A protein G-based, antibody-free lateral flow assay for IgG detection

Immunoglobulin G (IgG), the most abundant antibody class, is essential for long-term immunity, making its detection vital for assessing infection status, disease phase, and vaccine-induced immunity. Conventional IgG detection methods, including enzyme-linked immunosorbent assays (ELISA) and lateral flow immunoassays (LFIA), typically rely on secondary antibodies. However, secondary antibody production, commonly done by extracting from the serum of large animals, is costly and complex, posing limitations on further applications employing the LFIA-based IgG detection method. In this study, we present a novel, versatile antibody-free LFIA (AF-LFIA) approach for IgG detection. By utilizing the inherent binding affinity between immunity protein (Im7) and its cognate partner, the colicin E7 DNase domain (CL7) to generate a reliable control signal and employing a modified polymerized Protein G as the IgG capture agent, our approach offers an alternative to antibodies and significantly reduces overall cost and time. Using the severe acute respiratory syndrome coronavirus-2 nucleocapsid IgG as a model analyte, a detection limit of 10 ng/mL was achieved. The high sensitivity of this AF-LFIA platform enables reliable detection of target IgG in both blood and saliva specimens. Furthermore, potential broad applicability is demonstrated by detecting IgG antibodies targeting human coronavirus-229E and Toxoplasma gondii immune mapped protein-1. Overall, this AF-LFIA method offers a cost-effective and efficient alternative for IgG detection, with significant potential for point-of-care testing during pathogen infection diagnostics.