RESUMO
Laboratory detection of specific foot-and-mouth disease virus (FMDV) is routinely carried out by ELISA and RT-PCR. Identification and serotyping of FMDV by ELISA requires polyclonal antibodies raised in rabbits and guinea pigs. The polyclonal antibodies have certain disadvantages such as batch to batch variation, inconsistent yields of antibodies and limited quantity of serum obtained from individual animals. This paper describes a method wherein monoclonal antibodies and chicken IgY were used in an antigen capture-ELISA for serotyping of thirty tongue epithelial samples and sixty tissue culture fluids. The results were compared with the routine antigen detection ELISA. The present study indicated that monoclonal antibodies and chicken IgY can substitute conventional polyclonal antibodies for routine serotyping of FMDV.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Febre Aftosa/classificação , Febre Aftosa/virologia , Sorotipagem/métodos , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Galinhas , Feminino , Febre Aftosa/imunologia , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Imunoglobulinas/imunologia , Imunoglobulinas/isolamento & purificação , Camundongos , Sensibilidade e EspecificidadeRESUMO
Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25µg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.