<p>Biotinylated antibodies are widely used in ELISA. However, their preparation typically requires labor-intensive purification steps to remove the excess biotinylating reagent. Here, we present a novel&#xa0;one-pot, purification-free&#xa0;strategy for preparing biotinylated antibodies, enabling a robust sandwich ELISA platform for the detection of the cardiac troponin I-C complex, a key biomarker for acute myocardial infarction. By introducing glycine (Gly) as a quenching agent to inactivate the residual biotinylating reagent (NHS-biotin esters) during the labeling process, our method eliminates the need for post-labeling purification while effectively reducing nonspecific background. Systematic optimization of labeling conditions yielded an assay with a detection limit of 1.08&#xa0;pM and a broad linear quantification range from 3.6&#xa0;pM to 1&#xa0;nM (<i>R</i>² = 0.99). The platform demonstrated high specificity, with recovery rates ranging from 91.5 to 101.8% and intra-assay variation below 2.9%. Furthermore, successful validation in a serum amyloid A detection model confirmed the generalizability of the Gly-mediated background suppression strategy. This cost-effective, streamlined ELISA workflow offers a powerful tool for rapid and reliable clinical diagnostics, with potential for broad application in biomarker detection.</p> Graphical abstract <p>This schematic contrasts the conventional and novel one-pot strategies for antibody biotinylation in ELISA. The traditional method requires a cumbersome purification step to remove excess biotin-NHS reagent after labeling. In contrast, our one-pot approach introduces a key innovation: an in-solution quenching reaction. The addition of a small molecule (e.g., glycine) efficiently inactivates residual biotin esters, allowing the biotinylated antibody to be used directly in subsequent steps, thereby streamlining the workflow.</p> <p></p>