Enzyme immunoassays with the miniature centrifugal fast analyzer

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Enzyme Multiplied Immunoassay Technique (EMIT) is widely used for therapeutic drug monitoring.
• Adapting EMIT procedures to automated analyzers is crucial for improving efficiency and precision.
• Phenytoin and phenobarbital are critical drugs requiring accurate serum concentration monitoring.

Purpose of the Study:

• To adapt and validate EMIT procedures for phenytoin and phenobarbital assays using a miniature Centrifugal Fast Analyzer.
• To evaluate the performance characteristics of the adapted assay, including reaction kinetics and precision.

Main Methods:

• Enzyme Multiplied Immunoassay Technique (EMIT) procedures were adapted for phenytoin and phenobarbital serum assays.
• Assays were performed on a miniature Centrifugal Fast Analyzer.
• Reaction rates were measured at specific time intervals (15-30 seconds) after reagent mixing.

Main Results:

• Reaction rates decreased continuously with time and were most sensitive to drug concentration immediately after mixing.
• Antibody excess was observed, with 75% inhibition of antibody-bound enzyme, explaining residual activity.
• The analyzer achieved a run-to-run coefficient of variation (CV) of 10% for phenobarbital and 9% for phenytoin, outperforming the manufacturer's quoted 15% CV.

Conclusions:

• The adapted EMIT method on the Centrifugal Fast Analyzer provides a precise and efficient means for serum phenytoin and phenobarbital measurement.
• The kinetic measurement within the initial 15-30 seconds post-mixing is a reliable indicator of drug concentration.
• This adaptation offers improved precision compared to standard EMIT procedures for these critical drug assays.