Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2 (original) (raw)
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Journal of Clinical Microbiology
We describe in this study a rapid enzyme immunoassay for the titration of neutralizing antibodies in serum against Semliki Forest virus. For this assay L cells were added to preincubated virus-antiserum mixtures to form monolayers. Six hours after infection by residual, nonneutralized virus, the monolayers were fixed, and the E2 glycoprotein of Semliki Forest virus on the surface of infected cells was quantified with an E2-specific, peroxidase-labeled monoclonal antibody (UM 5.1). The serum antibody titer was defined arbitrarily as the inverse value of that dilution of serum associated with a 25'% inhibition of control absorbance values. These titers of both early and later mouse immune sera were similar to those determined in simultaneously performed 50% plaque reduction tests. The results indicate that the enzyme immunoassay (duration, 9 h) is reliable and compares favorably with the conventional plaque reduction test (duration, 25 h) in rapidity, ease of performance, and objectivity.
Journal of virology, 1984
Fifteen monoclonal antibodies (MAs) directed against either the E1 or E2 glycoprotein of Semliki Forest virus (SFV) were characterized by immunoglobulin subclass, pI traject, hemagglutination inhibition, neutralization of infectious virus, and protection against virulent infection in mice. All MAs except UM8.4 (immunoglobulin M [IgM]) belonged to various subclasses of IgG and predominantly to IgG2a, but all were unique as indicated by their banding patterns in isoelectric focusing. Competitive binding assays with these MAs revealed the presence of at least six distinct antigenic determinants (epitopes) on the E1 glycoprotein and five epitopes on the E2 glycoprotein. Two of the epitopes on E1, as defined by the properties of the MAs, were associated with hemagglutination inhibition (E1c and E1d), three were associated with neutralization (E1a, E1b, and E1f), and five were associated in various degrees with protection (E1a, E1b, E1c, E1e, and E1f) of mice against virulent SFV infectio...
A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers
Journal of Immunological Methods, 1979
A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays using peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods.
Mechanisms of monoclonal antibody-mediated protection against virulent Semliki Forest virus
Journal of virology, 1985
Both neutralizing and nonneutralizing immunoglobulin G2a monoclonal antibodies (MAs) directed against the E2 glycoprotein of Semliki Forest virus (SFV) protected mice prophylactically and therapeutically against virulent SFV infection. The neutralizing MAs, however, conferred protection to mice at lower doses than did nonneutralizing MAs. The antibody-dependent, complement-mediated cytolysis of SFV-infected L cells was effectuated by both kinds of antibodies, but again neutralizing MAs were more effective. Removal of the Fc part of the neutralizing MA UM 5.1 by pepsin digestion resulted in a 100-fold reduction of the neutralization titer (10(4) versus 10(6)) and a complete loss of its capacity to mediate antibody-dependent, complement-mediated cytolysis. Passive protection of infected mice occurred only after administration of relatively high doses of F(ab')2 of MA UM 5.1 (30.0 micrograms versus 0.1 microgram). F(ab')2 fragments prepared from the nonneutralizing MA UM 4.2 ha...
The molecular pathogenesis of Semliki Forest virus: a model virus made useful?
The Journal of general virology, 1999
The effect of infection with Sindbis virus and its temperature-sensitive mutants on cellular protein and DNA synthesis. Virology 71, 593-597. Atkins, G. J. (1977). Cytopathogenicity of temperature-sensitive mutants of Sindbis virus. FEMS Microbiology Letters 2, 51-55. Atkins, G. J. (1983). The avirulent A7 strain of Semliki Forest virus has reduced cytopathogenicity for neuroblastoma cells compared to the virulent L10 strain.
Cellular immunity against Semliki Forest virus in mice
Infection and immunity, 1979
Intracutaneous immunization of BALB/c mice with purified inactivated Semliki Forest virus resulted in cellular immunity without detectable antibodies. The animals were protected against subcutaneous challenge, from which the challenge virus spreads slowly. After intraperitoneal challenge, which permits a rapid virus spread, the protection was marginal. Stimulation of the intraperitoneal cell population with thioglycolate before challenge resulted in complete protection. The protection could be transferred to normal mice with peripheral lymph node cells, but not with spleen cells. The course of the infection in immunized and normal mice was also studied. Semliki Forest virus does not multiply in peritoneal cells in vivo. In immunized mice part of the challenge virus in the peritoneal cavity was rapidly eliminated and viremia was reduced. After challenge, immunized mice produced less antibody than normal mice.
Isolation and Preliminary Characterization of Semliki Forest Virus Mutants with Altered Virulence
Journal of General Virology, 1980
SUMMARY Four mutants of Semliki Forest virus (SFV) strain LIo showing altered virulence in weanling mice were selected following mutagenesis with N-nitro-N-methyl-N'- nitrosoguanidine. Intraperitoneal (i.p.) injection of wild-type (wt) virus resulted in the death of all mice given more than 30 p.f.u. Protection of mice surviving injec- tion with lower doses of virus was not obtained. A proportion of mice