Development of an ELISA for an effective potency determination of recombinant rabies human monoclonal antibody.

Rapid Fluorescence Focus Inhibition Test (RFFIT) is the most widely used cell-based assay to measure the potency of recombinant human rabies monoclonal antibodies. Nonetheless, RFFIT assay is time-consuming and it requires well-equipped biosafety level 2 facility, virulent live rabies virus cultures, permissive cell lines, and well-trained manpower. Therefore, the development of alternative methods to the RFFIT has been encouraged by the World Health Organization (WHO) expert working groups to overcome these barriers. An In-vitro ELISA test has been developed as an alternative to the RFFIT assay, for quantifying the rabies monoclonal antibody (mAb) potency using inactivated rabies virus vaccine (Rabivax-S). It is based on the specific interaction between the antigen and the antibody, that induces neutralizing antibody response to rabies virus. The ELISA was validated involving accuracy and precision within 20 % coefficient of variance. The validation has been done by 4PL standard curve with linearity r² ˃ 0.98 and LLOQ of 0.3 μg/mL indicating high assay sensitivity. The specificity of the assay was ascertained by challenging with another homologous non-rabies humanized mAb, which does not show binding with the rabies virus. The indirect ELISA developed here, is precise, robust, and accurate to quantitate the potency of rabies monoclonal antibody. It is highly sensitive and has a broad range of detection. It is easy to perform, and it has a short turnaround time (results available in few hours). Furthermore, it is cost effective and can be performed with low-cost resource setting, as there is no requirement of handling the live cells and live virus and also BSL-2 Facility.