Development and Qualification of a Nipah Virus Glycoprotein-Specific IgG ELISA for the Assessment of Human Antibody Responses.

Background/Objectives: Nipah virus (NiV) is a highly pathogenic zoonotic virus with fatality rates exceeding 70% and causes recurring outbreaks in South and Southeast Asia. Reliable serological assays are critical for outbreak surveillance, diagnosis, and evaluation of vaccine-induced immune responses. This study aimed to develop and qualify an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant NiV glycoprotein G for the detection of virus-specific IgG antibodies in human serum. Methods: An indirect ELISA was developed and optimized for antigen concentration, blocking conditions, and serum dilution. The assay performance was evaluated using convalescent human sera from Bangladesh, along with the World Health Organization (WHO) International Standard for anti-Nipah virus antibodies, maintained and distributed by the National Institute for Biological Standards and Control (NIBSC). Analytical validation was conducted in accordance with ICH Q2 (R2) guidelines, including assessments of sensitivity, specificity, Precision, Linearity, and detection limits. Results: The assay demonstrated 100% sensitivity and specificity relative to reference sera. Intra-assay coefficients of variation ranged from 0.36% to 5.73%, and inter-assay variation was 4.16%, indicating high precision. The ELISA showed excellent Linearity (R2 > 0.995). The lower limit of detection was 0.51 IU/mL, and the lower limit of quantification was 0.98 IU/mL. Conclusions: The developed ELISA is a BSL-2-compatible, robust, and scalable platform suitable for serosurveillance and the assessment of vaccine-induced immunity in endemic regions. Calibration against an international standard supports its applicability for standardized antibody measurement. This assay provides a practical tool for NiV outbreak response and vaccine evaluation.