Desarrollo de una elisa para la detección de anticuerpos IgM anti.ipnv en salmón
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Archivos
Fecha
2002
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
Título de la revista
ISSN de la revista
Título del volumen
Editor
Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
Las pérdidas económicas generadas por el virus de la necrosis
pancreática infecciosa (IPNV) han conllevado al desarrollo de técnicas para la
rápida detección del patógeno y al control de la enfermedad mediante el uso de
vacunas. En este contexto, se estudió el desarrollo de un ELISA que permitiría
evaluar la respuesta inmune de los peces inmunizados.
El primer objetivo fue caracterizar un panel de anticuerpos
monoclonales anti-lgM de salmónidos, del cual se seleccionó el anticuerpo
3H7/E1 . Este anticuerpo fue exitosamente conjugado con peroxidasa ,
constituyendo uno de los componentes importantes en el desarrollo del ELISA.
Además se determinó la concentración óptima de virus necesario para la
activación de la placa, y la dilución del anticuerpo monoclonal anti-lgM
conjugado a peroxidasa. Para la versión indirecta del ELISA, también se evaluó
la dilución adecuada de un anti-lgG de ratón conjugado a peroxidasa.
Para evaluar el ensayo se utilizó un panel de 49 sueros provenientes
de peces vacunados con una virina del virus, y de 50 sueros de peces
controles no vacunados. Al asumir un valor de corte ("cut-off') para el ensayo
de ELISA de 0,3 0 .0 . a 450 nm, el grupo inmunizado presento un 89% de
lecturas por sobre dicho valor, lo que sería equivalente a peces positivos. En el
grupo control sólo un 44% de los sueros presentaba títulos de lgM altos contra
el virus. Sin embargo, sueros que poseían altas lecturas de anticuerpos antiIPNV
reaccionaron positivamente en placas activadas con Piscirickettsia
sa/monis. Para determinar la especificidad de las lgM, estas fueron absorbidas
con el virus, P. salmonis y otros patógenos de peces. Al absorber las lgM con los otros patógenos de peces se encontró que los títulos contra el virus o contra
P. salmonis no fueron afectados. Sin embargo, los resultados de absorción con
el virus o con P. salmonis indicaron que existe reacción cruzada de los sueros
entre ambos patógenos, lo que sugiere que las lgM presentes en el suero son
inespecíficas o reaccionan con epítopos de algunos antígenos que son
comunes al virus y a P. salmonis.
The economic loss generated by the infectious necrosis pancreatic virus (IPNV) has lead to the development of techniques for fast detection of the pathogen and to control the disease by vaccination. In this context, the development of an ELISA which may allow us to evaluate the immune response of immunised fish has been studied. The first objective was to characterize a panel of monoclonal anti-salmon lgM antibodies, from which the 3H7/E1 antibody was selected. This antibody was successfully conjugated with peroxidasa, being one of the important component for the development of the ELISA. Furthermore, the optimal concentration of virus needed to activate the plate and the optimal dilution of the monoclonal anti-lgM antibody conjugated with peroxidasa was determinated. For the indirect version of the ELISA, we also evaluated the optimal dilution of a mouse anti-lgG antibody conjugated with peroxidasa. To evaluate the assay we used a panel of 49 fish sera that were vaccinated whit a virine vaccine against IPNV, and 50 sera of non immunized control fish. A "cut-off' value of 0,3 0.0. at 450 nm was assumed. The vaccinated group showed a 89% of lectures over that value, considering these fish as positives. In the control group, only 44 % of the serum presented high titres against the virus. However, serum that have positives lectures of antiIPNV antibodies reacted positively in ELISA plate activated with Piscirickettsia sa/monis. To determinate the lgM specifity, these sera were absorbed with the virus, P. sa/monis and other fish pathogens. When the serum was absorbed with the other fish pathogens, we found that the titres against the virus or against P. salmonis were not affected . Nevertheless, the results of the absorption with the virus or with P. salmonis showed that cross-reaction of the serum between these two pathogens exists, and suggesting that the lgM present in the serum are either non specific or reacted with sorne epitopes of antigen common to the virus and P. salmonis.
The economic loss generated by the infectious necrosis pancreatic virus (IPNV) has lead to the development of techniques for fast detection of the pathogen and to control the disease by vaccination. In this context, the development of an ELISA which may allow us to evaluate the immune response of immunised fish has been studied. The first objective was to characterize a panel of monoclonal anti-salmon lgM antibodies, from which the 3H7/E1 antibody was selected. This antibody was successfully conjugated with peroxidasa, being one of the important component for the development of the ELISA. Furthermore, the optimal concentration of virus needed to activate the plate and the optimal dilution of the monoclonal anti-lgM antibody conjugated with peroxidasa was determinated. For the indirect version of the ELISA, we also evaluated the optimal dilution of a mouse anti-lgG antibody conjugated with peroxidasa. To evaluate the assay we used a panel of 49 fish sera that were vaccinated whit a virine vaccine against IPNV, and 50 sera of non immunized control fish. A "cut-off' value of 0,3 0.0. at 450 nm was assumed. The vaccinated group showed a 89% of lectures over that value, considering these fish as positives. In the control group, only 44 % of the serum presented high titres against the virus. However, serum that have positives lectures of antiIPNV antibodies reacted positively in ELISA plate activated with Piscirickettsia sa/monis. To determinate the lgM specifity, these sera were absorbed with the virus, P. sa/monis and other fish pathogens. When the serum was absorbed with the other fish pathogens, we found that the titres against the virus or against P. salmonis were not affected . Nevertheless, the results of the absorption with the virus or with P. salmonis showed that cross-reaction of the serum between these two pathogens exists, and suggesting that the lgM present in the serum are either non specific or reacted with sorne epitopes of antigen common to the virus and P. salmonis.
Notas
Tesis (Ingeniero en Acuicultura)
Palabras clave
Salmones, Test de Elisa, Detección de anticuerpos