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Related Experiment Videos

Engineering a regulatable enzyme for homogeneous immunoassays

D Legendre1, P Soumillion, J Fastrez

  • 1Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Place L. Pasteur, Louvain-la-Neuve, Belgium.

Nature Biotechnology
|January 27, 1999
PubMed
Summary
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Engineered enzymes act as biosensors by modulating activity upon antibody binding, enabling sensitive detection of analytes like prostate-specific antigen (PSA) in immunoassays.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunotechnology

Background:

  • Traditional immunoassays often require labeled detection molecules.
  • Developing novel signaling systems for homogeneous immunoassays is crucial for sensitive analyte detection.
  • Phage display technology offers a powerful platform for protein engineering.

Purpose of the Study:

  • To engineer phage-displayed TEM-1 beta-lactamase enzymes with antibody-binding capabilities for homogeneous immunoassays.
  • To create hybrid enzymes whose activity is modulated by the binding of monoclonal antibodies (Mabs).
  • To develop a method for generating signaling molecules for analyte detection without epitope identification.

Main Methods:

  • Genetic insertion of random peptide libraries into TEM-1 beta-lactamase loops to create hybrid enzymes.

Related Experiment Videos

  • Utilizing antibiotic resistance conferred by beta-lactamase activity for clone selection.
  • Biopanning engineered libraries on immobilized Mabs against prostate-specific antigen (PSA) or streptavidin.
  • Characterizing enzyme activity modulation upon Mab or streptavidin binding.

    • Main Results:

    • Successfully generated hybrid enzymes with specific binding sites for Mabs and streptavidin.
    • Demonstrated that enzyme activity is regulated by Mab or streptavidin binding, with dissociation constants in the 10(-9) to 10(-6) M range.
    • Achieved sensitive detection of PSA at a minimal concentration of 10(-9) M in a competitive assay.
    • Identified that Mabs recognize mimotopes, with no sequence similarity to PSA fragments in the engineered inserts.

    Conclusions:

    • Engineered phage-displayed enzymes can function as regulated signaling molecules in homogeneous immunoassays.
    • This approach allows for the sensitive detection of analytes through antibody-mediated modulation of enzyme activity.
    • The method provides a versatile platform for developing biosensors without prior knowledge of the target epitope.