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Related Experiment Video

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Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells
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Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s.

Jun Ye1,2, Jie Xu2, Chuanmeng Zhang2

  • 1Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China.

Frontiers in Immunology
|February 10, 2023
PubMed
Summary

A new fluorescence resonance energy transfer (FRET)-based immunoassay allows for the quantitative detection of activated C1s in serum. This method offers a sensitive and specific tool for diagnosing complement-related diseases.

Keywords:
C1sC2C4FRETcomplementimmunoassay

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Area of Science:

  • Biochemistry
  • Immunology
  • Assay Development

Background:

  • C1s activation is implicated in the pathogenesis of various diseases.
  • Accurate detection of C1s activation is crucial for clinical diagnosis and treatment monitoring.

Purpose of the Study:

  • To develop a fluorescence resonance energy transfer (FRET)-based immunoassay for the quantitative detection of activated C1s in human serum.
  • To evaluate the performance characteristics of the developed immunoassay.

Main Methods:

  • Preparation and selection of FRET-based fluorogenic peptides sensitive to C1s enzymatic activity.
  • Immune capture of C1s using anti-C1s-conjugated magnetic microbeads.
  • Quantification of activated C1s enzymatic activity using fluorescent quenched substrate assays.

Main Results:

  • The immunoassay demonstrated a linear quantification range for activated C1s enzymatic activity from 0.096 to 10 μmol·min⁻¹·mL⁻¹ in serum.
  • High accuracy (90-110% recovery) and precision (CV < 10%) were achieved.
  • Excellent specificity was observed, with minimal cross-reactivity (<0.5%) with related enzymes and no significant interference from common serum components, although anticoagulants showed inhibitory effects.

Conclusions:

  • An anti-C1s-based FRET immunoassay for activated C1s detection in serum samples has been successfully established.
  • This assay provides a valuable tool for exploring the role of C1s activation in the pathogenesis, diagnosis, and treatment of complement-related diseases.