Vol. 16, No. 1JOURNAL OF CLINICAL MICROBIOLOGY, July 1982, p. 34-400095-1137/82/070034-07$02.00/0

Detection by Radioimmunoassay and Enzyme-LinkedImmunosorbent Assay of Coronavirus Antibodies in Bovine

Serum and Lacteal SecretionsL. RODAK,1'2t L. A. BABIUK,12* AND S. D. ACRES2

Department of Veterinary Microbiology' and Veterinary Infection Disease Organization,2 University ofSaskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO

Received 11 January 1982/Accepted 18 March 1982

The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosor-bent assay (ELISA), and a serum neutralization assay (SN) for detectingantibodies to bovine coronavirus in serum and colostrum were compared.Although there proved to be a good correlation among all three assays (r = 0.915and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved tobe at least 10 times more sensitive than neutralization tests. By using thesetechniques, it was possible to detect a time-dependent decrease in antibody levelsin bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of12 herds in Saskatchewan, and 109 of 110 animals tested, had antibody to bovinecoronavirus. There was no elevated antibody response in serum or lactealsecretions of cows vaccinated once or twice with a commercially availablemodified live rota-coronavirus vaccine. In addition to being more sensitive thanSN, ELISA and RIA proved to have other advantages for measuring antibodylevels to bovine coronavirus and therefore warrant wider use as tools in diagnosticvirology.

Coronaviruses are a group of single-strandedRNA viruses with characteristic club-shapedprojections on the surface of their lipid enve-lope. In addition to sharing biochemical andbiophysical properties, they also appear to sharean affinity for mucosal surfaces, particularlythose of the respiratory and gastrointestinaltracts (10-12, 14). Most bovine coronavirus iso-lates grow poorly in tissue culture. Bovine cor-onavirus is often a cause of gastroenteritis inanimals between 5 and 30 days of age (2, 16, 17).Even though bovine coronavirus is often diffi-cult to isolate directly from feces, a number ofisolates have been adapted to grow in vitro andto produce various degrees of cytopathic effects(3, 13, 16, 18). Using one of these isolates hasallowed us to compare the sensitivity of twoimmunodiagnostic tests, radioimmunoassay(RIA) and enzyme-linked immunosorbent assay(ELISA), with serum-neutralizing assays (SN)to detect antibody titers in the serum, colos-trum, and milk of cattle. Additionally, we corre-lated the sensitivity of the tests with the qualityof reagents used in the respective immunoas-says.

It has been reported for coronaviruses ofother species that ELISA can be used for detec-

t Permanent address: Veterinary Research Institute, 621 32Brno, Czechoslovakia.

tion of virus (8) and that it is more sensitive thanSN (9). The present report demonstrates theprevalence of coronavirus antibody in cattleafter natural infection and after vaccination witha commercially available, modified live vaccine.The use of RIA and ELISA as rapid diagnosticand epidemiological tools for studies of corona-viruses in cattle is discussed.

MATERIALS AND METHODSCells and viral antigens. Georgia bovine kidney cells

were cultured in Eagle minimal essential medium asdescribed previously (1). Each liter of medium wassupplemented with 2 mmol of glutamine, 10 ml ofnonessential amino acids (GIBCO Laboratories, no.114), 3.5 mg of gentamicin, and 2.5 g of sodiumbicarbonate. For growth, the medium contained 10%fetal bovine serum. Confluent cultures were washedtwice with Hanks balanced salt solution before infec-tion with bovine coronavirus (obtained from D. Brian,University of Tennessee, Knoxville). After adsorptionof virus for 1 h at 37°C, Eagle medium plus 2% bovineserum albumin fraction V was added. When cytopath-ic effects were evident, 3 to 4 days later, the cultureswere subjected to three freeze-thaw cycles beforeremoval of the cells by centrifugation at 2,500 x g for20 min. The virus present in the supernatant fluid waspelleted at 82,500 x g for 2.5 h at 4°C. The pelletedvirus antigen was suspended in phosphate-bufferedsaline (PBS) and stored at -70°C. Control antigen wasprepared in an identical manner from uninfected cells.Before being used for coating plates, the antigen was

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SENSITIVITY COMPARISON OF RIA AND ELISA 35

diluted 200-fold in 0.1 M carbonate-bicarbonate buffer(pH 9.6).

Preparation of 251I-labeled rabbit antibovine immu-noglobulin. Commercially available immunoglobulin G(IgG) fraction of rabbit antibovine IgG (Cappel Labo-ratories, lot number 15205) containing 25% proteinwith anti-IgG activity was used. For use in RIA, 1 mgof protein was labeled with 1251 by the chloramine-Tmethod (6), and unbound 1251 was removed by passingthe material through Sephadex G-25. In the stocksolution of labeled protein, 98.4% of the activity wasprecipitable with 10%o trichloroacetic acid. The finaltest solution of 125I-labeled antibody was prepared bydiluting the stock solution with PBS containing 5%normal rabbit serum and 0.2% Tween 20 to a finalactivity of 5 x 105 cpm/ml (8.3 x 103 dpm/ml).

Peroxidase-labeled conjugates and substrate. Twoconjugates were used. One was purified by affinitychromatography, and the other was not. The affinity-purified peroxidase-conjugated rabbit antibovine IgG(Zymed Laboratories, lot number 10723) was diluted1:1,000 in PBS containing 5% normal rabbit serum and0.2% Tween 20 before use. The other conjugate ofnon-purified rabbit antibovine IgG (Cappel Labora-tories, lot number 14984) was diluted 1:200 in the samediluent before use.

Substrate was prepared by dissolving 8 mg of 5-aminosalicylic acid in 10 ml of distilled water. Thesolution was adjusted to pH 5.0 with 0.1 M NaOH, and1 ml of 0.05% H202 in 0.1 M phosphate-citric acidbuffer (pH 5.0) was added before use.RIA procedure. Flat-bottomed polystyrene wells

(Removastrip Imulon, Dynatech Laboratories, Inc.)placed in a Removawell strip holder (Dynatech) werecoated with 0.1 ml of diluted viral or control antigenand incubated overnight at room temperature. Thewells were then washed three times with PBS andincubated with 0.1-ml volumes of diluted test sera orcolostral whey. Each sample dilution was added totriplicate wells coated with viral or control antigen orboth. After incubation for 2 h at room temperature in ahumidified chamber, the wells were washed threetimes with PBS and incubated an additional 2 h with0.1-ml volumes of 125I-labeled rabbit antibovine IgG.After a final three washes in PBS, the radioactivity incounts per minute bound in individual wells wasdetermined with an automatic gamma counter (SearleRadiographics, Inc., model 1185). The average activi-ties and standard deviations of triplicate wells coatedwith viral or control antigen and incubated with thesame sample dilution were determined, and a V/C ratio(ratio of average activities bound in wells with viraland control antigens) was calculated. Samples wereconsidered positive up to the highest dilution giving aV/C ratio greater than 2. The V/C ratio obtained afterincubation without serum or with negative serum wasalways approximately 1. Under such conditions, V/Cratios higher than 2 are highly significant (usually P s0.001) by Student's t test.ELISA procedure. The preparation of coated plates

and antibody dilutions for ELISA were identical tothose for RIA. After incubation with antibody, 0.1-mlvolumes of peroxidase-labeled rabbit antibovine IgGwere added to each well. After a 2-h incubation, theplates were washed three times and incubated for anadditional 60 min with 5-aminosalicylic acid. Theenzyme-mediated reaction was measured at 450 nm

with the aid of an ELISA plate reader (Titertek, FlowLaboratories, Inc.). The average absorbance, standarddeviation, and VIC ratio of triplicate wells coated withviral or control antigen were calculated. The highestsample dilution giving a V/C ratio greater than 2 wasconsidered positive. Reaction of the coronavirus anti-gen with monospecific bovine antibody to bovineherpesvirus and rotavirus always gave a V/C ratio lessthan 2.SN procedure. Neutralization titers were obtained

by preparing serial dilutions of the serum of thecolostral whey and reacting 0.5-ml volumes with 0.5ml of coronavirus containing 1,000 50%o tissue cultureinfective dose units for 1 h at 37°C. The virus-serum orvirus-whey mixture was then added to quadruplicatewells of a microtiter plate containing Georgia bovinekidney cells. After adsorption for 1 h, the fluids wereremoved and replaced with fresh Eagle medium plus2% bovine serum albumin and incubated an additional4 days. Since the cytopathic effect seen is generallynot specific, the endpoints were determined by hemad-sorption. This was achieved by layering a 1% suspen-sion offresh mouse erythrocytes on the monolayer for60 min. Erythrocytes were removed by washing, andthe antibody titers were computed by the Karbermethod (7), using the dilution that reduced hemadsorp-tion in excess of 90%o as the endpoint.

Evaluation of a commercial coronavirus vaccine. Amodified live-virus vaccine for prevention of diarrheacaused by bovine coronavirus is available commercial-ly (Calf-Guard, Norden Laboratories); however, noserological studies on the vaccine have been reported.We used the ELISA procedure developed here tomeasure the immune response of cows to the vaccine.Pregnant beef cows were vaccinated intramuscularlywith 3 ml of vaccine according to the manufacturer'sinstructions. One group of cows was vaccinated onceapproximately 6 to 9 weeks before parturition. Asecond group was given two vaccinations 3 weeksapart during the last trimester of gestation. A thirdgroup of unvaccinated cows served as controls. Cowswere bled at the time of the first vaccination and 3 and6 weeks later. Cows in the control and single-vaccina-tion groups were milked within 1 h, 24 h, 5 days, and20 days after calving. Cows in the double-vaccinatedgroup were milked within 1 h, 36 h, 5 days, and 21 daysafter calving. Whey was prepared by using commercialrennin and stored at -20°C until used for analysis.

RESULTSSpecificity and sensitivity of assays. Viral and

control antigens for RIA and ELISA assays aresuitable only if the activities or absorbancesresult in a VIC ratio of approximately 1 afterreaction with negative serum in the case of viralantigen, or with any serum reacted with controlantigen. In our preliminary experiments, theantigens were prepared to achieve this goal (datanot shown).The sensitivity of RIA and ELISA methods

generally depends not only on the quality of viraland control antigens, but also on the quality of125I-labeled or peroxidase-conjugated antibod-ies. This was illustrated in our studies by the useof two different commercially available peroxi-

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36 RODAK, BABIUK, AND ACRES

TABLE 1. Comparison of reagents for detection ofantibodies to coronavirus

Titera

ELISASample no.ELISA

RIAb Purified UnpurifiedIgG IgG

Serum 71 109,350 36,450 1,350Serum 73 12,150 12,150 1,350Colostrum 72 450 150 50Colostrum 4 1,350 450 50

a Values represent the highest dilution which pro-vided a V/C ratio of greater than 2.

b Antibody used for RIA was unpurified and ob-tained from the same company as unpurified IgG forELISA.

dase-labeled rabbit antibovine IgG conjugates(Table 1). With a conjugate purified by affinitychromatography, we obtained titers 3 to 25 timeshigher than those obtained with an unpurifiedconjugate.A comparison of the RIA and ELISA methods

using the same commercially available unpuri-fied antibody demonstrated that RIA was atleast 10 times more sensitive than ELISA. How-ever, the sensitivity of ELISA approached thatof RIA if chromatographically purified reagentwas used in the ELISA and unpurified antibodywas used in the RIA (Table 1). Similar resultswere obtained on numerous occasions, evenafter the serum was frozen and thawed threetimes, indicating that the test was reproducibleand could use sera that had been subjected to anumber of freeze-thaw cycles.

All samples to be examined were dilutedthreefold in the range of 50 to 100,000. Through-out the positive range, the V/C ratio for eachdilution was higher than 2, but the actual V/Cratio values gave a sigmoidal curve similar to aprozone effect often observed in immunologicalassays (Fig. 1). These results demonstrate thatthe approximate titer cannot be extrapolatedfrom one V/C value obtained using a low serumdilution, since at high concentrations ofimmunoglobulin there is higher nonspecificbinding and thus lower V/C ratios. Furthermore,the actual shape of the curve is influenced by thestarting concentration of immunoglobulins in thesample. In the case of serum, concentrations of1:50 and higher allowed a complete analysis ofthe sigmoidal curve (Fig. 1), whereas in colos-trum taken at the time of parturition, in whichthe concentration of total immunoglobulin washigh, the positive part of the curve becameevident only after dilution of the colostrumbeyond 1:450 (Fig. 2). Therefore, if only lowerdilutions were tested, the colostrum might beconsidered negative for coronavirus antibody.

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FIG. 1. Detection of coronavirus antibodies in bo-vine serum by RIA. ( ) Activity bound to corona-virus positive-antigen-coated plates; (--*) activitybound to control plates; (-) ratio of virus to controlplates.

In contrast, by using milk obtained 5 days afterparturition when the level of immunoglobulin islower, it was possible to observe only the de-creasing part of the curve.Comparison of RIA, ELISA, and SN titers in

serum and colostrum. For a comparison of thethree assays, a total of 32 serum and colostrumsamples were examined by ELISA, RIA, or SN.A high degree of correlation between SN andRIA titers (correlation coefficient r = 0.915)(Fig. 3) and between RIA and ELISA (r = 0.964)(data not shown) was achieved. Although theactual values used for ELISA to determine thecorrelation coefficient were obtained with theaid of an ELISA reader, comparable resultscould be obtained by visual examination (Fig. 4).The actual titer for serum A as determined withthe ELISA reader was 12,150; for serum B itwas 450 (Fig. 4). Thus, the advantage of ELISAover the other two assays is the speed withwhich the assay can be performed and readwithout the need for expensive equipment.

Further evidence that the tests were all mea-suring specific antibody to coronavirus was ob-

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SENSITIVITY COMPARISON OF RIA AND ELISA 37

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tion. (-) Activity in virus coated plates; (- -)activity in control plates; (-) ratio of virus to controlplates.

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tained by determining coronavirus antibodiespresent in colostrum at different times postpar-turition. Very high levels of antibody were pres-ent at the time of parturition, as detected by allthree methods, and rapidly declined thereafter(Fig. 5). Similarly, before suckling, calf serumtiters were negative, but within 24 h of birth andsuckling, these titers approached those of thedam (data not shown).

Survey of prevalence of coronavirus in cattle.Using ELISA, we screened 12 different herds ofcattle situated within a 300-mile radius of eachother in western Canada for antibodies to coro-navirus. Antibodies were found in all of theherds, and only 1 of 110 animals was found notto have antibody, suggesting that the virus isubiquitous in cattle populations in western Can-ada. A survey of this type, using over 100 serumsamples, could be performed in one day. Thus,the sensitivity and speed of ELISA warrantfurther use of this technique in diagnostic labo-ratories.

Serological and lactogenic response to vaccina-tion. Serum antibody titers to coronavirus didnot change in cows given one or two injectionsof the modified live-virus vaccine (Table 2). Inaddition, there were no significant differences incolostrum or milk titers between cows in eitherof the vaccinated groups and the control group.

Similarly, SN tests on serum and lacteal se-cretions from cows in the double-vaccinationgroup showed no increase in titer as comparedwith control cows (data not shown).

DISCUSSIONRIA and ELISA are widely used methods for

diagnosis of various infections, especially viraldiseases of humans and animals. These methodsgenerally exceed the sensitivity of conventionalmethods used in diagnostic laboratories, butspecificity and sensitivity are very dependent onthe quality of the antigens. Any attempts to usethese assays must take this into consideration.For this reason, these assays were first intro-duced for use with viruses which grow well inculture (4, 5, 20).Bovine coronaviruses generally are difficult to

adapt to grow in cell culture and produce recog-nizable cytopathic effects (2, 3, 13, 18). We wereable to obtain an isolate of a bovine coronavirusthat grew in culture and thus could be used inRIA and ELISA for diagnosis of this agent. Tocompare the sensitivity of these assays with thatof conventional assays, we compared the anti-body levels in serum and colostrum obtained byRIA and ELISA with those obtained by SN. Theresults demonstrated a high correlation betweenthe tests (r = 0.915 for RIA-SN; r = 0.964 forRIA-ELISA). In most cases, ELISA titers were

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38 RODAK, BABIUK, AND ACRES

SERUM A SERUM B

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antigen (C-Ag) incubated with various dilutions of serum as described in the text.

higher than those obtained by SN. Furthermore,using the same antibody in the indicator system,the RIA gave titers at least 10 times higher thanELISA titers. This is comparable to what hasbeen reported in other systems (19). However,once the proper antigens are available, the sensi-tivity of the assays can be increased even moreby using chromatographically purified antibody(Table 1), and a further increase in sensitivity

can be achieved by using luminometry withELISA (15). Although the sensitivity of RIA andELISA in our study was only 10 to 100 timesthat of SN, it should be pointed out that thesensitivity of the SN test for comaviruses isrelatively high compared with that for otherviruses.Another advantage of RIA and ELISA over

SN, in addition to sensitivity and speed, is the

t Colostral wheyIz No 4-8

0 2 4 5 10 20 30

Colostral wheyNo 72

0 2 4 5 10 20 30

DAYS AFTER PARTURITIONFIG. 5. Coronavirus antibodies in milk at different times postparturition as measured by RIA (U), ELISA

(A), and SN (0).

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SENSITIVITY COMPARISON OF RIA AND ELISA 39

TABLE 2. Coronavirus antibody titer (by ELISA) in serum and lacteal secretions of cows given a modifiedlive virus vaccine

Titer in:

Serum, at time after injection of Lacteal secretions, at time after calvingGroup Cow vaccine (days)

0 3 wk 6 wk Part- 0 1-1.5 5 20-21urition

Control 1 4,050 4,050 4,050 36,450 4,050 4,050 1502 4,050 4,050 4,050 12,150 4,050 450 1503 4,050 4,050 1,350 12,150 4,050 450 1504 4,050 4,050 4,050 36,450 12,150 1,350 1505 4,050 4,050 4,050 12,150 12,150 1,350 150

GMTa 4,050 4,050 3,263 18,855 6,286 1,082 150

Single vaccination 6 1,350 1,350 1,350 12,150 12,150 150 507 4,050 4,050 4,050 36,450 12,150 1,350 1508 4,050 4,050 1,350 12,150 4,050 1,350 4509 4,050 4,050 4,050 12,150 1,350 450 15010 4,050 4,050 4,050 12,150 4,050 150 150

GMT 3,263 3,263 2,616 15,040 5,035 450 150

Double vaccination 11 4,050 4,050 4,050 4,050 36,450 4,050 150 5012 4,050 4,050 4,050 4,050 12,150 12,150 150 013 1,350 1,350 1,350 1,350 12,150 450 450 5014 1,350 1,350 1,350 1,350 12,150 450 150 5015 4,050 4,050 4,050 4,050 36,450 1,350 450 50

GMT 2,616 2,616 2,616 2,616 18,855 1,679 233 24a GMT, Geometric mean titer.

fact that these techniques do not require sterilesamples. Thus, samples that are submitted toregional diagnostic laboratories in unsterile vialscan still be assayed by RIA and ELISA. Fur-thermore, if samples are not processed properlybefore submission, they may be toxic to tissueculture cells. The use of RIA and ELISA canalso alleviate this problem. Finally, the proce-dures can be automated so that numerous sam-ples can be assayed at once and the results areavailable within a few hours whereas SN forcoronavirus takes at least 6 days for results to beobtained. Because of automation, the reproduc-ibility of the results by ELISA and RIA is muchgreater than for SN. Since it is difficult to readthe rather nonspecific endpoints in SN tests withbovine coronavirus, titers may not be reproduc-ible; such is not the case with nonsubjectivetests such as RIA and ELISA.The prevalence of coronavirus antibodies in

cattle suggests that this agent is very widespreadand may be an important pathogen in cattle inwestern Canada. By using sensitive methods ofdetection for both virus antigen and antibody, itshould be possible to understand more fully theepidemiology of coronavirus and its role in neo-

natal diarrhea as well as to determine the level ofcoronavirus antibody in colostrum and milk thatwill protect calves from enteric disease underdifferent systems of management. Furthermore,these assays will greatly assist us in the evalua-tion of serological responses to vaccination. Thecommercial modified live vaccine failed to causesignificant increases in coronavirus titers of theserum, colostrum, or milk of cows which weregiven one or two doses of vaccine, and antibodytiters declined just as rapidly in vaccinated as incontrol cows. These results indicate that thisvaccine does not stimulate the development ofprotective antibody titers in the milk of cowsand may be of limited value in preventing coro-navirus diarrhea in the field.The finding that RIA was the most sensitive

assay for detection of coronavirus antibodymakes it the most suitable assay system forresearch laboratories equipped with gammacounters and where a high degree of sensitivityis required to measure small amounts of antigenor antibody. In contrast, ELISA, although lesssensitive, can be carried out in every laboratorysince the results can be evaluated even with thenaked eye. Furthermore, the fact that all serum

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40 RODAK, BABIUK, AND ACRES

samples that were positive by RIA were alsopositive by ELISA should make the ELISA thetest of choice in routine laboratory testing ofsera for bovine coronavirus.

ACKNOWLEDGMENT

We appreciate the financial support provided by the Inter-national Atomic Energy Agency in the form of a fellowship toL.R.

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12. Mebus, C. A., L. E. Newman, and E. L. Stair. 1975.Scanning electron, light, and immunofluorescent micros-copy of intestine of gnotobiotic calf infected with calfdiarrheal coronavirus. Am. J. Vet. Res. 36:1719-1725.

13. Mebus, C. A., E. L. Stair, M. B. Rhodes, and M. J.Twiehaus. 1973. Neonatal calf diarrhea: propagation, al-teration and characteristics of a coronavirus-like agent.Am. J. Vet. Res. 34:1719-1725.

14. Mebus, C. A., E. L. Stair, M. B. Rhodes, and M. J.Twiehaus. 1973. Pathology of neonatal calf diarrhea in-duced by a coronavirus-like agent. Vet. Pathol. 10:45-64.

15. Pronovost, A. D., A. Baumgarten, and G. D. Hsiung.1981. Sensitive chemiluminescent enzyme-linked immu-nosorbent assay for quantification of human immunoglob-ulin G and detection of herpes simplex virus. J. Clin.Microbiol. 13:97-101.

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