Competitive flow injection enzyme immunoassay for steroids using a post-column reaction technique

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunochemistry

Background:

• Steroid bioanalysis is crucial for clinical diagnostics and research.
• Traditional immunoassays can be time-consuming and require complex sample preparation.
• Developing rapid, sensitive, and flexible analytical methods is essential for efficient steroid monitoring.

Purpose of the Study:

• To develop and evaluate two competitive flow injection enzyme immunoassays (FI EIAs) for steroid bioanalysis.
• To implement a novel post-column reaction system for enhanced signal generation.
• To assess the flexibility and performance of the developed immunoassay system.

Main Methods:

• Development of two competitive FI EIAs: pre-incubation and displacement assays.
• Utilized a post-column reaction system where unbound analyte reacted with enzyme substrate.
• Amperometric or fluorometric detection of enzyme products (p-aminophenol or 4-methyl umbelliferol).
• Optimization of flow rates, reaction coil, and affinity column parameters.
• Evaluation of signal precision and sample throughput.

Main Results:

• The pre-incubation assay achieved high sample throughput (20 h⁻¹) with excellent precision (1% RSD for cortisol, 2% RSD for budesonide).
• The displacement assay demonstrated effective cortisol detection with a standard curve (4-20% signal precision).
• The same instrumental setup was used for both assays, highlighting system flexibility.
• Minimal modification (affinity column replacement) was required to switch between assay types.

Conclusions:

• The developed competitive FI EIAs are flexible, sensitive, and rapid tools for steroid bioanalysis.
• The post-column reaction strategy offers an efficient alternative to conventional immunoassay signal generation.
• These methods provide precise quantification of steroids at low concentrations.
• The adaptable system facilitates diverse applications in steroid monitoring and research.