Calculating rabies virus neutralizing antibodies titres by flow cytometry.
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.
Bordignon
Juliano
J
Departament of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianópolis, SC, 88040-900, Brazil.
Comin
Fabiano
F
Ferreira
Sílvia Córdoba P
SC
Caporale
Graciane M M
GM
Lima Filho
José Hermênio Cavalcante
JH
Zanetti
Carlos Roberto
CR
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D004285:Dogs; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D009500:Neutralization Tests; D011818:Rabies / Q000517:prevention & control; D011819:Rabies Vaccines / Q000276:immunology*; D011820:Rabies virus / Q000276:immunology*; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D011819:Rabies Vaccines