Abstract

The development of robust immunoassays depends on high-affinity antibodies; however, rational design strategies remain challenging. Here, we present a structure-guided computational framework for antibody optimization and demonstrate its application in a clinical-grade chemiluminescence assay for C-reactive protein (CRP). Based on key topological residues identified from the cryo-EM structure of the CRP–HCAb complex, we rationally designed mutations within the complementarity-determining regions (CDRs). Molecular dynamics simulations predicted improved binding stability for the optimized variants, which was further validated by peptide-based binding assays. Upon recombinant expression, the engineered antibodies—HCAb3-CDR1-MT and HCAb4-CDR2-MT—exhibited enhanced specificity and affinity. When configured into a sandwich chemiluminescence immunoassay, the system showed excellent precision (CV < 5%) and strong resistance to interference from common serum components. In a clinical validation study involving 120 human serum samples, the assay demonstrated a strong correlation (R 2  > 0.98) with a commercial method and outperformed it in repeatability when resolving discrepant samples. Collectively, this work establishes an integrated pipeline from atomic-level structure to functional clinical assay, offering a generalizable strategy for developing next-generation diagnostic antibodies.