Antibody-Initiated Loop-Mediated Isothermal Nucleic Acid Amplification (ai-LAMP) as a New Biosensor for Antigen Detection

Abstract

Highly sensitive protein detection is critical in research and healthcare diagnostics but remains limited to resource intensive environments. In contrast, lateral flow immunoassay (LFIA)-based diagnostics are popular point-of-care tests due to their simplicity and user-friendly format but lack sensitivity for detecting the low concentrations of proteins that are present early in infections and among asymptomatic individuals. To overcome these limitations, we developed a novel biosensor platform utilizing antibody-initiated loop-mediated isothermal amplification (ai-LAMP) with an LFIA readout for protein detection. This platform integrates protein-specific antibody-antigen binding with the robust signal amplification of LAMP. By conjugating pairs of antibodies to overlapping DNA strands, the presence of a target protein brings the DNA strands into proximity, completing a DNA target to directly initiate the LAMP reaction without any additional ligation step. This approach facilitates the rapid detection of low concentration proteins with a clear visual readout and can be performed in 3 steps from sample to answer. Using ai-LAMP we detected HIV-1 p24 on commercially available LFIAs at a limit of detection (LOD) of 20 fM (0.53 pg/mL), demonstrating 46x improvement over existing HIV p24 LFIAs. The use of ai-LAMP eliminates the need for sophisticated laboratory equipment to detect protein targets in low concentrations, paving a new way for rapid and accessible biomarker detection in clinical and research settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/678293v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5b587forg.highwire.dtl.DTLVardef@10f3587org.highwire.dtl.DTLVardef@1833eeborg.highwire.dtl.DTLVardef@197d1bf_HPS_FORMAT_FIGEXP M_FIG C_FIG