Production, characterization and application of monoclonal antibodies against the virus of infectious bronchitis

A bronquite infecciosa das galinhas (BI) ?? uma doen??a aguda, altamente contagiosa, causada por um Coronav??rus que infecta principalmente c??lulas dos aparelhos respirat??rio e genito-urin??rio das galinhas. A import??ncia econ??mica dessa doen??a decorre da diminui????o na produ????o e qualidade interna e externa dos ovos, da redu????o da eclodibilidade, da diminui????o da efici??ncia alimentar e do ganho de peso e do aumento da mortalidade e da condena????o de carca??as ao abate, al??m dos gastos com medicamentos para debelar infec????es bacterianas secund??rias. A confirma????o do diagn??stico de BI ?? baseada no isolamento viral, em conjunto com a sorologia. O uso de anticorpos monoclonais (AcMs) tem colaborado e facilitado o diagn??stico desse tipo de enfermidade, por??m seu uso ainda ?? limitado, pois os AcMs utilizados s??o importados, elevando o custo de tais metodologias. Este trabalho relata a produ????o, caracteriza????o e aplica????o de AcMs contra o v??rus da bronquite infecciosa das galinhas (VBI). Para a produ????o dos AcMs, camundongos Balb/c foram imunizados intraperitonealmente com o VBI cl??ssico (M41) local ou com o variante. Foram gerados dois hibridomas secretores de AcMs anti-VBI do isotipo IgM a partir da fus??o entre os linf??citos B de um camundongo imunizado com o VBI variante e c??lulas de mieloma. Foi observado, na caracteriza????o por ELISA indireto e Western blot, que os AcMs reconhecem o VBI cl??ssico e variante, sendo as rea????es com o VBI variante mais intensas. O AcM 2G4 parece ser espec??fico para a prote??na S1. A aplicabilidade dos AcMs foi avaliada atrav??s da t??cnica de imuno-histoqu??mica em tecidos de aves inoculadas experimentalmente com o VBI cl??ssico. Os AcMs anti-VBI apresentaram o mesmo padr??o de marca????o que o AcM utilizado como controle positivo, ou seja, marcaram predominantemente c??lulas inflamat??rias. Este resultado pode ser explicado pelo fato de que, em casos mais severos da infec????o por VBI, o ant??geno pode ser localizado tamb??m em c??lulas inflamat??rias. Concluiu-se que os AcMs obtidos apresentam potencial para uso no imunodiagn??stico para BI.The infectious bronchitis (IB) is an acute and highly contagious disease caused by a coronavirus that infects mainly cells of the respiratory and genitourinary of chicken. The economic importance of this disease results from decreased production, such as internal and external quality of eggs, reduced hatchability, feed efficiency and weight gain, increased mortality, condemnation of carcasses at slaughter, and also with drugs spending to suppress secondary bacterial infections. The diagnosis of IB is based on virus isolation, together with serology. The use of monoclonal antibodies (MAbs) has been collaborated to diagnosis of this type of disease, but their use are still limited because the MAbs used are imported, increasing the cost of such methodologies. This work reports the production, characterization and application of MAbs against the virus of infectious bronchitis (IBV). For the production of MAbs, Balb/c mice were immunized intraperitoneally with classic IBV (M41) or variant IBV. We generated two hybridomas secreting MAbs anti-IBV (IgM isotype) from the fusion of B-lymphocytes from a mouse immunized with the variant IBV and myeloma cells. Following the characterization by ELISA and Western blot, it was observed that MAbs recognize the IBV classic and variant, showing more intense reactions with IBV variant. The AcM 2 G4 seems to be specific for protein S1. The applicability of the MAbs was assessed using the immunohistochemistry technique on histological tissues of birds inoculated experimentally with classic IBV. These MAbs anti-IBV had the same pattern of label of the MAb used as positive control, labeling predominantly inflammatory cells. This result can be explained by the fact that in severe cases of infection with IBV, the antigen may be located also in inflammatory cells. It was concluded that the MAbs obtained have potential for use in immunodiagnostic for IB