Flow cytometry model for the detection of neutralizing antibodies against of IFN-β Modelo de detección de anticuerpos neutralizantes contra IFN-β mediante citometría de flujo

Introduction. Interferon beta (IFN-β) 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-β treatment efficacy. Materials and methods. Two different tumor cell lines (K562 and U937) were cultivated with human recombinant IFN-β1a at different concentrations and time in order to measure the expression of intracellular ISG15, an inducible molecule in the IFN-β1a signaling cascade. Blood was obtained from non-immunized and IFN-β1a 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-β1a stimulated cell lines and ISG15 expression was evaluated by flow cytometry. Results. K562 cell was selected with a dose of 1000 IU of IFN-β1a, 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-β1a. 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-β1a stimulated cells (p=0.008).Immunized rabbit sera decreased the expression of ISG-15 in K5652 cells stimulated with IFN-β1a, 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. Introducción. El interferón beta (IFN-β) se usa para tratamiento de la forma recaída-remisión de la esclerosis múltiple. Sin embargo, el uso de proteínas recombinantes como medicamentos, pueden generar la producción de anticuerpos, disminuyendo así la efectividad del tratamiento. Objetivo. Estandarizar una técnica de detección de anticuerpos neutralizantes contra IFN-β mediante citometría de flujo. Materiales y métodos. Se cultivaron dos líneas tumorales humanas (U937 y K562) con IFN-β1a humano recombinante y mediante citometría de flujo se determinó la expresión de la proteína ISG15 intracelular. Los sueros fueron obtenidos de un conejo Nueva Zelanda pre- y post-inmunización con 100.000 UI de IFN-β1a en adyuvante de Freund. Para la detección de anticuerpos neutralizantes, se estimularon células K562 con IFN-β1a pre-incubado