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<p>Preclinical screening for β-amyloid 42 (Aβ42), a key biomarker of Alzheimer’s disease (AD) with an extremely low concentration in blood, faces major technical challenges. Herein, two high-affinity monoclonal antibodies (mAbs) 2E7 and 3B6 were obtained through hybridoma screening. Molecular docking and epitope identification demonstrated that the complementarity-determining regions (CDRs) of mAbs 2E7 and 3B6 targeted the N-terminal (2E7) and C-terminal (3B6) of Aβ42, respectively, forming a non-competitive epitope binding mode. Based on the T4 phage display system, a dual-display T4 phage (T4@scFv@HRP) integrating 81 single-chain fragment variables (scFv) of mAb 2E7 and 203 horseradish peroxidase (HRP) was constructed. Subsequently, a phage-linked immunosorbent assay (PLISA) was developed using mAb 3B6 as the capture element and the T4 phage displaying both scFv of 2E7 as the target recognition and HRP as the signal amplification element. The assay results for Aβ42 by PLISA showed excellent linearity in the range of 1.56–100 pg/mL (<i>R</i>² = 0.9926) with a limit of detection (LOD) of 0.89 pg/mL, which was 17-fold more sensitive than the conventional sandwich enzyme-linked immunosorbent assay (ELISA) (LOD = 15.51 pg/mL). The PLISA, established in this study, provides an innovative immunoassay method capable of analyzing low-abundance proteins in the blood. It can be considered an improved version of ELISA, as it theoretically can be used to detect any target peptide or protein by replacing the displayed antibodies on the phage.</p>

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Phage-linked Immunosorbent Assay for Ultrasensitive Detection of Aβ42

  • 期刊论文

  • 发表日期：

    2025年10月17日

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  • Runbo Tian,
  • Qihui Zhang,
  • Haibing Zhu,
  • Yaping Liu,
  • Jing Jin,
  • Zhanjun Yang,
  • Xin Zhou

摘要

Preclinical screening for β-amyloid 42 (Aβ42), a key biomarker of Alzheimer’s disease (AD) with an extremely low concentration in blood, faces major technical challenges. Herein, two high-affinity monoclonal antibodies (mAbs) 2E7 and 3B6 were obtained through hybridoma screening. Molecular docking and epitope identification demonstrated that the complementarity-determining regions (CDRs) of mAbs 2E7 and 3B6 targeted the N-terminal (2E7) and C-terminal (3B6) of Aβ42, respectively, forming a non-competitive epitope binding mode. Based on the T4 phage display system, a dual-display T4 phage (T4@scFv@HRP) integrating 81 single-chain fragment variables (scFv) of mAb 2E7 and 203 horseradish peroxidase (HRP) was constructed. Subsequently, a phage-linked immunosorbent assay (PLISA) was developed using mAb 3B6 as the capture element and the T4 phage displaying both scFv of 2E7 as the target recognition and HRP as the signal amplification element. The assay results for Aβ42 by PLISA showed excellent linearity in the range of 1.56–100 pg/mL (R² = 0.9926) with a limit of detection (LOD) of 0.89 pg/mL, which was 17-fold more sensitive than the conventional sandwich enzyme-linked immunosorbent assay (ELISA) (LOD = 15.51 pg/mL). The PLISA, established in this study, provides an innovative immunoassay method capable of analyzing low-abundance proteins in the blood. It can be considered an improved version of ELISA, as it theoretically can be used to detect any target peptide or protein by replacing the displayed antibodies on the phage.