in the above manner contained 105 to 106 plaqueforming virus preparation was used without further manipulation

Transcription

1 INFECTION AND IMMUNITY, June 1978, p /78/0020-tE60$02.00/0 Copyright 1978 Americn Society for Microbiology Vol. 20, No. 3 Printed in U.S.A. Enzyme-Linked Immunosorbent Assy for Mesurement of Serologicl Response to Respirtory Syncytil Virus Infection RICHARDSON,`* ROBERT H. YOLKEN,' ROBERT B. BELSHE,' ENA CAMARGO,' HYUN W. KIM,2 AND ROBERT M. CHANOCK' Lbortory of Infectious Diseses, Ntionl Institute ofallergy nd Infectious Diseses, Bethesd, Mrylnd 20014,'nd Children's Hospitl Ntionl Medicl Center, Wshington, D.C LINDA S. Received for publiction 31 October 1977 An enzyme-linked immunosorbent ssy ws pplied to the detection of serum ntibodies ginst respirtory syncytil virus. The end points of the vrious ser tested in the ssy were pproximtely 100 times higher thn in the complementfixtion test nd 2 to 4 times higher thn in the plque reduction test. In ddition, the immunosorbent ssy ppered to be more efficient thn the plque reduction nd complement-fixtion techniques for detecting serologicl response in young infnts (1 to 6 months old) with serious respirtory syncytil virus lower respirtory disese. The simplicity, sensitivity, nd rpidity of the enzyme-linked immunosorbent ssy mke it useful tool for immunologicl studies with respirtory syncytil virus. The immunologicl response to respirtory syncytil (RS) virus infection hs been mesured in the pst by the detection of rise in complement-fixtion (CF) ntibodies in the convlescent serum of the infected individul (1). Filure to detect CF ntibody rise in young infnts during convlescence from severe respirtory disese cused by RS virus hs vriously been scribed to immunologicl immturity of the infnt or to the presence of preexisting mternl ntibodies which msk n immune response or impir the bility of the infnt to mount n immunologicl response (5-7). More recently, the plque reduction test hs been used for ssying RS virus neutrlizing ntibodies in humn ser. This technique is more sensitive thn the CF test; however, the difficulty of performing the plque ssy, s well s the longer time necessry to crry out the test, hs limited its generl use (4). We report here sensitive nd rpid ssy for serum ntibodies to RS virus, utilizing the enzyme-linked immunosorbent ssy (ELISA) technique, tht should hve wide pplicbility in dignostic nd seroepidemiologicl investigtions. tug/ml), nd mphotericin (10 ug/ml). When cytopthic effects of the virus were mximl (3 to 4 dys), the cells nd fluid were hrvested together by quick freezing nd stored t -70'C. Virus suspensions mde in the bove mnner contined 105 to 106 plqueforming units of RS virus per ml. A dilution of this virus preprtion ws used without further mnipultion in the ELISA test to provide both internl nd surfce virl ntigens for interction with ntibodies. Enzyme conjugte. Got nti-humn immunoglobulin G, y-chin-specific serum ws obtined from Antibodies Inc., Dvis, Clif. The globulin frction ws prepred by sodium sulfte precipittion nd coupled to lkline phosphtse (Sigm type VII) by the method of Engvll nd Perlmn (3) using 5 mg of enzyme to 2 mg of immunoglobulin. The ctivity nd specificity of the enzyme-linked ntiglobulin ws tested s previously described (11). ELISA procedure. The method used ws modified from Voller et l. (8). The optiml dilutions of regents were determined by checkerbord titrtion. The ntigen (RS A2 virus grown in HEp-2 or GMK cells) ws diluted 1:10 in crbonte buffer (ph 9.8), nd 75,ul ws dded to the wells of round-bottom polyvinyl microtiter pltes (Cooke Lbortory Products, Alexndri, V.). Uninfected cells treted in the sme mnner s virus-infected cells served s control. Pltes coted with ntigen were then stored for t lest 14 h t 40C MATERIALS AND METHODS in moist chmber. Additionl storge t 40C for t lest 3 months did not result in mesurble loss of CF nd plque reduction ssys. Procedures ctivity. hve previously been described for the CF nd plque At the time of testing, the pltes were wshed three reduction ssys (2, 4). times in solution of phosphte-buffered sline contining polysorbte (Tween 20) t concentrtion of Antigen for ELISA. The A2 strin of RS virus (4) ws grown in HEp-2 or primry Africn green monkey 0.5 ml/liter (PBS-Tween). Fourfold dilutions of serum kidney (GMK) cells (Flow Lbortories, Rockville, were mde in the ntigen-coted pltes using PBS- Md.) mintined in Egle miniml essentil medium Tween supplemented with 1% fetl clf serum nd supplemented with 2% gmm clf serum, M 10% uninfected cell suspension. The clf serum nd glutmine, penicillin (200 U/ml), streptomycin (200 control cell suspension were dded to the diluent to 660

2 VOL. 20, 1978 reduce the nonspecific binding of the test serum (9). The finl volume of diluted serum in ech well ws 75 pl. After n overnight incubtion t 40C, the pltes were wshed three times with PBS-Tween, nd 75- pl smple of 1:400 dilution of the enzyme-linked globulin frction of got nti-humn immunoglobulin G serum (mde in the sme diluent s used for the dilution of ptients' ser) ws dded. This ws llowed to rect for 2 h t 370C. The pltes were gin wshed three times with PBS-Tween, nd 75 ul of p-nitrophenyl phosphte substrte (Sigm 104), diluted to contin 1 mg in 1 ml of diethnolmine buffer (ph 9.8), ws dded (8). After 15 min of incubtion t 370C, the mount of yellow color produced by the ction of the enzyme (bound to the solid phse) on the substrte ws mesured in colorimeter, which determined the bsorbnce t 400 nm through the bottom of the microtiter plte (10). The ELISA titer of ech serum ws determined by compring the bsorbnce vlue of ech serum dilution with the vlue of known positive humn serum stndrd diluted 1:1,600. In ech cse the bsorbnce vlue of the serum in the control well ws subtrcted from the vlue obtined in the RS ntigen-coted wells. The 1:1,600 dilution of the stndrd serum ws considered the end point in the ELISA test since it hd n bsorbnce vlue of pproximtely 0.35, while the bsorbnce vlue of the serum t 1:6,400 dilution ws only Therefore vlues greter thn 0.35 were considered positive. The stndrd serum t 1:1,600 dilution ws run in ech plte to eliminte plte-to-plte vrition. Serum specimens. Acute nd convlescent ser were obtined from ptients dmitted to the Children's Hospitl Ntionl Medicl Center of the District of Columbi with severe RS virus respirtory disese. The dignosis of RS virus disese ws estblished by isoltion of RS virus from the orophrynx nd/or fourfold rise in RS virus serum CF ntibodies during convlescence. Ech of the 1- to 3-month-old infnts whose ser were used in the study shed RS virus. RESULTS Rectivity with control ntigens. Initilly, the strting serum dilution for the ELISA ws 1:20; however, rectivity of mny ser with control cell ntigens mde it necessry to use higher strting serum dilution, i.e. 1:100, to void this rectivity. Even t 1:100 n occsionl serum rected with control cell ntigens to the sme extent s the positive serum control with RS virus ntigens. Both cute- nd convlescent-phse ser from 5 of 91 ptients tested exhibited such rectivity. In four of these serum pirs, the RS virus rectivity ws t lest fourfold greter in titer thn the control cell rectivity, nd thus the ser were considered stisfctory for evlution by ELISA. The cute-phse serum of the remining serum pir rected to similr degree with control cell ntigens nd RS virus ntigens t 1:100 dilution yet ws not positive with either 1:400 dilution of serum. ELISA FOR RS VIRUS SERUM ANTIBODIES 661 Thus, the titer of this serum ws <1:400. The titer of the convlescent serum in this cse ws 1:6,400. Therefore, even though definite endpoint titer could not be given to the cute-phse serum, seroresponse in this child ws evident. (This set of pired ser is not included in Fig. 1, 2, or 3, but is included in Tble 1.) Comprison with other serologicl tests. Acute- nd convlescent-phse ser from 91 infnts nd children hospitlized with RS virus lower respirtory trct disese were ssyed by the CF, plque reduction, nd ELISA techniques to define the reltive efficiency of the three methods. Previously the dignosis of RS virus disese hd been estblished by virus isoltion nd/or fourfold or greter rise in serum CF ntibody titer. The ELISA test proved more sensitive thn CF for detecting RS virus serum ntibody, since 40 of 55 cute-phse ser with CF ntibody titer of <1:4 hd RS ntibody ctivity t serum dilution of 1:100 or greter in the ELISA test (Fig. 1). In no instnce ws CF ntibody detected without concomitnt ELISA ntibody. In generl there ws concordnce between the plque reduction nd ELISA techniques for detection of RS serum ntibodies, lthough the ltter method usully yielded higher titer (Fig. 2). Fifteen of the cute-phse ser which lcked >25,60r I-. z wc 6,400 k 1,600 F 400 k 100 F * 0 so m s A *16L Pon U < >256 COMPLEMENT FIXATION ANTIBODY TITER (recpocl) FIG. 1. Comprison oj RS virus ntibodies mesured by ELISA nd CF using end-point titrtion. Both the cute (0) nd convlescent (0) ser from infnts nd young children with severe respirtory disese were mesured.

3 662 RICHARDSON ET AL. > 25,600 : CO z CL w 6,400 1, IW r _- 8 to.1 A LI 8A olood * nd plque reduction for detection nd mesurement of RS virus ntibodies (Fig. 2). Ech of 14 ser which lcked detectble ELISA ntibody (<1:100) lso lcked detectble plque reduction lb A * I ntibody. Also, every serum tht possessed plque reduction ntibody ws lso positive when tested by ELISA. Efficiency of ELISA for serodignosis of * MIo oca RS virus infection. The efficiency of the CF, plque reduction, nd ELISA techniques for serodignosis ws compred using 91 pired ser from infnts nd children with RS virus disese co Omof the lower respirtory trct (Tble 1). In the 1- to 3-month-old ge group, RS virus ws isolted from the orophrynx of ech of the ptients, yet only 1 of 49 develped significnt AS 0 serum CF ntibody rise to RS virus. One of 47 young infnts hd fourfold rise in plque reduction ntibody. Heprinized plsm ws collected from two of the children in this ge group insted of serum; heprinized plsm could not be tested for plque reduction ntibodies to RS 200 4w 16 >3200 virus, since heprin inctivtes the virus (Chn- I < ock nd Richrdson, unpublished dt). In con- PLAQUE REDUCTION ANT 'IBODY TITER (reciprocl) FIG. 2. Comprison of R.' trst to the results obtined with the other tech- 5 virus ntibodies me-. sured by ELISA nd the plque reduction ssy. niques, 25 of the 49 infnts hd significnt Both the cute (0) nd con vlescent (0oser from ntibody rise when tested by ELISA, indicting infnts nd young children with severe respirtory the greter efficiency of the ELISA technique disese were mesured. for detection of n RS virus serologicl response during erly infncy, the period when this virus is most importnt s cuse of serious lower detectble plque reduction ntibody hd nti- respirtory trct disese. This greter efficiency body demonstrble by ELISA. In no instnce of the ELISA technique ws lso evident to ws plque reduction nt: ibody ctivity present lesser degree in the 4- to 6-month ge group. In without corresponding EL ISA ntibody ctivity. infnts over 7 months old, the number of signif- ELISA ntibody de- icnt ntibody rises detected by ech of the In only two instnces wrs tected during convlescesnce without concomi- three methods ws similr. tnt plque reduction nt;ibody. Age distribution of cute-phse serum Reproducibility of ELISA. Three ser were ELISA ntibody titers. RS serum ntibody A. v_ 31L--T _ A tested by EL1SA T TC1 A mne * - s- times A. over period of 3 months to evlute the reproducibility of the ssy. With one serum there ws no vribility in titer from test to test; of the remining two ser, one vried in titer (fourfold) only once in nine tests, while the other serum vried in titer (fourfold) on two of nine occsions. Specificity of ELISA. To test the specificity of the ELISA technique for detecting RS virus serum ntibodies, 12 sets of pired ser from ptients with greter thn fourfold rise in ntibody level ginst prinfluenz 1, 2, or 3 virus, denovirus, mesles virus, or influenz A virus were tested by the ELISA technique. In ech cse, the ntibody titer ginst RS virus remined constnt, indicting the specificity of the ELISA for the detection of RS virus-specific ntibodies. Additionl evidence for specificity of the RS virus ELISA ws provided by the generl concordnce between the results of this ssy INFECT. IMMUN. TABLE 1. Serologicl response ofptients with RS virus infections s mesured by CF, plque reduction, nd ELISA techniques No. with increse' in ntibody titer by Age indicted technique/no. tested (months) of ptients CF Plque re- ELISA duction 1 to 3 1/49 1/47b 25/49 4 to 6 9/19 11/19 18/19 7 to 12 9/10 9/10 9/10 13to24 6/9 8/9 8/9-25 3/4 3/4 3/4 Totl 28/91 32/89 63/91 Fourfold or greter rise. b Two children in this ge group could not be tested by the plque reduction technique, since heprinized plsm ws collected insted of serum nd heprin inctivtes RS virus. Ech of these children hd serologicl response detectble by ELISA.

4 VOL. 20, 1978 levels s determined by the ELISA technique in the cute-phse ser of ptients with respirtory disese showed n ge distribution similr to tht described previously for plque reduction ntibodies (5) (Fig. 3). All 1- to 3-month-old infnts possessed ELISA ntibodies, wheres mny older infnts nd young children lcked detectble ntibodies. This suggested tht the young infnts' ntibodies were pssively cquired from the mother. This pttern provided further evidence tht the ELISA technique mesured specific RS virus ntibodies. DISCUSSION The ELISA technique ppers to be specific nd sensitive method for detection of RS virus serum ntibodies. The test hs the dvntge of mesuring higher titers of ntibody thn the CF nd plque reduction techniques, the current methods of ssying serum ntibodies to RS virus. In ddition, heprinized plsm cn be tested by ELISA, but not by the plque reduction technique since heprin inctivtes RS virus Ṫhe lowest dilution of serum tht could be tested by ELISA ws determined by the rectivity of serum with control cell ntigens. Most ser tested (172 of 182) did not exhibit such rectivity t dilution of 1:100. Becuse of the sensitivity of the RS virus ELISA, it ws still possible to test stisfctorily ech of 91 pirs of ser for evidence of seroresponse. In no instnce did rectivity with control cell ntigens prevent detection of seroresponse. Of specil interest is the bility of the ELISA r 400 H ish '.5 ELISA FOR RS VIRUS SERUM ANTIBODIES 663 technique to detect serologicl response to RS virus in pproximtely 50% of 1- to 3-month-old infnts infected with the virus. In contrst, other serologicl techniques re reltively inefficient in this ge group, the trget popultion for most serious disese. It ppers tht the mjority of young infnts re cpble of n immunologicl response to RS virus. Although the presence of preexisting mternl ntibody my obscure or suppress this response, it does not completely block it. Thus, the bility of the ELISA technique to detect n immune response in the young infnt should id in serodignostic nd seroepidemiologicl studies of RS virus infection during erly life. It ws surprising tht the ELISA detected serologicl response in 25 of 49 infnts 1 to 3 months of ge while the plque reduction test detected only one response. Possibly this my be explined by the fct tht the plque reduction test mesures only those serum ntibodies which neutrlize virus nd thereby render the virus incpble of inititing plque (i.e., ntibodies to one or more surfce ntigens), wheres the ELISA mesures neutrlizing ntibodies s well s ntibodies tht re directed ginst internl ntigens. The ltter ntigens re prticulrly bundnt in RS virus-infected tissue culture. All three tests pper to be eqully cpble of detecting n immunologicl response in the older group of individuls tested (>7 months old). However, the simplicity nd rpidity of the ELISA test suggest tht it my offer n dvntge in this ge group s well. L0 100 H * U. 16. * X6 l * * * AGE OF INDIVIDUAL TESTED (months) * IS I L~~~~~~~~~~~~L~~~~~~J~~.0% I112 '13 FIG. 3. RS virus ntibody titer s mesured by ELISA in the cute-phse serum of infnts nd young children with RS virus respirtory disese by ge.

5 664 RICHARDSON ET AL. ACKNOWLEDGMENT This study ws supported by Public Helth Service grnt AI from the Ntionl Institute of Allergy nd Infectious Diseses. LITERATURE CITED 1. Chnock, R. M., L. Chmbon, W. Chng, F. Gon- Vlves Ferreir, P. Ghrpure, L. Grnt, J. Htem, I. Imm, S. Klr, K. Lim, J. Mdlengoiti, L Spence, P. Teng, nd W. Ferreir WHO respirtory disese survey in children: serologicl study. Bull. W.H.O. 37: Chnock, R. M., H. W. Kim, A. J. Vrgosko, A. Delev, K.M. Johnson, C. Cumming, nd R. H. Prrott Respirtory syncytil virus. I. Virus recovery nd other observtions during 1960 outbrek of bronchiolitis, pneumoni, nd minor respirtory diseses in children. J. Am. Med. Assoc. 176: Engvll, E., nd P. Perlmn Enzyme-linked immunosorbent ssy, ELISA. III. Quntittion of specific ntibodies by enzyme-linked nti-immunoglobulin in ntigen-coted tubes. J. Immunol. 109: Mills, J., J. E. Vn Kirk, P. F. Wright, nd R. M. Chnock Experimentl respirtory syncytil virus infection of dults. J. Immunol. 107: Prrott, R. H., H. W. Kim, J. 0. Arrobio, D. S. Hodes, B. R. Murphy, C. D. Brndt, E. Cmrgo, nd R. M. Chnock Epidemiology of respirtory syncytil virus infection in Wshington, D.C. II. Infection nd disese with respect to ge, immunologic sttus, INFECT. IMMUN. rce nd sex. Am. J. Epidemiol. 98: Prrott, R. H., A. J. Vrgosko, H. W. Kim, C. Cumming, H. Turner, R. J. Huebner, nd R. M. Chnock Respirtory syncytil virus. II. Serologic studies over 34-month period of children wih bronchiolitis, pneumoni, nd minor respirtory diseses. J. Am. Med. Assoc. 176: Ross, C. A. C., I. W. Pinkerton, nd F. A. Assd Pthogenesis of respirtory syncytil virus disese in infncy. Arch. Dis. Child. 46: Voller, A., D. Bidwell, nd A. Brtlett Microplte enzyme immunossys for the immunodignosis of virus infections, p In N. R. Rose nd H. Friedmn (ed.), Mnul of clinicl immunology. Americn Society for Microbiology, Wshington, D.C. 9. Yolken, R. H., H. B. Greenberg, M. H. Merson, R. B. Sck, nd A. Z. Kpikin Enzyme-linked immunosorbent ssy for detection of Escherichi coli het-lbile enterotoxin. J. Clin. Microbiol. 6: Yolken, R. H., H. W. Kim, T. Clem, R. G. Wytt, A. R. Klic, R. M. Chnock, nd A. Z. Kpikin Enzyme linked immunosorbent ssy (ELISA) for detection of humn reovirus-like gent of infntile gstroenteritis. Lncet ii: Yolken, R. H., R. G. Wytt, H. W. Kim, A. Z. Kpikin, nd R. M. Chnock Immunologicl response to infection with humn reovirus-like gent: mesurement of nti-humn reovirus-like gent immunoglobulin G nd M levels by the method of enzymelinked immunosorbent ssy. Infect. Immun. 19: Downloded from on October 17, 2018 by guest