Calculating rabies virus neutralizing antibodies titres by flow cytometry

Abstract

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.

Titulação de anticorpos contra o vírus da raiva em cães, em Campo Grande, MS, na Campanha Anti-Rábica de 2003

“…Em estudo comparativo entre o RFFIT e o Fluorescent Antibody Vírus Neutralization (FAVN), não foi encontrada diferenças significativas entre o uso deles para detecção de anticorpos no soro sanguíneo 6 8 . Comparando o uso do Flow Cytometry (CF) com o Simplified Fluorescent Inibition Test (SFIMT) e o Enzyme-Linked Immunosorbent Assay (ELISA), os autores encontraram correlação de 0,7712 e 0,6702, respectivamente, indicando que o CF pode ser usado para análises de títulos de anticorpos neutralizantes em soro 5 .…”

Raiva: uma breve revisão

“…No Brasil, Piza et al [111] avaliaram um teste de ELISA para a detecção de anticorpos em pessoas vacinadas. Em outro estudo de pesquisadores brasileiros, avaliaram a citometria de fluxo para a detecção de anticorpos contra o VR [16].…”

Measures of Rabies Immunity

“…As with ELISA techniques, some steps of the virus neutralization test can be automated, especially when performed using micro-titer plates, for example, the addition of media to the plates, serial dilution of the serum samples, addition of virus and anti-rabies virus conjugate, as well as some of the more tedious steps such as plate washing, allowing the application of these methods to high-throughput testing. Using this modified CVS-11 in combination with flow cytometry to detect residual virus present after incubation with serum, reportedly increases the sensitivity because each cell is individually assessed for viral infectivity, creating a more precise percentage of viral inhibition (Bordignon et al, 2002). No significant difference was noted when comparison was made between the rabies virus antibody levels reported by automated and non-automated reading, but the automated reading resulted in lower sensitivity.…”