Influenza A Neuraminidase Antibody Assay with Sensitized Erythrocytes

Transcription

1 APPLuD MicouowGY, Jn. 1973, p Copyright Americn Society for Microbiology Vol. 25, No. 1 Printed in U.SA. Influenz A Neurminidse Antibody Assy with Sensitized Erythrocytes J. L. HOLSTON, JR., I AND W. R. DOWDLE Respirtory Virology Unit, Center for Disese Control, Helth Services nd Mentl Helth Administrtion, U.S. Deprtment of Helth, Eduction, nd Welfre, Atlnt, Georgi Received for publiction 21 September 1972 Erythrocytes sensitized with purified neurminidse (Hong Kong) ntigens were used for ssy of influenz A neurminidse ntibodies. The neurminidse indirect hemgglutintion test ws equl to the neurminidse hemgglutintion-inhibition (enhncement) test nd ppered to be better thn the neurminidse inhibition test for detection of fourfold or greter ntibody rises in pired ser from influenz ptients or vccinees. It ws better thn both tests for detection of neurminidse ntibody. The neurminidse indirect hemgglutintion test is simple to perform nd hs the dvntge of direct ntigenntibody ssy. The recognition tht the surfce of the influenz virus is composed of second mjor ntigen, the neurminidse (N), in ddition to the hemgglutinin (H) ntigen, hs stimulted efforts to develop prcticl methods for ssying neurminidse ntibodies. The neurminidse inhibition test (NI), in which ntibody titer is expressed in terms of inhibition of enzyme ctivity (14), hs proved useful, but even with modifictions (5) it is often imprcticl for smll lbortories or for lrge-scle studies. The neurminidse-hemgglutintion-inhibition (N-HI) enhncement test (6), in which ntibody titer is determined indirectly through interference with hemgglutintion, is simple to perform, but its dependence upon four rectnts (virus, ntibody, nti-ntibody, nd erythrocytes) mkes it complex test system (W. R. Dowdle, D. Srtenu, nd C. B. Reimer, J. Immunol., in press). In this report (submitted by J. L. Holston, Jr., in prtil fulfillment of the requirements for the Doctor of Public Helth degree in Lbortory Prctice from the University of North Crolin, Chpel Hill, N.C.) we described serologicl test which uses glutrldehyde-fixed nd tnnic cid-treted erythrocytes sensitized with neurminidse ntigen. The test is simple nd sensitive nd hs the dvntge of direct ntigenntibody ssy. MATERIALS AND METHODS Seed viruses. Influenz strins A/ 'Present ddress: Stte Deprtment of Public Helth, Montgomery, Al NWS/33(HON1), A/Hong Kong/8/68(H3N2), A/ Aichi/2/68(H3N2), nd B/Msschusetts/3/66 were from the reference virus collection of the World Helth Orgniztion (WHO) Interntionl Influenz Center for the Americs (IlCA), Atlnt, G. Recombinnt virus A/NWS/33(HO)-Hong Kong/8/68(N2) ws prepred ccording to the method of Webster (13). Recombinnt virus A/equine/Prgue/1/56 (Heql)-Hong Kong/16/68(N2) ws furnished by E. D. Kilbourne, Mount Sini School of Medicine, New York. Prent viruses nd recombinnt strins re nmed ccording to the system of nomenclture recommended by WHO (15). Reference ntiser. Chicken ntiser prepred ginst A/NWS(HON1) nd the recombinnt viruses A/Prgue(heql)-HK(N2), A/HK(H3)-NWS(N1), nd A/NWS/33(HO)-Tiwn/1/64(N2) were obtined from the reference ntiser collection of the IICA. Rbbit ntiser to isolted type A ribonucleoprotein, isolted type A mtrix protein, nd purified Hong Kong neurminidse (N2) were furnished by G. C. Schild, Ntionl Institute for Medicl Reserch, London. Guine pig ntiser to type A RNP were obtined from the IICA reference serum collection. Humn ser. Humn serum specimens were from vccine study conducted mong inmtes t the Georgi Stte Prison, Reidsville, G. "Vccine" ser consisted of pired serum specimens, the first drwn 1 week before the dministrtion of vccine nd the second 28 dys lter. Vccinees received either 300 or 3,000 chick cell-gglutintion (CCA) units of A/Jpn/170/62(H2N2) or 300 or 3,000 CCA units of A/Aichi/2/68(H3N2). "Illness" ser consisted of pired ser from volunteer controls. Ech subject hd been nturlly infected with Hong Kong influenz s shown by fourfold or greter serum ntibody rise in the hemgglutintion-inhibition (HI) test. The detils of this study re published elsewhere (9, 12). 97

2 98 HOLSTON AkpND DOWDLE HI test. Ser were ssyed for HI ntibodies ccording to the Center for Disese Control (CDC) stndrdized Microtiter procedure (4). Neurminidse ssy. Neurminidse ctivity of virus preprtions ws ssyed s described by Webster nd Pereir (14). Smples to be ssyed were prepred in seril 0.5-log10 dilutions. A 0.05-ml mount of ech dilution ws dded to n equl volume of fetuin (24 mg/ml) substrte nd buffer, ph 5.9, nd incubted for 1 hr t 37 C. The opticl density (OD) of the colored orgnic extrct ws red t 549 nm ginst regent blnk (fetuin nd sline) on Beckmn DU-2 spectrophotometer. Neurminidse inhibition test. Antineurminidse serum titers were lso determined ccording to the method described by Webster nd Pereir (14). Virus concentrtions with neurminidse ctivity of OD 0.5 to 0.9 per 0.05 ml were mixed with equl volumes of seril 0.5-log1o dilutions of ntiser, nd incubted t 37 C for 30 min. Fetuin (24 mg/ml) nd buffer, ph 5.9, were then dded, nd the mixture ws incubted for 1 hr t 37 C. The OD of the orgnic extrct ws red t 549 nm ginst regent blnk (fetuin nd sline). Antineurminidse titers were clculted by plotting the corrected (from norml serum controls) OD redings ginst the dilution fctor of ntiserum. The NI titer of serum ws expressed s tht dilution (per milliliter) which inhibited 50% of the enzyme ctivity. N-HI test. The N-HI test for the ssy of ntineurminidse ws performed in Microtiter with the recombinnt A/Prgue(Heq1)-HK(N2). The test ws similr to tht described by Kendl et l. (6). Ser were serilly diluted in duplicte in twofold increments in ml of phosphte-buffered sline (PBS) (ph 7.2). Equl volumes of virus suspension contining 4 hemgglutinin (HA) units were dded to ech row of diluted ser. After 30 min of incubtion t room temperture, ml of 1:20 dilution of rbbit ntihumn immunoglobulin G with n E vlue of 105 (10) ws dded to the first row nd 0.25 ml of PBS to the second row of ech duplicte titrtion. After 30 min of incubtion t room temperture, 0.05 ml of 0.5% chicken erythrocytes ws dded to ech row. Test ptterns were red fter 1 hr t room temperture. Complement fixtion test. The complement fixtion (CF) test ws performed by the Microtiter method (2). Immunodiffusion test. The micro doubleimmunodiffusion (ID) test employed ws similr to tht previously described by Schild (11). Ten percent sodium dodecyl sulfte (SDS) (in wter) ws dded to ntigen wells to disrupt the test virus suspension nd permit migrtion of structurl components through the gel medium. Neurminidse indirect hemgglutintion test. Neurminidse indirect hemgglutintion (NIHA) tests were performed with sheep erythrocytes stbilized by glutrldehyde fixtion s described by Bing et l. (1). Glutrldehyde (50% biologicl grde) ws diluted to 1% with solution contining 1 prt of N2HP04 (ph 8.2), 9 prts of 0.15 M NCl, nd 5 prts of distilled wter. Before glutrldehyde nd APPL. MICROBIOL. diluent were mixed, they were cooled in n ice bth for 10 min to void clouding. Sheep erythrocytes, wshed twice in 0.15 M NCl nd pcked for 10 min t 350 x g, were diluted to 2% with the cold 1% glutrldehyde nd mixed on mgnetic stirrer for 30 min t 4 C. The fixed cells were wshed five times with 0.15 MNCl nd diluted to 1.5% with 0.1 MPBS, ph 7.2. Tnnic cid (regent grde), diluted to 1: 100,000 in PBS (ph 7.2), ws mixed with equl volumes of the 1.5% glutrldehyde-fixed cells nd incubted t 37 C for 15 min. The tnnic cid-treted cells were wshed with PBS (ph 7.2) nd resuspended to 1.5% with PBS (ph 5.5). Optiml dilutions of ntigen for sensitiztion were determined s follows. Test ntigens were diluted in 0.5-ml volumes in twofold series with PBS (ph 5.5). Equl volumes of tnned cells were dded to ech dilution, mixed well, nd incubted t room temperture for 60 min. Control tnned cells (0.5 ml) were mixed with n equl volume of PBS (ph 5.5) nd incubted for the sme period. Both sensitized nd control cells were centrifuged t 150 x g for 5 min nd resuspended in 0.1 M glycine (in PBS, ph 7.2). After 15 min t room temperture, the cells were wshed in 0.15 M NCl nd diluted to 0.5% suspension with 1: 100 dilution of norml horse serum (NHS). Ser to be tested were inctivted t 56 C for 30 min nd dsorbed with 50% sheep erythrocytes (0.2 ml/1 ml of serum) t 4 C for 60 min. Seril twofold dilutions of the ntiser were prepred in Microtiter "U" pltes contining 0.05 ml of NHS (1: 100). Equl volumes of sensitized cells were dded to the test wells. The pltes were incubted for 2 hr t room temperture, nd the hemgglutintion pttern ws exmined. The optiml ntigen dilution ws defined s the lowest ntigen dilution which produced the highest specific ntibody titer. Isoltion of neurminidse. The procedures used were similr to those described by Lver (7). Recombinnt virus A/NWS(HO)-HK(N2) ws grown in 11-dy embronted eggs, nd the llntoic fluid hrvests were clrified by low-speed centrifugtion (350 x g). The virus ws pelleted by centrifugtion t 100,000 x g for 2 hr nd purified by centrifugtion t 43,000 x g for 90 min on 10 to 40% sucrose grdient. Finl virus concentrtes contining 106 to 107 HA units/ml were disrupted t room temperture by dding (dropwise) 10% SDS in wter. Disruption ws judged to be complete when the oplescence of the suspension clered. This usully required 0.05 ml of 10% SDS per 0.1 ml of virus. Electrophoresis of SDS-disrupted virus ws performed on 5 by 36 cm cellulose cette membrnes in tris(hydroxymethyl)minomethne-ethylenediminetetrcetic cid-boric cid buffer, ph 8.0, contining 0.1% SDS. One-tenth milliliter of the SDSdisrupted virus ws divided eqully mong four membrnes nd electrophoresed for 6 hr t 180 v. Severl smll (5 mm) horizontl strips were cut from the membrne for loction nd identifiction of protein bnds. One or more strips were stined with procion brillint blue dye (0.5% [w/v I in hydrochloric cid, 1% [w/v] in methnol). Other strips were plced

3 VOL. 25, 1973 INFLUENZA A NEURAMINIDASE ANTIBODY ASSAY on gr gels longside strips sturted -with either nti-neurminidse (N2), -hemgglutinin (HO), -type A nucleoprotein or, -type A mtrix-.protein rbbit ntiser. A precipitin line with nti-n2 corresponded to the bnd closest to the cthode. There ws no serologicl evidence of contmintion of this bnd with other virl structurl proteins. The N2 bnd ws locted on the membrne by mtching stined strips. The membrne ws cut verticlly nd its contents were eluted overnight t 4 C in 0.75 ml of distilled wter. SDS ws precipitted by reducing the temperture to 0 C for 2 to 3 hr, nd-the precipitte ws removed by centrifugtion (350 x g) in the cold. The remining SDS ws precipitted by dding 1 drop of sturted KCl (in wter). After 2 hr t zero C, the precipitte ws removed nd the superntnt ws dilyzed for 24 hr ginst PBS (ph 7.2) t 4 C. This product is referred to s the N2 ntigen. Preprtion of ntiser. Antiser were prepred in femle New Zelnd White rbbits (6 to 8 lb.; c. 2.7 to 3.6 kg) by intrmusculr injections of ntigen mixed with n equl volume of Freund's complete djuvnt. Rbbits were injected t 3-week intervls nd bled 3 weeks fter the third injection. Protein determintions. Protein concentrtions were determined by the method of Lowry et l. (8) with dsorption redings t 750 nm. RESULTS Antigenic purity of the isolted neurminidse. Rbbit ntiserum ginst the electrophoreticlly isolted neurminidse ntigen hd n NI titer of 1:400,000 with the recombinnt A/Prgue(Heql)-HK(N2). NI tests showed the ntiserum to be free of ntibodies to the prent A/NWS(HO) neurminidse, nd HI tests showed it to be free of ntibodies to HA. HI ntibodies to the H3 ntigen were bsent, lthough low-level inhibition of hemgglutintion by recombinnt viruses possessing the N2 component of the prent A/HK(H3N2) ws observed. When the ID test ws used, single precipitin line ws formed by N2 ntiser ginst SDS-disrupted A/Aichi virus. This line hd complete serologicl identity with tht formed with reference purified nti-n2 rbbit serum prepred in nother lbortory (World Influenz Centre, London). Control ntiser to the remining three virl structurl proteins produced precipitin lines of non-identity to those with the purified N2 ntiser. The rbbit N2 ntiserum ws lso negtive for RNP ntibodies by CF tests. Preprtion of N2-sensitized erythrocytes. Glutrldehyde-fixed nd tnnic cidtreted sheep erythrocytes were sensitized with the electrophoreticlly isolted N2 ntigens. The optiml dilution for sensitiztion ws determined by titrtion of humn ser (NI titer: 1,380) ginst ech sensitized ntigen preprtion. Glutrldehyde-fixed erythrocytes treted with tnnic cid dilutions rnging from 1: 20,000 through 1: 400,000 were eqully sensitive for dsorption of N2 ntigen. Optiml dilutions for most N2 preprtions rnged from 1:4 to 1: 6, nd corresponded to n OD reding of pproximtely 0.15 in the stndrd neurminidqe test (1 hr of incubtion with fetuin). N-IHA titers decresed shrply when erythrocytes were sensitized with N2 preprtions of n OD less thn 0.1. The ttchment of neurminidse to the erythrocyte under these conditions ws confirmed by wshing sensitized erythrocytes three or more times nd testing for neurminidse ctivity. One milliliter of 0.5% sensitized erythrocytes yielded n verge OD vlue of in the stndrd neurminidse test. Since purified N2 preprtions in PBS rpidly lost enzyme ctivity, only freshly prepred (immeditely fter dilysis) N2 suspensions were used. The N2 ntigen ppered to be stble fter it becme ttched to the erythrocytes. Sensitized erythrocytes could be stored t 4 C for t lest 4 months (lst time evluted) without loss of sensitivity to ntibody or ltertion in erythrocyte settling ptterns. Specificity of gglutintion of N2-sensitized erythrocytes. The specificity of the N-IHA test ws evluted with selected recombinnt nd component specific ntiser. Agglutintion ws observed only with ser contining ntibodies to the N2 component (Tble 1). Humn pired ser with fourfold or greter HI ntibody rises to A/Jpn/305/57, B/Msschusetts/3/66, nd A/Hong Kong/8/68 lso were exmined for gglutintion of N2 sensitized erythrocytes (Tble 2). Pired ser collected in 1957 from persons experiencing first infections with Asin viruses (H2N2) showed no evidence of ntibody to Hong Kong N2 by N-IHA. Pre- nd postinfection ser collected in from persons infected with influenz B hd sttionry titers by the N-IHA test. All five pired ser from Hong Kong infections hd fourfold or greter increses in N-IHA titers. Further evlution of the specificity of the N-IHA test ws undertken by dsorbing humn ser with sensitized nd nonsensitized erythrocytes. Five-tenths of milliliter of pcked erythrocytes ws dded to 1 ml of predetermined dilution (1: 20-1: 30) of serum. The serum-erythrocyte mixture ws gitted intermittently. Ser were dsorbed for 18 hr t 4 C. CF ntibody titers to the ribonucleopro- 99

4 100 HOLSTON AND DOWDLE APPL. MICROBIOL. TABLE 1. Specificity of the neurminidse indirect hemgglutintion (N-IHA) test: rection with influenz component-specific ntiser N-IHA titer I_ Non- Control tizeld steni nel-.. test cells Antiser Sensi- Non- trol tiell Guine pig-nti-type A ribonucleoprotein.<10 <10 64 Rbbit nti-type A ribonucleoprotein... <20 <20 + Rbbit nti-type A mtrix protein <20 <20 +b Rbbit nti-a/nws (HON1).. <10 <10 630c Rbbit nti-type A purified N2 (Hong Kong).1,280 <10 1,700" Rbbit nti-nws (HO)-Tw <10 2,560C (N2)...i2,560, Complement fixtion titer. b Precipitin line in immunodiffusion test. c Neurminidse inhibition titer. TABLE 2. Specificity of the neurminidse indirect hemgglutintion (N-IHA) test: rection with pired ser from humns infected with A/Jpn/305/57, B/Mss/3/66, or A/Hong Kong/8/68 influenz viruses Humn ser Infecting virus N-IHA titer Group I A/Jpn P-131 0/0b P-132 0/0 P-133 0/0 P-135 0/0 P-138 0/0 Group Ih B/Mss RU /160 RU /160 RU /160 RU /40 RU /40 Group Hj A/Hong Kong / /5, /2, / /1,280 Pired ser from persons with greter thn fourfold rises in hemgglutintion-inhibition titer to: I, A(H2N2), winter ; II, influenz B, winter ; III, A(H3N2), winter b Zero mens less thn 10. tein nd HI titers to A/NWS were not ltered by dsorption of ser with N2-sensitized erythrocytes. N-IHA nd NI titers to the N2 ntigen were reduced threefold or greter (Tble 3). Evlution of the N-IHA test. Forty-one cute nd convlescent ser from subjects confirmed by HI to hve been infected with A/HK/68 were tested by N-IHA, nd the results were compred with those obtined by NI nd N-HI tests. Ninety-three percent of the serum pirs were shown by t lest one of the three tests to hve fourfold or greter rise in neurminidse ntibody titer. Of the totl ntibody rises, the N-IHA test detected 83%, the N-HI 68%, nd the NI 54% (Tble 4). Pre- nd postvccintion ser from A/Jpn/ 170/62 nd A/Aichi/2/68 vccinees (confirmed by HI to hve been successfully vccinted) were lso tested by N-IHA, N-HI, nd NI. Of the pired ser from the 25 A/Jpn/62 vccinees, 84% were found by t lest one of the three tests to hve fourfold or greter rise in ntibody titer. The N-IHA detected 76%, the N-HI 64%, nd the NI 44% (Tble 4). Of the pired ser from the 25 A/Aichi/68 vccinees, 96% were found by t lest one test to hve fourfold or greter rise in ntibody titer. The N-IHA gin detected 76%, the N-HI 88%, nd the NI 64% (Tble 4). The percent greement between the N-IHA nd the N-HI tests ws similr with ll three study groups (Tble 5). The percent greement between the N-IHA nd the NI tests ws significntly better with the A/Aichi/68 vccine group thn with the illness or the A/Jpn/170/ 62 vccine group (P > 0.05). The N-IHA, N-HI, nd NI tests were lso evluted for detection of ntibody in prevccintion nd preillness ser (Tble 6). One hundred precent of the totl 91 ser were positive for ntibody by N-IHA. The numbers positive by the N-HI nd NI test were considerbly less, 25 nd 22%, respectively. TABLE 3. Specificity of the neurminidse indirect hemgglutintion (N-IHA) test: dsorption of humn ser with N2-sensitized erythrocytes Test Humn Ndopto HIc A/ ser Adopin A/Prgue CF NWS/33 HK (N2) (Oi Tnned cells < 10 Sensitized 20 < < 10 cells Tnned cells < 10 Sensitized 30 < < 10 cells Neurminidse inhibition. b Complement fixtion, ribonucleoprotein. c Hemgglutintion inhibition.

5 VOL. 25, INFLUENZA A NEURAMINIDASE ANTIBODY ASSAY TABLE 4. Comprison of neurminidse indirect hemgglutintion (N-IHA), neurminidse hemgglutintion inhibition (N-HI), nd neurminidse inhibition (NI) tests for detection of fourfold or greter rises in neurminidse ntibody titers in pired ser Illness Vccinees Test A/Jpn A/Aichi No. % No.%No. % No. % Any (t lest one) N-IHA N-HI NI All Only N-IHA N-IHA nd N-HI N-HI N-HIndNI N-IHA nd NI NI None Subjects nturlly infected with A/Hong Kong/8/68(H3N2) nd from volunteers immunized with A/Jpn/170/62(H2N2) nd A/Aichi/2/68(H3N2) vccines. TABLE 5. Percent greement between the neurminidse indirect hemgglutintion (N-IHA) nd the neurminidse hemgglutintion-inhibition (N-HI) tests nd the N-IHA nd the neurminidse inhibition (NI) tests for detection of fourfold or greter rises in neurminidse ntibody titers in pired ser Tests Percent greement between tests (no. greed/no. tested) Illness A/Jpn A/Aichi N-IHA vs. N-HI (29/41) 71% 18/25) 72% (18/25) 72% N-IHA vs. NI (23/41) 56% 15/25) 60% 22/25) 88% Subjects nturlly infected with A/Hong Kong/8/68(H3N2) nd from volunteers immunized with A/Jpn/170/62(H2N2) nd A/Aichi/2/68(H3N2) vccines. DISCUSSION All evidence suggests tht erythrocytes sensitized with isolted N2 ntigen were gglutinted only by neurminidse ntibody. Hemgglutintion did not occur in the presence of high-titered ntibody to the recombinnt hemgglutinin, type A ribonucleoprotein, TABLE 6. Positive preiliness nd preimmuniztion neurminidse ntibody titers by neurminidse indirect hemgglutintion (N-IHA), neurminidse hemgglutintion-inhibition (N-HI), nd neurminidse inhibition (Ni) tests Test Totl ser No. % Any (t lest 1) N-IHA N-HI NI All Titers of t lest 10 by N-IHA nd N-HI nd t lest 60 by HI were considered positive. or mtrix protein ntigens. In ddition, dsorption of humn influenz convlescent ser with N2-sensitized erythrocytes removed only neurminidse ntibody. The N-IHA test ws t lest equl to the N-HI test nd seemed to be better thn the NI test for detecting fourfold or greter ntibody rises in pired ser from infected ptients or vccinees. It ws clerly better thn both tests in its bility to detect N2 ntibody. N-IHA titers were positive in ll 1968 prevccine nd pre-epidemic ser. Evidence tht these titers were not rtifctul is suggested by the bsence of N-IHA titers in ser collected in 1957, the time of first ppernce of the N2 ntigen. The high incidence of N2 ntibodies in 1968 ws not unexpected. Influenz A strins which circulted in the yers immeditely preceding the Hong Kong influenz epidemic possessed N2 ntigens which were closely relted to those of the Hong Kong virus (3). Whether the greter number of titers detected by N-IHA test indictes it is less strin specific thn the NI test remins to be determined. It should be noted, however, tht n improved NI test which hs greter sensitivity thn the present NI test is now vilble (World Helth Orgniztion Committee, Bull. W.H.O., in press). The N-IHA test is simple. It requires only sensitized erythrocytes nd cn be performed in ny lbortory. The stbility (t lest 4 months t 4 C nd presumbly longer t -70 C) of the glutrldehyde-fixed nd sensitized erythrocytes mkes possible the use of the sme regents for multiple tests. Furthermore, the N-IHA test should permit, in theory, direct ssy of ntibody rective with ntigens ssocited with the neurminidse structure. The one disdvntge of the test is the specilized equipment required for the initil virus preprtion nd isoltion of the N2 nti-

6 102 HOLSTON AND DOWDLE gen. Even so, the test proved to be resonbly economicl. A 0.1-ml mount of the purified recombinnt (10" HA units) yielded sufficient N2 ntigen to sensitize enough glutrldehydefixed erythrocytes to test 400 ser through full rnge of dilutions. Our experience with the N-IHA test suggests tht it should be useful ddition to the present methodology for the ssy of N ntibody. ACKNOWLEDGMENTS We grtefully cknowledge the dvice nd encourgement given by Mrion T. Colemn nd the ble ssistnce of Wilm Yrbrough, Judy Glphin, Avis Sisson, nd Rlph Pike. This reserch ws supported by reserch grnt CC from the Center for Disese Control, Atlnt, G. ADDENDUM IN PROOF After our work ws completed, single-rdil-diffusion test for ssy of influenz neurminidse ntibodies ws described by other workers (16). LITERATURE CITED 1. Bing, D. H., J. G. M. Weynd, nd A. B. Stvitsky Hemgglutintion with ldehyde-fixed erythrocytes for ssy of ntigens nd ntibodies. Proc. Soc. Exp. Biol. Med. 124: Csey, H. L Adpttion of LBCF method to microtechnique. In Public Helth Service Monogrph no. 74. U.S. Govt. Printing Office, Wshington, D. C. 3. Colemn, M. T., W. R. Dowdle, H. G. Pereir, G. C. Schild, nd W. K. Chng The Hong Kong/68 influenz A2 vrint. Lncet 2: Hierholzer, J. C., M. T. Suggs, nd E. C. Hll Stndrdized virl hemgglutintion nd hemgglutintion-inhibition tests. II. Description nd sttisticl evlution. Appl. Microbiol. 18: APPL. MICROBIOL. 5. Kendl, A. P., nd C. R. Mdeley A comprtive study of influenz virus neurminidses, using utomted techniques. Biochem. Biophys. Act 185: Kendl, A. P., E. Minuse, nd F. M. Dvenport An improved procedure for mesuring neurminidse ntibodies by hemgglutintion-inhibition. Z. Nturforschung 27: Lver, W. G Structurl studies on the protein subunits from three strins of influenz virus. J. Mol. Biol. 9: Lowry, 0. H., A. L. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: Mostow, S. R., S. C. Schoenbum, W. R. Dowdle, M. T. Colemn, nd H. S. Kye Studies with inctivted influenz vccines purified by zonl centrifugtion. I. Adverse rections nd serologic responses. Bull. W.H.O. 41: Reimer, C. B., D. J. Phillips, S. E. Mddison, nd S. L. Shore Comprtive evlution of commercil precipitting ntiser ginst humn IgM nd IgG. Lb. Clin. Med. 76: Schild, G. C Evidence for new type-specific structurl ntigen of the influenz virus prticle. J. Gen. Virol. 15: Schoenbum, S. C., S. R. Mostow, W. R. Dowdle, M. T. Colemn, nd H. S. Kye Studies with inctivted influenz vccines purified by zonl centrifugtion. II. Efficcy. Bull. W.H.O. 41: Webster, R. G Antigenic hybrids of influenz A viruses with surfce ntigens to order. Virology 42: Webster, R. G., nd H. G. Pereir A common surfce ntigen in influenz viruses of humn nd vin sources. J. Gen. Virol. 3: World Helth Orgniztion Committee A revised system of nomenclture for influenz viruses. Bull. W.H.O. 45: Schild, G. C., M. Henry-Aymrd, nd H. G. Pereir A quntittive, single-rdil-diffusion test for immunologicl studies with influenz virus. J. Gen. Virol. 16: