Comparison of a Microneutralization Test in Cell Culture and

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1 JOURNAL OF CLINICAL MICROBIoLoGy, Feb. 1976, p Copyright C 1976 Americn Society for Microbiology Vol. 3, No. 2 Printed in U.SA. Comprison of Microneutrliztion Test in Cell Culture nd Virus Neutrliztion Test in Embryonted Eggs for Determining Infectious Bronchitis Virus Antibodies1 R. E. WOOLEY,* J. BROWN, R. B. DAVIS, J. L. BLUE, AND P. D. LUKERT Deprtment of Medicl Microbiology nd Poultry Disese Reserch Center, College of Veterinry Medicine, University of Georgi, Athens, Georgi Received for publiction 6 October 1975 A microneutrliztion test (MNT) system utilizing cytopthic effect end points ws effective in determining neutrliztion indexes for infectious bronchitis virus ntibodies. The system is reproducible within 1 index unit t the 95% level of probbility. Comprison of the MNT to tests in eggs resulted in positive correltion (B = 0.81), which ws significnt (P > 0.01). The quntittive doseresponse reltionship of the MNT is liner (P > 0.005), with the 95% prediction limits fitting between one 10-fold dilution. In recent yers, microculture methods for virus titrtion nd serologicl procedures hve come into frequent use. The ppliction of microculture hs been reported in the study of rboviruses (2), trnsmissible gstroenteritis virus (18), mesles virus (9), poliovirus (8), Newcstle disese virus (19), respirtory virus seroepidemiology (14), nd in other serologicl investigtions (4, 6, 15). Methods utilizing microculture re generlly s sensitive nd fr more economicl thn mcroculture methods (6, 19). Lbortory procedures used to detect infectious bronchitis virus (IBV) ntibodies include neutrliztion in embryonted chicken eggs (13) nd plque reduction (11). These procedures re both cumbersome nd expensive (1). The objective of this study ws to develop virus neutrliztion test (VNT) for IBV ntibodies utilizing microcultures s the indictor system to detect un-neutrlized virus nd to compre the micromethod with the stndrd VNT in embryonted chicken eggs with regrd to sensitivity, reproducibility, nd correltion of titers. MATERIALS AND METHODS Medi. Growth medium for cell cultures consisted of Hnks blnced slt solution supplemented with 0.25% lctlbumin hydrolyste, 10% fetl clf serum, nd 10% tryptose phosphte broth. A solution of methyl--d-glucoside, sterilized by filtrtion, ws dded to the growth medium to finl concentrtion of 1.5%; this solution ws used to block IBV inhibitors present in bovine serum (12). Gentmicin ws dded to concentrtion of 100,ug/ml. Virus dilutions were prepred in Hnks blnced slt solution. I Mnuscript no Cell cultures. Chicken kidney cell cultures were used for virus propgtion. Minced kidneys from 1- dy-old chicks were trypsinized for 15 min t 37 C. Two hundred milliliters of 0.25% trypsin ws sufficient to disperse the cells from four to five chick kidneys. The dispersed cells were filtered through sterile guze nd the trypsin ws then inctivted by the ddition of 20 ml of clf serum. The cells were sedimented by centrifugtion (Interntionl Centrifuge, Universl Model UV, Interntionl Equipment Co., Needhm Heights, Mss.) t 500 x g for 20 min, nd 1 ml of pcked cells ws suspended in 800 ml of growth medium. Source of virus. The Beudette strin (IBV-42), dpted to chicken embryonic kidney cells (10), ws used throughout the investigtion. Virus stocks mintined t titer of pproximtely 107 plqueforming units/ml by frequent pssge in chicken kidney cell cultures were stored t -70 C. Ser. Pooled ser used in the experiments were obtined from 30 flocks. The chickens selected included broilers (6 to 7 weeks old), broiler breeders (22 to 66 weeks old), nd commercil lying hens (23 to 63 weeks old). All 30 flocks hd been vccinted ginst infectious bronchitis by the following methods: broilers received the Msschusetts strin of IBV by the Bek-o-vc method t 1 dy of ge; broiler breeders nd lying hens were dministered the Mschusetts nd Connecticut strins of IBV in the drinking wter t 1, 4, nd 14 weeks of ge. Three negtive serum smples were obtined from unvccinted, specific-pthogen-free chickens housed t the Poultry Disese Reserch Center, University of Georgi. The 33 serum smples were employed in the microneutrliztion test (MNT) nd the VNT in embryonted chicken eggs. MNT. Microculture pltes (IS-FB-96-TC; 0.4 ml/ well; Linbro Chemicl Co., New Hven, Conn.) were used for determintion of neutrliztion indexes (NIs) (constnt serum-virus dilution method). Ser were diluted 1/10 in Hnks solution, nd the IBV stock virus (107plque-forming units/ml) ws 149

2 ~r--_ I 150 WOOLEY ET AL. used in 10-fold dilutions rnging from undiluted to The virus dilutions (0.025 ml) were dded to n equl volume of the 1:10 serum dilution nd llowed to rect in the microculture wells for 30 min t 26 C. Ech virus-serum mixture ws plced into four replicte wells, nd ech plte ws used for three serum smples (Fig. 1). Virus nd cell controls were included in seprte plte (Fig. 2). After ddition of the virus nd virus-serum mixtures to pproprite wells nd completion of the rection time, 0.20 ml of chicken kidney cell suspension ws dded per well. The pltes were then seled with cler mylr sheet with n dhesive bck (35 PSM, Linbro Chemicl Co., New Hven, Conn.), covered with cler polystyrene top (53, Linbro Chemicl Co., New Hven, Conn.), nd incubted t 37 C for 96 h. The incubtion period used in the test ws determined by preliminry trils to give mximl sensitivity in the shortest period of time. After incubtion, the medium ws removed nd the cells were fixed with 10% neutrl formlin for 3 to 5 min. The formlin ws decnted, nd the fixed cells were stined with 1% crystl violet for 30 min. The stined cells were exmined for cytopthic effects by gross exmintion with n pproprite light source. Control monolyers nd virus-negtive monolyers ppered solid blue, wheres virus-infected monolyers were mottled nd lighter in color (see Fig. 1 nd 2). End points were determined by compring the solid blue virus-negtive monolyers nd the lighter, mottled virus-infected cells. End points were expressed s the highest dilution of virus or virus-serum mixture tht hd the mottled, lighter-stined cell monolyer. NIs were clculted by compring the virus control nd virus-serum mixture end points. The NI ed10-2st C -3 ;F;1 lo- rnged from 1 to 5. A NI of 1 ws considered -1 becuse 1/10 serum dilution ws used. A NI of 5 ws considered to be.5 becuse it ws the mximum reding. Antibody titer is expressed in MNT index numbers where neutrliztion of 10-fold virus dilutions re represented by the numbers 1, 2, 3, 4, nd 5. The reproducibility of the NI ws determined by repeting the test with the 33 serum smples three times t seprte intervls. VNT. This test in embryonted chicken eggs is the stndrd reference for the exmintion of poultry biologics (13). Procedures for performing the test re well documented (1). In this test, the NI is the difference between the log titer of the virus control end point nd the titer of the virus-serum mixture end point. This difference represents the logrithim of the NI of the serum. Sttisticl nlysis. Assessment of the ccurcy of the MNT requires knowledge of the true neutrlizing ntibody titer in the cytopthic effects microculture system (19). Since this informtion is not vilble, the question of the ccurcy of the test system cnnot be nswered directly. However, if results of test system re consistently reproducible, ccurcy cn be inferred. This is becuse the true MNT titer remins the sme, nd only the humn nd mechnicl vritions inherent in ny test system need to be considered to nswer questions concerning reproducibility. The "best" estimtes of vrition re the men nd the stndrd devition of the men (16). With these sttistics, "reproducibility" level cn be defined. An cceptble level of reproducibility of mny serologicl tests is, by custom, commonly referred to s being "within" one 10-fold dilution. SER UM SAMPL :' >10-5iE3 _: CCNELLo%% J. CLIN. MICROBIOL. FIG. 1. Microculture plte showing position of wells used for the virus dilutions nd ser for the MNT.

3 VOL. 3, 1976 INFECTIOUS BRONCHITIS VIRUS ANTIBODIES 151 CELL VIRUS CONTROL CONTROL z 0 -J (I) Dn loo 100X l 0-5 I I 6 0 t A.. A.. -, FIG. 2. Microculture plte showing position of wells used for the virus nd cell controls in the MNT. Accepting this empiricl vlue s stndrd of reproducibility two hypotheses re dvnced: (i) IBV ntibody titers s mesured by the MNT re reproducible "between" one 10-fold dilution (Fig. 3); (ii) IBV ntibody titers s mesured by MNT re reproducible "within" one 10-fold dilution (Fig. 3). By definition, then, the MNT will be considered reproducible if the clculted confidence intervl of the mens of the vrious MNT does not exceed 2 (between) or 4 (within). The confidence intervl is bsed upon the t distribution clculted t the 95% level. The definition of"between" one 10-fold dilution is tht it must be less thn two 10-fold dilutions, i.e., plus or minus less thn one 10-fold dilution. In similr fshion, "within" one 10-fold dilution mens tht it must be less thn four 10-fold dilutions, i.e., plus or minus one 10-fold dilution but not two 10-fold dilutions. After the completion of the first three trils, fourth tril ws conducted. The purpose of the fourth tril ws to test the hypothesis tht the IBV ntibody titer expressed by MNT ws not reproducible within 1 index unit of ny of the indexes of the first three trils for the ser in question. The hypothesis ws tested with goodness-of-fit test (16). To estblish nd estimte the direct ssocition between the MNT nd the VNT, the product-moment correltion coefficient ws clculted (3, 17). In this test, the null hypothesis is tht correltion is zero. To estblish nd estimte the dependence of ntibody titer clculted by either the MNT or VNT "BETWEEN" ONE DILUTION e 4 "WITHIN" ONE DILUTION FIG. 3. Schemtic representtion of virus dilution tubes illustrting the rnge between nd within one 10-fold virus dilution utilizing NI numbers from 1 to 5. methods upon serum dilution, test of regression ws reckoned for ech (17). This test for regression included n nlysis of vrince, clcultion of the 95% confidence limits round the regression line, clcultion of the 95% prediction limits for smple of size 1, nd determintion if the clculted 95% prediction limit for smple size of 1 ws either "between" or "within" one 10-fold dilution. Both the MNT nd VNT rection systems consist of decresing virus nd constnt serum. For the test of correltion (regression), the rection system of diluting virus nd constnt serum is mintined. In this test of regression, one serum with NI in MNT tht equled 5 ws chosen rndomly for dilution. However, the test serum ws diluted twofold from 1/ 10 to 1/1,280. Ech dilution (eight smples) ws employed s the constnt serum. I

4 152 WOOLEY ET AL. RESULTS The NIs for the 33 ser re given in Tble 1. The ser from the specific-pthogen-free chickens were negtive. Seventeen of the ser completely neutrlized the IBV t index number 5 (undiluted virus) in the three trils nd were therefore not menble to further nlysis. The determintion of reproducibility ws ccomplished with the remining 13 ser. As shown in Tble 2, the MNT ws reproducible for ech of these ser within 1 index unit t the 95% level of probbility. The results of the fourth tril were similr to the first three trils in tht the three ser from the specific-pthogen-free chickens remined negtive nd the 17 ser tht completely neutrlized the IBV t NI 5 continued to TABLE 1. IBV ntibody titer expressed by the NIs of the MNT nd the VNT MNT replicte Serum Men VNT Ob b b Logrithmic number. The NI is more properly expressed s the ntilog of the number in question, but by common usge the log number is usully described s the NI. b Smples from specific-pthogen-free chickens. do so. The NIs for the remining ser of tril 4 re given in Tble 3. In this instnce, hypothesis hs been proposed tht the NI of ny of the ser of tril 4 would not be within 1 index unit of ny of the indexes of the first three trils. None of the ser met this criterion. A x2 of 13.0 ws clculted from these results. As compred with tbulr x (1) of 7.879, the null hypothesis ws rejected. Determintion of correltion between MNT nd VNT ws done utilizing the 13 ser with NIs of less thn 5. The results of this test re given in Tble 4. In testing the hypothesis tht the correltion between MNT nd VNT ws zero, it ws determined tht the correltion ws This positive correltion is significnt (P > 0.001). IBV ntiserum no. 3 ws chosen rndomly to test the effect of serum dilution upon the two tests. The results of three trils for ech test re presented in Tble 5. The quntittive doseresponse reltionships of the MNT is given in Tble 6 nd grphiclly in Fig. 4. First, the dose-response reltionship ws liner (P > 0.005), nd the clculted F for devitions from liner regression ws less thn 1. Therefore, the hypothesis tht devition from regression is less thn zero is ccepted. The 95% prediction limits if the smple size ws 1 ws clculted. All of the clculted 95% prediction limits fit inside "between" one 10-fold dilution. Since clculted x2 = s compred with tbulr x2 (0.005) = 7.879, tht this could hve hppened by chnce lone seems improbble. In the clcultions concerned with the doseresponse reltionships of the VNT, it ws estblished tht the response ws not liner. Therefore, the hypothesis tht devition from regression is less thn zero ws rejected. DISCUSSION J. CLIN. MICROBIOL. The lbortory procedures presently used to detect IBV ntibodies re cumbersome, expensive, nd time-consuming. The development of microculture method for determining the NI of IBV-vccinted birds hs eliminted these disdvntges. With the use of microculture, smples from individul birds, rther thn pooled-flock smples, cn be used for determintion of NI, due to the smll smple volumes required. With the use of multistrin vccines in the poultry industry, it my be desirble to use strins other thn the test strin used in this study. Presently, numerous other IBV strins hve been dpted to chicken kidney cells (7) nd chicken embryo kidney cells (5) nd my be menble to microculture methods. It ws stted tht for the MNT to be consid-

5 VOL. 3, 1976 TABLE 2. INFECTIOUS BRONCHITIS VIRUS ANTIBODIES 153 Determintion of reproducibility of MNT by t distribution to clculte 95% confidence intervl round men NI Serum sm- Men index Stndrd error Confidence Confidence inple unit of men intervlb tervl rnge Reproducibility level Within 1 in- dex unitd Between 1 index unite Yese No Yes No Yes Yes Yes Yes Yes No Yes No Yes Yes Yes No Yes No Yes Men of three trils. b 2 Degrees of freedom (4.303). c Plus or minus less thn one dilution. The clculted confidence intervl rnge must be less thn 2 to be considered reproducible (see Fig. 3). dplus or minus one dilution but not two dilutions. The clculted intervl rnge must be less thn 4 to be considered reproducible (see Fig. 3). e The confidence rnge does not exceed the pproprite stted reproducibility level. Therefore, the MNT is reproducible. TABLE 3. Testing hypothesis tht IBV ntibody titer s expressed by MNT is not reproducible within 1 index unit (goodness-of-fit test) Serum MNT index tril MNT Within index one dilusmple tril 4 tionr Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Is the NI of tril 4 within one dilution of ny of the NIs of the first three trils of the serum in question? Within one b Hypothesis b Yes 13 p=0.50 Pn = 6.5 No 0 =0.50 qn = 6.5 n = 13 Clculted X2 is 13.0 s compred with tbulr X (1) The evidence is sufficient to reject the null hypothesis nd ccept the lterntive tht MNT is reproducible within 1 index unit. TABLE 4. Reltionship (product-moment correltion) of MNT nd VNT for IBV serum ntibody titer Serum Men MNT NIb VNT NI Correltion coefficient = 0.81 (P > 0.01); 95% confidence limits, Li = 0.47 nd L2 = b Three trils. ered reproducible, the stndrd of reproducibility must be "within" one 10-fold dilution. At the 95% confidence intervl rnge, ll of the ser exmined hd NIs tht met this criterion (Tble 2). Only three replictions were used in rriving t smple men nd stndrd devition. This ws deliberte vigorous ssessment of reproducibility. Slight vrition in ny of the NI end points either sequentilly (2, 3, 4) or differing by 100-fold dilution (2, 2, 4) would hve

6 154 WOOLEY ET AL. TABLE 5. Effect of serum dilution on ntibody titer forib V when titers re expressed s NIs ofmnt nd VNT Serumb dilu- MNT NI in tril: VNT NI in tril: tion / / / / / / /640 NDc 1 1 1/1,280 ND 1 1 Three trils for ech test. b IBV ntiserum no. 3 ws chosen rndomly for these trils. c ND, Not determined. ment tht the stndrd of reproducibility could not hve been met. These devitions did not occur, so it would pper tht extrneous vrition is miniml in the MNT. Underlying the estblishment of stndrd of reproducibility is the requirement of virologists nd serologists tht relible results, within certin defined rnge, cn be scertined by conducting test once. For this reson, fourth tril ws conducted to nswer the hypothesis tht the ntibody titer would not be within 1 index unit of ny of the three indexes previously determined for ech serum. In ech instnce, this hypothesis could not be upheld. Since the probbility tht this could hve hppened by chnce lone (P > 0.005) ws remote, the lterntive tht the ntibody titer ws within 1 index unit of ny of the three indexes previously determined ws ccepted. A requirement of ny test is specificity. The specificity of the MNT ws tested by the blind inclusion of three ser derived from pthogen-free chickens. All were negtive. The MNT nd VNT re positively correlted (B = 0.81, P > 0.01). This is not surprising since the sme ntiserum is used to neutrlize the sme strin of IBV. The difference between the tests consists of the indictor systems used. In the MNT, this is monolyer of chicken kidney cells, nd in the VNT it is n entire chicken embryo. In both of the test systems, specificity ws evidenced by the fct tht none of the ser from specific-pthogen-free chickens gve neutrliztion. The dose-response reltionship ofthe two test systems indicted gret differences in sensitivity. First, the dose-response reltionship of the MNT is liner (P > 0.005). Dt of the NI in Tble 5 indicte tht t the extremes of serum TABLE 6. Quntittive dose-response reltionships ofmnt for IBV Anlysis of Vrince Source of vrition df F Probbility Among groups (dilu tions) Liner regression Devitions from 4 1 Not signifiregression cnt Within groups 11 Totl b 16 J. CLIN. MICROBIOL. Regression coefficient (B) = ; 95% confidence limits for B: Li = nd L2 = b Totl represents degrees of freedom mong groups (dilutions) nd within groups. 95% Prediction Limits if Smple Size is One L-ogrithm Stnde-95% Predic-,Bten of dilution Stndrd er tion limits "Betwen' reciprocl ror Y 0.05 (13) one dilution Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes "Between" one dilution f Hypothesis I Yes 11 = 0.50 Pn = 5.5 No 0 q = 0.50 An = 5.5 n= 11 x2 = (11 5.5)2/(0.50 x 0.50 x 11) (significnt, tbulr x2 [0.005] = 7.879). dilution some sigmoidl tendency in the regression is evident; therefore, the nlysis of vrince excludes the responses for serum dilutions 1/10 nd 1/1,280. Since no evidence of devition from linerity is found, the men squre ctully being less thn tht for within dilutions (error), the linerity over dilution rnge of 1/ 20 to 1/640 is effectively liner (Tble 6). In test systems of this type, one primry interest of virologists nd serologists is wht sort of credence does one give to the results of single test. For this reson, the 95% prediction limits for smple the size of 1 were clculted. In turn, the question ws sked if these prediction limits fit inside the criterion of being "between" one 10-fold dilution. All of the clculted 95% prediction limits fitted inside of the

7 VOL. 3, 1976 INFECTIOUS BRONCHITIS VIRUS ANTIBODIES L.bF X X < ~2.2 _2. t 2<0 A\ =.05 cc 2.0 Wm "Between" one o F2 95% prediction o 1.8 limits (smple size O X 95% confidence limits > 1.6 of regression line NEUTRALIZATION INDEX =Y FIG. 4. Quntittive dose-response reltionship of the NI including the 95% confidence limits of the regression line, the 95% predictionbetween" limits if the smple size ws 1, nd the rnge of one 10-fold dilution. "between" one 10-fold dilution. To determine the significnce ofthis finding, goodness-of-fit test ws done. Clculted x2 ws s compred with tbulr x2 (0.005), which ws (Tble 6 nd Fig. 4). Tht this difference could hve resulted from chnce lone is improbble. Therefore, the credence tht biologist cn give to the results of single test of this type should be high. The dose-response reltionship of the VNT ws not liner. Therefore, the issue of reproducibility could not to be exmined. However, the lck of sensitivity of this test is consistent with the findings of other investigtors (1). In previous studies (10), the NIs were much higher when embryonted eggs were used for the detection of IBV-neutrlizing ntibodies s compred with chicken embryo kidney cell monolyers. However, when the serum ws diluted, the embryonted egg ws ffected dversely s n indictor. It ws believed tht the virus-serum mixtures continued to rect in cell cultures forming more stble union. This is in contrst to the technique wherein the virusserum mixture is inoculted into the llntoic cvity of the embryonted egg: the llntoic fluid cts s diluent (pproximtely 1/100) nd stops the rection, fvoring dissocition (11). In summry, the MNT for the detection of IBV ntibodies is simple to perform, specific, nd reproducible within defined criterion. LITERATURE CITED 1. Cunninghm, C. H Immunologic methods in vin reserch: neutrliztion test. Avin Dis. 17: DeMdrid, A. T., nd J. S. Porterfield A simple microculture method for the study of group B rboviruses. Bull. W.H.O. 40: Finney, D. J Sttisticl method in biologicl ssy. Chrles Griffin & Co., London. 4. Fuccillo, D. A., L. W. Ctlno, Jr., F. L. Moder, D. A. Debus, nd J. L. Sever Minicultures of mmmlin cells in new plstic plte. Appl. Microbiol. 17: Gillette, K. G Plque formtion by infectious bronchitis virus in chicken embryo kidney cell cultures. Avin Dis. 17:

8 156 WOOLEY ET AL. 6. Helmke, R. J., R. L. Heberling, nd S. S. Klter Technique for virl neutrliztion ntibody surveys in primry microcultures. Appl. Microbiol. 20: Hopkins, S. R Serologicl comprisons of strins of infectious bronchitis virus using plgue-purified isolnts. Avin Dis. 18: Kende, M., nd M. L. Robbins Titrtion nd neutrliztion of poliovirus in micro tissue culture under incresed crbon dioxide. Appl. Microbiol. 13: Kriel, R. L., H. Wulff, nd T. D. Y. Chin A microneutrliztion test for determintion of ntibodies to rubeol virus. Proc. Soc. Exp. Biol. Med. 130: Lukert, P. D Comprtive sensitivities of embryonting chickens eggs nd primry chicken embryo kidney nd liver cell cultures to infectious bronchitis virus. Avin Dis. 9: Lukert, P. D A plque reduction method for the detection of neutrlizing ntibodies for infectious bronchitis virus. Avin Dis. 10: Lukert, P. D Avin infectious bronchitis virus chrcteristics on n inhibitor found in serum. Arch. J. CLIN. MICROBIOL. Gesmte Virusforsch. 49: Ntionl Acdemy of Sciences Methods for exmining poultry biologics nd for identifying nd quntifying vin pthogens. Ntionl Acdemy of Sciences, Wshington, D.C. 14. Rosenbum, M. J., E. A. Edwrds, nd E. J. Sullivn Micro-methods for respirtory virus seroepidemiology. Helth Lb. Sci. 7: Sever, J. L Appliction of microtechnique to virl serologicl investigtions. J. Immunol. 88: Snedecor, G. W., nd W. G. Cochrn Sttisticl methods, 6th ed. The Iow Stte University Press, Ames. 17. Sokl, R. R., nd F. J. RohIf Biometry. W. H. Freemn & Co., Sn Frncisco. 18. Witte, K. H Micro-color test for ssy of trnsmissible gstroenteritis virus-neutrlizing ntibodies. Arch. Gesmte Virusforsch. 33: Wooley, R. E., J. Brown, J. B. Grtzek, S. H. Kleven, nd T. A. Scott Microculture system for detection of Newcstle disese virus ntibodies. Appl. Microbiol. 27: Downloded from on July 15, 2018 by guest