Practical enzyme immunoassay for plasma cortisol using beta-galactosidase as enzyme label

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Cortisol measurement is crucial for diagnosing adrenal function disorders.
• Existing immunoassays may have limitations in sensitivity or specificity.
• Development of novel enzyme immunoassay (EIA) methods is needed for improved cortisol detection.

Purpose of the Study:

• To develop and validate a highly sensitive and specific enzyme immunoassay for cortisol detection.
• To evaluate the performance of novel cortisol haptens conjugated with beta-galactosidase.
• To assess the clinical applicability of the developed EIA method.

Main Methods:

• Enzyme immunoassay development using beta-galactosidase as the enzyme label.
• Synthesis and conjugation of cortisol derivatives (CT-MB and CHS-MB) as haptens.
• Evaluation of enzyme-coupling efficiency, immunoreactivity, and cortisol displacement.
• Assessment of sensitivity, cross-reactivity, accuracy, precision, and correlation with RIA.

Main Results:

• Highly efficient enzyme-coupling (>90%) with full enzyme activity retention.
• CT-MB-beta-galactosidase conjugate demonstrated high immunoreactivity and excellent cortisol displacement.
• Achieved a maximum sensitivity of 1 microgram/dl with a 20-microliter sample.
• Demonstrated satisfactory accuracy, precision, and correlation with radioimmunoassay (RIA).
• Low cross-reactivity observed for cortisol-21-acetate (150%), cortisone (10%), and corticosterone (7%).

Conclusions:

• The developed enzyme immunoassay using CT-MB-beta-galactosidase conjugate offers high sensitivity and specificity for cortisol measurement.
• Modification of the cortisol derivative linkage significantly enhanced assay sensitivity.
• The method is accurate, precise, and correlates well with RIA, indicating its suitability for clinical applications.