Resumen

ESTRADA, Eric et al. Restoration in Immunoblotting of Neisseria meningitidis proteins denatured by reducing agents and heat. Vaccimonitor [online]. 2000, vol.9, n.3, pp.19-23. ISSN 1025-028X.

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.

Palabras clave : Immunoblotting; Neisseria meningitidis; detergents; outer membrane proteins; renaturation; denatured proteins.

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