVP-2) (Cercopithecineherpes�irus 16), whose natural hosts are baboons

(Papio spp.), Simian Agent 8 (SA8) (Cercop-ithecine herpes�irus 2), whose natural hosts areAfrican green monkeys (Cercopithecus aethiops)and the two human herpesviruses, herpes simplex-1 (HSV-1) (Human herpes�irus 1) and herpes sim-plex-2 (HSV-2) (Human herpes�irus 2). Anadditional alphaherpesvirus from langurs (Pres-bytis spp.) was isolated recently, but was notclassified officially and named (Eberle andHilliard, 1995). In this communication, this viruswas named provisionally ‘herpesvirus langur’ orHVL.

* Corresponding author. Tel.: +1-404-651-4098; fax: +1-404-651-0821.

E-mail address: [email protected] (D. Katz).

0166-0934/02/$ - see front matter © 2002 Published by Elsevier Science B.V.

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D. Katz et al. / Journal of Virological Methods 103 (2002) 15–2516

Current serological methods for the diagnosisof herpes B virus infections and differentiationfrom other alphaherpesviruses include enzyme-linked immunoassays (ELISA) and Western blots(WB). These methods use immobilized virus-in-fected cell lysates as antigens to detect antibodiesin monkey or human sera. The methods are effec-tive without any modification for diagnosing her-pes B virus infection in macaques, since thisspecies is infected naturally only by herpes B virusand not by any other recognized viruses of theSimplexvirus genus in the alphaherpesvirussubfamily.

Differential diagnosis of alphaherpesvirus infec-tions is complicated in monkeys other thanmacaques or humans that may be infected inad-vertently with herpes B virus and may have co-ex-isting antibodies to their alphaherpesvirusesoccurring naturally (Van Hoosier and Melnick,1961; Kalter et al., 1978). In these cases, identifi-cation of herpes B virus specific antibodies iscomplex, but possible by using comparative WBand competition ELISA (cELISA) techniques(Katz et al., 1986a,b; Hilliard and Ward, 1999).For quality control purposes, alphaherpesvirusantigen optimization for ELISA and WB assayshave been carried out by tedious box-titrations.

Antigen detection immunoassays similar tothose described in this paper have been used fornearly two decades (Adler-Storthz et al., 1983;Clayton et al., 1985; Verano and Michalski,1995). In some cases, the assays have been re-placed by the more sensitive polymerase chainreaction (PCR), once developed for a specificvirus (Slomka et al., 1998). The main objective ofthis study was to develop simple quantitativeanalysis of ‘virus-antigen capture’ type of directenzyme linked immunoassays (dELISAs) for qual-ity control and standardization of the antigensused in serological assays. The same principle wasused for all dELISAs. ‘Capture antibodies’ forcoating plates and IgG for preparation of theperoxidase-IgG conjugates were all derived fromsera of naturally infected hosts. Five dELISAswere constructed for five of the six major cross-re-acting herpes viruses, which are currently used fordiagnosis, i.e. BV, HVP-2, HVL, HSV-1 andHSV-2. A dELISA for SA8 was not developed

because of difficulties obtaining sufficient anti-body positive sera from African green monkeys.Data describing development steps and perfor-mance of the five dELISAs, along with examplesfor their potential use in diagnosis of BV andother alphaherpesvirus infections, are presented.

2. Materials and methods

2.1. Preparation of antibody positi�e pools toalphaherpes�iruses

Herpes B antibody positive sera (anti-herpes Bvirus) were collected from rhesus monkeys (M.mulatta), herpesvirus papio 2 antibody positivesera (anti-herpesvirus papio 2) from baboons, her-pesvirus langur antibody positive sera (anti-her-pesvirus langur) from langurs and HSV-1 andHSV-2 antibody positive sera (anti-HSV-1 andanti-HSV-2, respectively) from humans. Sera frommacaques, baboons and humans were titrated bytesting serial 2-fold dilutions of individual sera intELISA (an antibody capture immunoassay)against homologous herpesviruses, essentially asdescribed previously (Katz et al., 1986a,b). Thetiter in ELISA units per 50 �l test sample (EU)was defined as the reciprocal dilution of a serumresulting in an optical density value (OD) thatwas equal to a cutoff value. The cutoff value usedwas twice the background OD value. The back-ground OD value was determined from the reac-tion of an antibody negative control serum at a1:100 dilution against the viral antigen. Sera withtiters of �10,000 ELISA EU were pooled. Thelangur serum pool was considerably smaller be-cause it was prepared from only five animals. Allother serum pools were prepared from 20 animals,at least.

2.2. Preparation of IgG fractions fromanti-herpes serum pools

IgG fractions were prepared from the anti-her-pes serum pools (anti-herpes B virus, anti-her-pesvirus papio 2, anti-hepesvirus langur,anti-HSV-1 and anti-HSV-2) using a similarpurification procedure for each of the sera. IgG

D. Katz et al. / Journal of Virological Methods 103 (2002) 15–25 17

was prepared from the serum pools by affinity-chromatography on protein-G columns, essen-tially as recommended by the manufacturer(Pharmacia, Sweden).

In a typical purification procedure, 6 ml of thedialyzed serum were treated in two consecutivepurification steps on a 1 ml ‘HiTrap’ protein Gcolumn. Bound IgG was eluted from the columnby 0.1 M glycine buffer, pH 2.7. Protein-richfractions from the two (or more purification steps)were pooled. Qualitative determination of theamount of protein removed from the protein Gcolumn after washing and elution steps was ac-complished by spotting 2 �l of the tested solutionon nitrocellulose paper, air drying for 5 min andimmersing in a 1% Ponceau red solution (Sigma,USA). After a few minutes, the paper was washedin distilled water. Red dots indicated the presenceof protein. Protein-IgG in the pooled fractionswas quantitated by reading the optical density ofthe pool at a wavelength of 280 nm. Proteinamount was based on the formula using the ex-tinction coefficient of the IgG at this wavelength(1 mg of IgG=1.4 optical density (OD) units).

2.3. Estimation of primate IgG binding to NUNCmicroplates by ELISA

ELISA procedures were essentially as describedfor tELISA (Section 2.1), except that instead ofcoating plates with viral antigens, plates werecoated with primate IgG or primate whole sera.Purified IgG preparations (50 �l) or whole pri-mate sera from which the IgG was purified wereadsorbed at different concentrations or dilutions(respectively) to plastic wells of NUNC mi-croplates. Adsorption was performed by incuba-tion overnight at room temperature in coveredplates in a humid chamber. After washing, a goatanti-human IgG peroxidase conjugate (Sigma)was added to the wells. Due to the high cross-re-activity between human and non-human primateIgG, this conjugate detected non-human primateIgG with high efficiency. After an additional threewashes, 100 �l of the 3,3�, 5,5�-tetramethylben-zidine (TMB) substrate (Sigma) was added toeach well for 20 min at room temperature. Thecolorimetric reaction was stopped and enhanced

by adding 50 �l of 1.0 M sulfuric acid to each ofthe TMB containing wells. Color intensity, inoptical density (OD) units, was evaluated in amicro-ELISA reader at a wavelength of 450 nm.Titration curves were constructed by plotting theOD×1000 values versus the log10 of IgG concen-trations or serum dilutions.

2.4. Preparation of IgG-horseradish peroxidaseanti-herpes�irus conjugates

The IgG pools obtained by protein-G affinity-chromatography were first concentrated using the‘Slide-A-Lyzer’ concentrating solution (Pierce,USA). The IgG concentrate was then dialyzed at4 °C for 24 h against 0.2 M carbonate–bicarbon-ate buffer, pH 9.3.

Labeling of IgG isolates with horseradish per-oxidase was performed using the HRP-EZ-Link™Plus Activated Peroxidase kit, according to themanufacturer’s protocol (Pierce).

Typically, 5 ml of IgG, at a concentration of 1mg/ml was labeled in each session. The reactionwas stopped by adding sodium cyanogen bromideand quenched by diethanolamine, according tothe manufacturer’s instruction. After dialysisagainst 2 l of phosphate buffered saline, pH 7.2(PBS) for a total of 48 h at 4 °C, IgG-peroxidaseconjugates were mixed with an equal volume ofglycerol and stored at −20 °C.

2.5. Standard herpes �irus antigens and uninfectedtissue culture control antigens

Herpes B virus (E2490), herpesvirus papio 2(X2980), herpesvirus langur (JKH), Simian agent8 (B264), HSV-1 (KOS) and HSV-2 (186) stockswere prepared in African green monkey kidney(vero) cell lines (ATCC) and grown in Dulbecco’sModification of Eagle’s Medium, supplementedwith 5% fetal calf serum. Infectivity titers weredetermined by a standard plaque assay. Antigenswere prepared by detergent solubilization usingTween40 and sodium deoxycholate (T/DOC), aspreviously described (Katz et al., 1986a). Controlantigens from mock-infected vero cell cultureswere prepared in an identical way. Antigens werestored in aliquots frozen at −70 °C.


2.6. dELISA reagents and general procedures

Coating of microtiter plates (Polystyrene-96-wells, MaxiSorb, Nunc, Denmark) with captureantibodies was carried out by adding 50 �l perwell of a predetermined optimal dilution (in PBS)of the anti-alphaherpesvirus serum. The plateswere incubated for 24 h at room temperature in ahumid chamber. After discarding the fluid fromthe wells, blocking buffer (2.5% dry skim milk(BLOTTO), DIFCO, USA in PBS) was added for60 min at 37 °C. Plates were then washed twicewith washing buffer, PBS+0.05% Tween (PBST)and stored dry at −70 °C, until used (up to 1year).

Before use, stored plates were washed once withPBST. For virus antigen titration, a series of six,2-fold dilutions in PBS, was prepared. Typically,herpes B virus dilutions started at 1:100 (all otherviruses at 1:25). Duplicate aliquots (50 �l) weretransferred to the coated wells. For determinationof background values, 50 �l of a 1:25 dilution ofthe negative control antigen were added to sixantibody-coated wells. The plate was then incu-bated (covered in a humid chamber) for 60 min at37 °C. After incubation, wells were washed threetimes with PBST. An optimal IgG-horseradishperoxidase homologous conjugate dilution (1:50)in 2.5% BLOTTO in PBST (50 �l) was then addedto the wells and incubated for another 60 min at37 °C.

After an additional three washes, 100 �l ofTMB substrate (Sigma) were added to each wellfor 20 min at room temperature. The colorimetricreaction was stopped and enhanced by adding 50�l of 1.0 M sulfuric acid to each of the TMBcontaining wells. Color intensity in OD units wasevaluated in a micro-ELISA reader at a wave-length of 450 nm. Titration curves were con-structed by plotting the OD×1000 values orpreferably their log (10) value versus the log (10)of the reciprocal of the dilution values. Onlyvalues within the linear part of the curve wereused. The antigen titer in ELISA units per 50 �ltest sample (EU) was determined from the titra-tion curve as the reciprocal of the dilution at thecutoff value. The cutoff value was calculated fromsix negative control wells as the mean OD×1000background value+3 S.D. of the mean.

Titers of ‘unknown’ samples that were run atone single dilution with the standard antigenstock could be determined from a ‘standardcurve’, provided that the sample generated an ODvalue within the linear range of the curve. Thestandard curve was constructed by plotting thelog10 EU values (on abscissa) versus the log10

OD×1000 values. The standard curve wasderived from the titration curve of the standardantigen. Log10 EU values were calculated by di-viding the titer of the standard antigen by thereciprocal of the dilutions that were used for thetitration. For determination of the final titer of asample, the dilution factor was taken intoaccount.

Determination of titers from titration or stan-dard curves could be done graphically or by usingthe linear regression function in Microsoft Excel(Microsoft Corporation, USA).

3. Results

3.1. IgG purification from anti-herpes serum poolsand preparation of peroxidase-IgG conjugates

IgG fractions were purified from sera byaffinity chromatography on protein-G affinitycolumns. Efficacies of IgG purification and anti-body yields were determined for four of the anti-herpes serum pools, anti-herpes B virus,anti-herpesvirus papio 2, anti HSV-1 and antiHSV-2. Titers for each of the four respectivehomologous virus-antibodies (herpes B virus, hep-esvirus papio 2, HSV-1 and HSV-2, respectively)were evaluated by tELISA titrations. The relativeantibody specific activity of each IgG preparationwas determined by relating the antibody titer inELISA units (EU) to the amount of protein (mg)in each preparation. Efficacy analysis was notcarried out for herpesvirus langur reagents be-cause of the low number of serum samplesavailable.

The serum pool titers obtained were of equalorder of magnitude, ranging from 15,000 to58,000 EU/50 �l (34,153 EU/50 �l�17,993 EU/50 �l). This titer range was typical of convalescentsera, as opposed to the titer of sera from hyper-


immunized animals that are usually two order ofmagnitudes higher.

The antibody specific activity of all four IgGpreparations were also similar, ranging from44,000 to 88,000 EU/mg (67,420 EU/mg�19,820EU/mg). The purification yield, namely the per-centage of serum antibodies present in the IgGfraction, was also similar in all four preparations,ranging from 31 to 54% (45�10%). The averageamount of IgG obtained from 1 ml serum was4.3�1.0 mg.

The antibody activity in EU per milligram ofIgG was calculated for the four preparations be-fore and after labeling with peroxidase. The per-centage of IgG antibodies found in the anti-herpesB virus, anti-herpesvirus papio 2, anti-HSV-1 andanti-HSV-2 peroxidase-conjugates, was 45, 53, 79and 80%, respectively. These results indicate somelosses or inactivation of the antibodies that mayhave been caused by the conjugation procedure.

Each of the five antibody conjugates was ti-trated directly on homologous antigen and con-trol antigen in ‘tELISA antigen coated plates’.Net OD values were calculated by subtracting thebackground values of the negative control antigen

from the values obtained on the homologous anti-gens. As shown in Fig. 1, the lowest conjugatedilution resulting in OD values that were withinthe linear section of all titration curves was 1:30.It was therefore decided to use conjugate dilutionsof 1:50 for all dELISA titrations.

3.2. Comparison of adsorption to NUNCmicroplates of IgG from purified preparationscompared to IgG from whole sera

The source of capture antibodies for coatingthe microplate wells for the dELISA could bepurified IgG preparations or whole sera. IgGpreparations were assumed superior to whole serafor coating plates because of lower protein con-centration and relatively higher antibody concen-tration. The amount of IgG that was needed tosaturate the NUNC microplate wells was evalu-ated by an ELISA for the detection of human IgG(Section 2.3). Purified IgG preparations from fourpools of antisera (rhesus anti-herpes B virus, hu-man anti-HSV-1, human anti-HSV-2 and baboonanti-herpesvirus papio 2) were adsorbed at differ-ent concentration to plastic wells of NUNC

Fig. 1. ELISA titrations of five anti-herpesvirus-peroxidase conjugates on homologous herpesvirus antigens (anti-herpes B virus-POon herpes B virus, anti-HSV1-PO on HSV-1, anti-HSV2-PO on HSV-2, anti-herpesvirus papio 2-PO on herpesvirus papio andanti-herpesvirus langur-PO on herpesvirus langur). Net OD values were calculated by subtracting background values from valuesobtained following reaction of viral antigens with the conjugates.


Fig. 2. Titration curves of herpes B virus, herpesvirus papio 2, herpesvirus langur, HSV-1 and HSV-2 antigens in their correspondentdELISAs.

plates. For comparison, dilutions of the wholesera from which the IgG was purified were alsoadsorbed to plastic wells. The results indicated anadsorption saturation of purified IgG starting atconcentrations within 1–2 �g/ml, but graduallyincreasing up to 10 �g/ml. The IgG adsorptionfrom whole sera reached a saturation plateau at aserum dilution of 1:20,000.

Since the average IgG concentration in humanand monkey sera is �10 mg/ml, the estimatedIgG serum concentration that adsorbs to the plas-tic wells at saturation was �0.5 �g/ml (10 mg/20,000). The approximate amount of IgGadsorbed to the plastic wells from whole serumwas therefore two to four times lower than theamount adsorbed from purified IgG preparations.

3.3. Optimizing antigen capture conditions fordELISA

The capacity of purified IgG preparations andwhole sera to serve as capture antibodies for theircorresponding viruses was tested using the fourviral antigens (herpes B virus, herpesvirus papio 2,

HSV-1 and HSV-2). IgG preparations and serawere adsorbed to microplate wells at differentconcentration or dilutions, respectively, as de-scribed above. Each of the four T/DOC treatedviral antigens at a 1:50 dilution were then tested,as described in Section 2.5. Background valueswere determined by parallel testing of a 1:50dilution of the negative antigen control. Resultsare briefly summarized below. Maximal antigenbinding on the IgG coated plates was obtainedwith 10 �g/ml IgG. The maximum antigen bind-ing activity of the sera was approximately at a1:20,000 dilution. Wells coated with purified IgGpreparations yielded higher OD values than wellsthat were coated with sera. However, there was noclear advantage of using purified IgG reagents forcoating wells, since the background in these wellswas also higher. It was therefore decided to usethe economical whole sera for coating plates in allfuture dELISA experiments. The dilution chosen(1:10,000) was two times lower than the dilutionat saturation.

Results of dELISA titrations for all five alpha-herpesviruses antigens (herpes B virus, herpesvirus


papio 2, herpesvirus langur, HSV-1 and HSV-2) inwhich 1:10,000 antiserum dilutions were used forcapture antibodies are shown in Fig. 2. A rela-tively higher (OD) reactivity was observed forherpes B dELISA. In addition, the slope of theherpes B titration curve was steeper than theslopes of all other titration curves. The cutoffvalues (background+3 S.D.) were relativelyhigher for HSV-1 and HSV-2 (120 and 100 OD×1000, respectively) than for herpesvirus papio 2,herpes B virus and herpesvirus langur (80, 60 and40, respectively). By using cutoff values, the titersfor each individual virus was estimated from thetitration curves to be 2000 EU for herpes B virus,2000 EU for herpesvirus papio 2, 900 for HSV-1,800 EU for HSV-2 and 800 EU for HVL. Asexpected, titer values were not only affected by thecolor intensity produced in the assay, but also bythe slope of the curves and background.

3.4. Antigen quantitation of �irus-stocks bydELISA using a standard cur�e

Quantitation of herpes B virus antigens bydELISA is shown in the next example. One of the

herpes B virus antigen stocks served as the stan-dard stock for the construction of a dELISAlog/log titration curve, as described in Section 2.This generated a straight line that enabled calcula-tion of the titer graphically or by using the linearregression function (Fig. 3(A); see formula and theR2 value). Using a cutoff value that was calculatedfrom the uninfected controls, a titer of 3597 EUwas obtained. Titers of 11 herpes B virus antigenstocks that were run in the same dELISA plate ata single dilution of 1:200 were calculated from areference ‘standard curve’ (Fig. 3B). The titers inEU per test (50 �l) or EU/ml and the proteinconcentration (mg/ml) of some of the herpes Bvirus antigen preparations are summarized inTable 1. The titration enabled the identification ofone low-titer stock (herpes B-DI1) and threegroups of stocks with titers clustering around adistinct mean: Group 1 (herpes B-STD, herpesB-HN2, herpes B-PT2 and herpes B-PT3; mean�S.D.=4035�424), group 2 (herpes B-HN1, her-pes B-HN3, herpes B-HN4, herpes B-DI4 andherpes B-DI5; mean�S.D.=6032�274) andgroup 3 (herpes B-DI2 and herpes B-DI3; mean�S.D.=7260�311).

Fig. 3. (A) Determination of herpes B virus antigen titer in ELISA units (EU) using a dELISA titration curve and a cutoff value.(B) Generation of a standard curve (EU versus OD) for the calculation of titers from one point dilutions of unknown samples. (Seealso text and Table 1).


Table 1Titer determination of 11 herpes B virus-antigen stocks using a 1:200 one point dilution and the standard curve in Fig. 2(B)

Titer (EU/50 ProteinDilution to obtain 1600OD×1000 at aBV stock Titer/ml (EU/ml) EU/mg(mg/ml)1:200 dilution EU/ml�l)

3597 71,940 12,2955.8Herpes B-STD 30432,3983.71:75120,520Herpes B-HN1 6026401

3776 75,520 1:47Herpes B-HN2 3145566 111,320 1:70Herpes B-HN3 406

345 6171 123,420 1:77 5.7Herpes B-HN4 21,581311Herpes B-PT2 4521 40,6931:57 2.290,420

Herpes B-PT3 2.6 32,8361:5384,980424933481423.6Herpes B-DI1 29,500192 1475

Herpes B-DI2 43,0383.51:94449 7480 149,6001:88140,8007040433Herpes B-DI3

Herpes B-DI4 405 6142 122,840 1:77 3.1 39,2963.1 39,9621:78125,160Herpes B-DI5 409 6258

The protein concentration values that were deter-mined for nine out of the 12 stocks enabled thecalculation of the specific virus antigen titer inELISA antigen units (EU) relative to the proteincontent of the preparation in milligrams (EU/mg).From the present experiment, we determined anantigen lot as acceptable for serological assays onlyif the EU/mg ratio was �10,000. Therefore, stockherpes B-DI1 fell below the acceptable limit forserological assays because of its low specific antigencontent (8142 EU/mg). All others were satisfactoryfor serological assays, but should be used at onepredetermined fixed titer. It was estimated that anantigen titer of 1600 EU/ml (80 EU/well/50 �l) wasoptimum for coating wells in tELISA microplates,thus each stock should be diluted differently ac-cording to its measured titer (see Table 1).

3.5. Reproducibility of dELISA

To determine the reproducibility of the dELISA,each virus titration was repeated on different days.The mean titers, the S.D. and the coefficient ofvariations obtained for each virus lot are summa-rized in Table 2. The results indicate high repro-ducibilities of the dELISAs for monkey viruses(herpes B virus and herpesvirus papio 2, CV=14and 12%, respectively) compared to lower repro-ducibilities of the dELISAs for human viruses(HSV-1 and HSV-2, CV=35 and 21%, respec-tively).

3.6. The detection limit of dELISA

During the preparation of five alphaherpesvirusantigen stocks (HSV-1, HSV-2, herpesvirus papio2, herpesvirus langur and herpes B virus), analiquot was removed before detergent solubiliza-tion to determine the amount of infectious units(PFU/ml) in each preparation. The stocks werethen titrated in homologous dELISAs for determi-nation of their antigenic content (titer) in ELISAunits (EU). Results, summarized in Table 3, indi-cate a detection limit (sensitivity) of dELISA thatranged from 1.4×105 PFU/ml for herpes B virusto 1.6×106 PFU/ml for herpesvirus papio 2 (aver-age=7.8×105 PFU/ml or 3×104 PFU per 50 �ltest volume). These values should be considered asrough estimations, since it may be expected (asconfirmed in the next experiment) that differentstocks may have different amounts of infectiveunits per antigenic mass, depending on cultureconditions.

3.7. Antigen quantitation of li�e and detergentsolubilized �irus preparations by dELISA

In this experiment, herpesvirus papio 2 was usedas a model to demonstrate the value of dELISA asa tool for studying and optimizing virus antigenproduction.

Vero cell cultures were infected with an her-pesvirus papio 2 stock at three different multiplic-


ities of infection (MOI): 0.1, 0.5 and 1.0, for eachof the four time periods: 18, 21, 24 and 27 h. Eachof the 12 preparations obtained containing liveHVP-2 was split into two portions. One was leftuntreated and the other was detergent solubilizedby the T/DOC technique. The 12 live preparationswere titrated by the standard plaque assay for thedetermination of infectivity titers (PFU/ml). All 24samples (live and detergent solubilized) were ti-trated by dELISA. Results indicated that at 18-hpost-infection, lower amounts of virus and thusantigen were produced when compared to otherincubation times. An increase in titer correlatedwith the increase in MOI in the 18-h samples. Thiscorrelation was not observed with the longer incu-bation times. The infectivity titers (PFU/ml) ob-tained at 21, 24 and 27 h post-infection were of thesame order of magnitude. The dELISA titers werealso similar, although some tendency for highertiters was observed at 24 and 27 h post-infection.

Interestingly, the average ratio of PFU/ml todELISA titers (EU) (‘detection limits’) of all livepreparations (6.4×105 PFU/EU�3.5×105) wassimilar to the T/DOC preparations (6.3×105 PFU/EU�2.6×105). This was predicted since the de-fective to infective particle ratio in the two dividedaliquots should be equal.

4. Discussion

Direct enzyme immunoassays (dELISAs) weredeveloped for the detection and quantitation of fivemembers of the alphaherpesvirus group. Theserapid (2.5 h) and simple assays enable standardiza-tion of antigen production and optimization ofserological procedures for differential laboratorydiagnosis of herpes B virus infections.

All assays are based on one simple and afford-able principle, i.e. all antibody reagents necessaryare derived from primates infected naturally ratherthan from hyper-immunized animals, thus reflect-ing measurement of viral antigens recognized dur-ing the course of a live virus infection. Thisprinciple is particularly suitable for identifying theoptimal antigen preparation for serological anti-body-detection diagnostic assays. Although conva-lescent sera contain significantly fewer antibodiesthan sera from hyper-immunized animals, theyhave the advantage of being less reactive to non-specific tissue culture components. In spite of therelatively lower amount of antibodies, a very (eco-nomical) high dilution of the sera (1:10,000) wasused for capturing antigen. Because of the relativelylow antibody titers of convalescent serum pools andthe losses that occur as part of the IgG purificationand labeling process, low dilutions of conjugateshad to be used (1:50). However, in practice, oneconjugate preparation, typically 5 ml, sufficed for5000 test wells or 300 stock virus antigen titrations.Moreover, when a single sample dilution is used (induplicates) for quantitation of antigen in a virusstock by comparison to a standard curve �2000titer determinations can be carried out with one (5ml) conjugate preparation.

By comparing the dELISA titration curves ob-tained for each of the five alphaherpesviruses, itseemed that the herpes B virus dELISA was moreefficient than the others, in spite of comparableantibody titers of the serum pools and conjugates,as measured by tELISA. The increased reactivity ofthe herpes B virus dELISA may be the result of ahigher antibody avidity in the antibody pool. It isalso possible that the herpes B virus stocks haverelatively more defective particles and thus, a larger

Table 2Reproducibility of the dELISA as determined for four alphaherpesvirus antigen stocks

No. of testsAntigen stock Average titer S.D. Coefficient of variation (%)

Herpes B 81899 145 29120378 124431 512Herpesvirus papio 2 23003 3510565 3708HSV-1 12999

HSV-2 12999 41863 21864


Table 3Detection limit (sensitivity) of dELISA for five alphaherpesviruses compared to the plaque infectivity assay

Antigen Titer (EU/50 �l)PFU*/ml PFU/ml/titer (sensitivity)

10,565HSV-1 12999 4.7×1055.0×109

HSV-2 12999 2.5×109 4186 6.0×105

Herpesvirus papio 2 2300 36477.0×109 1.6×106

9461.0×109 1.1×106Herpesvirus langur 3 12006981Herpes B 41400 1.4×1056.6×108

* PFU, plaque forming units.

antigenic mass than the other viruses. Preliminaryresults indicated differences between tELISA anddELISA when virus cross-reactivities were stud-ied by each of the two assays. Surprisingly, anddifferent from the tELISA results, the herpes Bvirus dELISA was more specific to herpes B viruswith only minor reactivities to other viruses.Other dELISAs seemed also to be less cross-reac-tive than their tELISA counterparts. Some of thedifferences observed may be due to the inherentdifferences between the two techniques. We in-tend to address this issue more carefully in aseparate study.

The average detection limit (sensitivity) of thedELISA, as determined for detergent solubilizedpreparations, was 7.8×105 PFU/ml. A similarsensitivity was demonstrated for live virus prepa-rations. Although this sensitivity range is pre-sumably not sufficient for direct detection ofherpes virus antigens in clinical specimens(Slomka et al., 1998; Slomka, 2000), it is perfectlysuited for our quality assurance needs, since mostof the stocks contain at least 109 PFU/ml.

Reasonable reproducibilities were achievedwith the herpes B virus and herpesvirus papio 2dELISAs. Lower reproducibilities were obtainedwith the HSV-1 and HSV-2 dELISAs. The reasonfor this difference is not well understood. It couldbe related to the higher and relatively unstablebackground values obtained with the human serapools. Our reproducibility results require furtherinvestigation, since a relatively small number ofrepetitions were examined.

By relating the dELISA titer to the totalprotein concentration of a virus, another impor-tant quality control parameter was determined,

i.e. the ‘specific virus antigen content’. Virus anti-gen stocks with similar titers but lower proteincontent (higher EU/mg values) are by definitionmore suitable for antibody detection assays(tELISA and WB) than those with higher proteincontent (lower EU/mg values) because they con-tain relatively less non-viral protein. This parame-ter is therefore valuable in enhancing theperformance of serological assays.

In an experiment where different culture condi-tions were compared, we demonstrated that incu-bation time and multiplicity of infection have aneffect on the number of infective particles pro-duced and on the antigenic mass as determinedby dELISA. Lower virus production was noticedat 18-h post-infection compared to the 21, 24 and27 h post-infection. No negative effect of deter-gent solubilization was observed, since similardELISA titers were obtained before and after thesolubilization step. Herpesvirus papio 2, whenused as a model, demonstrated the value ofdELISA as a tool for optimizing antigen produc-tion, as well as virus titration before inactivation.Similar optimization experiments for the produc-tion of better antigen preparations will be carriedout with all other members of the alphaher-pesvirus group.

Acknowledgements

We thank Marty Wildes, Diane Castro andKim Bush from the National BV Resource BVLaboratory, for assembling the collected sera em-ployed in this study. This work was supported byNIH award R01-R03163 and P40-RR05162.


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Alphaherpesvirus antigen quantitation to optimize the diagnosis of herpes B virus infection