Rotavirus specific antibodies in fetal bovine serum and commercial preparations of serum albumin

Copyright © 1984,AmericanSociety forMicrobiology

Rotavirus-Specific Antibodies

in Fetal

Bovine Serum

and

Commercial

Preparations of Serum Albumin

PAULA. OFFIT,l2* H. FRED CLARK,1'2 ALEX H.

TAYLOR,'

R. GUENTERHESS,3 PETERA. BACHMANN,3

AND STANLEY A. PLOTKIN1,2

The Wistar Institute of Anatomy and

Biology'

and Divisionof lnfectious Diseases, The Children'sHospital of Philadelphia,2 Philadelphia, Pennsylvania 19104, andInfectiousandEpidemic Diseases, Veterinary Faculty, Institutefor

MedicalMicrobiology, University ofMunich,8000Munich22, FederalRepublic of Germany3

Received 29 March1984/Accepted 14 May 1984

Rotavirus-specific antibodiesweredetected in fetal bovineserum,bovineserumalbumin,and humanserum

albuminby radioimmunoprecipitation with the NCDV strain of bovine rotavirusasthedetecting antigen. Fetal bovinesera neutralized bovine rotavirus inaplaquereduction neutralization test to titers of 1:20orgreater. Immunoglobulins purifiedfrom fetal bovineserumby protein A-agarose affinity chromatography precipitated rotavirus antigens but did not neutralize bovine rotavirus. Rotavirus antibodies in fetal bovineserumandin purified serum albumin preparations may interfere with diagnostic assays for the detection of rotavirus antigens orantibodies.

Theprevalence of rotavirus-specific antibodies in thesera of domestic and laboratory animals is extensive (27, 32). Investigators performing serological studies of rotaviruses mustthereforepayscrupulous attention toavoiding theuse of adult animal sera containing rotavirus antibodies. It has generally been assumed that theriskofencountering natural-ly occurring antibodies in serological reagentscanbe avoid-ed by the use of fetal bovine serum (FBS), bovine serum albumin (BSA), orhuman serum albumin(HSA). However, thefinding of neutralizing activityincommercialFBS (9)led

us tofurther investigate FBS as well as BSA and HSA for the presence of rotavirus antibodies. We have found rota-virus-specific antibodies by radioimmunoprecipitation (RIP) incommercial preparations ofFBS, BSA, and HSAaswell as in serum samples obtained under our supervision from precolostral calves. The incidence of such antibodies in these reagents and their identification as immunoglobulins

with specific antirotavirus activity are described in this report.

MATERIALS AND METHODS

Cells andviruses. Fetal rhesus monkey kidney cells (MA-104) were grown in BHK cell medium (22) supplemented

with 10% FBS, 100 U of penicillin per ml, and 100 ,ug of streptomycin perml.

Thebovine rotavirus strain NCDV, adapted togrowth in tissueculture at theNorden Laboratories (Lincoln, Nebr.),

was generously provided by Robert Yolken (Baltimore, Md.). The Wa strain of human rotavirus was obtainedfrom RichardWyatt (Bethesda, Md.).

Viral growth, purification, and quantitation were per-formed aspreviously described (26).

Serum preparations. Commercial lots of FBS were pur-chased from Flow Laboratories, Inc. (McLean, Va.) MA Bioproducts (Walkersville, Md.), Biocell (Carson, Calif.), GIBCO L-aboratories (Grand Island, N.Y.), and Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Crystalline bovine albumin was purchased from Sigma Chemical Co. (St. Louis, Mo.; stockno.A 7030) andCalbiochem-Behring (La Jolla, Calif.; stock no. 12657). Albumin solutions (25%

* Correspondingauthor.

[wt/vol])

were prepared in phosphate-buffered saline (PBS)

for use in these studies. HSA was obtained from Cutter

Laboratories(Berkeley, Calif.; Plasbumin-25,stockno. 684-20). Blood samples were obtained under our supervision (Institute for Medical Microbiology, Munich, Federal

Re-public ofGermany)fromtheexternaljugularvein of

cesari-an-derived Holstein-Friesian or Fleckvieh calves before colostral feeding. All serum preparations were treated at 56°C for 30 min.

Serumpreparations adsorbedwithStaphylococcusaureus Cowan 1werereacted with 10 mg of the bacterium per ml of serum. After incubation at 0°C for 1 h, bacteria were

removed by centrifugation for2 min at 12,800 x g. Superna-tantportions were used for furthertesting.

RIPofrotavirus proteins. Purified double-shelled NCDV

virions were labeled with 1251 by the chloramine-T method

(10).

125I-labeled

virus preparations (60,000 trichloroacetic acid-precipitable cpm/pI) were adsorbed with S. aureus

Cowan1 by adding500

RlI

of10%S. aureusCowan1to 1.0 ml oflabeled virus. After incubation for 15 min at0°C, the

bacteriawereremoved bycentrifugation for2 min at 12,800 x g. The adsorbed supernatant fluids were then divided into

5-Rl

portions to which 50 ,ul ofthe serum preparation was

added. After incubation for 18 h at 4°C, 80 ,ul of 10% S. aureusCowan1 wasadded to each serum-virus mixture and held at 0°C for 1 h. The bacteria were then pelleted and washed four times with PBS containing0.1% sodium

dode-cyl sulfate (SDS) and 0.5% Triton X-100. The adsorbed,

labeledproteins were recovered by suspending the bacterial pellets in 20 ,ul of sample buffer containing 0.25 M

Tris-hydrochloride (pH 6.8), 20% glycerol, 1% SDS, 2% 2-mercaptoethanol, and 0.003% phenol red and boiling the

suspension for 2 min. The bacteria were pelleted, and the supernatantfluids were applied to SDS-polyacrylamide gels. Discontinuous SDS-PAGE. Discontinuous SDS-polyacryl-amidegel electrophoresis (PAGE) was performed by using a 10% acrylamide separating gel as previously described (19). Inexperimentsdesigned to determine the molecular weight of nonreduced purified bovine immunoglobulin, 2-mercap-toethanolwas omittedfrom the samplebuffer.

Electrophoresiswasperformed at 30 mA per gel.

Molecu-lar weightstandards(Bio-RadLaboratories, Richmond,

Cal-if.)weredetectedby stainingwith silver nitrate as described 266

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ROTAVIRUS ANTIBODIES IN FBS 267

1 2 3 4 5 6 7 8 9 101112131415161718

116-94- ,

88

:

P-

la*.,

I*

--

I*.,*

*111

~

84

6

1

₆₁-

is

_w

41- -

0

*-

_b

Om*

M

FIG. 1. 125I-labeled, purified, double-shelled NCDV virions

wereseparated by electrophoresis in10%SDS-polyacrylamide gels and visualized by fluorography (lane 1). Numbers refer to the molecular weights (in thousands) ofthe viral polypeptides. RIP analysis of100, 10, and5%FBS from Flow (lanes 2to4), Biocell (lanes5to7),andMABioproducts (lanes8to10),25and 2.5%BSA

fromSigma (lanes11and12)and Calbiochem-Behring(lanes 13 and 14), 25, 2.5, and 1.25% HSA from Cutter Laboratories (lanes15 to

17), and PBS control (lane 18) with the NCDV strain of bovine rotavirusasthe detecting antigen.

by Merril et al. (25). Fluorograms were prepared as

de-scribedby Laskey and Mills (20).

PRN assay. The plaque reduction neutralization (PRN)

assay was a modification of the technique described

previ-ously by Matsunoetal. (23). A virus suspension containing 500PFUof bovine rotavirus(NCDV)permlwasmixed with

anequal volume of serial fivefold dilutions ofserum

prepara-tions. The serum-virus mixture was incubated in a water bath at 37°C for 30 min. Confluent monolayers of MA-104 cells in 6-well plates were washed twice with PBS. The

serum-virus mixture (0.2 ml)wasthen inoculated onto MA-104 cells andincubated for 30 minat 37°C. The plateswere

again washed twice with PBS, and 2.5 ml of overlay medium consisting of 0.5% purifiedagar(agarose;Seakem) and 13 Fg

oftrypsin (Flow) per ml inEagle minimal essentialmedium wasadded. The cultureswereplaced inahumidified

incuba-torfor4 daysat 37°C in5%CO2.Asecondoverlaymedium containing 0.5% purifiedagarand0.03%neutral red in Earle balanced salt solutionwasthenadded, andtheplaqueswere

countedca. 5h later. A greater than 50% reduction in viral plaques was considered to be a positive result at a given serumdilution.

Protein A-agarose affinity chromatography. Affinity chro-matography was carried out on columns (2 by 11 cm) of protein A-agarose (Boehringer Mannheim). FBS (MA Bio-products)waspassed throughthecolumnataflowrateof 25 ml/h at 25°C. The column was then washed with

Britton-Robinson(5) buffer (pH 7.0),and when the absorbance ofthe effluent at 280 nm was zero, the adsorbed proteins were

elutedwithalinearpH gradientfrom 7.0to3.0.All fractions with anabsorbance greaterthan 0.002 werepooled, and the pH wasadjusted to 7.0by the addition of 0.5 M Na2HPO4 solution (pH 7.4). The pooled fractions were then

concen-trated 30-foldthroughacollodionbag (Schleicher& Schuell

Inc., Keene, N.H.) with an exclusion molecular weight of

75,000.

Toestimate the concentration of elutedimmunoglobulins,

it was assumed that 1.4 absorbance units at 280 nm repre-sented a concentration of 1.0mglml (33).

RESULTS

125I-labeling

of structural rotavirus polypeptides by RIP.

Figure1(lane 1) shows the SDS-PAGE pattern of iodinated rotavirus structural polypeptides. Proteins with molecular weights of 116,000, 94,000, 88,000, 84,000, 41,000, and

37,000wereidentified.Theseproteinsarecomponents of the

inner and outer capsid of bovine rotavirus (26). The

37-kilodalton protein is the major outer capsid glycoprotein associated with viral neutralization (11).

Analysis of commercial serum preparations by RIP.

Com-mercialpreparations of FBS, BSA, and HSA were found by RIPtocontainantibodies directed against rotavirus structur-al proteins with molecular weights of 116,000, 94,000,

88,000, 84,000, and 41,000 (Fig. 1). Rotavirus atitibodies were detected in 5% FBS, 2.5% BSA, and 1.25% HSA solutions (Fig. 1). Twelveadditional lots of FBS were found tocontainrotavirus-specific antibodiesat similar

concentra-tions (datanot shown).

Identification of immunoglobulins as source of RIP activity. (i) Protein A-agarose affinity chromatography of FBS. FBS

(50 ml)found previously to have rotavirus-specific antibod-ies by RIP (Fig. 1, lanes 8 to 10) was passed through a

protein A-agarose column,followedby alinearpHgradient

elution. Fractions with an absorbance greater than 0.002 were eluted between pH 6.15 and 5.41, with the peak

absorbance fraction eluted at pH 5.8. Eight micrograms of eluted immunoglobulinsperml of FBSwererecoveredfrom

pooled and concentrated fractions.

(ii) Discontinuous SDS-PAGE of protein A-purified immu-noglobulins. The results of SDS-PAGE of purified

immuno-globulinstreatedwithreducing ornonreducingsamplebuffer and stained with silver nitrate are illustrated in Fig. 2.

Unreduced immunoglobulinsweredemonstratedbyasingle band with a molecular weight of 150,000, and reduced

immunoglobulins were cleaved into heavy and light chains

1 2

150

-50-

-

28-FIG. 2. SDS-PAGE ofimmunoglobulinspurifiedfrom FBS

treat-ed withreducing (lane 1)ornonreducing (lane2)samplebufferand stained with silvernitrate. Numbers refertothemolecularweights

(inthousands) of visualizedproteins. VOL. 20, 1984

I

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demonstrated by bands withmolecularweightsof50,000and 28,000,respectively.

(iii) Analysis of purified bovine immunoglobulins by RIP.

Purified immunoglobulins at a concentration of 20 ,ug/ml were found to have rotavirus-specific antibodies by RIP

directed against proteins with molecular weights of116,000, 94,000, 88,000, 84,000, and 41,000. Rotavirus-specific

anti-bodies were not detected by RIP of protein A-agarose-adsorbed FBS.

Analysis of serum preparations by PRN. Three commercial

lots ofFBS shownbyRIP(Fig.1,lanes2to 4, 5 to 7,8to 10) tocontainrotavirus-specific antibodiesweretestedby PRN. Allthreelots ofFBS testedat aconcentration of 5%caused a ¢50%plaque reduction of bovine rotavirus strain NCDV.

Preparationsof12.5% BSA and 12.5% HSA did not neutral-izebovineorhuman (strain Wa) rotaviruses.

Roleof bovine immunoglobulins in rotavirus inactivation by FBS. To determine whetherrotavirus-specific immunoglob-ulinswereassociatedwith therotavirus-neutralizing activity of FBS, a 5% solution of three different lots of FBS was

adsorbed with S. aureus Cowan 1. The adsorbed solutions

were assayed for rotavirus-specific antibodies by RIP and

forneutralizing activity byPRN. Norotavirus-specific anti-bodies were detected in the adsorbed solutions by RIP; however,

rotavirus-neutralizing

activity was detected at

concentrations identicaltothosefoundinunadsorbed

prepa-rations.

Conversely,

immunoglobulins

purified

from FBS did not

neutralize NCDV rotavirusat aconcentration of200 p.g/ml. Source of bovine immunoglobulin in FBS. To determine whether the immunoglobulins detected in commercial FBS

were the result of either colostral feeding or mixing of

maternal with fetal sera, 10 cesarian-derived calves were

bled under oursupervision before colostral

feeding.

All 10 sera werefoundtohave

rotavirus-specific

antibodiesbyRIP at concentrations similar to those found in commercial lots ofFBS (datanot

shown).

Serum specimens from both mother and neonate were

obtainedduring onecesariandelivery. Neonatal and mater-nal sera(testedatconcentrations of5and

0.002%)

contained antibodies directedagainst rotavirusproteins withmolecular

weights of116,000, 94,000,

88,000,

84,000, and41,000(Fig. 3). At a higher concentration of maternal serum (0.2%), antibodies directed against the 37-kilodalton major outer

capsid glycoproteinwerealso demonstrable(Fig. 3). Neona-tal and maternal seraneutralized rotavirus strain NCDV by PRN at dilutions of1:40and 1:830,respectively.

DISCUSSION

We have found that antibodies directed against rotavirus

proteins are consistently present in FBS. Sato et al. (28) detected hemagglutination-inhibiting antibodies to bovine rotavirus in the sera of 27 of 29 precolostral calves at

dilutionsof 1:2to 1:128. Using a complement-fixation assay, Estes etal. (9) were unable todetect rotavirus antibodies in commercialpreparations of FBS. However, they found that

inclusionofFBS into viral growth medium decreased

rota-virus infectivity 5- to 15-fold. Our studies with S. aureus Cowan 1-adsorbed FBS and immunoglobulinspurified from

FBSclearly show that antirotavirus activity detected by RIP is due to immunoglobulins. The antibodies detected in our

studiesofFBS, BSA, and HSA may interfere with standard assays for antibody or antigen detection (e.g.,

radio-immunoassay,enzyme-linked immunosorbentassay) orwith assays which evaluate the viral structural specificities of

1

234567

116

94

88

84

- a

7

0

4

61-41 ,_

37-'._

FIG. 3. "25I-labeled,purified, double-shelled NCDV virions sep-arated by electrophoresis in 10% SDS-polyacrylamide gels and visualized by fluorography (lane 1). Numbers refer to the molecular weights (in thousands) of viral polypeptides. RIP analysis of 100, 10 and 5% neonatal (lanes 2 to 4) and 0.2, 0.02, and 0.002% maternal (lanes 5 to 7) bovine serum obtained during cesarian delivery with the NCDV strain of bovine rotavirus as the detecting antigen.

polyclonal or monoclonal antibody preparations (e.g., RIP,

Western blot analysis).

Purifiedimmunoglobulins recovered from FBSbyprotein Aaffinitychromatography did notappear to be responsible for the observed neutralization of rotavirusesby FBS. This

finding is consistent with the fact that these immunoglob-ulinsare not directed against the 37-kilodalton major outer

capsid glycoproteinassociated with viral neutralization(11).

Antibodies directed against inner capsid and outer capsid

proteinsweredetected in maternalserum at serumdilutions of 1:50,000 and 1:500, respectively. The concentration of

rotavirus-specific antibodies in FBS was ca. 2,500-fold less than thatdetectedin maternalserum; ourinabilitytodetect antibodies directed against the 37-kilodalton outer capsid

proteinin neonatalsera mostlikelyreflects these

concentra-tion differences. Rotavirus neutralization associated with

FBS maybetheresult of eitherserum

antiprotease

activity

(9) or of bovine immunoglobulins which do not bind to

protein A.

To our knowledge, there are no published reports

docu-menting the presence of rotavirus-specific antibodies in commercial albumin preparations. The detection of these

antibodies may beexplainedby (i) the extensive prevalence of rotavirus antibodies in random and convalescent sera obtained from both animals and humans (7, 23, 27, 32) and

(ii) the indication (suppliers catalog) that these albumin

preparations may contain trace amounts of globulin. The detection of antibodies in HSA directed against bovine rotavirus proteins is consistent with previous reports of shared antigenic determinants on both the inner and outer

capsidsofrotaviruses ofbovine, human,andother mamma-lianorigins (26).

Transplacental transfer of immunoglobulins presumably

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ROTAVIRUS ANTIBODIES IN FBS 269

does not occur in cattle (4). Thisfact is difficult toreconcile

with our consistent finding of antirotavirus immunoglobulins in FBS. Several investigators have detectedimmunoglobulin

G in FBS by radial immunodiffusion at concentrations of 13 to 859 ,ug/ml; maternal immunoglobulin G was detected in maternal serum at a concentration of ca. 50mg/ml (2, 8, 12-14, 17, 18, 29). We have detected rotavirus-specific antibod-ies in maternal serum at concentrations 2,500- and 20-fold greater than precolostral calf serum by RIP and PRN, respectively. Our results are consistent with the

immuno-globulin concentration differences detected between fetal and maternal seraby radial immunodiffusion. Thefinding of rotavirus-specific antibodies in 10 sera collected under our supervision from precolostral calves excludes the

possibili-ties that antibodies in commercial sera were the result of either (i) mixing of maternal with fetal sera during or after collection or (ii) collection ofsera after colostral feeding.

The bovine fetus is able to respond to certain antigenic

stimuli beginning at ca. day 118 of gestation (31). Bovine viral diarrhea, infectious bovine rhinotracheitis, and blue-tongueviruses have been foundto crosstheplacentalbarrier and to induce a specific immune response by the fetus(1, 3,

6, 14-16, 21, 24, 30). Wyattet al.(34) demonstrated that the bovine fetus can generate a vigorous immune response in utero tobovine rotavirus; when seven calveswere inoculat-ed with the bovine rotavirus NCDV 2 to 14 weeks before delivery, neutralizing antibodies were detected in cord se-rum at dilutions of 1:370to 1:17,975. However, viremia and fetal disease have never been documented in rotavirus

infections. Since rotavirusesare notknownto causeinutero

infection, ourconsistent detection of

rotavirus-specific

anti-bodies in 5% FBS

by

RIPsuggests that

immunoglobulins

are

passively transferred across an intact bovine

placenta

in small but detectable

quantities

before

partuition.

ACKNOWLEDGMENTS

This workwassupportedby Public Health Servicegrant F 32 Al-06733from the National InstitutesofHealthtoP.A.O.and inpartby

the Hassel Foundation and the Merieux Institute.

WethankWalterGerhardfor supportofthis work. We also thank Charles Hackett, Jan Tuttleman, and Jon Yewdell for helpful

discussions and careful reading of the manuscript.

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