ARTICULOS ORIGINALES

 

Restauración en el Inmunoblotting de proteínas de Neisseria meningitidis dañadas por calor y agentes reductores.

 

 

Restoration in Immunoblotting of Neisseria meningitidis proteins denatured by reducing agents and heat.

 

Eric Estrada, Rolando Ochoa, Xenia Ferriol, Ana García, Juan Carlos Martínez, Franklin Sotolongo.

Instituto Finlay. Centro de Investigación-Producción de Vacunas y Sueros. Ciudad de La Habana, Cuba. E-mail: [e-mail]

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RESUMEN

En este trabajo se utilizaron cinco detergentes para restaurar las proteínas de membrana externas (PME) del meningococo dañadas por el efecto del calor y de agentes reductores utilizados en el Inmunoblotting. La acción de los detergentes fue evaluada en la solución de lavado, en el diluente de la muestra y del conjugado. Las bandas de proteínas, reconocidas por la IgG del suero, fueron identificadas usando un conjugado anti IgG humana peroxidasa. Los antígenos reconocidos por el control positivo se corresponden con las proteínas P1, P3, P4 y P5 atendiendo a su peso molecular. Además, fueron reconocidas bandas de 80, 70, 24 kDa y otra con peso mayor a 150 kDa. En general el reconocimiento de todas las PME, excepto esta última de alto peso molecular (APM), se vieron favorecidas con la utilización del Tween 20, con el que se logró un incremento del número y la intensidad de las bandas así como la disminución de los fondos con respecto al resto de detergentes evaluados (Empigen BB, Triton X-100, Nonidet NP-40 y CHAPS). El reconocimiento de la proteína de APM (>150 kDa) se vio afectado por la presencia de detergente como el Tween 20 y Empigen BB. Los lavados con Tween 20
constituyeron los pasos más importantes en la renaturalización de los sitios de unión de la IgG a las PME.

Palabras claves: Inmunoblotting, Neisseria meningitidis, detergentes, proteínas de membrana externa, renaturalización, proteínas desnaturalizadas.

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ABSTRACT

Five different detergents for restoration of IgG antibody binding were studied in the washing steps and in the buffer solution used for the dilution of the conjugate and samples. Binding of human serum IgG antibodies to outer membrane protein (OMP) was detected with class-specific peroxidase-conjugated human antibodies. The positive control reacted with those proteins whose molecular weight corresponded approximately to P1, P3, P4 and P5. Proteins of 80 kDa, 70 kDa and 24 kDa and another protein of >150 kDa were also recognized. Our studies indicated that Tween 20 was the best detergent for restoration of OMP antigenicity, except for the 150 kDa protein. Tween 20 achieved a greater number and intensity of bands and a lower background with respect to Empigen BB, Triton X-100, Nonidet NP-40 and CHAPS. The use of detergents affected the antigenicity of the 150 kDa protein. Washing with Tween 20 were the most important steps for restoration of IgG antibody binding sites of heat-denatured OMPs.

Key words: Immunoblotting, Neisseria meningitidis, detergents, outer membrane proteins, renaturation, denatured proteins.

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