PubMed Health⌕ Search

Biomedical subjects

J M Escribano

Publications and source records attributed to J M Escribano.

At least 55 records · Page 3Linked to original sources
PubMed1992

Evaluation of two ELISA kits for the detection of Aujeszky's disease antibodies in pigs.

This work describes the evaluation of two commercial ELISA kits for the detection of gI antibodies against Aujeszky's disease. A collection of experimental sera from infected pigs, field sample sera, and sera from pigs vaccinated with seven different modified gI-negative commercial vaccines were used to evaluate each test. Both ELISA kits showed good reproducibility, and specificity, but differences could be appreciated in sensitivity when sera obtained at early stages of infection was analysed. These results also indicated that both kits could be used in conjunction with the seven vaccines evaluated in this study.

Animals↗
PubMed1992

Flow cytometric analysis of African swine fever virus-induced plasma membrane proteins and their humoral immune response in infected pigs.

African swine fever (ASF) virus-induced plasma membrane proteins may contribute to the protective immune response against the disease since they can be involved in the antibody-mediated lysis of infected cells. In this study we describe the regulation of ASF virus-induced plasma membrane protein expression and its antibody induction in pigs after viral infection by flow cytometric analysis. More than 80% of infected cells contained viral antigens on the surface membranes at 6 hr postinfection (hpi), and the relative amount of viral antigen expression was increased at 12 and 20 hpi. The kinetics of individual viral protein expression on cell surfaces was studied by a collection of monospecific antibodies directed against the six viral plasma membrane proteins p12, p15, p16, p23.5, p30, and p35. Most of these proteins were expressed at 6 hpi, with the exception of p35, which was first detected at 12 hpi. The percentage of cells expressing each antigen at different hpi was also determined. The immune response against virus-induced plasma membrane proteins in pigs infected with an attenuated ASF virus strain was studied. Antibodies against viral epitopes exposed on plasma membranes reached a plateau at 20 days postinfection (dpi). The relative amount of antibodies induced during infection with these specificities was not directly related to the antibody titer of the sera. Sera obtained at 20 and 40 dpi contained antibodies against most of the viral plasma membrane proteins and were most efficient in recognition of viral antigens exposed on the surface of infected cells at early times.

African Swine Fever↗
PubMed1992

Characterization of p30, a highly antigenic membrane and secreted protein of African swine fever virus.

We have identified and characterized a 30-kDa phosphoprotein (p30) of African Swine Fever Virus (ASFV) that is synthesized, membrane localized, and released into the culture medium at early times after infection. Sequence analysis of the p30 open reading frame predicts a highly antigenic protein with putative phosphorylation, glycosylation, and membrane attachment sites.

African Swine Fever Virus↗
PubMed1992

Cell culture propagation modifies the African swine fever virus replication phenotype in macrophages and generates viral subpopulations differing in protein p54.

We have detected 86 African swine fever (ASF) virus-induced proteins in infected pig macrophages by two-dimensional electrophoresis. No differences among protein patterns of wild-type viruses could be observed by this methodology. However, during cell culture adaptation and propagation we have characterized changes in the molecular weight of the ASF virus specified protein p54, which show direct correlation with both size and number of viral subpopulation variants generated during cell culture propagation. Passages in culture appear to select for viral subpopulations that specify p54 proteins with higher molecular weights than the wild-type virus. The virus propagation in cell culture also affected its replication phenotype in pig macrophages decreasing the viral titers in these cells between passage 44 and 81. Nevertheless, the changes observed in p54 did not imply differences in biological properties, such as infectivity, virulence or host cell range among viral clones isolated, each one specifying for only one p54 form with different molecular weight. This protein becomes then a valuable quantification marker to follow evolution and generation of ASF virus diversity in vitro.

African Swine Fever↗
PubMed1992

Epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus.

The epitope specificity of the protective immune response against swine transmissible gastroenteritis (TGE) has been investigated by using circulating and secretory antibodies. This study was carried out with sows vaccinated with TGEV or the antigenically related porcine respiratory coronavirus (PRCV). TGEV vaccination of sows resulted in greater lactogenic protection of suckling piglets against TGEV challenge and a higher secretory immune response than PRCV vaccination did. These differences in the immune response were conditioned by the route of antigen presentation as a result of the different tropism of each virus. Epitopes on S protein, and in particular those contained in its antigenic site. A, were more immunogenic than epitopes on N and M proteins in both groups of vaccinated sows, as determined by a competitive radioimmunoassay. Minor differences in antibody response against the previously defined antigenic subsites Aa, Ab, and Ac were also detected, with subsite Ab being the most antigenic in both TGEV- and PRCV-immune sows. These findings suggest that antigenic site A on S protein, involved in virus neutralization, is the immunodominant site in pregnant sows that confer lactogenic protection. They also validate, in experiments with secretory antibodies, the antigenic maps made with murine monoclonal antibodies. Therefore, this antigenic site should be considered for vaccine or diagnostic development.

Animals↗
PubMed1992

A sensitive dot immunobinding assay for serodiagnosis of African swine fever virus with application in field conditions.

The present work describes a simple dot immunobinding assay (DIA) for African swine fever virus (ASFV) antibody detection that can be used under field conditions. The assay uses nitrocellulose strips dotted with a cytoplasmic soluble antigen (CS-P) of ASFV. The nitrocellulose strips are adhered to a plastic handle. The test serum samples react with the CS-P, and antibodies are detected using a protein A-peroxidase conjugate. Both incubations are carried out at 20 C. The efficacy of the DIA as a screening test for ASFV was compared to an enzyme-linked immunosorbent assay (ELISA) and an immunoblotting (IB) test using 343 sera collected from natural African swine fever epizootics and from inapparent ASFV carriers. The DIA had comparable sensitivity to both reference techniques, and all samples positive in the ELISA and IB test were also positive in the DIA. False-positive reactions were not detected when whole blood or poorly preserved serum samples were tested by DIA. Some poorly preserved sera that were positive initially by the ELISA were no longer ELISA positive in a later run, although they were positive in IB and DIA. These positive DIA and IB test results could be caused by the differences in antibody epitope binding.

African Swine Fever↗
PubMed1991

Proteins specified by swine transmissible gastroenteritis virus: identification of non-structural proteins by two-dimensional electrophoresis.

Four virus-induced non-structural proteins with apparent molecular weights of 11-14 kilodaltons (kDa) were identified by two-dimensional electrophoresis in cells infected by TGEV. Differences in the number of non-structural proteins were observed among virulent and attenuated TGEV strains as well as with two antigenically related feline and canine coronaviruses.

Animals↗
PubMed1990

Antigenic homology among coronaviruses related to transmissible gastroenteritis virus.

The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested.

Animals↗
PubMed1990

Evaluation of sensitivity of different antigen and DNA-hybridization methods in African swine fever virus detection.

ELISA, immunodot and DNA hybridization methods have been adapted to detect African swine fever virus (ASFV), and their sensitivities were compared using virus obtained from cell cultures. About 2.3 x 10(2) 50% hemadsorbing doses (HAD50) of virus were detected with ELISA sandwich using an anti-ASFV IgG biotinylated followed by avidin-peroxidase. The immunodot technique showed similar sensitivity, detecting about 4.6 x 10(2) HAD50 of virus. ASFV-DNA was detected using radioactive DNA probes and molecular hybridization. The maximal viral detection capacity of this technique was about 1.8 x 10(3) HAD50. The antigenic and DNA detection of ASFV during the infection of animals with virulent and attenuated viruses, was also studied. For this purpose, sera and red blood cells from several infected pigs were obtained at different days post-inoculation. The virus was detected at the third day after infection by the three methods. However, ASFV-DNA detection was more efficient than antigenic detection at nine days post-inoculation, when antigen detection failed, because immunocomplexes with circulating viruses were formed in the subacute infection.

African Swine Fever↗
PubMed1990

Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed.

The peplomer protein (S) and the transmembrane protein (M) of transmissible gastroenteritis virus (TGEV) of swine were identified by iodination and serologically on the surface of infected cells. Of a total of 4 monoclonal antibodies (mAb) directed against four antigenic sites of S protein (Correa et al., 1988), 3 specific for sites A, B and D attached to the plasma membrane of infected cells, as disclosed by indirect immunofluorescence and by complement-mediated cytolysis. Four of the mAbs assayed were specific for the viral protein M and two of them gave plasma membrane immunofluorescence and mediated cytolysis in the presence of complement. The viral nucleoprotein N could not be demonstrated on the surface of infected cells either by iodination or employing 3 mAbs against this protein. Finally, a time course infection experiment demonstrated that S and M proteins were expressed on the surface of infected cells at 4 h after infection, before infective virus was released from infected cells.

Animals↗
PubMed1990

Comparison of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus.

A radioimmunoprecipitation assay (RIPA) has been developed for detection of antibody to African swine fever virus (ASFV) and compared with the immunoblot assay with regard to sensitivity and specificity. Two hundred seven field sera, obtained from pigs in Spain from different geographic areas between 1975 and 1986, that were positive by ASFV enzyme-linked immunosorbent assay (ELISA) were also analysed by immunoblot assay and RIPA. By serum dilution experiments, the RIPA appeared at least as sensitive as the ELISA and immunoblotting tests, although ELISA and RIPA detected antibodies to ASFV earlier in natural infection than did the immunoblot assay, as disclosed by animal inoculation studies. The most antigenic ASFV-induced proteins in natural infection detected by RIPA were the viral proteins p243, p172, p73, p25.5, p15, and p12 and the infection proteins p30 and p23.5. In the immunoblot assay, the proteins that were most reactive with the same sera were the viral protein p25.5 and the infection proteins p30, p25, and p21.5. Only 1 serum, from an animal infected with ASFV, was negative by immunoblot assay but showed a positive result by RIPA. A modification of conventional RIPA was performed using a dot transference of immunoprecipitated proteins to a nitrocellulose filter. This modification simplified the conventional RIPA procedures by eliminating the electrophoresis of immunoprecipitated proteins without affecting sensitivity and specificity. The ease of use, specificity, and the sensitivity comparable to that of the immunoblot assay make the RIPA a useful confirmatory assay for sera that yield conflicting results in other ASFV antibody assays.

African Swine Fever↗
PubMed1990

Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody.

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% has positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5 IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of ASFV-infected pigs.

African Swine Fever Virus↗
PubMed1989

African swine fever virus-induced proteins on the plasma membranes of infected cells.

The African swine fever virus-induced proteins on plasma membranes of infected cells have been studied by two different procedures, iodination and incubation of infected cells labeled with [35S]methionine with a specific antiserum, obtained from pigs immunized with a monkey stable cell-adapted African swine fever virus. The combined use of both procedures identified proteins IP56, IP51, IP35, IP34, IP31, IP30, IP25.5, IP23.5, IP16, IP15, IP14, and IP12 as viral antigens exposed on the surface of infected cells. Proteins IP16, IP15, and IP14 were recognized by the immune serum from survivor pigs, obtained after challenge with homologous virulent virus, but not by the immune serum from the same pigs immunized only with the cell-adapted virus.

African Swine Fever Virus↗
PubMed1989

[The prevalence of antibodies to influenza virus and respiratory coronavirus in fattening pigs in Spain].

The presence of antibodies to two influenza viruses of the type A (H1N1 and H3N2) and to a porcine respiratory coronavirus was investigated in a study lasting a year. 735 blood serum samples were collected from 79 closed pig fattening farms in the province Segovia (Spain). Hemagglutination inhibition was used with influenza viruses. The percentage of positive results was 78.5% and 62.5% respectively for the serotypes H1N1 and H3N2. A clear reduction in the spread of antibodies was observed in the autumn. The ELISA technique was used with the porcine respiratory coronavirus. As antigen we used the antigenically related transmissible porcine gastroenteritis virus. Using this technique 87% of the sera were positive. Some of these sera with representative ELISA values were confirmed by means of serum neutralisation and radioimmune precipitation of the viral proteins. The incidence of these antibodies remained unchanged over the whole year of the investigation.

Animals↗
PubMed1989

Antibodies to bovine serum albumin in swine sera: implications for false-positive reactions in the serodiagnosis of African swine fever.

Antibodies to bovine serum albumin were detected in swine sera by use of an immunoblotting technique. Such sera had false-positive reactions, as determined by results of African swine fever virus serodiagnostic techniques when bovine serum albumin was a contaminant in the soluble cytoplasmic antigen obtained from infected cells cultured in the presence of bovine serum. The soluble cytoplasmic antigen obtained from cell cultures infected with African swine fever virus in the presence of porcine serum did not react with the false-positive sera and, therefore, was used for African swine fever virus serodiagnostic methods, with 0% false-positive results.

African Swine Fever↗
PubMed1989

Detection of African swine fever virus antibodies by immunoblotting assay.

An immunoblotting assay has been adapted to detect antibodies against African swine fever virus. The electrophoretic transfer of proteins and the immunoreaction conditions were optimized, using 4 mA/cm2 of current intensity and 10 micrograms of soluble cytoplasmic antigen of infected cells per strip. Filters of polyvinylidene difluoride showed the highest capacity for protein absorption, but nitrocellulose filters showed lower backgrounds. The specificity and the pattern of the proteins induced by African swine fever virus that react with the antisera were determined in immunoblotting assay, IP30 being the most reactive protein.

African Swine Fever Virus↗
PubMed1983

Proteins specified by African Swine Fever virus. IV. Glycoproteins and phosphoproteins.

African Swine Fever virus infected MS cells labeled with radioactive 14C-amino acids, 32Pi or [3H]-glucosamine were examined by high resolution sodium dodecylsulfate polyacrylamide gel electrophoresis and showed 43 infected cell polypeptides. Twenty-one of these proteins were present in the nuclear fraction of infected cells. At least 22 of the infected cell polypeptides induced antibodies during natural infections in swine. The pattern of infected cell polypeptides modified by incorporation of showed prosthetic groups that at least 8 polypeptides were phosphorylated and at least three specific viral glycoproteins (A, B and C) were detected by immunoprecipitation. The most highly glycosylated polypeptide corresponds to the structural viral protein VP51.

African Swine Fever Virus↗