Development and validation of a higher-throughput, automated FAMA assay for varicella-zoster virus antibody detection, offering a scalable alternative to the classic 12-well slide method.

The classical fluorescent antibody to membrane antigen (FAMA) slide assay is the gold standard for varicella-zoster virus (VZV) antibody detection, yet it suffers from low throughput, subjective interpretation, labor-intensive operation, and poor data traceability. To address these limitations, a 96-well plate FAMA assay was developed and preliminarily validated based on the classical FAMA principle for quantitative VZV antibody detection. VZV-infected MRC-5 cells were used as substrate to prepare higher-throughput FAMA plates, and clinical samples were tested using an automated system. Cut-off determination, comprehensive method validation, and consistency comparison with the classical FAMA slide assay were performed. The established higher-throughput FAMA assay demonstrated comparable performance to the classical method in terms of signal specificity and sensitivity under the tested conditions while improving throughput and reducing turnaround time. Automated interpretation and storage ensured objective and traceable results, with an overall concordance of 88% (r = 0.886) with the classical FAMA slide assay. This pilot study suggests that the higher-throughput FAMA assay has the potential to serve as an alternative to the classical FAMA slide assay for large-scale VZV serological surveys and varicella/zoster vaccine immunogenicity evaluation, providing a more efficient and objective tool for vaccine and drug development.IMPORTANCEThe gold-standard antibody test for varicella-zoster virus (VZV), the membrane antigen fluorescent antibody (FAMA) method, is labor-intensive, subjective, and poorly suited for large-scale use. To overcome these limitations, we developed and preliminarily validated a higher-throughput, automated 96-well FAMA method. This novel approach retains good sensitivity and specificity while enabling rapid, objective, and traceable results. Pilot validation demonstrated 88% agreement (r = 0.886) with the classic method, with acceptable performance across specificity, selectivity, precision, linearity, stability, and durability. This scalable method shows potential to meet clinical requirements and could serve as an alternative to manual procedures for large-scale antibody detection, supporting more efficient vaccine development and immune monitoring against VZV.