Vol. 17, No. 6JOURNAL OF CLINICAL MICROBIOLOGY, June 1983, p. 1081-10910095-1137/83/061081-11$02.00/0

ViralSingle

Antibody Screening System That Uses a StandardizedDilution Immunoglobulin G Enzyme Immunoassay with

Multiple AntigensJONAS BLOMBERG,* INGRID NILSSON, AND MARGARETA ANDERSSON

Section of Virology, Department of Medical Microbiology, University of Lund, Solvegatan 23, S-223 62Lund, Sweden

Received 20 January 1983/Accepted 3 March 1983

We present an enzyme-linked immunosorbent assay (ELISA) system for thesimultaneous determination of immunoglobulin G antibodies directed againstseveral viruses. Antibodies to up to eight different viruses could be determined forthree different sera on one microtitration plate. After subtraction of the absor-bance values obtained with the control antigens, the viral antigen absorbancieswere expressed as percentages of the absorbance obtained with a pooledimmunoglobulin standard. This value, the relative antibody activity, was rapidlycalculated by means of a computer directly connected to the ELISA photometerand was stored on magnetic disks, thereby facilitating seroepidemiologicalstudies. The reproducibility of the relative antibody activity was calculated to atbest ±3.6% (standard deviation) in an intraassay test and to at worst ±20.4%(standard deviation) in an interassay test. Each serum was analyzed only at adilution of 1/75. The sensitivity of this single-dilution ELISA (SD-ELISA) methodfor the detection of titer rises was compared with those of conventional methods,mostly complement fixation but also hemagglutination inhibition. A total of 142 of155 (92%) paired sera showing fourfold complement fixation or hemagglutinationinhibition rises also showed significant results in SD-ELISA. A total of 22 of 57(39%) significant relative antibody activity rises were significant in complementfixation or hemagglutination inhibition. Overall, up to twice as many significanttiter rises could be detected with SD-ELISA. Most of these seemed to have asound correlation with clinical data. The specificity of SD-ELISA was found to besimilar to that of complement fixation, with some cross-reactions occurringbetween herpes simplex and varicella-zoster virus antigens and between parain-fluenza viruses. We have found SD-ELISA to be a valuable clinical virologicaltool that supplements conventional serology.

Even in large laboratories a broad repertoireof virus serology often cannot be performeddaily, due to the necessity of collecting enoughsamples for a specific analysis to justify theeffort. Inevitably, this leads to delays in report-ing. The time-honored complement fixation (CF)test, which is the mainstay of virological serolo-gy, requires careful calibration of the biologicalreagents, is relatively cumbersome, and must bejudged as semiquantitative. Additionally, thedemand for rapid determination of viral immuni-ty in recently or occupationally exposed personsis increasing, and CF is not suitable for this.More efficient analytical procedures are needed.The enzyme-linked immunosorbent assay

(ELISA) is winning acceptance as a valuabletool for the diagnosis of viral disease. The tech-nique is relatively rapid and sensitive and inmany cases allows the detection of antibodies ofboth immunoglobulin M (IgM) and IgG classes

(3, 5, 9, 13, 15, 19, 20, 22). The advent of high-speed photometric reading of microplate ELISAresults together with computer processing of thedata (20) as part of a laboratory informationsystem with automatic printout of laboratoryreports (4) has made the ELISA methodologyeven more attractive. Additionally, in severalstudies (14, 22) ELISA analysis at a single serumdilution, as opposed to the more common use ofseveral dilutions, has been found practicable.Obviously, this approach increases the analyti-cal capacity considerably. An evaluation of thesingle-dilution test in a routine clinical virologi-cal setting with a broad spectrum of antigens isneeded.

In this report, we describe an ELISA systemthat uses an array of antigens for the simulta-neous determination of IgG antibodies againstseveral common viruses. The system is intendedprimarily for comparisons of acute- and conva-

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1082 BLOMBERG, NILSSON, AND ANDERSSON

lescent-phase sera in the diagnosis of viral infec-tions. Although IgG ELISA has been advocatedfor clinical virology in many publications (seeabove), we found that we had to overcome anumber of difficulties to achieve a dependableand practical routine procedure. It was neces-sary to reduce unspecific adsorption to the solidphase and the reactivity of sera with controlantigens and to enhance reproducibility by stan-dardization of viral antibody activity. By testingsera at a single dilution in duplicate, simplicitycould be combined with precision.

MATERIALS AND METHODSViruses. Some of the viral ELISA antigens were

prepared in our laboratory. The prototype herpessimplex virus (HSV) strain F was kindly provided byBernhard Roizman, University of Chicago, Chicago,Ill. It was grown overnight in African green monkeykidney (GMKAH1) (17) cells. These and other celllines used were found to be free of mycoplasmacontamination by the large-specimen broth culturemethod (2). The LEC strain of measles virus and theJerryl Lynn strain of mumps virus were grown inGMKAH1 cells. The cytomegalovirus strain AD169and adenovirus type 12 were grown in a strain ofhuman embryonic lung (HEL) cells maintained in ourlaboratory.

Antigen preparation. Cells grown in Dulbecco modi-fied Eagle medium containing 10% fetal bovine serum(Flow Laboratories, Inc., Rockville, Md.) in rollerflasks were harvested at the time of maximum viralcytopathic effect by scraping and were collected bylow-speed centrifugation. At 1 day before harvest,monolayers were washed twice and the medium waschanged to serum-free Dulbecco modified Eagle medi-um. At harvest, virus- and mock-infected cells werewashed once with phosphate-buffered saline (PBS; 8.0g of NaCl, 0.2 g of KH2PO4, 1.4 g of Na2HPO4 * 2H20,0.2 g of KCl, and 10 mg of sodium Merthiolate perliter) and disrupted by sonication. Cellular debris wasthen removed by centrifugation at 1,000 x g for 10min. The supernatant fluid was dispensed in 0.1-mlvolumes and stored at -60°C after protein determina-tion by UV spectrophotometry. The supernatant wassubsequently used as ELISA antigen. Working dilu-tions were determined by checkerboard titrationsagainst negative- and positive-control sera.Commercial ELISA antigens. Appropriate negative-

control antigens from uninfected cultures were alwayspurchased together with the viral antigens, many ofwhich were intended primarily for use in the CF test.Varicella-zoster virus antigen (Ag 770217) produced inHEL cells was purchased from the National Bacterio-logical Laboratory, Solna, Sweden. Influenza A2(strain Hongkong), influenza B, parainfluenza types 1and 2, respiratory syncytial virus, and Mycoplasmapneumoniae antigens were from Orion Oy, Helsinki,Finland. The parainfluenza type 3 antigen was fromMicrobiological Associates, Bethesda, Md.Original ELISA protocol. At the outset of this work,

the following procedure was used for coating andwashing. A 100-,ul portion of the antigen diluted inPBS containing 10 mM sodium azide instead of 10 mgof sodium Merthiolate per liter was allowed to adsorbto wells in a Nunc Immunoplate II (Nunc AS, Ros-

kilde, Denmark) overnight at 4°C. After a wash withPBS containing 0.05% Tween 20 (PBS-Tween) withazide, anti-IgG enzyme conjugate in PBS-Tween withazide was added to each well and reacted for 1 h atambient temperature. After an additional wash withPBS-Tween with azide, the enzyme reaction was runfor 1 h at ambient temperature. Otherwise, the sameprocedures, chemicals, and equipment as describedbelow were used.SD-ELISA. Except when stated otherwise, the fol-

lowing procedure was followed for the final single-dilution ELISA (SD-ELISA). A 100-,ul portion of viralor control antigen diluted in PBS was added to a 96-well microtitration plate (Nunc Immunoplate II) andleft to adsorb at 4°C overnight. After a wash with PBS-Tween (Multiwash; Skatron AS, Heggtoppen, 3401Lier, Norway), 200 1±l of a blocking solution contain-ing 4% (wt/vol) bovine serum albumin (BSA) (fractionV; United States Biochemical Corp., Cleveland, Ohio)and 0.1% gelatin (Difco Laboratories, Detroit, Mich.)in PBS was added to each well, left for 6 h at roomtemperature, and subsequently left overnight at 4°C.At this stage, plates could be frozen at -20°C andstored for several months. Before use the plates werewashed with PBS-Tween. Sera were diluted in 4%(wt/vol) BSA-0.1% (wt/vol) gelatin-0.05% (vol/vol)Tween 20 containing 1% (vol/vol) each of two cellularcontrol antigen preparations, GMKAH1 cells (1 to 2mg of protein per ml) and chorioallantoic membrane (2to 4 mg of protein per ml) (both were from the NationalBacteriological Laboratory, Solna, Sweden). The serawere allowed to stand in the dilution fluid for 10 to 20min at room temperature to permit blocking of anticel-lular antibodies before addition to the ELISA plate.For routine antibody determination, 100 ±I1 of a stan-dard serum dilution of 1/75 was applied in duplicatewells. The plate containing serum dilutions was thengently shaken on a microtiter plate shaker (BellcoGlass, Inc., Vineland, N.J.) for 1 h at room tempera-ture. After a wash with PBS-Tween, 100 1±1 of an anti-human IgG-alkaline phosphatase conjugate (Orion Oy)diluted 1/100 in PBS-Tween containing 0.1% gelatinwas added and incubated at room temperature withshaking for 1 h. Finally, after washing and removal ofextraneous PBS-Tween by beating the plate upsidedown against paper towels (residual PBS was found toinhibit the enzyme reaction [data not shown]), 100 1±lof a substrate solution containing 1 mg of p-nitro-phenyl phosphate (Sigma Chemical Co., St. Louis,Mo.) per ml in 10% diethanolamine buffer (pH 9.8) wasadded to each well. After 30 min at 37°C the plateswere read in a Titertek Multiskan (Flow) colorimeterat 405 nm.The procedures described herein were in most cases

based on optimization through experimentation (seebelow).CF and HI tests. The micro CF test was performed

according to the method of Sever (24), as modified byCasey (8). Viral antigens for CF were purchased fromthe National Bacteriological Laboratory, Solna, Swe-den. A microtiter modification of the hemagglutinationinhibition (HI) test for measles antibodies (18) wasused.

Calculation of ELISA results. (i) Calculation of RAA.The mean absorbance of the duplicate wells containingthe appropriate control antigen was subtracted fromthe mean of the absorbance values from duplicate

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MULTIPLE VIRAL ANTIBODY SCREENING BY ELISA 1083

wells containing viral antigen. To obtain the relativeantibody activity (RAA) this differential absorbancewas calculated for both the serum being analyzed anda standard commercial IgG preparation (containing165 mg of human IgG [Gammaglobulin, AB Kabi,Stockholm, Sweden] per ml) diluted 1/1,200 with dilu-tion buffer. The serum value was then expressed as a

percentage of the value of the standard. Absorbancevalues were directly processed by computer programs(READPROG and ELISAPR) written in MBASIC.This system should be easily adaptable to any micro-computer using CP/M or CP/M-compatible operatingsystems.

(ii) Calculation of reproducibility. The variance wascalculated for the following comparisons: betweenduplicate wells, between RAA values of three sera onthe same or different microplates, and between RAAvalues of the same sera determined on two differentoccasions with the same or different batches of viralantigens (block II [see below]). Absorbancies of lessthan 0.05 were not included.

(iii) Calculation of significance of titer rises. A titerchange was considered significant when the differencebetween RAA values was larger than four times thestandard deviation from the means of the extinctionvalues of duplicates of acute- and convalescent-phasesera, after subtraction of control antigen values. Thisarbitrary convention was based on our experiencefrom routine analysis. The calculation was made auto-matically by the computer.

Organization of antigen blocks. Two sets of antigenswere used. Block I, for acute respiratory diseases,contained the following antigens, in this order: influen-za A, influenza B, parainfluenza type 1, parainfluenzatype 3, influenza and parainfluenza control, parainflu-enza type 2, respiratory syncytial virus, parainfluenzatype 2 and respiratory syncytial control, M. pneumo-niae, adenovirus type 12, and HEL control. Block II,

for meningoencephalitis and fever of unknown origin,contained the following antigens: HSV, measles,mumps, GMKAH1 cellular control, cytomegalovirus,varicella-zoster, adenovirus type 12, and HEL cellularcontrol. Most of the work was performed with blockII.

Reanalysis of CF and HI titer rises by SD-ELISA.Before the introduction of an antigen into the analysisof patient sera on a semiroutine basis concurrentlywith conventional methods, it was tested against acollection of serum pairs showing CF or HI titer rises.

Reanalysis of SD-ELISA RAA rises by CF and HI. Incooperation with the Department of Infectious Dis-eases of the University of Lund, SD-ELISA (mostlywith antigen block II) was performed on request onroutine clinical samples (380 serum pairs) during aperiod of 6 months. All RAA rises reported as signifi-cant were reanalyzed by means of CF or HI.

RESULTS

Reduction of nonspecific binding. Initially,binding to control antigens was a severe prob-lem. By adjustments in methodology, this back-ground activity was gradually diminished. Todemonstrate the effect of the major modifica-tions, a control experiment was done (Fig. 1). Amicrotiter plate was divided into PBS-treated,

GMK cell-treated, and HEL cell-treated areas of24 wells each. Each area was then subdividedinto four areas. Each subdivision was subjectedto one of the following four treatments. (i)Blocking of residual protein-binding capacity bythe addition of 4% (wt/vol) BSA and 0.1%(wtlvol) gelatin in PBS-Tween after antigen at-tachment greatly reduced the binding to mock-or cellular-antigen-treated plastic (Fig. 1B). (ii)A further improvement was obtained by theaddition of 4% BSA and 0.1% gelatin to theserum dilution fluid (Fig. 1C). However, thegeneral background absorbance (including blankwells) often rose to prohibitively high values inproportion to the age of the dilution fluid. (iii)Azide, the most unstable and reactive compo-nent, was therefore substituted with 10 mg ofMerthiolate per liter in the dilution fluid. Afterthis change, low background reactivity was con-sistently observed (Fig. 1D). (iv) In principle, alarge enough concentration of control antigen inthe dilution fluid should completely block thebinding of anticellular antibodies in the virus andcontrol antigen wells. Consequently, a series ofexperiments was designed to investigate thispossibility (data not shown). Extracts ofGMKAH1 cells, HEL cells, and chorioallantoicmembrane were tested at different concentra-tions alone and in combination with each other,BSA, Tween 20, and gelatin. In the presence of4% BSA, 0.1% gelatin, and 0.05% Tween 20 inthe serum dilution fluid, a reduction of antibodybinding by a certain control antigen by admix-ture of the same antigen was seen primarily withGMKAH1 cell and chorioallantoic membraneextracts and to a lesser extent with HEL cellextracts at around 0.1 mg of protein per ml. Atlower concentrations, an enhanced binding tocontrol antigen was sometimes seen, suggestingan antibody-antigen equilibrium with binding ofcomplexes to the antigen on the solid phase. Wesubsequently added GMKAH1 and chorioallan-toic membrane control antigens to the serumdilution fluid at a concentration of 0.1 mg ofprotein per ml (Fig. 1E).

Antibody titrations. The IgG standard wastitrated on all antigens to study the possibleoccurrence of prozone phenomena. Of the anti-gens chosen for evaluation, only a toxoplasmo-sis antigen showed a clear prozone (data notshown). It was not used further. After the intro-duction of all modifications to reduce back-ground activity, control antigens gave low orundetectable optical densities even at high anti-body concentrations (Fig. 2). With most anti-gens a specific antibody reaction could be seeneven at high dilutions of the standard. In theHSV and adenovirus systems adjusted for rou-tine SD-ELISA use, the standard immunoglob-ulin preparation still gave detectable reactions at

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A B C D EFIG. 1. Reduction of nonspecific binding by modifications of the SD-ELISA procedure. A microtitration

plate was incubated with PBS or sonic extracts ofGMK and HEL cells. The three surfaces created in this waycorrespond to the first, second, and third bars, respectively, in each treatment group. Modifications of theoriginal procedure (see text) were as follows: A, no blocking after coating with antigen, and dilution of sera inPBS-Tween with azide; B, same as A, but a blocking step (4% [wt/vol] BSA and 0.1% gelatin in PBS-Tween withazide overnight) was added; C, same as B, but 4% BSA and 0.1% gelatin were added to the serum dilution fluid;D, same as C, but azide was substituted with 10 mg of Merthiolate per liter; E, same as D, but GMKAH1, HEL,and chorioallantoic membrane preparations (1% [vol/vol] each) clarified by low-speed centrifugation were addedto the serum dilution fluid. Each bar represents the mean of the absorbance values from six wells.

a dilution of 1/32,000 (5 ,ug of IgG per ml). At thedilutions chosen for routine tests (1/75 for sera,1/7.5 for cerebrospinal fluids, and 1/1,200 for theimmunoglobulin standard), the microbial anti-gens generally gave optical densities that were10 to 100 times higher than those of the controlantigens. An antigen was deemed unsuitable forfurther use if absorbance values of less than 0.2were obtained with the immunoglobulin stan-dard in our routine ELISA procedure.

Standardization of results by calculation of

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RAA. Due to the difficulty of standardizingantigenic activity and maintaining exact timing,we found it desirable to include a standardantiserum on each ELISA microplate. Commer-cial IgG, diluted 1/1,200 to correspond to aserum containing 10 mg of IgG per ml, wasapplied to the two left-most rows of the plate.This concentration was chosen to obtain opticaldensities of 0.2 to 0.8 for all antigens, thusincreasing the accuracy of the RAA calculation.

Reproducibility of results. Table 1 presents the

200 160012800200 160012800200 160012800200 160012800200 1600128002200 160012800RECIPROCAL DILUTION

FIG. 2. Titration of the immunoglobulin standard on viral antigens. The lower curves of each panel are theresults obtained with the respective control antigen (uninfected cells). Abbreviations: MEAS, measles virus;CMV, cytomegalovirus; VZV, varicella-zoster virus; AD, adenovirus; INF A and INF B, influenza virus types Aand B, respectively; PAR 1, PAR 2, and PAR 3, parainfluenza virus types 1, 2, and 3, respectively; RS,respiratory syncytial virus; MP, M. pneumoniae.

,,. 9 HSV MEAS CMV VZV AD INF A

10 - .l-t=k., . -. ' 40,.,., ., ,,..,.,k . ,- ~ -iINF B PAR PAR 2 PAR 3 RS MP.

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MULTIPLE VIRAL ANTIBODY SCREENING BY ELISA 1085

TABLE 1. Reproducibility of relative and absoluteabsorbance values

Values Conditionsb No. ofExpta Vomalues a

obser- SD(%Excompared Plate sion Batch vations

1 Absorbance S S S 104 ±5.22 RAA S S S 42 ±3.63 RAA D S S 42 ±8.54 RAA D D S 42 ±16.05 RAA D D D 42 ±20.4a Six block II antigens were used. Duplicate wells

from routine analyses of 21 sera (104 pairs) werecompared (experiment 1). Two sera were diluted anddispensed eight times each into wells of the sameELISA plate (experiment 2) and into wells of anotherELISA plate (experiment 3). The same sera werereanalyzed on another day (experiment 4) and on athird occasion with a plate from a different coatingevent (experiment 5).

b S, Same; D, different.

results of experiments in which a small numberof sera were reanalyzed in different ways. Thestandard deviations of the absorbance values ofduplicate wells were relatively small (5%). RAAvalues which were calculated from the means of

RAA400'

300

200'

100

20

>20c420

duplicate wells had an even smaller standarddeviation (4%). However, much larger devi-ations (9%) resulted when the comparison wasmade between RAAs from different plates, andeven higher deviations (16%) were obtained be-tween different runs (interassay variation). Thelargest standard deviation (20%) was foundwhen plates prepared from different antigenbatches were compared.

Distribution of titer values. Figure 3 depictsthe distribution of RAA values for children 0.5to 10 years of age and for an adult population 30to 70 years of age. RAA values of 0 to 10 oftencould not be distinguished from random varia-tions in the viral and control antigen wells andwere thus designated negative. RAA values of10 to 20 were sometimes of uncertain signifi-cance, depending on the precision of work dur-ing the test or on diminishing strength of aparticular antigen. Such values were conse-quently designated borderline. All antigens gavea higher percentage of positive RAA values inthe sera from adults than in those from children.

Correlation of SD-ELISA with CF and HI. (i)CF and HI titer rises reanalyzed by SD-ELISA. Acollection of sera showing viral antibody titerrises by our routine CF serology was used for a

90tHSV MEAS CMV

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17 38 22 29 15 17 10 18 16 13

11 6 7 9 6 2 8 1 5 6

FIG. 3. Distribution of RAA values in sera submitted to our laboratory for routine serology. Values fromchildren 0.5 to 10 years of age (a) and adults 30 to 70 years of age (b) are shown. The means ofRAA values higherthan 20 and their standard deviations (bars) are shown at the right of each column. The number of patients with(RAA 2 20) and without evidence for possession of antiviral antibody is shown below each column.Abbreviations are as in the legend to Fig. 2.

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TABLE 2. CF and HI fourfold titer rises analyzedby SD-ELISAa

No. ofAntigen significantRAA rises/total

no.

HS ................................

Measles ............................

Cytomegalovirus ....................

Varicella-zoster .....................

Adenovirus .........................

Influenza A.........................Influenza B .........................

Parainfluenza type 1 .................

Parainfluenza type 3 ................Respiratory syncytial virus ...........

M. pneumoniae .....................

23/2511/1227/2814/1415/167/98/125/65/5

12/1315/15

All antigens...... 142/155 (92%)

a The CF method was used for all antigens; formeasles antigen, HI was also used. With HSV antigen,one case of a primary genital (type II) and one case of aneonatal (type I) HSV infection did not exhibit asignificant herpes RAA rise. In both cases IgM herpesantibodies were detectable by the use of an anti-IgMconjugate in SD-ELISA. With measles antigen, onecase of measles judged as typical by clinical criteriawas RAA negative in both acute- and convalescent-phase sera taken 2 and 9 days after the debut of theillness. In both sera, the HI titer was unaffected byadsorption with Cowan strain staphylococci, whichwas evidence for a large excess of non-IgG antibodiesin the sera. With the other antigens, the followingcases exhibited no or insignificant RAA rises: fever ofunknown origin (one case) (cytomegalovirus); tonsilli-tis (one case) (adenovirus); clinically typical influenza(two cases) and acute laryngitis (one case) (influenzaA); influenza syndrome (three cases) and laryngitis(one case) (influenza B); and upper respiratory infec-tion (one case) (parainfluenza type 1). Four of sixparainfluenza type 1 titer rises had a smaller parainflu-enza type 3 rise, and three of five parainfluenza type 3titer rises had a smaller parainfluenza type 1 rise.

comparison of the specificity and sensitivity ofIgG SD-ELISA as compared with CF for thediagnosis of acute viral infection (Table 2). Inmost cases, sera showing a significant (at leastfourfold) titer rise in CF clearly showed rises inELISA values (Fig. 4). However, especially inthe case of the influenza viruses (3 of 9 influenzaA and 4 of 12 influenza B rises), the RAAsometimes did not change significantly. Thereason for this inability of SD-ELISA to detectall titer rises is unknown. However, in somecases (Table 2), interference by an excess ofnon-IgG viral antibodies was suggested by anIgM-specific ELISA, indirect immunofluores-cence, or differential adsorption of IgG by pro-tein A-containing staphylococci (1). In any case,SD-ELISA was able to distinguish a significanttiter change in more than 90% of the significant

titer rises seen with conventional serology. BySD-ELISA, antibodies were often found inacute-phase sera, in which CF often yielded anegative result (Fig. 4). All serum pairs weretested with either block I or block II. By thismeans it was found that 5 of 25 HSV titer risesalso had a significant rise against varicella-zostervirus and that 1 of 14 varicella-zoster (a varicellaencephalitis) titer rises also had a significantHSV RAA rise. In 7 of 11 parainfluenza titerrises there was a simultaneous rise against types1 and 3. No other probable cross-reactions werenoted. However, in five cases, two titers rosesimultaneously (two cases with influenza B andM. pneumoniae; one case each with M. pneumo-niae and respiratory syncytial virus, cytomega-lovirus and HSV, and measles and adenovirus),and in one case, three rises (parainfluenza type1, parainfluenza type 3, and M. pneumoniae)were detectable. A fourfold titer rise in CFcorresponded to a two- to threefold rise in RAA.Especially prominent rises were seen with respi-ratory syncytial virus and M. pneumoniae. In nocase did the serum have to be diluted more than1/5 to correct for an unmeasurably high enzy-matic activity.

(ii) RAA rises reanalyzed by CF or HI. Allsignificant RAA rises obtained with block IIduring a period of 6 months (57 rises in 48 serumpairs from 47 patients) were reanalyzed by CF ormeasles HI (Table 3). Only around 40% of theRAA rises were detectable as fourfold titer risesby CF or HI. With the inclusion of twofold CFor HI titer rises, this number was increased toaround 50%. RAA rises confirmed by neither atwofold nor a fourfold CF titer rise were dividedinto the following four categories on the basis ofevidence for the rise obtained from clinical andlaboratory data: (i) conclusive evidence (vacci-nation against the agent or confirmatory serolo-gy other than SD-ELISA; (ii) strong evidence(clinically typical disease); (iii) weak evidence(suggestive clinical data); and (iv) no evidence.Of 27 RAA rises unconfirmed by CF, 9 weresupported by conclusive evidence, 13 by strongevidence, and 2 by weak evidence. Three riseswere not supported by clinical or laboratorydata.The mean interval between acute- and conva-

lescent-phase sera was 12.2 (±6.3 [standarddeviation]) days for RAA rises unconfirmed byCF and 16.4 (±7.1) days for RAA rises con-firmed by CF. The average increase in RAA was1.9 (±0.6) times for unconfirmed rises and 2.5(+0.8) times for confirmed rises.

(iii) Titer comparisons with single sera. Scatterplots of CF versus ELISA titer of 102 unpairedsera from 71 patients without suspected viraldisease and from 31 blood donors are shown inFig. 5. Although the spread often was wide, the

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MULTIPLE VIRAL ANTIBODY SCREENING BY ELISA 1087

RECIPROCAL DILUTION (CF)FIG. 4. Correlation of RAA values with CF titers. RAA is plotted against CF titer for acute- and

convalescent-phase sera with known CF titer rises. Serum pairs are connected with a line. Abbreviations are asin the legend to Fig. 2. For technical reasons, only five to nine representative titer rises per antigen are displayed.

correlation was good for all antigens. Often,samples negative by CF were weakly or moder-ately positive by ELISA. CF-positive sera wereall positive by SD-ELISA.

(iv) Temporal comparison of conventional se-rology and SD-ELISA in four cases. In frequentlysampled cases, an SD-ELISA titer rise some-times was detectable before the rise in CF titer(Fig. 6). From the RAA level in the first serumsample and the kinetics of the rise, it can bededuced that case 1 (Fig. 6A) was a primarycytomegalovirus infection, whereas case 2 (Fig.6B) was a reactivation. Case 3 (Fig. 6C) was acase of herpes simplex encephalitis, and case 4(Fig. 6D) had three different infections (typicalmeasles, mumps with meningoencephalitis, andtonsillitis) during a period of 1 month, all ofwhich could be demonstrated as RAA rises inSD-ELISA block II. Although the possibility ofnonspecific polyclonal reactivation cannot beentirely excluded, this seems unlikely. Nochange was seen to other block II antigens.

(v) Mumps and parainfluenza type 2 antigens.Throughout the test period described in thisreport, mumps antigen was included in block II,and parainfluenza type 2 antigen was included inblock I. However, it was found that RAA valuesobtained with these antigens were somewhatunreliable for the determination of both past andpresent infection. The mumps antigen reacted,although weakly, with some (2 of 10) sera show-ing rises in parainfluenza virus type 1 and 3titers. This antigen was retained for screeningpurposes, and results are not reported here(except in Fig. 6). The parainfluenza type 2antigen failed to yield RAA rises in any of threeserum pairs showing a CF titer rise. The results

obtained with this antigen were consequentlynot further reported.

DISCUSSIONThe goal of the present investigation was to

devise a rational virus serological procedure

TABLE 3. SD-ELISA (block II) RAA rises in CFor HI

Reana- No. of No. ofAntigen lyzed fores/ftold twofold rises!

by: rssttl total no.no.

HSa CF 0/1 0/1Measlesb HI 6/20 8/20Cytomegalovirusc CF 8/22 12/22Varicella-zoster" CF 6/10 7/10Adenoviruse CF 2/4 3/4

All antigens 22/57 (39o) 30/57 (53%)a This case offever of unknown origin had a simulta-

neous RAA rise with respect to both HSV and cyto-megalovirus antigens. In CF, however, a twofold risewith respect to only cytomegalovirus was seen.

b Patients included 6 Virivac vaccination controls,11 cases of clinically diagnosed measles, and 3 cases offever with concurrent exanthema. Two of the vacci-nated children who did not respond in HI within 3weeks were found to have seroconverted in HI at 1month postvaccination. These cases were not countedas titer rises in the table.

c Patients included 4 kidney transplant patients withfever, 12 cases of heterophile-negative mononucleosis,and 6 cases of fever of unknown origin.

d Patients included four cases of herpes zoster andsix cases of chicken pox.

e Patients included four cases of upper respiratoryinfection.

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1088 BLOMBERG, NILSSON, AND ANDERSSON

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55 0 0 8 5 5 0OOO ' 1248 5512480 ~ ~ ~ EIRCLO IE F

FIG.5Scattrplotof RA versu CF tter fo sera rom paients ithou evidece of cute vral inectio(71sea) r ro blo oos(1sr) brvain r si h eedt i.2

which could supplement or replace existingmethods. The results indicate that SD-ELISAwith simultaneous screening of many viral anti-gens has many of the desired features. However,in a relatively small percentage of cases, it didnot detect titer changes that could be demon-strated by other procedures. On the other hand,in a larger percentage of cases, it detectedsignificant titer changes that were not consid-ered significant by CF or other conventionalprocedures.

Theoretical aspects of SD-ELISA. Titer deter-mination by stepwise dilution to an endpoint isgenerally used in virus serology. However, sev-eral recently developed sensitive assay systemsuse only one serum dilution. The most importantprerequisite for a quantitative single-dilution as-say is an unambiguous relationship betweenreaction intensity and antibody activity. Toavoid saturation of the binding sites on theantigen, relatively potent antigen preparationshave to be used. A dilution method is inherentlynot as affected by blocking phenomena.We found it possible to use a single serum

dilution for the determination of antibody activi-ty against many different antigens. RAA is alinear transformation of absorbance whichranges from 0 to 200 in most cases and rarelyreaches above 1,000. In our experience RAA isacceptable to clinicians (11, 12).

Practical aspects of SD-ELISA. The SD-

ELISA antigen block approach permits tailor-making of test combinations to fit several clini-cal situations. A fairly complete report can besent to the clinician within 1 day, obviating theneed to have many specimens from differentpatients to justify performing a test with a specif-ic antigen.The microbial ELISA antigens used in this

work were clarified sonic extracts of virus-infected cells or commercial CF and ELISAantigens. Although higher purity may be desir-able for further increases in sensitivity andspecificity, we, like others (9, 15, 19, 22), havefound that these readily available antigens areoften satisfactory in ELISA for clinical virologi-cal purposes. Presently, it is beyond the meansof the average virological laboratory to maintaina stock of high-purity ELISA antigens.The measures taken to reduce background

activity, including addition of control antigen inthe dilution fluid, provided an absorbance ofbelow 0.01 to 0.04 for most sera from patients.This greatly enhanced the sensitivity of the testand reduced the requirement for high-potencyantigen.

Specificity. In numerous reports (5, 9, 15, 19,20, 22, 27) ELISA has been noted for highsensitivity, but its specificity has been much lessstudied. The simultaneous analysis of severalantibodies in this study permitted a comprehen-sive surveillance for cross-reactions between

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MULTIPLE VIRAL ANTIBODY SCREENING BY ELISA

:F RAA

200

100

n60 0 20 40 60DAYS POST HEART SURGERY

CF

-40

20

0

0 10 20 30 0 20 40 60DAYS OF ILLNESS DAYS POST EXANTHEMA

FIG. 6. Sequential viral serological surveillance with SD-ELISA. Each panel represents one patient. (A) A54-year-old male who underwent a coronary bypass operation. He developed fever, lymphocytosis, and malaise24 days later, which waned 50 to 60 days after surgery. (B) A 60-year-old male who developed mild fever andmalaise 31 days after coronary surgery. These symptoms subsided around 60 days after surgery. (C) A 58-year-old male with encephalitis who became unconscious on day 4. A brain biopsy taken on day 3 was found tocontain HSV type I antigen by an ELISA method (26). Acyclovir treatment was instituted on day 3. Althoughmentally impaired, the patient survived. (D) An 11-year-old male who at admission (day 0) had an exanthematypical of measles. On day 21 (first arrow) he contracted a sore throat, and on day 32 (second arrow) hedeveloped a meningoencephalitis. Mumps virus was isolated from a cerebrospinal fluid (CSF) sample obtainedon day 33. Due to limitations of space, only the RAA values are shown. Titer rises were eventually confirmed byCF (results for adenovirus [AD]: 1/5, 1/5, 1/40, and 1/10) and by hemolysis in gel (16) (results for mumps virus[MU]: 6, 6, and 10.3 mm). HI (results for measles virus [MEAS]: 1/20, 1/20, 1/20, and 1/20) could not confirm theSD-ELISA RAA rise. However, this diagnosis was likely on clinical grounds.

antigens within a block (25).Prerequisites for an acceptable specificity in

SD-ELISA with relatively crude viral antigenpreparations are to always include control anti-gens prepared in parallel with the viral antigen

and to have a low background of nonspecificbinding to the solid phase. Both conditions weremet in the ELISA system presented in thispaper. The following observations were madewith regard to specificity. (i) SD-ELISA titer

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1090 BLOMBERG, NILSSON, AND ANDERSSON

rises generally were confined to antigens givingCF titer rises. (ii) Clinical findings in patientswith SD-ELISA rises nearly always were com-patible with infection caused by the respectiveagents or with exposure to them (Table 3). Most(22 of 27) RAA rises not confirmed by CF wereconclusively or strongly supported by clinicaldata or independent virus serology. Althoughthe proportion of additional titer rises found bySD-ELISA probably will vary among labora-tories, our results do indicate that most or all ofthe rises correspond to an immune responsecaused by a specific infection. (iii) The agedependency of SD-ELISA RAAs suggests thatthey measure antibodies to agents acquired dur-ing childhood or adolescence. (iv) Control anti-gens gave flat and low titration curves with thestandard immunoglobulin preparation. In con-trast, viral antigens yielded 2- to 1,000-fold-higher extinction values up to high immunoglob-ulin dilutions. (v) Prozone phenomena were notfound by checkerboard titration against standardsera. (vi) Cross-reactions were sometimes en-countered (see above) (21, 23). On the whole,however, cross-reactions were a minor problem.(vii) Of the 978 sera tested with SD-ELISAblock II, only 32 (3.3%) showed anticellularantibody titers high enough to break through theexcess of cellular antigens used in the serumdilution fluid. Most of these sera, however, stillgave useful although less precise results whenthe optical density of the control wells wassubtracted from that of the viral antigen wells,which is part of the RAA calculation. Largenegative values due to oversubtraction wererare.

Sensitivity. Not all CF or HI titer rises wererecognizable in SD-ELISA (Table 3). Interfer-ence with virus-specific IgG binding by non-IgGantibodies may have been the reason in somecases. Another cause may have been the occur-rence of low-affinity IgG antibodies (6, 7) earlyduring the course of the humoral immune re-sponse. These may interfere with the binding ofhigh-affinity antibodies but be washed awaybefore the color development step in ELISA. Ineither case a further titration of the antiserummight have led to a recognition of a titer rise. Tocontrol for this possibility during routine serolo-gy, another serum dilution would have to beincluded in the test. This would, however, seri-ously increase the labor and costs of the ELISAsystem. On the other hand, we found (Table 3)that SD-ELISA detects far more titer rises thanCF does. The net increase was around 40%. Thesensitivity of SD-ELISA probably could be fur-ther increased by including determination ofantiviral IgM and IgA. It is also possible thatmore RAA rises could have been detected withthe use of stronger antigens.

Precision. The relatively high precision of theRAA determination (standard deviation as lowas ±4%) leads to the possibility of discriminatingbetween RAAs that differ by only 16% (4 x 4).This level of precision cannot always be main-tained during routine work. In any case, theincrease in discriminatory power over that of thestandard CF test (fourfold titer rise) is substan-tial and might permit detection of antibody titerrises in sera taken only a few days apart duringacute disease. This aspect has to be furtherinvestigated.

Conclusions. We found SD-ELISA to be aconvenient and sensitive alternative to CF. Aswith any new technique, the results often willhave to be controlled by established techniquesuntil more experience has been accumulated.For virological laboratories contemplating anextension of services, the final choice of meth-odology has to depend on resources, individualexperience, and the local diagnostic tradition.For SD-ELISA to achieve general acceptance, abroad variety of quality-controlled and reason-ably priced antigens has to be made available.

LITERATURE CITED

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2. Barile, M. F. 1973. Mycoplasma contamination of cellcultures: incidence, source, prevention and problems ofelimination, p. 729-735. In P. F. Kruse, Jr., and M. K.Patterson, Jr. (ed.), Tissue culture. Methods and applica-tions. Academic Press, Inc., New York.

3. Bidwell, D. F., A. Bartlett, and A. Voller. 1977 Enzymeimmunoassay for viral diseases. J. Infect. Dis.136(Suppl.):274-278.

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8. Casey, H. L. 1965. Adaptation of LBCF method to microtechnique. Public Health Monogr. 74:31-34.

9. Castellano, G. A., G. T. Hazzard, D. L. Madden, andJ. L. Sever. 1977. Comparison of the enzyme-linked im-munosorbent assay and the indirect hemagglutination testfor detection of antibody to cytomegalovirus. J. Infect.Dis. 136(Suppl.):357-340.

10. Chia, W. K., and L. Spence. 1979. Quantitative determi-nation of cytomegalovirus IgG antibody by enzyme-linkedimmunosorbent assay (ELISA). Can. J. Microbiol.25:1082-1086.

11. Cremer, N. E., C. K. Cossen, C. V. Hanson, and G. R.

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SheUl. 1982. Evaluation and reporting of enzyme immuno-assay determinations of antibody to herpes simplex virusin sera and cerebrospinal fluid. J. Clin. Microbiol. 15:815-823.

12. de Savigny, D., and A. Voller. 1980. The communicationof ELISA data from laboratory to clinician. J. Immunoas-say 1:105-128.

13. Engvall, E., and P. Perlmann. 1972. Enzyme-linked im-munosorbent assay, ELISA. III. Quantitation of specificantibodies by enzyme-labelled anti-immunoglobulin inantigen-coated tubes. J. Immunol. 109:129-135.

14. Feigner, P. 1978. Stepless antibody determination with thestick-ELISA technique. Results expressed as multiple ofnormal activity (MONA). Zentralbi. Bakteriol. Parasi-tenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 242:100-105.

15. Forghani, B., and N. J. Schmidt. 1979. Antigen require-ments, sensitivity, and specificity of enzyme immunoas-says for measles and rubella viral antibodies. J. Clin.Microbiol. 9:657-664.

16. Grillner, L., and J. Blomberg. 1976. Hemolysis-in-gel andneutralization tests for determination of antibodies tomumps virus. J. Clin. Microbiol. 4:11-15.

17. Gunalp, A. 1965. Growth and cytopathic effect of rubellavirus in a line of green monkey kidney cells. Proc. Soc.Exp. Biol. Med. 118:85-90.

18. Katz, S. L., and J. F. Enders. 1969. Measles virus, p. 523-525. In E. H. Lennette and N. J. Schmidt (ed.), Diagnos-tic procedures for viral and rickettsial infections, 4th ed.American Public Health Association, New York.

19. Kleiman, M. B., C. K. L. Blackburn, S. E. Zimmerman,and M. L. V. French. 1981. Comparison of enzyme-linkedimmunosorbent assay for acute measles with hemaggluti-

nation inhibition, complement fixation, and fluorescent-antibody methods J. Clin. Microbiol. 14:147-152.

20. Leinikki, P. O., and S. Passila. 1977. Quantitative, semi-automated, enzyme-linked immunosorbent assay for viralantibodies. J. Infect. Dis. 136(Suppl.):294-299.

21. Lennette, E. H., F. W. Jensen, R. W. Guenther, and R. L.Magoffin. 1963. Serologic responses to parainfluenza vi-ruses in patients with mumps virus infection. J. Lab. Clin.Med. 61:780-788.

22. Loon, A., J. T. M. van Logt, and J. van der Veen. 1981.Enzyme-linked immunosorbent assay for measurement ofantibody against cytomegalovirus and rubella virus in asingle serum dilution. J. Clin. Pathol. 34:665-669.

23. Schmidt, N. J., E. H. Lennette, and R. L. Magoffin. 1969.Immunological relationship between herpes simplex andvaricella-zoster viruses demonstrated by complement fix-ation neutralization and fluorescent antibody tests. J.Gen. Virol. 4:321-328.

24. Sever, J. L. 1962. Application of a microtechnique to viralserological investigations. J. Immunol. 88:320-329.

25. Sever, J. L., R. L. Huebner, G. A. Castellano, and J. A.Bell. 1963. Serological diagnosis "en masse" with multi-ple antigens. Am. Rev. Respir. Dis. 88:342-358.

26. Vestergaard, B. F., and 0. Jensen. 1980. Diagnosis andtyping of herpes simplex virus in clinical specimens by theenzyme linked immunosorbent assay (ELISA), p. 391-394. In A. Nahmias (ed.), The human herpes viruses.Elsevier/North-Holland Publishing Co., New York.

27. Voller, A., D. Bidwell, and A. Bartlett. 1980. Enzyme-linked immunosorbent assay, p. 359-371. In N. R. Roseand H. Friedman (ed.), Manual of clinical immunology,2nd ed. American Society for Microbiology, Washington,D.C.

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