Beyond the limits of conventional 'endpoint' ELISA and rescuing the signal with lag k-ELISA

Conventional colorimetric e-ELISA assays are widely used for quantifying antibodies in serum but are often labour-intensive, costly, and require serial dilutions for high-titre samples to ensure accuracy. To address these limitations, we propose the lag k-ELISA, a novel method for assessing antibody concentrations against SARS-CoV-2 in blood samples. This technique simplifies workflows while reducing time and labor compared to traditional ELISA. In this study, lag k-ELISA was applied to 169 sera for anti-S1 IgG and 79 samples for anti-S1 IgM assessment. Distinct analytical workflows were developed to process kinetic signal data, allowing for the description of Ab content. Key signal metrics-including time at OD minima, k-rates, and signal kinetics-proved effective in distinguishing samples by dilution factor or unitage. The assay's performance was systematically evaluated under different operating conditions, such as stirring, readout time, and temperature, which significantly impacted assay outcomes and needed to be optimised to enhance the resolving power of lag k-ELISA. Our findings demonstrate that lag k-ELISA is a robust, efficient complement to traditional ELISA methods, offering reliable results with reduced labour, time, and costs. This technique is particularly advantageous for high-titre samples, streamlining antibody quantification workflows without compromising accuracy.