A simple method for separating unbound and bound cortisol in a radioimmunoassay

Area of Science:

• Endocrinology
• Biochemistry
• Analytical Chemistry

Background:

• Accurate measurement of cortisol levels is crucial for diagnosing adrenal disorders.
• Existing radioimmunoassay methods can involve complex separation steps.

Purpose of the Study:

• To develop and validate a simplified radioimmunoassay for cortisol.
• To improve the efficiency of separating antibody-bound from unbound cortisol.

Main Methods:

• Utilized a toluene: water scintillation fluid mixture for phase partitioning of cortisol at 0-5°C.
• Employed liquid scintillation spectrometry to quantify unbound [3H]cortisol in the organic phase.
• Validated assay performance through standard curve analysis, cross-reactivity testing, and comparison with existing methods.

Main Results:

• Achieved high regression coefficients (0.980–0.999) for standard curves.
• Demonstrated low cross-reactivity (<3%) with most tested steroids.
• Reported good intra-assay (8%) and inter-assay (13%) variability, sensitivity of 1.4 ng/ml, and 97.5% recovery.
• Correlated well (r=0.90) with a competitive binding assay.

Conclusions:

• The described radioimmunoassay offers a simplified and effective method for cortisol determination.
• This assay provides reliable quantification of cortisol with improved separation efficiency.
• The method is suitable for clinical applications requiring accurate glucocorticoid level assessment.