Calculating rabies virus neutralizing antibodies titres by flow cytometry Detecção e quantificação de anticorpos anti-rábicos neutralizantes pela técnica de citometria de fluxo

• Published in: Revista do Instituto de Medicina Tropical de São Paulo
• Main Authors: Juliano BORDIGNON, Fabiano COMIN, Sílvia Córdoba P. FERREIRA, Graciane M. M. CAPORALE, José Hermênio Cavalcante LIMA FILHO, Carlos Roberto ZANETTI
• Format: Article in Journal/Newspaper
• Language: English
• Published: Universidade de São Paulo (USP) 2002
• Subjects: Rabies virus; Flow cytometry; Neutralizing antibodies; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1590/S0036-46652002000300007; https://doaj.org/article/a2cf63236da04c09bf0379d0571da8a5

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity . A titulação de anticorpos neutralizantes contra o vírus rábico (AcN) pós-imunização é um parâmetro aceito como indicador de eficácia da vacina e do sucesso do processo de imunização. Este estudo descreve um teste de neutralização realizado em cultura de células, analisado através da técnica de citometria de fluxo. Diluições seriadas de amostras de soro foram misturadas in vitro com vírus rábico e adicionados a células ...