%0 Journal Article
%T Flow cytometry model for the detection of neutralizing antibodies against of IFN-¦Â Modelo de detecci¨®n de anticuerpos neutralizantes contra IFN-¦Â mediante citometr¨ªa de flujo
%A Juan Carlos Villa-Camacho
%A Juan Camilo Vargas-Zambrano
%A John Mario Gonz¨¢lez
%J Biom¨¦dica
%D 2012
%I Instituto Nacional de Salud
%X Introduction. Interferon beta (IFN-¦Â) is a treatment for relapsing remitting multiple sclerosis (RRMS). However, the therapeutic use of recombinant proteins induces a humoral immunologic response resulting in the induction of binding (BAbs) or neutralizing (NAbs) antibodies against the biological product. The presence of neutralizing antibodies has been associated with decreased IFN-¦Â treatment efficacy. Materials and methods. Two different tumor cell lines (K562 and U937) were cultivated with human recombinant IFN-¦Â1a at different concentrations and time in order to measure the expression of intracellular ISG15, an inducible molecule in the IFN-¦Â1a signaling cascade.  Blood was obtained from non-immunized and IFN-¦Â1a immunized (100,000 IU) New Zealand rabbit. The presence of BAbs was evaluated by ELISA. For NAbs detection, sera 1:20 dilution were added to the IFN-¦Â1a stimulated cell lines and ISG15 expression was evaluated by flow cytometry. Results. K562 cell was selected with a dose of 1000 IU of IFN-¦Â1a, and a 1:100 dilution for the primary antibody and a 1:200 dilution for the secondary antibody as the best conditions for the assay. ISG15 expression was compared between cells alone or cultivated with IFN-¦Â1a. Mean fluorescence intensity (MFI) for ISG-15 expression median was 198 arbitrary units (AU) with interquartile ranges of 173-231 AU for non-stimulated cells and 430 UA with interquartile ranges of 316-611.5 UA for IFN-¦Â1a stimulated cells (p=0.008).Immunized rabbit sera decreased the expression of ISG-15 in K5652 cells stimulated with IFN-¦Â1a, whereas non-immunized rabbit sera did not. Conclusions. This rabbit model demonstrates that ISG-15 expression evaluated with flow cytometry can be used as a detection assay for NAbs. Introducci¨®n. El interfer¨®n beta (IFN-¦Â) se usa para tratamiento de la forma reca¨ªda-remisi¨®n de la esclerosis m¨²ltiple. Sin embargo, el uso de prote¨ªnas recombinantes como medicamentos, pueden generar la producci¨®n de anticuerpos, disminuyendo as¨ª la efectividad del tratamiento. Objetivo. Estandarizar una t¨¦cnica de detecci¨®n de anticuerpos neutralizantes contra IFN-¦Â mediante citometr¨ªa de flujo. Materiales y m¨¦todos. Se cultivaron dos l¨ªneas tumorales humanas (U937 y K562) con IFN-¦Â1a humano recombinante y mediante citometr¨ªa de flujo se determin¨® la expresi¨®n de la prote¨ªna ISG15 intracelular. Los sueros fueron obtenidos de un conejo Nueva Zelanda pre- y post-inmunizaci¨®n con 100.000 UI de IFN-¦Â1a en adyuvante de Freund. Para la detecci¨®n de anticuerpos neutralizantes, se estimularon c¨¦lulas K562 con IFN-¦Â1a pre-incubado
%K Anticuerpos neutralizantes
%K c¨¦lulas K562
%K citometr¨ªa de flujo
%K esclerosis m¨²ltiple
%K interfer¨®n beta
%K ISG-15.
%U http://www.revistabiomedica.org/index.php/biomedica/article/view/637