Measurement of Parainfluenza-3 Virus Antibody by the

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1975, p Copyright C 1975 Americn Society for Microbiology Vol. 2, No. 3 Printed in U.S.A. Mesurement Prinfluenz-3 Virus Antibody by the Single Rdil Hemolysis Technique M. PROBERT* AND S. M. RUSSELL Virology Deprtment, Wellcome Reserch Lbortories, Lngley Court, Beckenhm, Kent BR3 3BS, Englnd Received for publiction 12 My 1975 A single rdil hemolysis ssy for prinfluenz-3 virus ntibody is described s simple, sensitive, ccurte, nd precise lterntive to hemgglutintion inhibition nd serum neutrliztion tests. A highly significnt correltion exists between single rdil hemolysis zone res nd hemgglutintion inhibition titers both serum nd nsl secretion smples. Antibody conversions equivlent to s little s twold rise in hemgglutintion inhibition titer re redily nd relibly detected in ser nd nsl secretions. Single rdil hemolysis nd hemgglutintion inhibition ctivity ws demonstrble in the immunoglobulin G frction hyperimmune prinfluenz-3 virus ntiserum. Initil serologicl studies with bovine prinfluenz-3 (Pi-3) virus utilized complement fixtion, serum neutrliztion (SN), nd hemgglutintion inhibition (HI) to mesure virus ntibody (1, 5-7). There ws good greement between the results these tests but HI ws found to be the most sensitive nd hs since been widely employed. However, greter sensitivity SN over HI hs been described in microtiter system (12). In both SN nd HI ssys, twold smple dilution series is usully employed nd the minimum ccepted significnt difference between ntibody titers is fourfold. The ccurcy nd resolution these ssys is thus limited by the size the dilution step used. It is lso difficult to chieve reproducible results from test to test with HI, nd SN suffers from the disdvntge being reltively complex nd lengthy ssy procedure. A single rdil hemolysis (SRH) method for mesurement influenz virus ntibody hs recently been described (13), nd this technique hs been extended nd dpted in the current work to provide simple, sensitive, nd precise lterntive ssy for Pi-3 virus ntibody. MATERIALS AND METHODS Viruses. Bovine Pi-3 virus ws grown in primry clf kidney cell cultures. Two isoltes were used. (i) "Compton" virus ws isolted t the Institute for Reserch on Animl Diseses, Compton, Berks, Englnd, in 1968 from n uninoculted clf kidney cell culture nd fully chrcterized s Pi-3 virus (Jnice C. Collins, Ph.D. thesis, Univ. Reding, Reding, 1971). It hs since undergone multiple pssges in 157 clf kidney cell cultures nd hs strong hemgglutinin ctivity, routinely reching titers 512 to 124 hemgglutinting units/.25 ml tissue culture fluid. (ii) J121 virus ws isolted from nturlly occurring cse clf respirtory disese (2) nd hs reltively wek hemgglutinin ctivity, routinely reching titers only 16 to 32 hemgglutinting units/.25 ml culture fluid. The infectivity titers the fluids re, however, high t 1 x 19 to 5 x 19 plque-forming units/ml. Ser nd nsl secretions. These were kindly provided by E. J. Stott nd were derived from field nd experimentl clves smpled in the course investigtion Pi-3 virus infection. Nsl secretions were collected by the method McKercher (1). All ser nd nsl secretions were inctivted t 56 C for 3 min before use. Erythrocytes. Adult bovine erythrocytes were collected in Alsevers solution nd stored t 4 C for up to 1 week. They were wshed three times in phosphtebuffered sline before use. SRH technique. The method followed ws essentilly tht described by Russell et l. (13). The erythrocyte concentrtion in the gels nd smple diffusion time were stisfctorily stndrdized on the bsis their results. Equl volumes pcked erythrocytes nd Anlr sodium periodte in sline were mixed nd left t room temperture for 1 min. M/6 periodte ws found to be optiml. Tissue culture fluid contining Pi-3 virus ws then dded to give t lest 13,5 hemgglutinting units (Compton isolte) or 1.3 x 19 plque-forming units (J121 isolte) per.1 ml pcked erythrocytes. The mixture ws left t room temperture for further 2 min to llow irreversible virus ttchment to receptors oxidized by the exposure to periodte. A smll mount hemolysis ws lwys observed t this stge. The treted cells were wshed

2 158 PROBERT AND RUSSELL three times in brbitone-buffered sline, finlly pcked, nd resuspended in brbitone-buffered sline to give 5% (vol/vol) suspension. Agrose (1%) (Indubiose A37, L'Industrie Biologique Frncise SA) in brbitone-buffered sline contining.1% sodium zide ws melted nd held t 45 C. A.1-ml mount the treted erythrocyte suspension,.2 ml guine pig serum (source complement), nd 2.7 ml the grose were rpidly mixed nd poured into n immunodiffusion plte (Hylnd Div., Trvenol Lbs Inc., Cost Mes, Clif.). Wells (3 mm dimeter) were punched in the gel nd ech well ws filled with 1 Al serum or nsl secretion. The gels were stored t 4 C overnight in moist box to llow rdil diffusion ntibody nd then incubted t 37 C for 2.5 h to develop zones complement-medited erythrocyte lysis round the ntibody-positive wells (Fig. 1). At lest three rdii t right ngles were mesured for ech zone with mgnifier incorporting "cross wire" grticule clibrted in.1-mm divisions (Grticules Ltd., Tonbridge, Kent). The men rdius ws used to clculte zone re which ws corrected by subtrction the well re. HI ntibody ssy. The microtiter method described by Rossi nd Kiesel (12) ws used except tht the pltes were incubted t 37 C for 45 min fter the ddition 1 drop (.25 ml) 1% humn "O" erythrocytes to the virus/serum mixture. The Compton isolte Pi-3 virus ws used in the ssy nd ll ser were bsorbed with humn erythrocytes for 1 h t room temperture before use. SN test. The microtiter method Rossi nd Kiesel (12) ws followed except tht secondry clf kidney cells were used. RESULTS Although initil experiments utilized the Compton isolte Pi-3 virus, the J121 isolte ws found to be more efficient in SRH nd ws therefore dopted for the routine ssy. J. CLIN. MICROBIOL. Effect serum dilution nd virus concentrtion on SRH zone re. Treting the erythrocytes with incresing mounts virus cused reduction in SRH zone re until concentrtion ws reched bove which no further decrese in zone re occurred (Fig. 2). This ws therefore the minimum concentrtion required to ensure reproducibility nd ws utilized in ll subsequent ssys with tht prticulr virus preprtion. Figure 2 lso shows tht n lmost liner reltionship exists between zone re nd the logrithm serum dilution. The grdients dose-response curves obtined with severl bovine ser were ll very similr. Precision nd resolution the SRH ssy. The vrition SRH zone re within nd between single gels from given btch ws ix 1-8/1nr 1:1 1: :64 Serum dilution FIG. 2. The effect serum dilution nd virus concentrtion on SRH zone re (J121 Pi-3 virus used in SRH test; ech point represents the men four replicte estimtions). rbc FIG. 1. Hemolysis zones produced by clf nsl secretions (left-hnd wells) nd ser (right-hnd wells) in gels contining J121 Pi-3 virus-sensitized bovine erythrocytes.

3 VOL. 2, 1975 SINGLE RADIAL HEMOLYSIS FOR PARAINFLUENZA 159 investigted nd the results re presented in Tble 1. It cn be seen tht the overll vrition is low mgnitude. As expected, the vrition in zone re between seprte ssys crried out on different dys ws greter (Tble 2), but the overll stndrd devition only just exceeded 1% the men in the cse the smllest zones. The ssy closely spced serum dilutions (Tble 3) demonstrted the bility the method to resolve reltive dilution 1:1.33 (men zone res significntly different by the Student's t test, P <.1). TABLE 1. Vrition in re hemolysis within nd between gels from single btch Gel hemolysis Men reb (mm2) devition Stndrd b Men res clculted from six replicte wells clf ntiserum per gel. Overll men re = mm2; overll stndrd devition = 1.63 mm2. TABLE 2. Vrition in re hemolysis between seprte ssys Clf serum Serum dilution 512/ Net 1.75b (5.4) 95.5 (3.94) (2.35) 1: (4.8) (4.31) 1: (3.14) 4.3 (2.83) 1: (2.48) 15.3 (2.6) b Overll men re (mm2) from four seprte ssys crried out on different dys; stndrd devition in brckets. Four replictes per serum dilution per ssy. TABLE 3. Resolving bility the SRH ssy Men re Serumb dilution hemolysisc Stndrd devi- (MM2) tion 1: : : : : : bclf serum 512/21 (Tble 2). c Mens four replictes, one replicte per gel. Correltion SRH with HI nd SN nd effect virus isolte on SRH zone re. A comprison ws mde between HI titers nd SRH zone res for severl groups clf ser. A highly significnt positive correltion between the results the two ssy methods ws obtined in group 3 ser (Fig. 3). Two ser with HI titers 2 nd 8 produced nonmesurble, diffuse zones hemolysis nd were scored s SRH zone negtive in Fig. 3. However, with the J121 isolte Pi-3 virus shrper mesurble zones were obtined with these ser (21.21 nd mm2, respectively). In further group 48 ser from experimentl clves there ws highly significnt correltion between the results HI nd SRH nd SN nd SRH (SRH/HI, r =.93; SRH/SN, r =.88, P <.1). There ws lso highly significnt correltion between the HI titers nd SRH zone res 48 prllel nsl secretion smples from the sme clves (r =.83, P <.1). HI titers nd SRH zone res in group 36 high-titer clf ser (29 ser with HI titers >16) showed highly significnt correltion which ws independent the virus isolte used for SRH (Compton isolte: r =.64; J121 isolte: r =.66, P <.1). A comprison the SRH zone res produced by the two virus isoltes (Fig. 4) demonstrted tht J121 virus-sensitized erythrocytes gve lrger zones. J121 ws therefore subsequently used routinely for Pi-3 SRH. Detection seroconversions. Upon considertion the precision the SRH ssy described bove, conservtive vlue 1 mm2 ws rbitrrily fixed s the minimum increse 5 Are / Lysis * mm2 41* r = 84 c1 2 8 Hi Titre FIG. 3. Correltion between SRH zone re nd HI titer in group 3O clfser (Compton Pi-3 virus used in SRH test).

4 16 PROBERT AND RUSSELL in zone re corresponding to positive seroconversion. In n experimentl Pi-3 tril, 15 clves were chllenged with Pi-3 virus nd 9 remined s unchllenged controls. Serum nd nsl secretion smples were tken t the time chllenge nd t 3 weeks postchllenge. Seroconversion the 15 virus-chllenged nimls ws detected by both SRH nd HI. Only 12 seroconversions were detected by SN. In ddition, conversion nsl secretions to ntibody positivity ws detected in 1 the chllenged nimls by HI nd in 12 by SRH. Frctiontion hyperimmune Pi-3 virus ntiserum. The globulin component hyperimmune clf Pi-3 virus ntiserum ws precipitted with 4% sturted mmonium sulfte nd the resuspended pellet ws dilyzed ginst brbitol-buffered sline nd pplied to Bio Gel A-1.5 million (Bio-Rd Lb., Richmond, Clif.) column. The eluted frctions were Are Lysis mm2 r = 93 FIG. 4. Correltion SRH zone res produced by the Compton nd J121 virus isoltes in group 36 clf ser. J. CLIN. MICROBIOL. tested for Pi-3 virus ntibody by HI nd SRH, nd the peks ctivity by both ssys corresponded to the loction immunoglobulin G (IgG) in the elution prile the serum (Fig. 5). This result lso reinforced the previous observtion close correltion between the HI nd SRH tests. SRH Zone re mm2 %Trnsmittnce t 28nm DISCUSSION The SRH technique (13) hs been successfully pplied to the ssy Pi-3 virus ntibody in serum nd nsl secretions. The ssy is t lest s sensitive s HI nd hs the mjor dvntge high precision nd ccurcy relted to the mesurement continuous vrible. It is simple nd rpid in comprison to SN tests which require 6 dys incubtion nd the use live tissue culture cells (12). Conversions to ntibody positivity in serum nd nsl secretions re redily detected nd smll increses in ntibody levels cn be relibly mesured. These highly significnt increses re much less thn the minimum 4-fold titer rises required in HI nd SN tests. Thus the SRH ssy is well suited to the rpid screening ser nd nsl secretions which cn be pplied to the gels undiluted to give n ccurte estimte totl ntibody content. Use control stndrd serum would minimize the lredy smll effects test-to-test vrition. From the results obtined with two seprte isoltes Pi-3 virus in the SRH test it is pprent tht, while the test does not provide evidence for n ntigenic difference, the isolte utilized my effect ssy sensitivity. The J121 isolte with wek hemgglutinin ctivity produced lrger, more distinct zones thn the Compton isolte which hs strong hemgglutinin ctivity. Therefore strins with wek hemgglutinin ctivity my be more suitble in the SRH ssy. It hs been shown tht in hyperimmune serum ntibody ctivity mesurble by HI nd SRH resides in the IgG frction. However, fur ')~~~~~~ ia I I 16 HI - 4 titre I Frction No. FIG. 5. Reltionship between protein concentrtion, SRH zone re, nd HI titer frctions hyperimmune clf Pi-3 virus ntiserum eluted from Bio Gel A-1.5 m.

5 VOL. 2, 1975 ther studies re in progress on the immunoglobulin species ctive in erly immune ser nd nsl secretions. Previous work (8, 9) showed residence lmost ll nti Pi-3 virus ctivity in the IgA frction, but since IgA does not fix complement (3) the ntibody detected by SRH in nsl secretions is thought to be IgG. Preliminry observtions suggest tht nsl secretions from young clves with high levels pssively derived colostrl ntibody (lrgely IgG,; 4, 11) produce cler SRH zones in contrst to incomplete hemolysis-produced smples from older clves with ctively cquired ntibody. Secretory IgA could hve blocking effect in the SRH test. We believe tht the SRH ssy for Pi-3 virus ntibody is significnt improvement on current methods ssy. It should mke contribution to the ccurcy, precision, nd discrimintion future serologicl studies with this virus nd my even provide informtion on different components the humorl immune response. ACKNOWLEDGMENTS We grtefully cknowledge the coopertion the stff the Institute for Reserch on Animl Diseses, Compton, in the current work, prticulrly E. J. Stott nd A. Collins for providing technicl ssistnce nd ccess to experimentl nd field mteril nd J. Brownlie for the serum frctiontion. LITERATURE CITED 1. Abinnti, F. R., A. B. Hoerlein, R. L. Wtson, nd R. J. Huebner Serologic studies myxovirus prinfluenz 3 in cttle nd the prevlence ntibodies in bovines. J. Immunol. 86: Betts, A. O., A. R. Jennings, A. R. Omr, Z. E. Pge, J. B. Spence, nd R. G. Wlker Pneumoni in SINGLE RADIAL HEMOLYSIS FOR PARAINFLUENZA 161 clves cused by prinfluenz virus type 3. Vet. Rec. 76: Bienenstock, J The physiology the locl immune response nd the gstro-intestinl trct, p In L. Brent nd J. Holborrow (ed.), Progress in immunology II, vol. 4. North-Hollnd Publishing Co., Amsterdm. 4. Butler, J. E Synthesis nd distribution immunoglobulins. J. Am. Vet. Med. Assoc. 163: Hmprin, V. V., F. V. Wshko, A. Ketler, nd M. R. Hillemn Lbortory nd field investigtions bovine myxovirus prinfluenz 3 virus nd vccine. Ill. Evlution n SF-4 (Shipping fever) virus vccine in cttle. J. Immunol. 87: Hoerlein, A. B., M. E. Mnsfield, F. R. Abinnti, nd R. J. Huebner Studies shipping fever cttle. I. Prinfluenz 3 virus ntibodies in feeder clves. J. Am. Vet. Med. Assoc. 135: Ketler, A., V. V. Hmprin, nd M. R. Hillemn Lbortory nd field investigtions bovine myxovirus prinfluenz 3 virus nd vccine. I. Properties the SF-4 (Shipping fever) strin virus. J. Immunol. 87: Morein, B Immunity ginst prinfluenz-3 virus in cttle. IgA in nsl secretions. Int. Arch. Allergy Appl. Immunol. 39: Morein, B Immunity ginst prinfluenz-3 virus in cttle; immunoglobulins in serum nd nsl secretions fter subcutneous nd nsl vccintion. Z. Immunitetsforsch. Exp. Klin. Immunol. 144: McKercher, D. G A simple method for obtining undiluted nsl secretions from cttle. Am. J. Vet. Res. 34: Porter, P Functionl heterogeneity the bovine immune system. J. Am. Vet. Med. Assoc. 163: Rossi, C. R., nd G. K. Kiesel Microtiter tests for detecting ntibody in bovine serum to prinfluenz 3 virus, infectious bovine rhinotrcheitis virus, nd bovine virus dirrhoe virus. Appl. Microbiol. 22: Russell, S. M., D. McChon, nd A. S. Bere A single rdil hemolysis technique for the mesurement influenz ntibody. J. Gen. Virol. 27:1-1.