Neutralizing antibodies (nAbs) prevent the entry of viruses into permissive cells. Since nAbs represent correlates of protection against the
Rabies lyssavirus, the presence of sufficient nAbs indicates effective vaccination. Accordingly,
Rabies lyssavirus-specific nAb titers need to be determined in routine diagnostics to identify individuals being at risk of
Rabies lyssavirus
infections due to insufficient immunity. The current gold standard for the quantification of
Rabies lyssavirus-specific nAbs is the rapid fluorescent focus inhibition test (RFFIT). However, RFFITs are expensive and labor-intensive since multiple microplate wells must be evaluated one-by-one by trained personnel through microscopic inspection, which limits the number of samples that can be processed. To overcome this disadvantage, we established a novel assay for
Rabies lyssavirus-specific nAbs relying on an in-cell-ELISA (icELISA)-based neutralization test (icNT). The icNT differs from the RFFIT in the readout phase, and can be automatically quantified in minutes using broadly available microplate readers. During the establishment, icNT parameters such as antibody concentrations, permeabilization procedures, blocking
reagents, infectious doses, and the duration of
infection were optimized. Afterwards, a dose-dependent detection of
Rabies lyssavirus neutralization was demonstrated using the WHO Standard
Rabies Immunoglobulin reference. A panel of 200 sera with known RFFIT titers revealed very good sensitivity and specificity of the icNT. Furthermore, the icNT showed very good intra- and inter-assay precision. By recognizing
Rabies lyssavirus-specific
antigens, the assay can be applied immediately to automatically quantify the concentration of
Rabies lyssavirus nAbs in routine diagnostics or for various basic research questions such as screening for
antiviral compounds.