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1、I eterinary Microbiology. 29 ( 199 ! ) 36 !-368 Elsevier Science Publishers B.V. Amsterdam Short Communication 361 Fixed-cell immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastroenteritis (TGEV) L.T. To a, S. Bernard b and I. LantieP aLabora
2、tory of Virology, lmtitute of Veterinary Research, Bach mai-Hiut6L Vidtr, am bLaboratoire de Pathologie lnfectieuse et dhnmunologie. 37380 Nou:illr. France (Accepted 4 March 199 ! ) ABSTRACT To, L.T., Bernard, S. and Lantier, !., 199 l. Fixed-cell immunoperoxidase technique for the study of surface
3、antigens induced by the coronavirus of transmissible gastroenteritis (TGEV). l,t. Micro- biol., 29: 361-368. An immunoperoxidase technique performed on the TGEV-infected cells was developed for detec- tion of virus-induced antigens. The presence of M antigen of TGEV on the surface of infected cells
4、was demonstrated by this technique. This finding is in contrast to the M protein of murine hepatitis coronavirus which migrates to the Golgi apparatus but is not traasported to the plasma membrane. The time coupe ofannearance M and S antigens on the surface of TGEV-mfected cell can _he_ studied by t
5、his technique. INTRODUCTION Three major structural proteins have been described for all coronaviruses, two glycoproteins (S and M ) and one phosphoryiated nucleoprotein (N) (for review, see Sturman, 1981; Garwes, 1982; Holmes et al., 1984; Laude et al., 1990). For mouse hepatitis virus (MHV), a well
6、 studied coronavirus, the M protein migrates to the Golgi apparatus, but is not transported to the plasma membrane as readily as is the S protein (Sturman and Holmes, 1983 ). For porcine TGEV, the presence of the virus envelope S antigen on the surface of infected cells was demonstrated by immunoflu
7、orescence (Laude et al., 1986), while the presence of M antigen on the plasma membrane has only been suspected by unspecified monoclonal antibodies (mAbs) (Welch and Saif, 1988). 0378- I 135/91/$03.50 ! 99 ! Elsevier Science Publishers B.V. All rights reserved. 362 L.T. TO ET AL. Recently, the expre
8、ssion of the TGEV envelope M protein on the surface of infected cells has been demonstrated by isotope labelling (Laviada et al., 1990). There has not been any published report concerning the presence of N antigen on the plasma membrane of infected cells. We describe in the present study a more conv
9、enient immunoperoxidase test (IPT) for the confirmation of the location ofTGEV M antigen at the cell surface. Moreover, this test, performed on 0.1% paraformaldehyde-fixed, TGEV-infected cells can be used not only for detecting but also for quantify- ing the expression oftwo viral M and S glycoprote
10、ins on the plasma membrane. MATERIALS AND METHODS l/ints, cells and monoclonal antibodies The swine testis (ST) cell line and Purdue-I 15 virus strain which have been described elsewhere (Laude et al., 1981 ) and three mAbs, anti-S (5 l-I 3), anti-M (25-22) (Delmas et al., 1986 ) and anti-N (22-6) (
11、Laude et al., 1986 ) were used in this study. Infection of cells Confluent monolayers of ST cells in 96-well, fiat-bottomed plastic plates (Falcon 3072, Becton Dickinson) were inoculated with 0.1 ml of virus sus- pension containing a multiplicity of infection (m.o.i) of either 2, 10 or 100. The unin
12、fected cells served as controls. After 30 min of incubation at 37C under 5.5% CO2, the inoculum in each well was removed from the cell mono- layer by two washes with Dulbeccos phosphate buffered saline (DPBS) and was replaced with o.1 mi of Eagies minimum essential medium containing 5% heat-inactiva
13、ted normal calf serum. The infected cells were then incu- bated at 37C under 5.5% CO2 for 18 hours unless otherwise specified. Fixation of cells with paraformaldehyde for detection of virus induced- antigens on surface Paraformaldehyde ( PFA ) powder (Prolabo-France) was dissolved in DPBS by heating
14、 at 80C and used as a fixative for the detection of virus-induced antigens on the surface of TGEV-infected cells. The desired concentration (0.1%) of the fixative was prepared by dilution in DPBS. The fixative solu- tion was freshly made just before use for each experiment. The monolayers were caref
15、ully washed twice with DPBS and the cells were fixed with the ap- propriate fixative at 4C for 30 min and then saturated with 5% skimmed milk in PBS without calcium and magnesium for 15 min at room temperature. Fixation of cells with 80% acetone for detection of virus-induced antigens in O,toplasm F
16、or the detection of virus-induced antigens in cytoplasm, the infected cells, after incubation for virus replication, were washed as for surface antigens and FIXED-CELL IMMUNOPEROXIDASE TECHNIQUE 363 then fixed with 80% acetone at -20C for 30 min. After fixation the cells were washed and then saturated as mentioned above. Detection of viral antigens by an immunoperoxidase test (IPT) The surface or cytoplasmic antigens induced by the virus were detected by an IPT as follow
