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Nov 13, 1984 - Rapid Detection of Bovine Herpesvirus Type 1 Antigens in Nasal .... herpesvirus type 1, pseudorabies virus, and canine herpes- virus, prepared ...

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1985, p. 375-380

Vol. 21, No. 3

0095-1137/85/030375-06$02.00/0 Copyright © 1985, American Society for Microbiology

Rapid Detection of Bovine Herpesvirus Type 1 Antigens in Nasal Swab Specimens with an Antigen Capture Enzyme-Linked Immunosorbent Assay JAMES K.

COLLINS,'*

ANN C. BUTCHER,' YOSHIO A. TERAMOTO,2

AND

SCOTT WINSTON2

Diagnostic Laboratory, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523,1 and Syngene Products and Research, Fort Collins, Colorado 805222 Received 27 August 1984/Accepted 13 November 1984

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of bovine herpesvirus type 1 (BHV-1) antigens in nasal swab specimens collected from infected animals. Development of the ELISA involved screening and selection of BHV-1-specific monoclonal antibodies for their ability to capture BHV-1 antigens and for their stability and activity after conjugation to horseradish peroxidase. Forty combinations of capture-conjugate monoclonal antibody pairs were screened for detection of nanogram amounts of purified BHV-1 by using a double-antibody-sandwich ELISA in which antigen and conjugated antibody were simultaneously added to antibody-coated wells. Of the 40 monoclonal antibody pairs, 4 were analyzed further and 1 was selected for routine application to clinical specimens. Of 129 nasal swab specimens collected during the first 10 days after experimental infection with BHV-1, 66 were found to be positive by both virus isolation and ELISA and 34 were positive for infectious virus but negative by ELISA. One specimen was positive by ELISA but negative by virus isolation, and the remaining 28 specimens were negative by both tests. Quantitation of the virus-containing specimens showed that the ELISA had a lower detection limit of 103-5 median tissue culture infective doses. The ELISA was judged to be highly useful for diagnosis of BHV-1 infections, since ail of the nasal swab specimens that were collected from 12 animals during the first 5 days of the infection, when the clinical signs were the most apparent, were positive.

Bovine herpesvirus type 1 (BHV-1) is associated with a wide spectrum of diseases of various severity, including rhinotracheitis, conjunctivitis, vaginitis, fetal infection, and, rarely, encephalitis (9, 11). The virus is a widespread cause of morbidity and mortality in situations such as feedlot and breeding operations, in which cattle are confined (9). Upper respiratory tract infections often severely compromise the natural defense mechanisms of the host, leading to life-threatening secondary bacterial pneumonia (21, 22). Diagnosis of BHV-1 respiratory tract infections involves isolation of the virus from nasal secretions or from lung or tracheal tissues collected postmortem (9). When BHV-1 is contributing to an outbreak of infection in a herd, the time involved in recognizing the symptoms, collecting and transporting the specimens to a laboratory, and conducting virus isolation can amount to 5 to 15 days. The need to begin treatment and preventative control measures occurs early in the course of BHV-1 outbreaks, and yet diagnosis is often made beyond this critical time. The need for more rapid diagnosis of BHV-1 infection is therefore dictated by (i) the rapid induction of conditions contributing to the development of pneumonia; (ii) the need to take appropriate preventative actions during an outbreak; and (iii) the widespread occurrence of BHV-1 during operations for which cattle are confined. A number of techniques have been developed for the rapid diagnosis of viral infections. These include fluorescent antibody or immunoperoxidase staining of cells from mucosal scrapings (5, 6, 19) or of tissue culture cells that have been inoculated for viral isolation (13, 15) and enzyme-linked immunosorbent assay (ELISA) with material collected from lesions or body fluids (2, 6, 8, 14, 20, 23). The ELISA has

*

been particularly useful in rapid diagnosis, since little time and equipment are required, results are obtained in a few hours, and in some cases the sensitivity of the test approaches that of virus isolation (10, 20). For specimens collected early in the course of the disease, when virus shedding is highest, ELISA procedures are highly useful. This report describes the development of an ELISA with monoclonal antibodies (MCAs) for detecting BHV-1 antigens in specimens collected from animals with upper respiratory tract infection. MATERIALS AND METHODS MCAs. MCAs that specifically bind to BHV-1 were developed and characterized as previously described (4). MCAs were precipitated from mouse ascites fluid and conjugated to horseradish peroxidase (HRPO) (4). Antigen-capture ELISA procedure. Ninety-six-well Immulon II microtiter plates (Dynatech Laboratories, Inc., AIexandria, Va.) were sensitized with various MCAs, as specified, at 0.5 ,ug/0.05 ml per well in coating buffer, 0.01 M sodium borate (pH 9.0). After adsorption at 37°C for 16 h, the plates were emptied and then blocked with 0.1 ml of 2% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) per well for 1 h at 37°C. The blocking solution was discarded, and 0.025 ml of purified BHV-1 or test sample was added. A multichannel pipettor was used to make serial dilutions in TEN buffer (0.05 M Tris, 0.001 M EDTA, 0.15 M NaCI [pH 7.2 to 7.4]) containing either 0.2% Triton X-100 (Sigma) (Triton-TEN) or 0.1% bovine serum albumin (BSATEN), as specified. Immediately after addition and dilution of the samples, 0.025 ml of HRPO-MCA diluted to the optimum concentration as described below was added. After a specified incubation time, the unbound sample-conjugate mixture was discarded and the plates were washed three

Corresponding author. 375

376

COLLINS ET AL.

times with TEN buffer containing 0.05% Tween 20 (Sigma). The bound HRPO-MCA was then detected by the addition of substrate, 0.05 ml of a 0.4-mg/ml solution of o-phenylenediamine, and 0.01% H202 in 0.05 M citrate-phosphate buffer (pH 5.0). After 15 min of incubation at 20°C, the reaction was stopped with 0.05 ml of 2.5 N H2SO4. The absorbance was measured at 490 nm with a MicroELISA Autoreader MR580 (Dynatech). Determination of capture-antibody concentration. For each MCA tested, microtiter plates were sensitized with 4, 2, 1, 0.5, or 0.25 p.g of antibody per well in borate buffer. Purified BHV-1 was added and diluted in Triton-TEN or BSA-TEN buffer, and an equal volume of HRPO-MCA was added. After a 1-h incubation at 37°C, the plates were washed, substrate was added, and absorbance was measured as described above. Determination of peroxidase-conjugated antibody concentration. The dilution of peroxidase-conjugated MCA used in the ELISA was determined by using a direct binding assay with BHV-1-antigen-coated wells as described previously (4). Briefly, HRPO-MCA was serially diluted and then added to wells coated with 400 ng of BHV-1. After a 1-h incubation, the wells were washed and substrate was added as described above. The titer was defined as the dilution of conjugate at which the absorbance decreased to approximately 50% of maximum. All HRPO-MCAs that were investigated for use in the antigen-capture ELISA had titers of 1:1,600 to 1:12,800. For optimization of the F2 conjugate in the G2-F2 antigen-capture assay (see below), the dilution of conjugated F2 antibody was determined by titrating the conjugate on wells coated with 0.5 ,ug of G2 antibody through addition of the F2 conjugate dilution and 100 ng of BHV-1. Determination of optimal coating buffer for binding capture antibody. Four MCAs were tested by dilution in four coating solutions and by sensitizing the plates with 4, 2, 1, 0.5, or 0.25 ,ug of antibody per well. The coating solutions were TEN buffer, 0.01 M sodium borate, 0.05 M carbonate-bicarbonate buffer (pH 9.6), and 0.02 M phosphate-buffered saline (pH 7.2). Adsorption was carried out at 37°C for 16 h, and the plates were blocked as described above. Purified BHV-1 was added and diluted, and the ELISA was performed as described above. Determinations of optimal time and temperature for the ELISA. Plates were sensitized with 0.5 ptg of G2 MCA per well and diluted in sodium borate. Purified BHV-1 was added and serially diluted in Triton-TEN, and an equal volume of a 1:1,000 dilution of peroxidase-conjugated F2 MCA was added. Plates were incubated for 0.5, 1, 2, 3, or 6 h at 4, 20, or 37°C and washed, and the ELISA was continued as described above. Specificity of the ELISA. With G2 MCA as the capture antibody and F2 MCA as conjugate, the following unrelated antigens were assayed for reactivity: (i) infected tissue culture cell extracts of BHV-2 (bovine herpes mammalitis virus), BHV-4 (Movar 31/61 and DN 599 strains), equine herpesvirus type 1, pseudorabies virus, and canine herpesvirus, prepared as described previously (J. K. Collins, G. A. Bulla, C. A. Riegel, and A. C. Butcher, Vet. Microbiol., in press); (ii) supernatant tissue culture fluid from cells infected with parainfluenza virus type 3, bovine viral diarrhea virus, and bovine respiratory syncytial virus; (iii) culture fluids from Haemophilus somnus and Pasteurella haemolytica cultures. Additional BHV-1 strains recovered from infectious outbreaks of respiratory disease (15 strains), abortions (7 strains), and conjunctivitis (3 strains) were tested in the

J. CLIN. MICROBIOL.

G2-F2 ELISA by using extracts of infected tissue culture cells. Competitive inhibition ELISA. Competitive inhibition with MCA was carried out as described previously (4). Experimental BHV-1 infections. Twelve BHV-1-seronegative, 6-month-old calves were challenged with a field isolate of BHV-1, strain DL-10902-82, as described previously (4). Briefly, on the day of transport to an isolation facility, the calves were inoculated in each nostril with 2 ml of 2 x 108 median tissue culture infective doses (TCID50) per ml of the virus; a model no. 152 compressed airpowered DeVilbiss atomizer (DeVilbiss Co., Somerset, Pa.) was used for the inoculations. To simulate feedlot conditions and the stress of transport, the ambient room temperature was kept at 90°F and the animals were deprived of water for the first 24 h. Clinical signs including rate of respiration, rectal temperature, character and severity of cough, amount and consistency of nasal and ocular exudates, and depression were monitored daily. One person evaluated the clinical signs throughout the course of the experiment on a subjective scale of 0 to 4+, with 4+ indicating the most severe sign. Monitoring of viral shedding was accomplished by inserting sterile cotton-tipped swabs 5 to 7 cm into the nostrils and scraping the mucosa. The swabs were immediately placed into 2 ml of cold viral transport medium (VTM) (Minimum essential medium containing Earle base: 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES] buffer, 125 mg of amphotericin B per liter, 100 mg of gentamicin per liter, 105 U of penicillin per liter, 2% fetal calf serum) and kept cold until frozen at -70°C. Virus isolation on nasal swabs. Quantitative virus titrations were performed by end-point dilution of VTM with eight replicate cultures on each of eight serial 10-fold dilutions. Ninety-six-well microtiter plate cultures of primary bovine embryonic lung cells were inoculated with 0.1 ml of the VTM dilution, and the wells were read for cytopathic effect after 3 days. The titer was calculated by the Karber method (3) and expressed as TCID50 per 0.025 ml of VTM. Virus was identified on the basis of characteristic cytopathic effect and reactivity of the VTM in the ELISA; if there was doubt about the virus identity because of lack of reactivity of VTM in the ELISA, the ELISA was performed on tissue culture medium from wells in which the isolated virus grew. RESULTS Selection of antibody for ELISA. Selection of MCAs for a simultaneous double-antibody-sandwich ELISA for BHV-1 involved several criteria. These included (i) titer, (ii) retention of specificity and stability after enzyme conjugation, (iii) recognition of distinct epitopes on the virus, and (iv) efficiency of antigen capture. A panel of 14 BHV-1-specific MCAs representing four antibody subclasses and having indirect ELISA titers ranging from 3.3 x 103 to 4.6 x 106 (4) were evaluated. All 14 MCAs were conjugated to HRPO and tested for direct ELISA titer with BHV-1-coated wells. Several had titers less than 2 x 102 or lost specificity for BHV-1, or both. Four MCAs (D9, F2, Gi, and H5) were selected for further evaluation because their specificity was retained and their sensitivity was greatest in detecting limiting levels of BHV-1 antigen by ELISA. Competitive binding analysis was performed with BHV-1coated wells in which unconjugated antibodies were tested for their ability to compete with the four selected HRPOMCAs (Table 1). B4, D9, H2, and Hl1 antibodies competed with the D9-conjugated antibody. Only the isologous MCA

VOL. 21, 1985

ELISA FOR DETECTION OF BHV 1 ANTIGENS

377

TABLE 1. Competitive inhibition of BHV-1 HRPO-MCA by unconjugated MCA HRPO-MCA B4

D9 +

ES pb

Hll

H2 +

F2

Competition

MCA

Gi

Cli

G2

H5

Bi

C6

E6

Ell

-

-

-

-

-

-

-

D9

+a

F2

-

-

-

-

-

+

-

Gi H5 Polypeptide groups

_

_

_

_

_

_

+

-

-

-

-

-

-

-

-

-

+

+

+

-

-

1

1

1

1

1

2

2

2

2

3

3

3

4

4

+

-a

+, MCA competed with HRPO-MCA; -, no competition. b P. MCA partially competed (50%) for the same epitope as HRPO-MCA. C Radioimmunoprecipitation group based on previous data (4). 1, gplO2, 96, 69, 55; 2, gp82; 3, a

competed with Gi and F2 conjugates. Bi, C6, and H5 MCA competed with the H5-conjugated antibody. Radioimmunoprecipitation data (4) have shown that MCA D9 has specificity for a group of glycoproteins of 102, 96, 69, and 55 kilodaltons, H5 has specificity for a 91-kilodalton protein, and Gi and F2 react specifically with an 82-kilodalton glycoprotein, but with different epitopes. MCA E6 and E11 react with a 115-kilodalton protein. Thus all of the viral components that were defined by the MCA panel were investigated as targets for the antigen-capture ELISA (Table

1). To evaluate the ability of the MCA to capture BHV-1 antigens, the many possible combinations of the 4 conjugated and 14 unconjugated MCAs were narrowed to representative combinations between each RIP or epitope group. For example, the capture ability of G2, a gp82 MCA, was tested with conjugated D9 (a gplO2, 96, 69, 55 group MCA), with F2 (a gp82 MCA conjugate) and with H5 (a p9l MCA conjugate). Over 40 capture-conjugate pairs were tested, and the sensitivity of each pair was measured with two antigendilution buffers (Table 2). F2 MCA was the most versatile conjugate, detecting BHV-1 with six different capture MCAs. D9 MCA worked well as a conjugate with only three different capture MCAs. Gi and H5 conjugates did not work with any of the tested MCAs (data not shown). Four capture-conjugate pairs (E5-D9, E5-F2, E11-F2, and G2-F2) were selected for additional testing and optimization on the basis of sensitivity, epitope combination, and range of activity. Optimization of antigen-capture ELISA. Serial dilutions of the four capture antibodies in four coating solutions were adsorbed to wells and tested with their appropriate conjugate pair by using two conjugate- and antigen-dilution buffers, Triton-TEN and BSA-TEN. Of the four coating solutions, sodium borate and TEN gave comparable results since absorbance values were similar and background was low, whereas carbonate-bicarbonate and PBS buffers gave unacceptable background in the ELISA (data not shown). Of the four pairs, G2-F2 worked best because it yielded the highest ELISA signal at the lowest capture antibody level (Fig. 1). The activity in the G2-F2 ELISA was best with Triton-TEN

p91; 4, pli5.

dilution buffer, whereas the other three pairs gave better results with BSA-TEN buffer. In addition, the other pairs gave much lower signals and required more antibody to capture BHV-1 antigen. All subsequent studies were carried out with G2 MCA as the capture antibody adsorbed to the microtiter wells in borate buffer, followed by the simultaneous addition of purified BHV-1 or test samples and peroxidase-conjugated F2 MCA diluted in Triton-TEN buffer. This pair gave the greatest sensitivity over the widest range of capture MCA dilutions (Fig. ld). To optimize time and temperature for the G2-F2 ELISA, three temperatures (4, 20, and 37°C) and five incubation periods (0.5, 1, 2, 4, and 6 h) were compared (Fig. 2). The sensitivity of the ELISA at 37°C was slightly lower than those at 4 and 20°C, which were not significantly different from each other. However, the time of incubation affected the sensitivity at all three temperatures. At 4 and 20°C there was a threefold increase in sensitivity between 1- and 2-h incubations, and at 37°C there was a threefold increase between 2- and 4-h incubations. Assays of 2 h at 20°C were chosen to achieve a high sensitivity and optimum convenience. Under these conditions, 1.5 ng of purified BHV-1 could be detected with an absorbance of 0.15 at 490 nm in the ELISA. Background values were 0.04 0.05 absorbance units. Specificity of the ELISA. The specificity of the G2-F2 ELISA was tested with bovine respiratory viruses including bovine viral diarrhea virus, parainfluenza virus type 3, and bovine respiratory syncytial virus, and with other herpesviruses including BHV-2, equine herpesvirus type 1, pseudorabies virus, canine herpesvirus, and two strains of BHV-4. Culture fluids from two bacterial species normally isolated from bovine respiratory tracts, H. somnus and P. haemolytica, were also tested in the ELISA. All gave absorbance readings of s0.09, demonstrating that these agents were not detected in the G2-F2 assay. The assay was also tested for its ability to detect field isolates of BHV-1 with samples acquired by the Colorado State University Diagnostic Virology Laboratory. Seven BHV-1 strains isolated from distinct respiratory disease outbreaks, three abortogenic strains isolated from fetuses, ±

TABLE 2. Amount of BHV-1 detected by using different capture-conjugate pairs in simultaneous double-antibody ELISA Amt (ng) of BHV-1 with capture MCA: MCA conjugate F2 Gi Cil G2 Bi E6 D9 B4 ES HMl 0 0 ND 0 0 0 D9 Triton-TEN NDa 0 0 0 30 30 0 ND 0 0 ND 0 BSA-TEN 0 s30 ND 12 0 0 0 0 F2 Triton-TEN 0 0 0 0 30

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