METHOD AND REAGENTS FOR ULTRASENSITIVE IMMUNOASSAY

Abstract

Disclosed are assays, reagents, and methods for ultrasensitive detection of target molecules. The assay comprises fusion proteins including binding moieties, such as antibodies, nanobodies (VHH/VNAR), or aptamers, linked to nonfunctional fragments of a protein. In the presence of a target molecule, the binding moieties recognize distinct target regions and bring the protein fragments into close proximity, reconstituting a functional protein that generates a detectable signal. Linkers connecting the fusion components may be flexible peptides or polypeptides that spontaneously form dimers, trimers, or tetramers, thereby providing multivalent fusion proteins with enhanced sensitivity. The reconstituted protein may produce luminescent, fluorescent, colorimetric, or spectroscopic signals detectable by microplate readers, handheld luminometers, or lateral flow devices. The invention encompasses solid-phase, homogeneous, and lateral flow assay formats for detecting viruses, bacteria, proteins, peptides, or small molecules, including GLRaV-3, SARS-CoV-2, PSA, and E. coli. The disclosed assays exhibit improved specificity, reduced background, and enhanced signal-to-noise ratios.

CPC Classifications