Glycoprotein Enzyme-Linked Immunosorbent Assay

JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1989, p. 2208-2213 0095-1137/89/102208-06$02.00/0 Copyright C 1989, American Society for Microbiology

Vol. 27, No. 10

Identification of Pseudorabies Virus-Exposed Swine with a gI Glycoprotein Enzyme-Linked Immunosorbent Assay MARK W. MELLENCAMP,t NANCY E. PFEIFFER,* BRIAN T. SUITER, JAMES R. HARNESS, AND WILLIAM H. BECKENHAUER Division of Biological Research and Development, Norden Laboratories, Inc., 601 West Cornhusker Highway, Lincoln, Nebraska 68521 Received 6 March 1989/Accepted 3 July 1989

A monoclonal antibody specific for the gI glycoprotein of virulent pseudorabies virus was produced and used gI was used in an enzyme-linked immunosorbent assay (ELISA) that identified and differentiated field virus-exposed animals from animals vaccinated with gI-deleted virus. The gI ELISA was evaluated by comparing it with the virus neutralization test and with a standard ELISA which does not distinguish between vaccinated and naturally infected animals. Pigs vaccinated with a gI-deleted vaccine were seropositive by the virus neutralization or standard ELISA but were seronegative in the gI ELISA. Nonvaccinated and vaccinated animals were detected as seropositive in the gI ELISA only after exposure to gI-containing field virus. Exposed animals were detected as early as day 7 and for as long as 141 days after field virus exposure. As little as 102.7 PFU of field virus was sufficient to seroconvert negative animals in the gI ELISA. Pseudorabies virus-seronegative animals which received multiple doses of gI-deleted vaccine remained seronegative in the gI ELISA. The use of this test to monitor swine for pseudorabies virus infection would offer significant benefits towards eradication of the disease. to affinity purify gI glycoprotein. The purified

Pseudorabies (Aujeszky's disease) presents severe economic problems to the swine industry. It is estimated this disease costs the swine industry millions of dollars each year from mortality, breeding problems, and reduced growth performance (6). Although efficacious federally licensed pseudorabies vaccines offer protection, efforts to control the disease are hampered by the difficulty in distinguishing vaccinated animals from animals exposed to standard field virus. Because of the highly contagious nature of pseudorabies, federal and state regulations govern the transport of infected swine. Transportation of animals, except to slaughter, requires the animals be free of infection as demonstrated by a serological test. Although a variety of methods exists for the detection of pseudorabies virus-seropositive animals, the virus neutralization (VN) and a licensed screening enzymelinked immunosorbent assay (ELISA) are the two most commonly used diagnostic assays (1, 20, 23). However, these tests do not differentiate vaccinated from field virusexposed animals. The VN and screening ELISA identify seropositive animals by detection of antibodies to antigens shared by both attenuated and virulent pseudorabies virus (PrV) strains. The major glycoproteins of PrV which serve as immunogens in vaccinated and/or field virus-infected animals are designated gI, gIt, gIII, and gVI (7, 9, 11, 27). The gX protein which is secreted from PrV-infected cells has also been shown to elicit an antibody response in infected swine (22), and the U.S. Department of Agriculture recently approved an ELISA based on the gX protein of PrV (8). However, this test requires seronegative animals be vaccinated with a complementary gX-deleted PrV vaccine in order to use the gX ELISA as a differential test. Most commercial attenuated PrV vaccines contain PrV strains with a deletion in the unique short region of the viral

genome (14, 16). This deleted sequence encodes for production of the gI glycoprotein (2, 13, 17). Since the majority of manufacturers of PrV vaccines utilize attenuated gI-deleted virus in their products and since all tested virulent field isolates of PrV have the gI protein (26), the presence of antibodies to gI in animal sera would appear to be the most

logical indicator of infection with standard field virus. Substantial research involving the use of gI as an indicator for PrV exposure has been done in Europe. Van Oirchot and associates have developed a competitive ELISA utilizing gI-specific monoclonal antibodies (MAbs) for the detection of anti-gI antibodies (26). Their results have shown that a gI-based diagnostic used in combination with a gI-deleted PrV vaccine is effective in controlling the spread of pseudorabies (25). The purpose of this report is to describe the use of affinity-purified gI glycoprotein as an ELISA antigen to differentiate vaccinated animals from animals infected with a field virus. The ability to accurately identify these two populations would greatly enhance efforts to eradicate pseudorabies in the United States.

MATERIALS AND METHODS Viruses and cell lines. Swine testicular (ST) and porcine kidney (PK-15) cells (Norden Laboratories, Lincoln, Nebr.) were maintained in Dulbecco modified Eagle medium supplemented with 5% fetal bovine serum. Virulent PrV strain Indiana Funkhauser (IND-F) (originally obtained from C. Kanitz, Purdue University, West Lafayette, Ill.) was used in experiments as the gI-containing virus. Our licensed PrV strain Bucharest (BUK) (Norden Laboratories, Inc., Lincoln, Nebr.) was used as the gI-deleted virus. Production of MAbs. Female 6- to 8-week-old BALB/c mice were immunized intraperitoneally with 100 ,ug of gradient-purified IND-F PrV emulsified in Freund complete adjuvant. A second immunization was given intraperitoneally 3 weeks later with the same amount of antigen in phosphate-buffered saline (PBS). Spleen cells from immunized mice were fused with NS1/Ag4 mouse plasmacytoma

Corresponding author. t Present address: Department of Microbiology, Miami University, Oxford, OH 45056. *

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cells 3 days after the last antigen dose (21). Hybridoma culture supernatants were screened by ELISA in 96-well microdilution plates (Nunc, Roskilde, Denmark) coated with gradient-purified IND-F and BUK PrV and sonicated noninfected PK-15 cells. Antigen specificity of the hybridomas was determined by radioimmunoprecipitation analysis (see below). Selected hybridomas were cloned twice by limiting dilution. Ascitic fluid was produced by intraperitoneal injection of 5 x 106 hybridoma cells into pristane-treated BALB/c mice (21). Radioimmunoprecipitation of PrV. Immunoprecipitation of radiolabeled PrV extracts was performed essentially as previously described (10). Samples (100 pil) of `4C-labeled IND-F or BUK PrV were mixed with MAb ascitic fluid (1:2,000) and incubated for 18 h at 4°C. Immune complexes were precipitated with 50 pul of a 10% suspension of Staphylococcus aureus Cowan 1 (10). Immunoprecipitates were electrophoresed on a 12.5% sodium dodecyl sulfate-polyacrylamide gel (12). After electrophoresis, gels were fixed, fluorographed (EN3HANCE autoradiography enhancer; Dupont, NEN Research Products, Boston, Mass.), and exposed to film (X-Omat AR; Eastman Kodak Co., Rochester, N.Y.) at -70°C. Indirect fluorescent-antibody assay. PK-15 and ST cells grown in Lab-Tek chamber slides (Miles Scientific, Naperville, Ill.) were infected with IND-F or BUK PrV at a multiplicity of infection of 0.1. Infected cells were fixed with acetone at a time postinfection when the viral cytopathic effects were present in 20 to 40% of the cell monolayer. Samples (50 ,ul) of MAb ascitic fluid (1:500) were added to each chamber of the slide and incubated for 30 min at 37°C. Slides were washed three times in PBS, and then 50-pul samples of fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (IgG) (H+L) (Kirkegaard and Perry, Gaithersburg, Md.) were added. After incubation for 30 min at 37°C, slides were washed as described above and air dried. Cells were counterstained with 0.002% Evans blue (Sigma Chemical Co., St. Louis, Mo.). Purification of gI glycoprotein. ST cells were infected at a multiplicity of infection of 1.0 with PrV IND-F and incubated for 18 to 24 h at 37°C. Viral fluids were treated with 0.1% Nonidet P-40 detergent (Sigma) at 4°C with stirring for 18 h. Cell debris was removed by centrifugation at 5,000 x g for 30 min, and the supernatant was clarified by filtration through a 0.45-,um filter (Nalge, Rochester, N.Y.). The gI-containing fluids (100 ml) were passed through a 2-ml agarose affinity resin to which 20 mg of gI MAb had been immobilized according to the directions of the manufacturer (AFC Medium, New Brunswick Scientific, Edison, N.J.). The column was washed with 100 ml of 0.1% Nonidet P-40 in PBS to remove nonspecifically bound material. Bound gI was eluted with 100 mM carbonate buffer, pH 10.6. Pre- and posteluted fractions were monitored for purity by reactivity to the PrV MAbs in an ELISA system. Wells of a microdilution plate (Nunc) were coated with dilutions (1:100) of column fractions in 10 mM borate buffer for 18 h at 4°C. Wells were washed and postcoated with 2% bovine serum albumin in PBS followed by the addition of each PrV MAb. Bound antibody was detected by the addition of horseradish peroxidase-conjugated anti-mouse IgG (-y-chain specific) (Kirkegaard and Perry). A405s were determined 15 min after addition of ABTS (2,2'-azino-di[3-ethyl-benzthiazoline sulfonate]) substrate (Kirkegaard and Perry). Western (immuno-) blot analysis. Column fractions collected from the affinity purification of the gI glycoprotein were evaluated for purity by a modification of the procedure

PSEUDORABIES VIRUS gI ELISA

2209

of Burnette (4). Samples were boiled for 7 min in electrophoresis sample buffer containing 0.2 M dithiothreitol and electrophoresed in a 12.5% sodium dodecyl sulfate-polyacrylamide gel (12). Separated proteins were electroblotted onto nitrocellulose paper (Schleicher & Schuell, Inc., Keene, N.H.) and then blocked in 20 mM Tris buffer (pH 7.2) with 150 mM NaCI and 5% nonfat dry milk for 18 h at room temperature. The nitrocellulose paper was probed with the PrV MAb (1:1,000 dilution of ascitic fluid) or swine serum (1:50) in the blocking buffer as described above for 1 h at room temperature. The nitrocellulose paper was washed three times in 20 mM Tris buffer (pH 7.2) with 150 mM NaCl and 0.2% Tween 20. Bound antibody was detected by the addition of alkaline phosphatase-conjugated anti-mouse IgG (,y-chain specific) or anti-swine IgG (H+L) (Kirkegaard and Perry). The nitrocellulose paper was then washed as described above, and proteins were visualized with 5-bromo3-chloro-3-indolyl phosphate and Nitro Blue Tetrazolium substrate (Kirkegaard and Perry). gI ELISA. Wells of a microdilution plate were coated with 100 pul of an optimal concentration of affinity-purified gI glycoprotein diluted in 10 mM borate buffer (pH 9.0). After incubation at 4°C for 18 h, plates were washed with PBS and postcoated for 30 min at 37°C with 10% nonfat dry milk. Swine serum samples and positive and negative serum controls (100 pul) were added to the wells. Plates were incubated for 1 h at 37°C and washed three times with PBS-0.05% Tween 20. Anti-swine IgG (H+L) horseradish peroxidase (1:1,000) (Kirkegaard and Perry) was added to wells, and plates were incubated for 1 h at 37°C. Plates were washed with PBS-0.5% Tween 20 as described above, and ABTS substrate solution was added to the wells. After incubation at room temperature for 15 min, A405 was determined at in an ELISA reader (Molecular Devices, Palo Alto, Calif.). Development of color indicated the animal had been exposed to field virus. Absorbance values were adjusted to a sample/control (S/C) ratio as follows: S/C = average unknown sample absorbance value/average negative control absorbance value. Serum samples with S/C values of 3.0 or greater were considered positive, whereas S/C values less than 3.0 were considered negative. The sensitivity of the assay was defined as the ability to correctly identify field PrV-exposed swine. Specificity was defined as the ability to differentiate vaccinated from field virus-exposed animals. Standard ELISA. A nondifferential ELISA was used for identification of seropositive animals and for comparison to the gI ELISA and VN assay. The standard ELISA was performed essentially as described above for the gI ELISA, except that plates were coated with an optimal dilution of pelleted IND-F or BUK PrV harvested from PK-15-infected cells. Results were reported as the reciprocal of the greatest serum dilution that gave an A405 reading that was 30% of the positive control 15 min after addition of the ABTS substrate. Virus-neutralizing antibody assay. Titers of swine sera to PrV were determined in 96-well tissue culture plates (Costar, Cambridge, Mass.). Twofold serial dilutions of heat-inactivated (56°C, 30 min) swine serum (50 pul per well) were incubated with 100 to 300 50% tissue culture infective doses (TCID50) (50 pul per well) of IND-F or BUK PrV for 2 h at 37°C. Samples (100 pil) of PK-15 cells (105 cells per ml) were added to all wells, and the plates were incubated at 37°C for 72 h. Neutralization titers were expressed as reciprocals of the highest dilutions showing no viral cytopathic effect. Vaccination and challenge of animals. Female 8- to 10week-old pigs were vaccinated two times intramuscularly at 3-week intervals with the PrV gI-deleted vaccines, PR-

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MELLENCAMP ET AL. 91

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A B A

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FIG. 1. Autoradiograph of immunoprecipitated PrV glycoproteins. The PrV MAbs against gI, gII, and gVI, and a medium control were used to precipitate ['4C]glucosamine-labeled extracts from gI-containing IND-F (lanes A) or gI-deleted BUK (lanes B) PrV. Positions of molecular weight standards are indicated on the left.

Vac-KV (inactivated BUK-PrV; Norden Laboratories) or experimental gI-deleted PrV vaccines (Norden Laboratories, unpublished data). Vaccinated and nonvaccinated control animals were challenged intranasally 3 weeks after the last vaccination with 106 5 TCID50 of IND-F PrV per pig. Other swine sera. Serum samples from animals challenged with a low dose (102.7 PFU) of field isolate PrV were a gift of K. Platt and M. J. Ginley, Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames. Serum samples from animals that received multiple doses of PrV gI-deleted vaccine were obtained from a certified PrV-negative herd that was routinely vaccinated every 6 months. Monitoring of unvaccinated sentinel animals assured the PrV-negative status of the herd. Serum samples from two nonvaccinated, PrV-infected herds were obtained from the Illinois State Veterinary Diagnostic Laboratory, Galesburg. RESULTS Mab specificity. Initial screening of hybridoma supernatants by ELISA identified antibodies reactive to both gIcontaining (IND-F) and gI-deleted (BUK) PrV as well as antibodies which reacted only with gI-containing PrV. Subsequently, three MAbs were chosen for further characterization on the basis of ability to immunoprecipitate specific PrV proteins (Fig. 1). Each MAb precipitated polypeptides of 135 kilodaltons, a complex containing 120-, 74-, and 60-kilodalton proteins, and a 50-kilodalton protein, respectively. These molecular weights and radioimmunoprecipitation patterns correspond to the gI, gît, and gVI proteins, respectively, and are similar to those reported by other investigators (2, 7). The gîI and gVI MAbs showed no difference in radioimmunoprecipitation when used to precipitate extracts from 14C-labeled gt-containing or gI-deleted PrV. However, the gI MAb reacted only with gI-containing PrV by ELISA and immunoprecipitation. Specificities of the PrV MAbs were also determined by indirect fluorescent-antibody assays. PrV-infected PK-15 cells produced strong fluorescence when reacted with the gîI and gVI MAb, with little difference between cells infected with gI-containing or gI-deleted PrV. The gI MAb produced strong fluorescence only in gI-containing infected cells. No fluorescence was observed in PrV-infected cells incubated

PRE COLUMN

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FIG. 2. Western blot analysis of gI MAb affinity column fractions. Precolumn fluids and eluted fractions were electrophoresed on a 12.5% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto nitrocellulose paper. Transferred proteins were visualized by probing with the gI MAb serum from a pig exposed to gI-containing PrV (Ex-pig) and with serum from a pig vaccinated with gI-deleted PrV (Vx-pig). Positions of molecular weight standards are indicated on the left.

with PBS as a control or when the MAbs were reacted with uninfected PK-15 cells. Purification of gI by immunoaffinity chromatography. The gI glycoprotein was purified by affinity chromatography with the gI-specific MAb described above. Detergent-disrupted viral proteins were analyzed by ELISA and Western blot assays before and after passage over the gI affinity column. All PrV MAbs reacted with precolumn viral fluids by ELISA. Passage of the viral fluids over the column removed most of the gI glycoprotein from the effluent and did not appear to affect concentrations of the other PrV proteins. The material specifically adsorbed to the immunoaffinity resin was selectively eluted at pH 10.6. Analysis of this high-pH eluant by ELISA demonstrated high concentrations of gI protein. Only very minor reactivity was observed with the gîI MAb, and no reactivity was seen with the gVI MAb. In addition, no reactivity was seen with the PBS control, indicating that the eluted material did not contain any mouse IgG. The absence of antibody in the eluted material demonstrated that the immunoaffinity resin is stable and that leaching of antibody from the column does not occur. As an additional criterion of purity, precolumn material and eluted protein were immunoblotted and probed with gI MAb or swine serum (Fig. 2). Polyclonal swine serum was used in place of the gIt and gVI MAb, which do not react on Western blots. A single band corresponding to the gI protein was observed in both precolumn material and eluted protein when probed with the gI MAb. Precolumn material probed with serum from an animal exposed to a virulent strain of PrV (IND-F PrV) reacted to gI and numerous other PrV proteins. However, only a single band corresponding to gI was observed in material eluted from the column. Serum from a gI-deleted PrV-vaccinated animal reacted predominantly with the gIî and gIV proteins in precolumn material (as estimated by the molecular weights of the reactive proteins) and showed no reactivity to the eluted gI protein. After confirmation by ELISA and Western blot that the protein obtained by immunoaffinity chromatography was gI protein, it was used as the antigen in the gI ELISA. Specificity of the gI ELISA. The ability of the gI ELISA to distinguish vaccinated from field virus-infected animals was used to determine test specificity. In the first study, serum samples from vaccinated and nonvaccinated animals challenged with a high dose (106 5 TCID50) of standard PrV were

VOL. 27, 1989

PSEUDORABIES VIRUS

TABLE 1. Specificity of the gI ELISA after two vaccinations with gI-deleted PrV preparations Vaccine groups

No. of animals

1 2 3 4 5

4 5 5 5 4

gI ELISA result (mean [range])b

0.41 0.26 0.47 0.25 0.21

Standard ELISA titerb

titer'

27 7 194 2