Rotavirus-Specific Antibodies in Fetal Bovine Serum and Commercial Preparations of Serum Albumin

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1984, p /84/ $02.00/0 Copyright 1984, American Society for Microbiology Vol. 20, No. 2 Rotavirus-Specific Antibodies in Fetal Bovine Serum and Commercial Preparations of Serum Albumin PAUL A. OFFIT,l2* H. FRED CLARK,1'2 ALEX H. TAYLOR,' R. GUENTER HESS,3 PETER A. BACHMANN,3 AND STANLEY A. PLOTKIN1,2 The Wistar Institute of Anatomy and Biology' and Division of lnfectious Diseases, The Children's Hospital of Philadelphia,2 Philadelphia, Pennsylvania 19104, and Infectious and Epidemic Diseases, Veterinary Faculty, Institute for Medical Microbiology, University of Munich, 8000 Munich 22, Federal Republic of Germany3 Received 29 March 1984/Accepted 14 May 1984 Rotavirus-specific antibodies were detected in fetal bovine serum, bovine serum albumin, and human serum albumin by radioimmunoprecipitation with the NCDV strain of bovine rotavirus as the detecting antigen. Fetal bovine sera neutralized bovine rotavirus in a plaque reduction neutralization test to titers of 1:20 or greater. Immunoglobulins purified from fetal bovine serum by protein A-agarose affinity chromatography precipitated rotavirus antigens but did not neutralize bovine rotavirus. Rotavirus antibodies in fetal bovine serum and in purified serum albumin preparations may interfere with diagnostic assays for the detection of rotavirus antigens or antibodies. The prevalence of rotavirus-specific antibodies in the sera of domestic and laboratory animals is extensive (27, 32). Investigators performing serological studies of rotaviruses must therefore pay scrupulous attention to avoiding the use of adult animal sera containing rotavirus antibodies. It has generally been assumed that the risk of encountering naturally occurring antibodies in serological reagents can be avoided by the use of fetal bovine serum (FBS), bovine serum albumin (BSA), or human serum albumin (HSA). However, the finding of neutralizing activity in commercial FBS (9) led us to further investigate FBS as well as BSA and HSA for the presence of rotavirus antibodies. We have found rotavirus-specific antibodies by radioimmunoprecipitation (RIP) in commercial preparations of FBS, BSA, and HSA as well 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 and viruses. 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 per ml. The bovine rotavirus strain NCDV, adapted to growth in tissue culture at the Norden Laboratories (Lincoln, Nebr.), was generously provided by Robert Yolken (Baltimore, Md.). The Wa strain of human rotavirus was obtained from Richard Wyatt (Bethesda, Md.). Viral growth, purification, and quantitation were performed as previously described (26). Serum preparations. Commercial lots of FBS were purchased 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.; stock no. A 7030) and Calbiochem-Behring (La Jolla, Calif.; stock no ). Albumin solutions (25% * Corresponding author. [wt/vol]) were prepared in phosphate-buffered saline (PBS) for use in these studies. HSA was obtained from Cutter Laboratories (Berkeley, Calif.; Plasbumin-25, stock no ). Blood samples were obtained under our supervision (Institute for Medical Microbiology, Munich, Federal Republic of Germany) from the external jugular vein of cesarian-derived Holstein-Friesian or Fleckvieh calves before colostral feeding. All serum preparations were treated at 56 C for 30 min. Serum preparations adsorbed with Staphylococcus aureus Cowan 1 were reacted with 10 mg of the bacterium per ml of serum. After incubation at 0 C for 1 h, bacteria were removed by centrifugation for 2 min at 12,800 x g. Supernatant portions were used for further testing. RIP of rotavirus 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 Cowan 1 by adding 500 RlI of 10% S. aureus Cowan 1 to 1.0 ml of labeled virus. After incubation for 15 min at 0 C, the bacteria were removed by centrifugation for 2 min at 12,800 x g. The adsorbed supernatant fluids were then divided into 5-Rl portions to which 50,ul of the serum preparation was added. After incubation for 18 h at 4 C, 80,ul of 10% S. aureus Cowan 1 was added to each serum-virus mixture and held at 0 C for 1 h. The bacteria were then pelleted and washed four times with PBS containing 0.1% sodium dodecyl sulfate (SDS) and 0.5% Triton X-100. The adsorbed, labeled proteins were recovered by suspending the bacterial pellets in 20,ul of sample buffer containing 0.25 M Trishydrochloride (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 supernatant fluids were applied to SDS-polyacrylamide gels. Discontinuous SDS-PAGE. Discontinuous SDS-polyacrylamide gel electrophoresis (PAGE) was performed by using a 10% acrylamide separating gel as previously described (19). In experiments designed to determine the molecular weight of nonreduced purified bovine immunoglobulin, 2-mercaptoethanol was omitted from the sample buffer. Electrophoresis was performed at 30 ma per gel. Molecular weight standards (Bio-Rad Laboratories, Richmond, Calif.) were detected by staining with silver nitrate as described 266

2 VOL. 20, , 88 : P- la*., I* -- ~ I*.,* * is w *-_bom* M I FIG I-labeled, purified, double-shelled NCDV virions were separated by electrophoresis in 10% SDS-polyacrylamide gels and visualized by fluorography (lane 1). Numbers refer to the molecular weights (in thousands) of the viral polypeptides. RIP analysis of 100, 10, and 5% FBS from Flow (lanes 2 to 4), Biocell (lanes 5 to 7), and MA Bioproducts (lanes 8 to 10), 25 and 2.5% BSA from Sigma (lanes 11 and 12) and Calbiochem-Behring (lanes 13 and 14), 25, 2.5, and 1.25% HSA from Cutter Laboratories (lanes 15 to 17), and PBS control (lane 18) with the NCDV strain of bovine rotavirus as the detecting antigen. by Merril et al. (25). Fluorograms were prepared as described by Laskey and Mills (20). PRN assay. The plaque reduction neutralization (PRN) assay was a modification of the technique described previously by Matsuno et al. (23). A virus suspension containing 500 PFU of bovine rotavirus (NCDV) per ml was mixed with an equal volume of serial fivefold dilutions of serum preparations. 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) was then inoculated onto MA- 104 cells and incubated for 30 min at 37 C. The plates were again washed twice with PBS, and 2.5 ml of overlay medium consisting of 0.5% purified agar (agarose; Seakem) and 13 Fg of trypsin (Flow) per ml in Eagle minimal essential medium was added. The cultures were placed in a humidified incubator for 4 days at 37 C in 5% CO2. A second overlay medium containing 0.5% purified agar and 0.03% neutral red in Earle balanced salt solution was then added, and the plaques were counted ca. 5 h later. A greater than 50% reduction in viral plaques was considered to be a positive result at a given serum dilution. Protein A-agarose affinity chromatography. Affinity chromatography was carried out on columns (2 by 11 cm) of protein A-agarose (Boehringer Mannheim). FBS (MA Bioproducts) was passed through the column at a flow rate of 25 ml/h at 25 C. The column was then washed with Britton- Robinson (5) buffer (ph 7.0), and when the absorbance of the effluent at 280 nm was zero, the adsorbed proteins were eluted with a linear ph gradient from 7.0 to 3.0. All fractions with an absorbance greater than were pooled, and the ph was adjusted to 7.0 by the addition of 0.5 M Na2HPO4 solution (ph 7.4). The pooled fractions were then concentrated 30-fold through a collodion bag (Schleicher & Schuell ROTAVIRUS ANTIBODIES IN FBS 267 Inc., Keene, N.H.) with an exclusion molecular weight of 75,000. To estimate the concentration of eluted immunoglobulins, it was assumed that 1.4 absorbance units at 280 nm represented a concentration of 1.0 mglml (33). RESULTS 125I-labeling of structural rotavirus polypeptides by RIP. Figure 1 (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 37,000 were identified. These proteins are components 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. Commercial preparations of FBS, BSA, and HSA were found by RIP to contain antibodies directed against rotavirus structural 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). Twelve additional lots of FBS were found to contain rotavirus-specific antibodies at similar concentrations (data not 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 antibodies by RIP (Fig. 1, lanes 8 to 10) was passed through a protein A-agarose column, followed by a linear ph gradient elution. Fractions with an absorbance greater than were eluted between ph 6.15 and 5.41, with the peak absorbance fraction eluted at ph 5.8. Eight micrograms of eluted immunoglobulins per ml of FBS were recovered from pooled and concentrated fractions. (ii) Discontinuous SDS-PAGE of protein A-purified immunoglobulins. The results of SDS-PAGE of purified immunoglobulins treated with reducing or nonreducing sample buffer and stained with silver nitrate are illustrated in Fig. 2. Unreduced immunoglobulins were demonstrated by a single band with a molecular weight of 150,000, and reduced immunoglobulins were cleaved into heavy and light chains FIG. 2. SDS-PAGE of immunoglobulins purified from FBS treated with reducing (lane 1) or nonreducing (lane 2) sample buffer and stained with silver nitrate. Numbers refer to the molecular weights (in thousands) of visualized proteins.

3 268 OFFIT ET AL. demonstrated by bands with molecular weights of 50,000 and 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 of 116,000, 94,000, 88,000, 84,000, and 41,000. Rotavirus-specific antibodies were not detected by RIP of protein A-agaroseadsorbed FBS. Analysis of serum preparations by PRN. Three commercial lots of FBS shown by RIP (Fig. 1, lanes 2 to 4, 5 to 7, 8 to 10) to contain rotavirus-specific antibodies were tested by PRN. All three lots of FBS tested at a concentration of 5% caused a 50% plaque reduction of bovine rotavirus strain NCDV. Preparations of 12.5% BSA and 12.5% HSA did not neutralize bovine or human (strain Wa) rotaviruses. Role of bovine immunoglobulins in rotavirus inactivation by FBS. To determine whether rotavirus-specific immunoglobulins were associated with the rotavirus-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 for neutralizing activity by PRN. No rotavirus-specific antibodies were detected in the adsorbed solutions by RIP; however, rotavirus-neutralizing activity was detected at concentrations identical to those found in unadsorbed preparations. Conversely, immunoglobulins purified from FBS did not neutralize NCDV rotavirus at a concentration of 200 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 our supervision before colostral feeding. All 10 sera were found to have rotavirus-specific antibodies by RIP at concentrations similar to those found in commercial lots of FBS (data not shown). Serum specimens from both mother and neonate were obtained during one cesarian delivery. Neonatal and maternal sera (tested at concentrations of 5 and 0.002%) contained antibodies directed against rotavirus proteins with molecular weights of 116,000, 94,000, 88,000, 84,000, and 41,000 (Fig. 3). At a higher concentration of maternal serum (0.2%), antibodies directed against the 37-kilodalton major outer capsid glycoprotein were also demonstrable (Fig. 3). Neonatal and maternal sera neutralized rotavirus strain NCDV by PRN at dilutions of 1:40 and 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 dilutions of 1:2 to 1:128. Using a complement-fixation assay, Estes et al. (9) were unable to detect rotavirus antibodies in commercial preparations of FBS. However, they found that inclusion of FBS into viral growth medium decreased rotavirus infectivity 5- to 15-fold. Our studies with S. aureus Cowan 1-adsorbed FBS and immunoglobulins purified from FBS clearly show that antirotavirus activity detected by RIP is due to immunoglobulins. The antibodies detected in our studies of FBS, BSA, and HSA may interfere with standard assays for antibody or antigen detection (e.g., radioimmunoassay, enzyme-linked immunosorbent assay) or with assays which evaluate the viral structural specificities of - a ,_ '._ J. CLIN. MICROBlOL. FIG. 3. "25I-labeled, purified, double-shelled NCDV virions separated 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). Purified immunoglobulins recovered from FBS by protein A affinity chromatography did not appear to be responsible for the observed neutralization of rotaviruses by FBS. This finding is consistent with the fact that these immunoglobulins are not directed against the 37-kilodalton major outer capsid glycoprotein associated with viral neutralization (11). Antibodies directed against inner capsid and outer capsid proteins were detected in maternal serum at serum dilutions of 1:50,000 and 1:500, respectively. The concentration of rotavirus-specific antibodies in FBS was ca. 2,500-fold less than that detected in maternal serum; our inability to detect antibodies directed against the 37-kilodalton outer capsid protein in neonatal sera most likely reflects these concentration differences. Rotavirus neutralization associated with FBS may be the result of either serum antiprotease activity (9) or of bovine immunoglobulins which do not bind to protein A. To our knowledge, there are no published reports documenting the presence of rotavirus-specific antibodies in commercial albumin preparations. The detection of these antibodies may be explained by (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 capsids of rotaviruses of bovine, human, and other mammalian origins (26). Transplacental transfer of immunoglobulins presumably

4 VOL. 20, 1984 does not occur in cattle (4). This fact is difficult to reconcile with our consistent finding of antirotavirus immunoglobulins in FBS. Several investigators have detected immunoglobulin 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. 50 mg/ml (2, 8, 12-14, 17, 18, 29). We have detected rotavirus-specific antibodies 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 immunoglobulin concentration differences detected between fetal and maternal sera by radial immunodiffusion. The finding of rotavirus-specific antibodies in 10 sera collected under our supervision from precolostral calves excludes the possibilities that antibodies in commercial sera were the result of either (i) mixing of maternal with fetal sera during or after collection or (ii) collection of sera 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 bluetongue viruses have been found to cross the placental barrier and to induce a specific immune response by the fetus (1, 3, 6, 14-16, 21, 24, 30). Wyatt et al. (34) demonstrated that the bovine fetus can generate a vigorous immune response in utero to bovine rotavirus; when seven calves were inoculated with the bovine rotavirus NCDV 2 to 14 weeks before delivery, neutralizing antibodies were detected in cord serum at dilutions of 1:370 to 1:17,975. However, viremia and fetal disease have never been documented in rotavirus infections. Since rotaviruses are not known to cause in utero infection, our consistent detection of rotavirus-specific antibodies in 5% FBS by RIP suggests that immunoglobulins are passively transferred across an intact bovine placenta in small but detectable quantities before partuition. ACKNOWLEDGMENTS This work was supported by Public Health Service grant F 32 Al from the National Institutes of Health to P.A.O. and in part by the Hassel Foundation and the Merieux Institute. We thank Walter Gerhard for support of this work. We also thank Charles Hackett, Jan Tuttleman, and Jon Yewdell for helpful discussions and careful reading of the manuscript. LITERATURE CITED 1. Baker, J. A., C. York, J. H. Gillespie, and G. B. Mitchell Viral diarrhea in cattle. Am. J. Vet. Res. 15: Boone, C. W., N. Mantel, T. D. Caruso, E. Kazam, and R. E. Stevenson Quality control studies on fetal bovine serum used in tissue culture. 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