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Rapid detection of equine herpesvirus type-1 antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay
Journal of Virological Methods, 0
39 (1992) 299-3 10
1992 Elsevier Science Publishers
299
B.V. / All rights reserved / 0166-0934/92/%05.00
VIRMET 01388
Rapid detection of equine herpesvirus type-l antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay R. Sinclair and J.A. Mumford Department
of Infectious Diseases, Animal Health Trust, Newmarket,
Suffolk
(UK)
(Accepted 24 April 1992)
Summary An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-l (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swabs from ponies which were experimentally infected with EHV-1. Of 72 nasal swabs collected, 32 were found to be positive by both virus isolation (VI) and ELISA, a further 15 samples were positive by VI alone, but none of the samples were positive by ELISA and negative by VI. This yielded an overall assay sensitivity of 68% and specificity of 100%. The assay proved useful for diagnosis since virus antigen was detected during the first four days post-infection which corresponded to the acute phase of disease when some clinical symptoms were apparent. In addition, the assay could be completed within one day when antibody coated plates were avaiIable. Equid herpesvirus type-l; Rapid diagnosis; ELISA; Nasal swab
Correspondence to: R. Sinclair, Department of Infectious Diseases, Animal Health Trust, P.O. Box 5, Newmarket, Suffolk, CB8 7DW, UK.
300
Introduction Equid herpes virus type-l is associated with a range of clinically different disease syndromes including respiratory disease, abortion, neurological disease and perinatal foal disease (Allen and Bryans, 1986). The virus is distributed worldwide and serological surveys have demonstrated outbreaks of disease in the majority of countries with appreciable horse populations (Matumoto et al., 1965). The disease is acquired by infection of the respiratory tract via aerosols, contaminated food, water, bedding or other formites. Current methods for the identification of EHV-1 rely upon growth of the virus using conventional tissue culture techniques from samples which may include nasal swabs, the leukocyte fraction of blood, or from tissues obtained during post-mortem examination, such as lung, liver, adrenal and thymus. This is followed by immunological screening using immunofluorescence or enzyme-immunofiltration tests (Mumford and Rossdale, 1980; Yeargan et al., 1985). However, diagnosis is complicated by the existence of three antigenically related viruses, equid herpesvirus type-2 (EHV-2), equid herpesvirus type-3 (EHV-3) and equid herpesvirus type-4 (EHV4). These viruses feature significant differences in their pathogenicity when compared to that of EHV-1. EHV-4 is generally associated with outbreaks of respiratory disease and on rare occasions, abortion, but is not usually associated with neurological disease (Allen and Bryans, 1985). EHV-3 causes an infection of the genital tract known as coital exanthema which is characterised by pustular lesions on the external genitalia and sometimes on the lips, nares, nasal mucosa and conjunctiva (Krosgrud and Onstad, 1971). EHV-2 is thought to be relatively non-pathogenic since it can be isolated from healthy animals as well as those with mild respiratory distress, although there is at least one report of foal mortality occurring through EHV-2 infection (Palli et al., 1978). The development of a rapid, sensitive and typespecific diagnostic test for EHV-1 would aid management of disease outbreaks and provide useful epidemiological data. At the present time, the availability of EHV-1 specific monoclonal antibodies has enabled improvements in diagnostic procedures, however, current methods for typing virus still rely upon prior isolation of virus in tissue culture, which may take up to 14 days. The objective of this study was to develop a rapid type-specific ELISA to detect EHV-1 antigen directly in nasal swabs. This type of assay has proven valuable in rapid diagnosis of other viral respiratory infections since quantitative results can be obtained within a few hours, and in some cases, the sensitivity of the test approaches that of virus isolation. For nasal swab specimens collected during the acute phase of the disease, when virus shedding is at its highest, an ELISA should prove a valuable aid to diagnosis.
301
Materials and Methods Viruses
EHV-1 and EHV4 strains were propagated in equine embryonic lung (EEL) cells grown in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum. All but one of the strains were low passage (< 5) field isolates obtained during naturally occurring British outbreaks. The exception was the prototype EHV-1 strain Army 183 which was isolated from a North American horse in 1941 (Jones et al., 1948) and was used for monoclonal and polyclonal antibody production. Polyclonal antiserum production
Antiserum to EHV-1 was prepared by immunisation of rabbits with 200 pg of purified whole virus antigen emulsified in Freund’s complete adjuvant. Three boost injections each of 200 lug prepared in Freund’s incomplete adjuvant were administered at 1 mth intervals and serum collected 10 days after the final boost. Monoclonal
antibody production
BALB/c mice were immunized by intraperitoneal injection with 100 pg of purified whole virus antigen, then boosted 4 wk later by intravenous injection with a further 25 pg on 4 consecutive days. Fusions were performed on the following day according to the protocol described by Campbell (1984). Hybridoma supernatants were screened by ELISA and an immunoflourescence test (Sinclair, 1990) and those producing antibody directed against EHV-1 cloned by limiting dilution and stored in liquid nitrogen. Ascitic fluid was prepared in Balb/c mice and purified by precipitation with ammonium sulphate (Campbell, 1984). Antibody isotypes were determined by ELISA using commercially available reagents. Immunofuorescence
test (IFT)
EEL cell monolayers were grown on eight well chamber slides and infected with EHV-1 or EHV-4 strains at a m.o.i. of 0.025. Monolayers were fixed approximately 24 h post-infection in PBS containing 1.34% w/v formaldehyde (20 min/RT) followed by PBS containing 1% v/v Nonidet P40 (10 min/RT). Monolayers were blocked with 4% w/v BSA in PBS for 30 mins at RT to reduce non-specific reactions, then incubated successively with (i) 1500 dilution of purified monoclonal antibody for 1 h at RT, (ii) 1:200 dilution of rabbit antimouse immunoglobulin for 90 min at 37°C and (iii) 1:40 dilution of fluorescein isothiocyanate conjugated goat anti-rabbit immunoglobulin for 30 min at 37°C. PBS containing 4% w/v BSA was used as diluent in each incubation step
302
and monolayers were washed 5 times in PBS between each incubation step. Monolayers were counterstained with 0.005% v/v Evans blue, and examined for fluorescence using a Lietz Orthoplan microscope. Antigen capture enzyme-linked
immunosorbent
assay
Monoclonal antibodies diluted in carbonate buffer (0.05 M NaHC03/ NazCOs, pH 9.6) were passively adsorbed to 96-well microtitre plates for 16 h at 37°C. Infected tissue culture supernatants, or test samples, were added to the wells for 6 h at 4°C. This was followed by (i) a 1:200 dilution of rabbit antiEHV-1 serum for 2 h at 37°C and then (ii) a 1:200 dilution of goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase for 2 h at 37°C. Bound viral antigen was detected by incubation with substrate solution (1 mg/ml pnitrophenylphosphate, 0.5 mM MgCl- 6 H20, 9.7% diethanolamine, pH 9.8) for 30 min at RT. The plates were washed 5 times with PBS containing 0.05% Tween 20 (PBST) between each incubation step and dilution buffer for both antisera was 2% BSA in PBST. The reaction was stopped by addition of 3 M sodium hydroxide and the absorbance read at 405 nm. Determination
of optimal MAb dilution
Assays were performed as recorded in the previous section, except that (i) microtitre plates were coated with serial 2-fold dilutions of capture antibody and (ii) test antigens were tissue culture supernatants containing either EHV-1 or EHV4 (lo4 TCIDso per well). Determination
of optimal dilution of polyclonal
antisera
The dilutions of peroxidase-conjugated antibody and anti-EHV-1 rabbit sera were determined using a direct binding checkerboard ELISA. EHV-1 antigen diluted to a concentration of 5 pg/ml in carbonate buffer (pH 9.6) and passively adsorbed to 96-well microtitre plates for 16 h at 4°C. The plates were then washed 5 times with PBST and serial 2-fold dilutions of anti-EHV-1 rabbit serum added for 2 h at 37°C. Plates were again washed 5 times with PBST, then serial 2-fold dilutions of peroxidase-conjugated anti-rabbit immunoglobulin added for 2 h at 37°C. The plates were then washed, substrate was added and absorbance was measured as described above. The optimum titre of each antibody was defined as the highest dilution of antibody which gave the maximum absorbance value. Experimental
infection of ponies with EHV-1
6 seronegative 2 yr-old Welsh mountain ponies were challenged with the low passage, British EHV-1 field isolate 592/85. Ponies were inoculated with undiluted tissue culture medium containing 6.6 loglo TCIDSO/ml using a
303
Brovon Midget Inhaler. 25 compressions were applied to each nostril and 7 compressions to each eye. Additionally, a sterile cotton nasal swab, immersed briefly in tissue culture medium, was inserted 10-15 cm into each nostril. After infection, virus shedding from the nasopharynx was monitored daily by insertion of swabs into the nostril followed by abrasion of the mucosae. The swabs were withdrawn and placed immediately into 2 ml of ice-cold viral transport medium, returned to the laboratory, centrifuged to remove cell debris and stored at -70°C. Titration of virus in nasal swabs Quantitative virus titrations were performed by microtitre assay. Briefly, semi-log,, dilutions of nasal swabs were added to 96-well plates in quadruplicate. A suspension of EEL cells in EMEM supplemented with 10% fetal calf serum were added and the plates incubated for 6 days at 37°C in a humidified incubator containing 5% CO;?. Wells were scored for cytopathic effect after 6 days and end-point dilutions calculated by the Karber (1931) method.
Results Selection of monoclonal antibody for ELBA Selection of monoclonal antibody for the antigen capture ELISA was based on several criteria. This included ability to differentiate between EHV-1 and EHV4, efficiency of antigen capture and recognition of a conserved epitope among a panel of field isolates. Eight MAbs derived from mice immunized with EHV-1 and having ELISA titres ranging from 5 x lo5 to 3 x lo4 were evaluated. Optimisation of the assay was performed in three phases. Firstly, the dilution of phosphatase-conjugated goat anti-rabbit immunoglobulin and rabbit anti-EHV-1 immunoglobulin were determined using a direct binding checkerboard ELISA. Microplates were coated with 500 ng/well of purified EHV-1 antigen and each antiserum titrated in two-fold serial dilutions. The highest dilution of each antibody which maintained maximum absorbance values was determined to be 1:200 for both antisera. In the second phase, the abilities of 8 monoclonal antibodies to selectively bind EHV-1 antigen (but not EHV-4) were evaluated. After coating the microtitre lates with serial 2-fold dilutions of each MAb, their abilities to capture 10B TCIDsO/well of EHV-1 or EHV4 vii-ions were compared (Fig. 1). Of the 8 MAbs tested, all except 8Ell (Fig. If) exhibited some potential for detection of EHV-1 antigens. However, MAbs 3E5 and 6Fll also cross-reacted with EHV4 antigens. This cross-reaction was confirmed by immunoflourescence tests using a panel of epidemiologically unrelated field isolates. Both MAbs exhibited positive fluorescence in equine cells infected with all EHV-1
,,4msLvL-.
woean, (a)
I
(h)
305
Abeorbance
(406nm)
0e71
7.0
6.0
6.0 TCID60/ml
4.0
3TO (-Log
2TO
1.0
10)
Fig. 2. Sensitivity of the antigen capture ELISA for detection of EHV-1 antigen in nasal swabs. After optimisation of assay conditions, four different monoclonal antibodies (MAb 4Cl1, 0; MAb 4Hl1, 0; MAb 8F8, A; MAb 8H8, 0) were compared for their ability to capture antigen. The values shown represent the mean absorbance for logic dilutions of infectious virus minus the absorbance of the ELISA zero determined on uninfected tissue culture supernatants.
and EHV4 isolates tested. These MAbs were therefore eliminated from further experiments. MAb 2E6 did not cross-react with the EHV4 isolate (MD) used for the capture ELISA but did cross-react with 8/12 EHV-4 field isolates in the immunofluorescence test and was also eliminated from further work. The four remaining MAbs were all EHV-1 specific in both the capture ELISA and immunofluorescence test. The optimal dilution of each MAb for the capture of EHV-1 virions was selected as the dilution which gave a maximum signal/noise ratio (i.e., ODds2 nm for EHV-1 divided by OD492 nm for EHV4). These were determined to be 1:1600 for MAbs 4Cll and 8H8, 1:400 for MAb 4H 11 and 1:lOO for MAb 8F8. For the third phase of assay development, the 4 remaining antibodies were compared for their efficiency of antigen capture using optimal antibody dilutions. The lower limit of virus detection for each MAb is shown in Fig. 2. c Fig. 1. Optimisation of antigen capture ELBA for type-specific detection of EHV-1 antigen. Serial two-fold dilutions of each MAb were passively adsorbed to microtitre plates and tested for their ability to capture 10’ TCIDso of EHV-1 (0) or EHV-4 (0) diluted in culture medium. (a) MAb 2E6; (b) MAb 3E5; (c) MAb 4Cll; (d) MAb 4Hll; (e) MAb 6Fll; (f) MAb 8Ell; (g) MAb 8F8; (h) MAb 8H8.
306
Abrorbanoe
(406nm)
TCIDCO/ml
0.0
7
0.8
6
0.7 6 0.6 0.6
4
0.6
4
0.4
3
0.4
3
0.3
0.3 2
0.2
2 0.2 1
0.1 0
0 0
1
2
3
Daye post-InfeCtIOn
Abeorbance
TCID60/ml
(406nm)
4
5
6
7
8
0 10 11 12
Daye post-infection
Absorbance 0.s ,
0.0
(406nm)
TCID60/ml
, _.,7
(C)l7 0.6 0.7 6 0.6 0.6 0.4 0.3
0.3
0.2
0.2
0.1
0.1
2
1
0
0
0 0
0123466789
1
2
4
6
6
7
8
9 10 1112
Days post-intectlon
Days port-infection
o.. jbeorbance
3
Abeorbance
WO6nm)
TCID6Ohnl
(406nm)
7
0.9
0.6 -
6
0.6
6
0.7
0.7 -
6
6 0.6
0.6 0.6 -
4
0.4
3
0.6
4
0.4
3
0.3
0.3
2
2 0.2
0.2 1
0.1
0
0 0
1
2
3
4
6
6
7
8
Daye poet-infection
# 10 11 12
1
0.1
0
0 0
1
2
3
4
6
6
7
8
Daye post-InfectIon
9 10 1112
The ELISA zero was determined from the absorbances from at least 3 replicate negative swabs. The predicted lower limit for a positive was then determined using the 95% prediction bound formulae where the ELISA zero equals the mean absorbance of the negative swabs + (SD x t9svo x (n- 1)/n), where SD is the standard deviation (n - 1) of n replicates of negative control absorbances and the t value at 95% probability for n - 1 degrees of freedom (Nerurkar et al., 1984a). Using this method of evaluation MAb 4C11,4Hll, 8F8 and 8H8 had lower limits of sensitivity of 4.0, 4.0, 5.0 and 3.0 logi0 of infectious virus respectively. Since the absorbance levels of 8H8 were highest over the complete range of virus dilutions, this antibody was selected for all further work. Experimental
EHV-I
infections
The antigen capture assay was compared with virus isolation as a method of monitoring the amount of virus shedding from the nasopharynx of six experimentally EHV-1 infected ponies (Fig. 3). ELISA absorbance values peaked on days l-2 post-infection and then declined to zero between 5-8 days post-infection. This pattern was similar to that of quantitative virus isolation except that the period of peak virus recovery and the duration of virus recovery were both slightly longer. Titers of infectious virus eeaked at days l-4 postinfection and declined to zero between days 8-12 post-infection. Both virus isolation and ELISA detected virus in the nasopharynx of all 6 ponies during the first 4 days post-infection. There was a general trend for ELISA absorbance to reflect infectious virus titre, particularly in samples containing at least 3.0 logi0 TCIDSO/ml (Fig. 4). This was confirmed by linear regression analysis performed on data sets for 34 nasal swabs, where the correlation coefficient r was computed to be 0.864. The ELISA detected virus in 99% of samples containing > 3.0 logi0 of infectious virus/ml, which was its approximate lower limit of sensitivity. The exception was a single sample containing 4.1 loglo TCIDSO/ml which was negative by ELISA. From a total of 72 nasopharyngeal swabs, 32 were positive by both VI and ELISA, a further 15 samples were positive by VI alone, but their were no samples which were positive by ELISA and negative by VI. This yielded an overall sensitivity of 68% and specificity of 100% for the antigen capture ELISA. Virus specificity of the antigen capture ELBA
The specificity of the antigen capture ELISA was tested on other equine viral pathogens including equine herpesvirus type-2, equine herpesvirus type-3, equine influenza virus and equine arteritis virus. Assays were negative for three different viral strains using positive nasal swabs or tissue culture medium. Fig. 3. Detection of antigen in nasal swabs from 6 ponies (a-f) with experimental EHV-1 infections. The antigen capture ELBA (0) was compared with virus isolation (0) from days O-12 post-infection. The ELISA absorbance values are shown minus the background absorbance of negative control swabs.
308
Absorbance 1.21 1.1
0
(495
nm)
q
t
’ 0
I
I
1
2
3
TCID50/ml Fig. 4. Comparison
of the ELISA
I
I
I
I
4
5
6
7
(-Log
10)
absorbance with the virus titre. Each dot represents assayed in both tests.
one nasal
swab
Discussion An antigen capture ELBA was developed for the detection of EHV-1 antigens in nasal swab specimens. The test format was that of a solid phase amplified sandwich assay in which a MAb was used to capture virus antigen. It was interesting to observe that seven out of eight MAbs each possessed the ability to capture virus antigen to the solid phase indicating that the majority of antibodies retained a degree of antigen binding capacity when passively adsorbed to plastic plates (Fig. 1). The failure of one antibody to bind antigen may have resulted from denaturation of the antibody during the adsorption process, which has been previously documented for certain MAbs (Kemeny and Challacombe, 1989; Kemeny, 1991). Alternatively, the failure to bind antigen could be explained by low antibody affinity or inappropriate antigen presentation in the virus. Of the four MAbs which were specific for EHV-1, 8H8 was selected as the best capture antibody based on higher absorbance values over the range of virus dilutions tested and the lowest limit of virus detection in infected tissue culture supernatants of approximately 3.0 log10 (Fig. 2). This was in close agreement with results from nasal swab specimens taken from experimentally infected ponies where the ELISA detected 99% of
309
swabs containing > 3.0 loglo of infectious virus. Other MAbs of interest were 3E5 and 6F11, which successfully captured EHV-1 antigen but also crossreacted with EHV4 antigen indicating that they both react with common epitopes (Fig. 1). Antigenic variation was only noted for a single MAb, designated 8El1, which reacted with a epitope common to EHV-1 and EHV4, but which demonstrated antigenic variability only among EHV-4 strains. In experimentally infected ponies, antigen was detected in all 6 animals from days l-4 post-infection, corresponding to the time of peak virus shedding, and in some ponies up to 7 days post-infection. The overall assay sensitivity was 68% when compared with virus isolation, which was similar to the sensitivity found with antigen capture ELISA’s for other viruses, such as herpes simplex virus type-l (Adler-Storthz et al., 1983; Morgan and Smith, 1984; Neurkar et al., 1984a) and bovine herpesvirus type-l (Collins et al., 1985). The antigen capture ELISA possessed several advantages over the conventional method of virus isolation in tissue culture. Firstly, the assay was rapid, requiring only one day to complete when antibody-coated plates were available, whereas virus isolation can take up to 14 days. This should aid management of disease outbreaks on studs or training stables affected with EHV-1, since management decisions can now be made during the early stages of infection. In addition, since absorbance values reflect the amount of infectious virus in the nasopharynx, identification and isolation of highly infectious animals may also reduce the chance of virus spread. Secondly, the assay does not necessarily require the presence of infectious virus. This may be important if infectivity is lost during transport of the virus to the laboratory, or, if the virus is exposed to neutralizing antibody produced locally in the nasopharynx. The main disadvantage of the ELISA is its lower sensitivity which would result in the production of some false negatives. However, in a field situation where several animals may be infected, group sampling of in-contact animals and of all animals with clinical signs may increase the chance of obtaining a positive. If clinical signs are recognised during the first four days of infection, this would further enhance the chance of a positive diagnosis. The most likely use of this assay will probably be in conjunction with virus isolation. The sensitivity of the assay may be increased, for example, by the use of biotinstreptavidin labelled antibodies and/or enzymes (Bayer and Wilchek, 1980; Nerurkar et al., 1984b) and refinement of the assay may lead to the development of a test which could be used in. the field. Acknowledgements
The authors are grateful to Miss B. Moult and Mr. D. Jessett for excellent technical assistance and to the Horserace Betting Levy Board for supporting this study.
310
References Adler-Storthz, K., Kendall, C., Kennedy, R.C., Henkel, R.D. and Dressman, G.R. (1983) Biotinavidin amplified enzyme immunoassay for detection of herpes simplex virus antigen. Laboratory Techniques in Biochemistry and Molecular Biology, Vol 13, Elsevier, Amsterdam. Allen, G.P. and Bryans, J.T. (1986). Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-l infections. In: Progress in Veterinary in Veterinary Microbiology and Immunology, vol 1, pp. 78-144. Edited by S. Pandy. Basel. Bayer, E.A. and Wilckek, M. (1980) The use of avidin-biotin complex as a tool in molecular biology. Methods Biochem. Anal. 26, 145. Campbell, A.M. (1984) Monoclonal antibody production. In: Laboratory Techniques in Biochemistry and Molecular Biology, Vol 13. Elsevier, Amsterdam. Collins, J.K., Butcher, A.C., Teramoto, A. and Winston, S. (1985) Rapid detection of bovine herpesvirus type-l antigens in nasal swab specimens with an antigen capture enzyme-linked immunosorbent assay. J. Clin. Microbial. 21, 375-380. Jones, T.C., Gleiser, C.A., Maurer, F.D., Hale, M.W. and Roby, T.O. (1948) Transmission and immunisation of equine influenza. Am. J. Vet. Res. 9, 243-253. Karber, G. (1931) Beitrag zur kollektiren behandlung pharmakologischer reihenversuche. Arch. Exp. Pathol. Pharmakol. 162, 48&487. Kemeny, D.M. (1991) A practical guide to ELISA. Pergamon Press, London. Kemeny, D.M. and Challacombe, S.J. (1989) ELISA and other solid phase immunoassays: technical and theoretical aspects. John Wiley, New York. Krosgrud, J. and Onstad, 0. (1971) Equine coital exanthema: isolation of virus and transmission experiments. Acta Vet. Stand. 12, 1-14. Matumoto, M. Ishizaki, R. and Shim& T. (1965) Serological survey of equine rhinopneumonitis virus infection among horses in various countries. Arch. Virusforsch. 50, 609-623. Morgan, M.A. and Smith, T.F. (1984) Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen. J. Clin. Microbial. 19, 73&732. Mumford, J.A. and Rossdale, P.D. (1980) Virus and its relationship to the ‘poor performance’ syndrome. Equine Vet. J. 12, 3-9. Nerurkar, L.S., Namba, M., Brashears, G., Jacob, A.J., Lee, Y. and Sever, J.L. (1984a) Rapid detection of herpes simplex virus in clinical specimens by use of a capture biotin-streptavidin enzyme-linked immunosorbent assay. J. Clin. Microbial. 20, 109-l 14. Nerurkar, L.S., Namba, M.J. and Sever, J.L. (1984b) Comparison of standard tissue culture, plus staining, and direct staining for detection of genital herpes virus infections. J. Clin. Microbial. 19, 631633. Palfi, V., Belak, S. and Molnar, T. (1978) Isolation of equine herpesvirus type 2 from foals showing respiratory symptoms. Zentralblatt Vet. 25, 165-167. Yeargan, M.R., Allen, G.P. and Bryans, J.T. (1985) Rapid subtyping of equine herpesvirus 1 with monoclonal antibodies. J. Clin. Microbial. 21, 694697.
39 (1992) 299-3 10
1992 Elsevier Science Publishers
299
B.V. / All rights reserved / 0166-0934/92/%05.00
VIRMET 01388
Rapid detection of equine herpesvirus type-l antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay R. Sinclair and J.A. Mumford Department
of Infectious Diseases, Animal Health Trust, Newmarket,
Suffolk
(UK)
(Accepted 24 April 1992)
Summary An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-l (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swabs from ponies which were experimentally infected with EHV-1. Of 72 nasal swabs collected, 32 were found to be positive by both virus isolation (VI) and ELISA, a further 15 samples were positive by VI alone, but none of the samples were positive by ELISA and negative by VI. This yielded an overall assay sensitivity of 68% and specificity of 100%. The assay proved useful for diagnosis since virus antigen was detected during the first four days post-infection which corresponded to the acute phase of disease when some clinical symptoms were apparent. In addition, the assay could be completed within one day when antibody coated plates were avaiIable. Equid herpesvirus type-l; Rapid diagnosis; ELISA; Nasal swab
Correspondence to: R. Sinclair, Department of Infectious Diseases, Animal Health Trust, P.O. Box 5, Newmarket, Suffolk, CB8 7DW, UK.
300
Introduction Equid herpes virus type-l is associated with a range of clinically different disease syndromes including respiratory disease, abortion, neurological disease and perinatal foal disease (Allen and Bryans, 1986). The virus is distributed worldwide and serological surveys have demonstrated outbreaks of disease in the majority of countries with appreciable horse populations (Matumoto et al., 1965). The disease is acquired by infection of the respiratory tract via aerosols, contaminated food, water, bedding or other formites. Current methods for the identification of EHV-1 rely upon growth of the virus using conventional tissue culture techniques from samples which may include nasal swabs, the leukocyte fraction of blood, or from tissues obtained during post-mortem examination, such as lung, liver, adrenal and thymus. This is followed by immunological screening using immunofluorescence or enzyme-immunofiltration tests (Mumford and Rossdale, 1980; Yeargan et al., 1985). However, diagnosis is complicated by the existence of three antigenically related viruses, equid herpesvirus type-2 (EHV-2), equid herpesvirus type-3 (EHV-3) and equid herpesvirus type-4 (EHV4). These viruses feature significant differences in their pathogenicity when compared to that of EHV-1. EHV-4 is generally associated with outbreaks of respiratory disease and on rare occasions, abortion, but is not usually associated with neurological disease (Allen and Bryans, 1985). EHV-3 causes an infection of the genital tract known as coital exanthema which is characterised by pustular lesions on the external genitalia and sometimes on the lips, nares, nasal mucosa and conjunctiva (Krosgrud and Onstad, 1971). EHV-2 is thought to be relatively non-pathogenic since it can be isolated from healthy animals as well as those with mild respiratory distress, although there is at least one report of foal mortality occurring through EHV-2 infection (Palli et al., 1978). The development of a rapid, sensitive and typespecific diagnostic test for EHV-1 would aid management of disease outbreaks and provide useful epidemiological data. At the present time, the availability of EHV-1 specific monoclonal antibodies has enabled improvements in diagnostic procedures, however, current methods for typing virus still rely upon prior isolation of virus in tissue culture, which may take up to 14 days. The objective of this study was to develop a rapid type-specific ELISA to detect EHV-1 antigen directly in nasal swabs. This type of assay has proven valuable in rapid diagnosis of other viral respiratory infections since quantitative results can be obtained within a few hours, and in some cases, the sensitivity of the test approaches that of virus isolation. For nasal swab specimens collected during the acute phase of the disease, when virus shedding is at its highest, an ELISA should prove a valuable aid to diagnosis.
301
Materials and Methods Viruses
EHV-1 and EHV4 strains were propagated in equine embryonic lung (EEL) cells grown in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum. All but one of the strains were low passage (< 5) field isolates obtained during naturally occurring British outbreaks. The exception was the prototype EHV-1 strain Army 183 which was isolated from a North American horse in 1941 (Jones et al., 1948) and was used for monoclonal and polyclonal antibody production. Polyclonal antiserum production
Antiserum to EHV-1 was prepared by immunisation of rabbits with 200 pg of purified whole virus antigen emulsified in Freund’s complete adjuvant. Three boost injections each of 200 lug prepared in Freund’s incomplete adjuvant were administered at 1 mth intervals and serum collected 10 days after the final boost. Monoclonal
antibody production
BALB/c mice were immunized by intraperitoneal injection with 100 pg of purified whole virus antigen, then boosted 4 wk later by intravenous injection with a further 25 pg on 4 consecutive days. Fusions were performed on the following day according to the protocol described by Campbell (1984). Hybridoma supernatants were screened by ELISA and an immunoflourescence test (Sinclair, 1990) and those producing antibody directed against EHV-1 cloned by limiting dilution and stored in liquid nitrogen. Ascitic fluid was prepared in Balb/c mice and purified by precipitation with ammonium sulphate (Campbell, 1984). Antibody isotypes were determined by ELISA using commercially available reagents. Immunofuorescence
test (IFT)
EEL cell monolayers were grown on eight well chamber slides and infected with EHV-1 or EHV-4 strains at a m.o.i. of 0.025. Monolayers were fixed approximately 24 h post-infection in PBS containing 1.34% w/v formaldehyde (20 min/RT) followed by PBS containing 1% v/v Nonidet P40 (10 min/RT). Monolayers were blocked with 4% w/v BSA in PBS for 30 mins at RT to reduce non-specific reactions, then incubated successively with (i) 1500 dilution of purified monoclonal antibody for 1 h at RT, (ii) 1:200 dilution of rabbit antimouse immunoglobulin for 90 min at 37°C and (iii) 1:40 dilution of fluorescein isothiocyanate conjugated goat anti-rabbit immunoglobulin for 30 min at 37°C. PBS containing 4% w/v BSA was used as diluent in each incubation step
302
and monolayers were washed 5 times in PBS between each incubation step. Monolayers were counterstained with 0.005% v/v Evans blue, and examined for fluorescence using a Lietz Orthoplan microscope. Antigen capture enzyme-linked
immunosorbent
assay
Monoclonal antibodies diluted in carbonate buffer (0.05 M NaHC03/ NazCOs, pH 9.6) were passively adsorbed to 96-well microtitre plates for 16 h at 37°C. Infected tissue culture supernatants, or test samples, were added to the wells for 6 h at 4°C. This was followed by (i) a 1:200 dilution of rabbit antiEHV-1 serum for 2 h at 37°C and then (ii) a 1:200 dilution of goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase for 2 h at 37°C. Bound viral antigen was detected by incubation with substrate solution (1 mg/ml pnitrophenylphosphate, 0.5 mM MgCl- 6 H20, 9.7% diethanolamine, pH 9.8) for 30 min at RT. The plates were washed 5 times with PBS containing 0.05% Tween 20 (PBST) between each incubation step and dilution buffer for both antisera was 2% BSA in PBST. The reaction was stopped by addition of 3 M sodium hydroxide and the absorbance read at 405 nm. Determination
of optimal MAb dilution
Assays were performed as recorded in the previous section, except that (i) microtitre plates were coated with serial 2-fold dilutions of capture antibody and (ii) test antigens were tissue culture supernatants containing either EHV-1 or EHV4 (lo4 TCIDso per well). Determination
of optimal dilution of polyclonal
antisera
The dilutions of peroxidase-conjugated antibody and anti-EHV-1 rabbit sera were determined using a direct binding checkerboard ELISA. EHV-1 antigen diluted to a concentration of 5 pg/ml in carbonate buffer (pH 9.6) and passively adsorbed to 96-well microtitre plates for 16 h at 4°C. The plates were then washed 5 times with PBST and serial 2-fold dilutions of anti-EHV-1 rabbit serum added for 2 h at 37°C. Plates were again washed 5 times with PBST, then serial 2-fold dilutions of peroxidase-conjugated anti-rabbit immunoglobulin added for 2 h at 37°C. The plates were then washed, substrate was added and absorbance was measured as described above. The optimum titre of each antibody was defined as the highest dilution of antibody which gave the maximum absorbance value. Experimental
infection of ponies with EHV-1
6 seronegative 2 yr-old Welsh mountain ponies were challenged with the low passage, British EHV-1 field isolate 592/85. Ponies were inoculated with undiluted tissue culture medium containing 6.6 loglo TCIDSO/ml using a
303
Brovon Midget Inhaler. 25 compressions were applied to each nostril and 7 compressions to each eye. Additionally, a sterile cotton nasal swab, immersed briefly in tissue culture medium, was inserted 10-15 cm into each nostril. After infection, virus shedding from the nasopharynx was monitored daily by insertion of swabs into the nostril followed by abrasion of the mucosae. The swabs were withdrawn and placed immediately into 2 ml of ice-cold viral transport medium, returned to the laboratory, centrifuged to remove cell debris and stored at -70°C. Titration of virus in nasal swabs Quantitative virus titrations were performed by microtitre assay. Briefly, semi-log,, dilutions of nasal swabs were added to 96-well plates in quadruplicate. A suspension of EEL cells in EMEM supplemented with 10% fetal calf serum were added and the plates incubated for 6 days at 37°C in a humidified incubator containing 5% CO;?. Wells were scored for cytopathic effect after 6 days and end-point dilutions calculated by the Karber (1931) method.
Results Selection of monoclonal antibody for ELBA Selection of monoclonal antibody for the antigen capture ELISA was based on several criteria. This included ability to differentiate between EHV-1 and EHV4, efficiency of antigen capture and recognition of a conserved epitope among a panel of field isolates. Eight MAbs derived from mice immunized with EHV-1 and having ELISA titres ranging from 5 x lo5 to 3 x lo4 were evaluated. Optimisation of the assay was performed in three phases. Firstly, the dilution of phosphatase-conjugated goat anti-rabbit immunoglobulin and rabbit anti-EHV-1 immunoglobulin were determined using a direct binding checkerboard ELISA. Microplates were coated with 500 ng/well of purified EHV-1 antigen and each antiserum titrated in two-fold serial dilutions. The highest dilution of each antibody which maintained maximum absorbance values was determined to be 1:200 for both antisera. In the second phase, the abilities of 8 monoclonal antibodies to selectively bind EHV-1 antigen (but not EHV-4) were evaluated. After coating the microtitre lates with serial 2-fold dilutions of each MAb, their abilities to capture 10B TCIDsO/well of EHV-1 or EHV4 vii-ions were compared (Fig. 1). Of the 8 MAbs tested, all except 8Ell (Fig. If) exhibited some potential for detection of EHV-1 antigens. However, MAbs 3E5 and 6Fll also cross-reacted with EHV4 antigens. This cross-reaction was confirmed by immunoflourescence tests using a panel of epidemiologically unrelated field isolates. Both MAbs exhibited positive fluorescence in equine cells infected with all EHV-1
,,4msLvL-.
woean, (a)
I
(h)
305
Abeorbance
(406nm)
0e71
7.0
6.0
6.0 TCID60/ml
4.0
3TO (-Log
2TO
1.0
10)
Fig. 2. Sensitivity of the antigen capture ELISA for detection of EHV-1 antigen in nasal swabs. After optimisation of assay conditions, four different monoclonal antibodies (MAb 4Cl1, 0; MAb 4Hl1, 0; MAb 8F8, A; MAb 8H8, 0) were compared for their ability to capture antigen. The values shown represent the mean absorbance for logic dilutions of infectious virus minus the absorbance of the ELISA zero determined on uninfected tissue culture supernatants.
and EHV4 isolates tested. These MAbs were therefore eliminated from further experiments. MAb 2E6 did not cross-react with the EHV4 isolate (MD) used for the capture ELISA but did cross-react with 8/12 EHV-4 field isolates in the immunofluorescence test and was also eliminated from further work. The four remaining MAbs were all EHV-1 specific in both the capture ELISA and immunofluorescence test. The optimal dilution of each MAb for the capture of EHV-1 virions was selected as the dilution which gave a maximum signal/noise ratio (i.e., ODds2 nm for EHV-1 divided by OD492 nm for EHV4). These were determined to be 1:1600 for MAbs 4Cll and 8H8, 1:400 for MAb 4H 11 and 1:lOO for MAb 8F8. For the third phase of assay development, the 4 remaining antibodies were compared for their efficiency of antigen capture using optimal antibody dilutions. The lower limit of virus detection for each MAb is shown in Fig. 2. c Fig. 1. Optimisation of antigen capture ELBA for type-specific detection of EHV-1 antigen. Serial two-fold dilutions of each MAb were passively adsorbed to microtitre plates and tested for their ability to capture 10’ TCIDso of EHV-1 (0) or EHV-4 (0) diluted in culture medium. (a) MAb 2E6; (b) MAb 3E5; (c) MAb 4Cll; (d) MAb 4Hll; (e) MAb 6Fll; (f) MAb 8Ell; (g) MAb 8F8; (h) MAb 8H8.
306
Abrorbanoe
(406nm)
TCIDCO/ml
0.0
7
0.8
6
0.7 6 0.6 0.6
4
0.6
4
0.4
3
0.4
3
0.3
0.3 2
0.2
2 0.2 1
0.1 0
0 0
1
2
3
Daye post-InfeCtIOn
Abeorbance
TCID60/ml
(406nm)
4
5
6
7
8
0 10 11 12
Daye post-infection
Absorbance 0.s ,
0.0
(406nm)
TCID60/ml
, _.,7
(C)l7 0.6 0.7 6 0.6 0.6 0.4 0.3
0.3
0.2
0.2
0.1
0.1
2
1
0
0
0 0
0123466789
1
2
4
6
6
7
8
9 10 1112
Days post-intectlon
Days port-infection
o.. jbeorbance
3
Abeorbance
WO6nm)
TCID6Ohnl
(406nm)
7
0.9
0.6 -
6
0.6
6
0.7
0.7 -
6
6 0.6
0.6 0.6 -
4
0.4
3
0.6
4
0.4
3
0.3
0.3
2
2 0.2
0.2 1
0.1
0
0 0
1
2
3
4
6
6
7
8
Daye poet-infection
# 10 11 12
1
0.1
0
0 0
1
2
3
4
6
6
7
8
Daye post-InfectIon
9 10 1112
The ELISA zero was determined from the absorbances from at least 3 replicate negative swabs. The predicted lower limit for a positive was then determined using the 95% prediction bound formulae where the ELISA zero equals the mean absorbance of the negative swabs + (SD x t9svo x (n- 1)/n), where SD is the standard deviation (n - 1) of n replicates of negative control absorbances and the t value at 95% probability for n - 1 degrees of freedom (Nerurkar et al., 1984a). Using this method of evaluation MAb 4C11,4Hll, 8F8 and 8H8 had lower limits of sensitivity of 4.0, 4.0, 5.0 and 3.0 logi0 of infectious virus respectively. Since the absorbance levels of 8H8 were highest over the complete range of virus dilutions, this antibody was selected for all further work. Experimental
EHV-I
infections
The antigen capture assay was compared with virus isolation as a method of monitoring the amount of virus shedding from the nasopharynx of six experimentally EHV-1 infected ponies (Fig. 3). ELISA absorbance values peaked on days l-2 post-infection and then declined to zero between 5-8 days post-infection. This pattern was similar to that of quantitative virus isolation except that the period of peak virus recovery and the duration of virus recovery were both slightly longer. Titers of infectious virus eeaked at days l-4 postinfection and declined to zero between days 8-12 post-infection. Both virus isolation and ELISA detected virus in the nasopharynx of all 6 ponies during the first 4 days post-infection. There was a general trend for ELISA absorbance to reflect infectious virus titre, particularly in samples containing at least 3.0 logi0 TCIDSO/ml (Fig. 4). This was confirmed by linear regression analysis performed on data sets for 34 nasal swabs, where the correlation coefficient r was computed to be 0.864. The ELISA detected virus in 99% of samples containing > 3.0 logi0 of infectious virus/ml, which was its approximate lower limit of sensitivity. The exception was a single sample containing 4.1 loglo TCIDSO/ml which was negative by ELISA. From a total of 72 nasopharyngeal swabs, 32 were positive by both VI and ELISA, a further 15 samples were positive by VI alone, but their were no samples which were positive by ELISA and negative by VI. This yielded an overall sensitivity of 68% and specificity of 100% for the antigen capture ELISA. Virus specificity of the antigen capture ELBA
The specificity of the antigen capture ELISA was tested on other equine viral pathogens including equine herpesvirus type-2, equine herpesvirus type-3, equine influenza virus and equine arteritis virus. Assays were negative for three different viral strains using positive nasal swabs or tissue culture medium. Fig. 3. Detection of antigen in nasal swabs from 6 ponies (a-f) with experimental EHV-1 infections. The antigen capture ELBA (0) was compared with virus isolation (0) from days O-12 post-infection. The ELISA absorbance values are shown minus the background absorbance of negative control swabs.
308
Absorbance 1.21 1.1
0
(495
nm)
q
t
’ 0
I
I
1
2
3
TCID50/ml Fig. 4. Comparison
of the ELISA
I
I
I
I
4
5
6
7
(-Log
10)
absorbance with the virus titre. Each dot represents assayed in both tests.
one nasal
swab
Discussion An antigen capture ELBA was developed for the detection of EHV-1 antigens in nasal swab specimens. The test format was that of a solid phase amplified sandwich assay in which a MAb was used to capture virus antigen. It was interesting to observe that seven out of eight MAbs each possessed the ability to capture virus antigen to the solid phase indicating that the majority of antibodies retained a degree of antigen binding capacity when passively adsorbed to plastic plates (Fig. 1). The failure of one antibody to bind antigen may have resulted from denaturation of the antibody during the adsorption process, which has been previously documented for certain MAbs (Kemeny and Challacombe, 1989; Kemeny, 1991). Alternatively, the failure to bind antigen could be explained by low antibody affinity or inappropriate antigen presentation in the virus. Of the four MAbs which were specific for EHV-1, 8H8 was selected as the best capture antibody based on higher absorbance values over the range of virus dilutions tested and the lowest limit of virus detection in infected tissue culture supernatants of approximately 3.0 log10 (Fig. 2). This was in close agreement with results from nasal swab specimens taken from experimentally infected ponies where the ELISA detected 99% of
309
swabs containing > 3.0 loglo of infectious virus. Other MAbs of interest were 3E5 and 6F11, which successfully captured EHV-1 antigen but also crossreacted with EHV4 antigen indicating that they both react with common epitopes (Fig. 1). Antigenic variation was only noted for a single MAb, designated 8El1, which reacted with a epitope common to EHV-1 and EHV4, but which demonstrated antigenic variability only among EHV-4 strains. In experimentally infected ponies, antigen was detected in all 6 animals from days l-4 post-infection, corresponding to the time of peak virus shedding, and in some ponies up to 7 days post-infection. The overall assay sensitivity was 68% when compared with virus isolation, which was similar to the sensitivity found with antigen capture ELISA’s for other viruses, such as herpes simplex virus type-l (Adler-Storthz et al., 1983; Morgan and Smith, 1984; Neurkar et al., 1984a) and bovine herpesvirus type-l (Collins et al., 1985). The antigen capture ELISA possessed several advantages over the conventional method of virus isolation in tissue culture. Firstly, the assay was rapid, requiring only one day to complete when antibody-coated plates were available, whereas virus isolation can take up to 14 days. This should aid management of disease outbreaks on studs or training stables affected with EHV-1, since management decisions can now be made during the early stages of infection. In addition, since absorbance values reflect the amount of infectious virus in the nasopharynx, identification and isolation of highly infectious animals may also reduce the chance of virus spread. Secondly, the assay does not necessarily require the presence of infectious virus. This may be important if infectivity is lost during transport of the virus to the laboratory, or, if the virus is exposed to neutralizing antibody produced locally in the nasopharynx. The main disadvantage of the ELISA is its lower sensitivity which would result in the production of some false negatives. However, in a field situation where several animals may be infected, group sampling of in-contact animals and of all animals with clinical signs may increase the chance of obtaining a positive. If clinical signs are recognised during the first four days of infection, this would further enhance the chance of a positive diagnosis. The most likely use of this assay will probably be in conjunction with virus isolation. The sensitivity of the assay may be increased, for example, by the use of biotinstreptavidin labelled antibodies and/or enzymes (Bayer and Wilchek, 1980; Nerurkar et al., 1984b) and refinement of the assay may lead to the development of a test which could be used in. the field. Acknowledgements
The authors are grateful to Miss B. Moult and Mr. D. Jessett for excellent technical assistance and to the Horserace Betting Levy Board for supporting this study.
310
References Adler-Storthz, K., Kendall, C., Kennedy, R.C., Henkel, R.D. and Dressman, G.R. (1983) Biotinavidin amplified enzyme immunoassay for detection of herpes simplex virus antigen. Laboratory Techniques in Biochemistry and Molecular Biology, Vol 13, Elsevier, Amsterdam. Allen, G.P. and Bryans, J.T. (1986). Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-l infections. In: Progress in Veterinary in Veterinary Microbiology and Immunology, vol 1, pp. 78-144. Edited by S. Pandy. Basel. Bayer, E.A. and Wilckek, M. (1980) The use of avidin-biotin complex as a tool in molecular biology. Methods Biochem. Anal. 26, 145. Campbell, A.M. (1984) Monoclonal antibody production. In: Laboratory Techniques in Biochemistry and Molecular Biology, Vol 13. Elsevier, Amsterdam. Collins, J.K., Butcher, A.C., Teramoto, A. and Winston, S. (1985) Rapid detection of bovine herpesvirus type-l antigens in nasal swab specimens with an antigen capture enzyme-linked immunosorbent assay. J. Clin. Microbial. 21, 375-380. Jones, T.C., Gleiser, C.A., Maurer, F.D., Hale, M.W. and Roby, T.O. (1948) Transmission and immunisation of equine influenza. Am. J. Vet. Res. 9, 243-253. Karber, G. (1931) Beitrag zur kollektiren behandlung pharmakologischer reihenversuche. Arch. Exp. Pathol. Pharmakol. 162, 48&487. Kemeny, D.M. (1991) A practical guide to ELISA. Pergamon Press, London. Kemeny, D.M. and Challacombe, S.J. (1989) ELISA and other solid phase immunoassays: technical and theoretical aspects. John Wiley, New York. Krosgrud, J. and Onstad, 0. (1971) Equine coital exanthema: isolation of virus and transmission experiments. Acta Vet. Stand. 12, 1-14. Matumoto, M. Ishizaki, R. and Shim& T. (1965) Serological survey of equine rhinopneumonitis virus infection among horses in various countries. Arch. Virusforsch. 50, 609-623. Morgan, M.A. and Smith, T.F. (1984) Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen. J. Clin. Microbial. 19, 73&732. Mumford, J.A. and Rossdale, P.D. (1980) Virus and its relationship to the ‘poor performance’ syndrome. Equine Vet. J. 12, 3-9. Nerurkar, L.S., Namba, M., Brashears, G., Jacob, A.J., Lee, Y. and Sever, J.L. (1984a) Rapid detection of herpes simplex virus in clinical specimens by use of a capture biotin-streptavidin enzyme-linked immunosorbent assay. J. Clin. Microbial. 20, 109-l 14. Nerurkar, L.S., Namba, M.J. and Sever, J.L. (1984b) Comparison of standard tissue culture, plus staining, and direct staining for detection of genital herpes virus infections. J. Clin. Microbial. 19, 631633. Palfi, V., Belak, S. and Molnar, T. (1978) Isolation of equine herpesvirus type 2 from foals showing respiratory symptoms. Zentralblatt Vet. 25, 165-167. Yeargan, M.R., Allen, G.P. and Bryans, J.T. (1985) Rapid subtyping of equine herpesvirus 1 with monoclonal antibodies. J. Clin. Microbial. 21, 694697.