Establishment and Clinical Validation of an In-Cell ELISA-Based Assay for the Rapid Quantification of Rabies Virus Neutralizing Antibodies

Abstract

Background: 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 absent or insufficient immunity. Methods: 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 by 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), which can be automatically quantified in minutes using broadly available microplate readers. Findings: 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 40 seronegative, 20 positive, and 20 highly positive sera with known RFFIT titers revealed excellent sensitivity and specificity of the icNT. Furthermore, the icNT showed excellent intra- and inter-assay precision. Interpretation: By recognizing Rabies lyssavirus-specific antigens, the assay can be applied immediately to automatically quantify to concentration of Rabies lyssavirus nAbs in routine diagnostics or for various basic research questions such as screening for antiviral compounds. Funding: The authors receive funding by Else-Kroner Promotionskolleg ELAN and the DFG. Declaration of Interest: None to declare. Ethical Approval: The assessment of existing samples for the improvement of diagnostic procedures has been approved by the ethics committee of the Medical Faculty of the University of Duisburg-Essen (#18-8309-BO).