Rapid Naked-Eye Detection of a Liver Disease Biomarker by Discovering Its Monoclonal Antibody to Functionalize Engineered Red-Colored Bacteria Probes

Abstract

Glycocholic acid (GCA) is a biomarker for liver diseases, but few facile naked-eye detection methods have been reported to detect it till now. To tackle this challenge, we first prepared a novel monoclonal mouse antibody (mAb) of GCA by a hybridoma technique. The anti-GCA mAb exhibited high specificity, making its cross-reactivity with seven structurally and functionally related GCA analogs negligible. Using this anti-GCA mAb and an engineered red-colored bacterial strain (Staphylococcus aureus, S. aureus), we developed a simple naked-eye visualized method for GCA detection. Toward this goal, S. aureus bacteria were turned red by 5-cyano-2,3-ditolyl tetrazolium chloride treatment and heat treated to an unculturable state, rendering the bacteria as an optical detection probe powerful in in vitro diagnostics. Through the natural binding ability of protein A on the surface of S. aureus and the Fc fragment of a mouse antibody, the anti-GCA antibody was simply conjugated onto S. aureus. Then, the engineered S. aureus served as a red-colored bioprobe for detecting GCA through a coagglutination test. In the presence of GCA, the bioprobes aggregated into dense red-colored eye-visible clusters, enabling the sensitive detection of GCA with a concentration of 0.05−0.10 μg/mL. This naked-eye visualization method only takes a few minutes to detect GCA and avoids the use of expensive equipment. It represents a rapid, convenient, and simple method for detecting GCA to diagnose liver diseases.