RESUMO
F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 microg of F1 antigen/disc, 3% w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.
Assuntos
Anticorpos Antibacterianos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/instrumentação , Peste/imunologia , Álcool de Polivinil/química , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/economia , Filtração/instrumentação , Cabras , CoelhosRESUMO
F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3 percent w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.
Assuntos
Animais , Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Peste/diagnóstico , Álcool de Polivinil/farmacologia , Yersinia pestis/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/sangue , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/instrumentação , Cabras , Peste/imunologia , CoelhosRESUMO
Soluble adult Schistosoma mansoni antigen preparation (SWAP) was covalently fixed onto polyvinyl alcohol-glutaraldehyde discs and an enzyme linked-immunosorbent assay (ELISA) was set up. The best conditions for the assay were established and it was found that small amount of antigen such as 1.5 micrograms was required. A comparison between this procedure and the conventional ELISA was proceeded. A reliable method of antigen immobilization was achieved and the low prices of the employed reagents are economically attractive.
Assuntos
Glutaral , Álcool de Polivinil , Esquistossomose/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática , Humanos , CoelhosRESUMO
Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 micrograms of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.