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Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates
T K Christopoulos1, E P Diamandis
1Department of Clinical Biochemistry, Toronto Western Hospital, Ontario, Canada.
Analytical Chemistry
|February 15, 1992
Summary
This study introduces a highly sensitive enzyme-amplified immunoassay using terbium fluorescence. The method detects low molecule counts, offering precision for automated, rapid diagnostic tests.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Immunoassays are crucial for detecting biomarkers.
- Enhancing sensitivity and speed in immunoassays remains a key challenge.
- Time-resolved fluorescence offers advantages in reducing background noise.
Purpose of the Study:
- To develop an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay.
- To utilize alkaline phosphatase (ALP) as a primary enzyme label.
- To employ terbium (Tb3+) chelate for sensitive detection.
Main Methods:
- Enzymatic amplification using alkaline phosphatase (ALP).
- Fluorogenic substrate cleavage to produce 5-fluorosalicylic acid (FSA).
- Formation of a fluorescent ternary complex (FSA-Tb3+-EDTA).
- Quantification via laser-induced microsecond time-resolved fluorometry.
Main Results:
- Detection of 10^6 molecules (monoclonal antibodies) or 1.5 x 10^5 molecules (polyclonal antibodies) of AFP.
- Assay demonstrated high precision (approx. 4%).
- Ultrasensitive detection achieved through enzymatic amplification and time-resolved fluorometry.
Conclusions:
- The developed immunoassay offers exceptional sensitivity and precision.
- The method is suitable for automated, rapid, and sensitive diagnostic applications.
- Time-resolved fluorescence with terbium chelates provides a robust detection platform.