Validation of Adapted Neutralization Assays Developed to Discriminate Anti-Rabies Virus Activity of Two Different Anti-Rabies Virus Monoclonal Antibodies Administered as a Combination

Assessment of rabies virus (RABV) neutralizing antibodies in subjects vaccinated or injected with anti-RABV immunoglobulins is central in determination of rabies protection. The rapid fluorescent focus inhibition test (RFFIT) is used for assessment of anti-RABV activity in serum. The current anti-RABV polyclonal preparations on the market pose difficulties in production and vary in quality. RABV neutralizing monoclonal antibodies (MAbs) are being evaluated as replacements. Different anti-RABV MAbs may neutralize different RABV isolates, thus two or more MAbs directed against different epitopes on the RABV glycoprotein are needed. It is therefore important to ensure neutralizing activity against all RABV isolates in sera of subjects injected with an anti-RABV MAb product consisting of two or more MAbs. The RFFIT, utilizing CVS-11 as challenge virus, cannot discriminate between the activities of different anti-RABV MAbs. We developed and validated two RFFIT methods enabling specific assessment of two different anti-RABV MAbs (CR57 and CR4098) in using two mutant CVS-11 strains resistant to either CR57 or CR4098 neutralization. The validation results demonstrate that both RFFIT assays using MAb resistant RABV are precise, accurate, linear, specific, and stable within the linear range of 0.025 IU/mL to 1.0 IU/mL. This method design can, therefore, be used to determine MAb specific anti-RABV activity in human serum samples.