Effect of fixation on the detection of transmissible gastroenteritis coronavirus antigens by the fixed-cell immunoperoxidase technique

Tô, Long-Thành; Bernard, Serge

doi:10.1016/0022-1759(92)90192-v

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…for the quantitation of viral antigens expressed in TGEVI cells), a formula dealing with the background of the TGEVI and MI cells has been used (66,68). For comparative purposes, another study expressed the degree of relative antibody binding to a specified viral determinant as a percentage of antibody binding to unfixed infected cells (67). In the present study, two values of the VI/MI ratio were used for the antibody screening test (e.g.…”

Section: Interpretation Of the Resultsmentioning

confidence: 99%

“…A previous study (67) established the effect of various fixatives and detergents on the detection of some TGEV antigenic determinants in infected cells, mainly by means of MAbs. When compared with glutaraldehyde and formaldehyde, 0.1% PFA was found to be the fixative of choice for the detection of these determinants on the membranes of infected cells.…”

Section: Cell Fixationmentioning

confidence: 99%

“…This is not the case for the development of a screening test, as it is not known which antibodies will be secreted by the hybrids, and it is therefore not possible to use one suitable fixative for the infected cells. Thus, it was decided to use 80% acetone to fix the infected cells, on the basis of previous results (67) showing that three structural antigens of TGEV were easily detected in 80% acetone-fixed TGEVI cells and that only anti-M and anti-S, but not anti-N antibody could be detected in infected cells fixed with aldehyde fixatives. These results demonstrated the importance of the fixation procedure with which an antigen could or could not be detected.…”

Section: Detection Of Anti-tgev Antibodiesmentioning

confidence: 99%

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Transmissible gastroenteritis coronavirus : the development and applications of the fixed-cell immunoperoxidase technique

Lt¹,

Bernard²,

Jm³

1993

Rev. Sci. Tech. OIE

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Since the first demonstration in 1971 that solid-phase enzyme-linked immunosorbent assays (ELISAs) could be used for the quantitative determination of antigens and antibodies, this method has been widely applied in serodiagnosis of parasitic and infectious diseases. In addition to the classic ELISA variants using antigen or antibody to coat the plastic plates, there has recently been growing interest in the application of fixed-cell ELISA to research and diagnostic work on viral diseases. The authors discuss the development and applications of this technique to basic research and diagnosis of transmissible gastroenteritis, a highly contagious disease of swine. The success of this technique, as the name suggests, is largely due to the use of a suitable fixative, which preserves the antigenicity of the neo-synthesised viral proteins, and the presence of optimal conditions for viral antigen synthesis. In addition, various parameters are optimised, and this is discussed with reference to transmissible gastroenteritis virus. These parameters would help veterinarians and research workers to develop this technique in their own laboratories.

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Section: Interpretation Of the Resultsmentioning

confidence: 99%

Section: Cell Fixationmentioning

confidence: 99%

Section: Detection Of Anti-tgev Antibodiesmentioning

confidence: 99%

See 1 more Smart Citation

Transmissible gastroenteritis coronavirus : the development and applications of the fixed-cell immunoperoxidase technique

Lt¹,

Bernard²,

Jm³

1993

Rev. Sci. Tech. OIE

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Site-specific alteration of transmissible gastroenteritis virus spike protein results in markedly reduced pathogenicity

Bernard

Laude

1995

Journal of General Virology

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The pathogenicity of neutralization-resistant mutants of the enteric coronavirus transmissible gastroenteritis virus (TGEV) was examined in the newborn piglet. The parental virus (Purdue-ll5 strain), as well as several mutants selected using monoclonal antibodies (MAbs) directed to antigenic sites A and B, caused an acute enteritis with 100 % mortality. By contrast, most of the site D (MAb 40.1) mutants exhibited a strongly reduced enteropathogenicity, leading to the survival of animals inoculated with up to 1000-fold the 100 % lethal dose of parental virus. Such a phenotypical change was correlated with point mutations or a small deletion, all located within the S gene sequence coding for the to Cys-155 segment of the mature polypeptide.These observations suggest that an N-terminal subregion of the S molecule is an essential determinant for pathogenesis in TGEV infection.

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Immunohistochemistry of Transmissible Gastroenteritis Virus Antigens in Fixed Paraffin-Embedded Tissues

et al. 1996

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Abstract. An immunohistochemistry technique was developed using fixed tissues to study the presence and location of transmissible gastroenteritis virus (TGEV) antigens in situ. Experimentally infected gnotobiotic and conventional pigs as well as pigs with natural TGEV infection were examined. The staining technique was based on detection of the major structural protein of TGEV, the nucleocapsid, by using a pool of 3 monoclonal antibodies. Formalin and periodate-lysine-paraformaldehyde (PLP)-fixed intestinal tissues from a gnotobiotic pig inoculated with virulent TGEV were used to determine optimal antibody concentrations and incubation times. The intestinal tissues remained in their respective fixatives for 6 months, and serial sections were removed at sequential times and embedded in paraffin blocks. PLP and 10% neutral buffered formalin were acceptable fixatives and preserved TGEV nucleocapsid antigenicity for up to 6 months. Formalin, in comparison with PLP as a fixative, was better for preserving original tissue morphology and provided better antigen detection. Conventional crossbred pigs were inoculated with virulent TGEV, and animals were euthanized on various postexposure days. Intestinal tissues were positive for TGEV nucleocapsid antigens on postexposure days 2, 4, and 8. The immunohistochemistry technique detected TGEV antigen in stored paraffin-embedded tissues from 14 naturally infected pigs previously confirmed as positive for TGEV using a direct immunofluorescence assay on intestinal mucosal smears, whereas 9 naturally infected pigs confirmed negative for TGEV antigen by the same immunofluorescence assay showed no staining consistent with the presence of TGEV antigen. Immunohistochemistry provides a method to detect TGEV and possibly other closely related coronaviruses such as porcine respiratory coronavirus in situ. A diagnostic test using the same fixed tissues processed for histopathology provides veterinary practitioners an alternative to delivering live pigs or refrigerated fresh intestinal samples containing infectious virus to a diagnostic laboratory. Investigators can utilize this technique to retrospectively screen fixed tissues for TGEV antigen.Transmissible gastroenteritis (TGE) virus (TGEV) belongs to the family Coronaviridae and causes enteritis in swine of all ages. Enteritis from TGEV results from destruction of villous enterocytes and villous atrophy of the small intestinal mucosa, especially within the jejunum and ileum.11 Clinical signs of TGE include anorexia, vomiting, diarrhea, and dehydration. Mortality in infected neonatal piglets approaches 100% because of severe diarrhea and dehydration.14 TGE annually costs the swine industry approximately $200 million through mortality and decreased production efficiency. ' During outbreaks of swine diarrhea, a presumptive diagnosis of TGEV can be made by evaluation of disease history and clinical signs. Histopathologic sections of formalin-fixed jejunum and ileum showing evidence of villous atrophy, although consistent with TGEV infect...

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Effect of fixation on the detection of transmissible gastroenteritis coronavirus antigens by the fixed-cell immunoperoxidase technique

Cited by 3 publications

References 16 publications

Transmissible gastroenteritis coronavirus : the development and applications of the fixed-cell immunoperoxidase technique

Transmissible gastroenteritis coronavirus : the development and applications of the fixed-cell immunoperoxidase technique

Site-specific alteration of transmissible gastroenteritis virus spike protein results in markedly reduced pathogenicity

Immunohistochemistry of Transmissible Gastroenteritis Virus Antigens in Fixed Paraffin-Embedded Tissues

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