Highly sensitive immunoassay based on E. coli with autodisplayed Z-domain

Area of Science:

• Biotechnology and Biomedical Engineering
• Immunodiagnostics
• Microbial Surface Display

Background:

• The Z-domain of protein A binds specifically to the Fc region of immunoglobulins (IgGs).
• Escherichia coli (E. coli) surface display systems offer potential for novel biosensor development.
• Conventional enzyme-linked immunosorbent assays (ELISA) are widely used but can be limited in sensitivity.

Purpose of the Study:

• To engineer E. coli to display the Z-domain of protein A on its outer membrane using Autodisplay technology.
• To evaluate the feasibility of using Z-domain displaying E. coli as a solid support in sandwich-type immunoassays.
• To assess the diagnostic potential of this E. coli-based immunoassay for rheumatoid arthritis (RA) by detecting C-reactive protein (CRP).

Main Methods:

• Autodisplay technology was employed to fuse and express the Z-domain of protein A on the E. coli outer membrane.
• Z-domain displaying E. coli was utilized as the solid support for immobilizing detection antibodies in a sandwich immunoassay format.
• C-reactive protein (CRP) was measured in serum samples from rheumatoid arthritis (RA) patients and healthy individuals.

Main Results:

• The E. coli-based immunoassay demonstrated significantly higher sensitivity and binding capacity compared to conventional ELISA.
• Enhanced sensitivity was attributed to controlled antibody orientation and the mobility of E. coli in the assay matrix.
• A cut-off value for RA diagnosis was established using 45 RA patient samples and 15 healthy samples, showing optimal sensitivity and selectivity.

Conclusions:

• Escherichia coli engineered with the Z-domain of protein A is a feasible and highly sensitive platform for sandwich-type immunoassays.
• This E. coli-based immunoassay system shows promise for medical diagnostics, particularly for conditions like rheumatoid arthritis.
• The technology offers advantages over traditional methods like ELISA due to improved sensitivity and specific antibody immobilization.