Homogeneous fluorescence-based immunoassay detects antigens within 90 seconds.

Area of Science:

• Biochemistry
• Immunotechnology
• Analytical Chemistry

Background:

• Homogeneous immunoassays offer advantages over heterogeneous assays, including reduced assay time and simplified procedures.
• Their ease of use facilitates rapid, on-site detection of various antigens, even by non-specialized personnel.

Purpose of the Study:

• To develop a novel homogeneous immunoassay for the rapid determination of antigens.
• To demonstrate the assay's proof of concept using a phosphorylation-specific antibody and a tailored peptide probe.

Main Methods:

• A phosphorylation-specific anti-human tau monoclonal antibody was labeled with an acceptor fluorophore.
• A corresponding peptide probe was labeled with a donor fluorophore, and its sequence was optimized for reduced antibody affinity.
• The assay involves spiking the analyte sample with a donor peptide probe, followed by the addition of the acceptor-labeled antibody, leading to fluorescence quenching.

Main Results:

• The fluorescence signal intensity of the donor peptide probe directly correlates with the concentration of the target antigen.
• Optimization of the donor peptide sequence enhanced the assay's response to the analyte antigen compared to the native epitope.
• The developed homogeneous immunoassay achieved semiquantitative analysis of antigens in just 90 seconds.

Conclusions:

• This homogeneous immunoassay provides a rapid and robust method for antigen detection.
• The assay's design allows for on-site analysis with minimal steps, suitable for detecting toxins and pollutants.
• The optimized peptide probe and fluorescence quenching mechanism enable sensitive and timely semiquantitative antigen determination.