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Farhima Akter1, Masayasu Mie, Eiry Kobatake

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This study introduces a novel fusion protein for enhanced immuno-rolling circle amplification (immuno-RCA). This method achieves ultrasensitive detection of cancer biomarkers like alpha-fetoprotein (AFP) in body fluids.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Ultrasensitive detection of low-level proteins in biological samples is crucial for early disease diagnosis.
  • Conventional immunoassays like ELISA face limitations in sensitivity and require stringent conditions for techniques like immuno-rolling circle amplification (immuno-RCA).

Purpose of the Study:

  • To develop a novel fusion protein to enhance signal amplification in immuno-RCA for ultrasensitive detection of cancer biomarkers.
  • To improve the efficiency and convenience of immunoassays by avoiding covalent antibody-DNA or antibody-biotin linkages.

Main Methods:

  • Genetically engineered a fusion protein incorporating the C2 domain of protein G and a biotin acceptor peptide (BAP).
  • Expressed and purified the fusion protein in Escherichia coli, verifying its binding affinity to IgG and streptavidin.
  • Applied the fusion protein in an immuno-RCA assay for the detection of alpha-fetoprotein (AFP) in a microplate format.

Main Results:

  • The novel BAP-C2 fusion protein effectively maintained antibody biological activity and binding affinity.
  • The developed immuno-RCA assay demonstrated specific and sensitive detection of AFP.
  • The method achieved significant signal enhancement compared to conventional approaches.

Conclusions:

  • The BAP-C2 fusion protein-based immuno-RCA offers a sensitive and convenient platform for cancer biomarker detection.
  • This approach holds promise for improving early cancer diagnosis through enhanced immunoassays.