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<p>This study demonstrates enhanced detection of Shiga toxin (Stx), a key virulence factor in <i>Shigella dysenteriae</i>-induced bloody diarrhea, through optimized surface plasmon resonance (SPR) biosensor design. We present a comparative evaluation of antibody immobilization strategies, revealing significant advantages of protein G-mediated oriented immobilization over conventional covalent attachment. The covalent (non-oriented) approach using 11-mercaptoundecanoic acid-modified chip showed moderate performance (K<sub>D</sub> = 37&#xa0;nM, LOD = 28&#xa0;ng/mL). In contrast, protein G-assisted orientation dramatically improved detection capabilities, achieving a 2.9-fold lower detection limit (9.8&#xa0;ng/mL) and 2.3-fold higher binding affinity (K<sub>D</sub> = 16&#xa0;nM). Control measurements with free antibody-antigen interactions established a baseline affinity (K<sub>D</sub> = 10&#xa0;nM), demonstrating that the oriented method preserves 63% of native binding efficiency versus only 27% in the covalent approach. Mechanistic studies attribute these improvements to protein G’s ability to maintain optimal antibody orientation, thereby: (1) maximizing paratope accessibility, (2) minimizing steric interference, and (3) preserving binding site functionality. The 57% reduction in K<sub>D</sub> relative to covalent immobilization confirms the method’s efficacy in maintaining antibody performance post-immobilization. These findings establish protein G-mediated orientation as the superior strategy for SPR-based Stx detection, offering substantial improvements in sensitivity and reliability for clinical diagnostics and food safety applications. The approach demonstrates particular promise for rapid, label-free detection of bacterial toxins in resource-limited settings.</p>

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Enhancing Shiga toxin detection using surface plasmon resonance a study of antibody immobilization strategies

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月16日

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  • Zahra Karbalaee,
  • Ali Hossein Rezayan,
  • Ramezan Ali Taheri,
  • Seyed Ali Mirhosseini,
  • Mohammad Barshan-Tashnizi

摘要

This study demonstrates enhanced detection of Shiga toxin (Stx), a key virulence factor in Shigella dysenteriae-induced bloody diarrhea, through optimized surface plasmon resonance (SPR) biosensor design. We present a comparative evaluation of antibody immobilization strategies, revealing significant advantages of protein G-mediated oriented immobilization over conventional covalent attachment. The covalent (non-oriented) approach using 11-mercaptoundecanoic acid-modified chip showed moderate performance (KD = 37 nM, LOD = 28 ng/mL). In contrast, protein G-assisted orientation dramatically improved detection capabilities, achieving a 2.9-fold lower detection limit (9.8 ng/mL) and 2.3-fold higher binding affinity (KD = 16 nM). Control measurements with free antibody-antigen interactions established a baseline affinity (KD = 10 nM), demonstrating that the oriented method preserves 63% of native binding efficiency versus only 27% in the covalent approach. Mechanistic studies attribute these improvements to protein G’s ability to maintain optimal antibody orientation, thereby: (1) maximizing paratope accessibility, (2) minimizing steric interference, and (3) preserving binding site functionality. The 57% reduction in KD relative to covalent immobilization confirms the method’s efficacy in maintaining antibody performance post-immobilization. These findings establish protein G-mediated orientation as the superior strategy for SPR-based Stx detection, offering substantial improvements in sensitivity and reliability for clinical diagnostics and food safety applications. The approach demonstrates particular promise for rapid, label-free detection of bacterial toxins in resource-limited settings.