An enzyme-linked immunosorbent assay for the detection of antibodies to avian reticuloendotheliosis virus using whole cell antigen

Research in Veterinary Science 1987, 43, 403-404

An enzyme-linked immunosorbent assay for the detection of antibodies to avian reticuloendotheliosis virus using whole cell antigen R. A. J. NICHOLAS, D. H. THORNTON, Central Veterinary Laboratory, Weybridge, Surrey

An enzyme-linked immunosorbent assay (ELISA) using reticuloendotheliosis virus-infected chick embryo fibroblasts as coating antigen is described for the detection of antibodies to reticuloendotheliosis virus in chicken sera, The ELISA was specific and during the early stages of infection more sensitive than an indirect fluorescent antibody test. VARIOUS tests have been reported for the detection of antibodies to avian reticuloendotheliosis virus. These include the agar gel precipitin test (Ianconescu 1977), the complement fixation test (Smith et al 1977), the indirect fluorescent antibody test (Aulisio and Shelokov 1969) and the enzyme-linked immunosorbent assay (ELISA) (Smith and Witter 1983). Conventionally, antigen in the ELISA is prepared from cell-free virus which has been concentrated from cell culture fluids and, more often than not, purified on gradients (Nicholas and Thornton 1986). Recently an ELISA has been described for Marek's disease virus which uses virus-infected cells to capture antibody (Cheng et al 1984). This report describes the application of this methodology to the detection of reticuloendotheliosis virus antibodies and compares it with the fluorescent antibody r test. The HPRS·I isolate of reticuloendotheliosis virus was used. The virus was isolated and plaque purified by Houghton Poultry Research Station then subcultured three times in chick embryo fibroblast cell cultures in this laboratory. Antigen for the ELISA was prepared as follows: five 100 ml bottles of chick embryo fibroblasts were each infected with O' 5 ml of reticuloendotheliosis virus containing 1()4'6 TCID50 while the cells were in suspension. After 24 hours the chick embryo fibroblasts were subcultured and reseeded at 350,000 cells ml- I on to new bottles. The cells were incubated for a further three days by which time the majority were infected as shown by the fluorescent antibody test. The culture fluids were discarded and the cells were treated with a solution of trypsin-versene. After the cells became detached, they were resuspended in Eagle's medium

supplemented with 5 per cent calf serum and 7· 5 per cent dimethyl sulphoxide at a concentration of3 x 106cells ml I. The cells were kept at - 70°C for 24 hours before storage in liquid nitrogen. For coating microtitre plates (Falcon pvc microtest III; Becton Dickinson), the cells were thawed rapidly, diluted in phosphate buffered saline (PBS, pH 7· 2) and centrifuged at 100 g for 10 minutes. They were resuspended at various concentrations in PBS and dispensed into plates in 50 ",I volumes. The plates were centrifuged as above and the supernatant discarded. The plates were dried at 37°C and stored at 4°C until used. Unless stated otherwise all volumes dispensed in the ELISA were 50 J.lI. . To determine the optimum number of cells necessary for the ELISA, microtitre plates were coated with reticuloendotheliosis virus-infected chick embryo fibroblasts and uninfected chick embryo fibroblasts at cell concentrations varying from 15,000 to 240,000 cells per well. After attachment the cells were incubated with duplicate 11100 dilutions of positive or negative anti-reticuloendotheliosis virus serum for one hour at 37°C and then washed five times with PBS containing O' 05 per cent Tween 20. A dilution of 112500 of rabbit anti-chicken IgG conjugated to horseradish peroxidase (Nordic) was added to all wells and the plates were incubated as before. After washing the enzyme substrate, ortho-phenylene diamine (4 mg dissolved in 10 ml in citratephosphate buffer 0·15 M pH 5·0 with 150 ",I of 10 volume hydrogen peroxide) was added. Colour was allowed to develop for five minutes and stopped with 2 M sulphuric acid. The optical densities were read on a Dynatech MR 600 microplate reader. Control chick embryo fibroblasts gave little background with either the positive or negative sera. Reticuloendotheliosis virus-infected chick embryo fibroblasts gave good discrimination between the sera, this being greatest at 30,000 cells per well. This cell concentration was chosen for routine use in the ELISA. Some measure of the consistency with which cells coat the wells was carried out by adding ai/lOa r

TABLE 1: Stability of reticuloendotheliosis virus whole cell antigen at three storage temperatures after three months as measured by standard reticuloendotheliosis virus antiserum

Temperature (OC)

23 4 -20

Titre of sera (10910) Weeks of storage

O'

2

4

6

8

10

12

14

3·81

3·20 3'51 3·51

3-51 3·51 3·81

3·20 3·20 3·51

NO

3·51 3·51 3-51

3'51 3·51 3·81

3·20 3·51 3'51

• Titre of serum on freshly prepared plates

403

3·51 3'51

R. A. J. Nicholas, D. H. Thornton

404

TABLE 2: Serological response of birds detected by ELISA and fluorescent antibody test Number of sera positive for REV antibody (mean 10910 titre) Weeks after infection

2 3 4

Test

17/20 (2·21) 11/20 (1·75)

ELISA FAT REV FAT

20/20 (3-54) 20/20 (3'13)

20/20 (3·88) 20/20 (3·85)

20/20 (3·76) 20/20 (3-64)

Reticuloendotheliosis Fluorescent antibody test

dilution of reticuloendotheliosis virus antiserum to plates from different sources. The ELISA was carried out as above. The coefficient of variation (v) for each type of plate was determined from the optical density values obtained. The lowest variation was seen with the Falcon pvc and Dynatech MI29A polystyrene plates (v = 6· 5 per cent); Nunc Immuno II and Dynatech MI29B polystyrene plates had v values of 9· 5 per cent and II per cent, respectively. To assess the specificity of the ELISA, antisera to the following viruses were titrated on the reticuloendotheliosis virus-infected chick embryo fibroblasts: avian adenovirus, infectious laryngotracheitis virus, infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, egg drop syndrome '76 virus, Marek's disease virus, avian encephalomyelitis virus and avian leukosis virus. No significant reaction was seen with any of these sera. To assess the stability of the ELISA, cell-coated plates were stored at 23°C, 4°C and - 20°C. At fortnightly intervals the plates were removed and used to titrate positive and negative control sera. Table I shows the stability of the ELISA over three months at the different temperatures. There was little decrease in titre of the positive control serum at 4°C, 23°C or - 20°C over the whole period. To compare the relative sensitivity of the ELISA with the fluorescent antibody test, 20 eight-week-old specific pathogen free chickens were inoculated intramuscularly with 1()3 TCIDSO of HPRS-I per bird. At weekly intervals of four weeks the birds were bled and their sera examined for antibodies to reticuloendotheliosis virus. Five birds were kept as uninoculated controls. For the ELISA, sera were titrated across the coated plates from 11100 to 116400. The titre of each test serum was given as the last dilution above the positive-negative cut-off point which was established as previously described (Nicholas et al 1986). The fluorescent antibody test was carried out as previously described (Nicholas and Thornton 1983). Briefly, reticuloendotheliosis virus-infected chick embryo fibroblasts were grown on multi well slides, washed in PBS, fixed in cold acetone and stored at - 20°C. When required the slides were thawed and washed in PBS. Test sera were diluted as above and 50 III placed on duplicate wells. The slides were incubated at 37°C for 20 minutes and washed in PBS. A dilution of 1/80 of rabbit anti-chicken IgG conjugated to fluorescein isothiocyanate was then applied to all slides for 20 minutes at 37°C. The slides were washed as before and mounted in Entellan (BDH, UK). The cells were examined with an ultraviolet microscope. Individual wells were scored for the presence or absence of specific fluorescence. The titre of the test sera was given as the last dilution showing fluorescence. Table 2 gives the results of the comparison between the

and fluorescent antibody test. The ELISA detected reticuloendotheliosis virus antibodies in more sera than the fluorescent antibody test seven days after infection and generally to higher titres. By 14 days all the inoculated birds were positive with both tests although ELISA tit res were still slightly higher. After 21 and 28 days there was good correlation between the tests with tit res being very similar. The results reported here confirm the finding of Cheng et al (1984) that virus-infected cells provide a convenient source of specific viral antigen for detecting antibody. The optimum number of infected cells needed in the reticuloendothelios virus ELISA was 30,000 cells per well but it should be noted that this will depend on the proportion of cells infected and this may vary from batch to batch. Therefore each batch of chick embryo fibroblasts should be tested before use. The stability of the cells was extremely good even after three months at ambient temperature. The reticuloendotheliosis virus ELISA was more sensitive than the fluorescent antibody test in the earlier stages of infection although titres became similar later on. In terms of ease of performance the ELISA is far superior to the fluorescent antibody test and is preferable for large scale screening of sera. Finally the use of whole cell antigen may be useful where the virus of interest does not occur in large quantities in a cell-free state but where the appropriate viral antigen is expressed on the cell surface.

ELISA

Acknowledgement We would like to thank Mr Goh Jfp Yin for his help with this work. References AULISIO, C. G. & SHELOKOV, A. (1969) Proceedings oj the Society for Experimental Biology and Medicine 130, 178-181 CHENG, Y.-Q., LEE, L. F., SMITH, E. J. & WITTER, R. L. (1984) Avian Diseases 28, 900-911 IANCONESCU, M. (1977) Avian Pathology 6,259-265 NICHOLAS, R. A. J. & THORNTON, D. H. (1983) Research ill Veterinary Science 34,377-379 NICHOLAS, R. A. J. & THORNTON, D. H. (1986) Veterinary Bulletin 56, 337-343 NICHOLAS, R. A. J., GODDARD, R. G., REAM, A. J .• HOPKINS, I. G. & THORNTON, D. H. (1986) Research in Veterinary Science 41, 420-422 SMITH, E. J., SOLOMON, J. J. & WITTER, R. L. (1977) Avian Diseases 21, 612-622 SMITH, E. J. & WITTER, R. L. (1983) Avian Diseases 27, 225-234

Received March 20, 1987 Accepted July 20, 1987