key: cord-0007659-xurttbt2
authors: Hohdatsu, T.; Eiguchi, Y.; Ide, S.; Baba, K.; Yamagishi, H.; Kume, T.; Matumoto, M.
title: Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies
date: 2002-11-13
journal: Vet Microbiol
DOI: 10.1016/0378-1135(87)90103-9
sha: c14ad3458d6a5fc567d85b3e4f479ef8ee8ac292
doc_id: 7659
cord_uid: xurttbt2

An enzyme-linked immunosorbent assay (ELISA) using a detergent-solubilized antigen of purified virus was developed for detection of antibody against porcine transmissible gastroenteritis (TGE) virus in swine serum. The ELISA demonstrated antibody responses in pigs immunized intramuscularly with the attenuated TO-163 strain of TGE virus and in pigs orally infected with the virulent Shizuoka strain of the virus. The results of the ELISA were well correlated with those of the neutralization test. These results indicate the usefulness of the ELISA as a serological tool for TGE virus antibody.

Transmissible gastroenteritis (TGE) is a viral disease of swine characterized by a short incubation period, high infectivity, diarrhea and vomiting, and a high mortality in young piglets. Various serological tests for the detection of TGE virus antibodies are available. They include the neutralization test (Harada et al., 1967; Bohl et al., 1972) , bentonite agglutination test (Sibinovic et al., 1966) , complement fixation test (Stone et al., 1976) , agar immunodiffusion test (Bohac et al., 1975; Bohac and Derbyshire, 1976; Stone et al., 1976) , indirect fluorescent antibody test (Benfield et al., 1978) , indirect haemagglutination test (Shimizu and Shimizu, 1977) , and immunoperoxidase test (Kodama et al., 1980) . The enzyme-linked immunosorbent assay (ELISA) has been shown to be a useful serological tool for viral infections. The present study was undertaken to develop an ELISA for detection and quantitation of TGE virus antibody in swine serum.

For preparation of viral antigen, the TO-163 strain of TGE virus (Harada et al., 1967; Furuuchi et al., 1975) was propagated in cultures of the CPK cell line derived from swine kidney (Komaniwa et al., 1981) . Infectious culture fluid was concentrated about 10-fold by ammonium sulfate precipitation, layered onto a discontinuous sucrose density gradient (10--60%) in a Beckman SW25.2 rotor and centrifuged at 24 000 r.p.m, for 2 h. The virus bands formed were collected, mixed with an equal volume of fluorocarbon (Wako Pure Chemicals, Tokyo) and vigorously shaken for 2 min. The water phase separated by centrifugation was layered onto a linear sucrose density gradient (10-60%) in a Beckman SW25.2 rotor and centrifuged at 24 000 r.p.m, for 4 h. Fractions with densities of 1.18--1.20 g ml -I were pooled and centrifuged at 77 000 × g for 1 h. The pellets were resuspended in a 1/500 volume of NTE buffer (0.1 M NaC1, 0.01 M Tris-HC1, pH 7.4, 0.001 M EDTA). The suspension was treated with 1% Triton X-100 at 4°C for 1 h and dialyzed against phosphate buffered saline (PBS) (0.1 M NaC1, 0.01 M phosphate buffer, pH 7.2). Supernatant fluid from CPK cell cultures was concentrated 10-fold by ammonium Sulfate precipitation and used as the control antigen.

For the ELISA, virus antigen prepared as above was appropriately diluted with carbonate buffer (0.05 M, pH 9.6) and delivered in 100-~1 volumes into wells of 96-well, flat-bottomed Microelisa plates (Dynatech Lab., U.S.A.). The plates were allowed to stand overnight at 4°C, washed four times with 0.85% NaC1 solution containing 0.02% Tween-20, and 100 pl of the test serum diluted 100-fold with PBS containing 10% calf serum and 0.05% Tween-20 was added to each well. After incubation at 37°C for 1 h, the plates were washed, and 100 gl of optimal dilution of horseradish peroxidase-conjugated rabbit antibody against porcine IgG (Miles Lab., U.S.A.) was added to each well. After incubation at 37°C for 1 h, each well was washed, received 100 pl of substrate solution and was incubated at 25°C for 20 min in a dark room. The substrate solution was prepared by dissolving 0-phenylenediamine dihydrochloride at a concentration of 0.1 mg m1-1 in 0.1 M citric acid--0.2 M Na2HPO4 buffer (pH 4.8) and adding 0.2 pl m1-1 of 30% H202. At the end of incubation, the reaction was stopped with 3 N H2SOa solution and absorbancy measurement was performed at a wave length of 492 nm. The value obtained by subtracting the absorbance of the control antigen from that of the viral antigen was taken as the ELISA value.

Neutralization (NT) tests were carried out by the serum dilution method in CPK cell cultures prepared in flat-bottomed microplates (Coming Glass Works, Coming, NY) . In wells of a transfer plate, 25 pl of each of serial 2fold dilutions of the serum inactivated at 56°C for 30 min was mixed with 25 pl of maintenance medium containing 200 TCIDs0 of virus. Four wells were employed for each serum dilution. The virus--serum mixtures were incubated at 37°C for 60 min, transferred into wells of a microplate containing CPK cell cultures, and incubated in an atmosphere of 5% CO2 in air at 37°C for 5 days. The antibody titre was expressed as the reciprocal of the antibody dilution showing 50% neutralization calculated by the method of K~ber.

In preliminary experiments with a swine antiserum with a NT titre of 256 against the TO-163 strain, specific reaction was observed when each well of the Microelisa plates received 10--2.5 pg protein in the virus antigen, as determined by Lawry's method. In the following experiments, 2.5 pg protein was delivered in each well.

Serum samples were collected from 78 specific pathogen-free pigs on a farm where no TGE outbreaks were experienced in the past. These serum samples had an average ELISA value of 0.131 with a standard deviation of 0.051. In order to give an ample safety margin, ELISA values of 0.284 (0.131 + 3 X 0.051) or higher were taken as positive. Serial 2-fold dilutions of four swine antisera having NT titres of 16 000, 2048, 256 and 32 were tested by the ELISA. The end point dilutions of these serum samples were 1:25 600, 1:5000, 1:600 and 1:400, respectively, being closely correlated with their NT titres.

Serum samples were collected at intervals from four initially seronegative, 2-month-old pigs inoculated with two intramuscular doses of about 107 TCIDs0 each of the TO-163 strain and tested by the ELISA and NT test. The results are summarized in Fig. 1 . The ELISA value and the NT titre increased gradually after the first inoculation, and rapidly after the second one.

Fifteen initially seronegative, 2-month-old pigs were infected by the oral route with the virulent Shizuoka strain of TGE virus (Harada et al., 1967) . Serum samples were collected before infection and 21 or 28 days after infection, and tested for ELISA value and NT titre. All the animals produced antibodies after infection, ELISA values and NT titres ranging from 0.64 to > 2.00 and from 2 to 256, respectively. ELISA values of individual pigs were closely correlated with their NT titres (Fig. 2) . The correlation coefficient was 0.911 (P > 0.01).

In the present study an ELISA using detergent-solubilized antigen of purified virus was developed for detection of antibody against TGE virus in swine serum. Nelson and Kelling (1984) reported an ELISA for TGE virus antibody in swine serum. Antibody responses were demonstrated by the ELISA and NT test in pigs immunized intramuscularly with the attenuated TO-163 strain and in pigs orally infected with the virulent Shizuoka strain. These results seem to indicate the usefulness of the ELISA as a serological tool. The ELISA may be applied with advantage to testing large numbers of sera for TGE virus antibody.

An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine

The detection of transmissible gastroenteritis viral antibody by immunodiffusion

The detection of transmissible gastroenteritis viral antigens by immunodiffusion

Antibody responses in serum, colostrum and milk of swine after injection or vaccination with transmissible gastroenteritis virus

Comparison between virulent and attenuated strains of transmissible gastroenteritis virus

Studies on transmissible gastroenteriris in pigs. III. Isolation of cytopathogenic virus and its use for serological investigation

Detection of antibody against transmissible gastroenteritis virus of pigs by indirect immunoperoxidase antibody test

Micromethod for performing titration and neutralization test of hog cholera virus using established porcine kidney cell strain

Enzyme-linked immunosorbent assay for detection of transmissible gastroenteritis virus antibody in swine sera

Micro-indirect hemagglutination test for detection of antibody against transmissible gastroenteritis virus of pigs

Bentonite agglutination test for transmissible gastroenteritis of swine

Partial characterization of the principal soluble antigens associated with the coronavirus of transmissible gastroenteritis by complement fixation and immunodiffusion

The authors thank Dr H. Koyama and Dr K. Ando of the University of Kitasato in Japan for useful suggestions in the development of this paper.