Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations

• Published in: Clinica chimica acta Vol. 378; no. 1; pp. 66 - 70
• Main Authors: Horie, Hitoshi, Kidowaki, Takeshi, Koyama, Yuhei, Endo, Tatsuji, Homma, Keiko, Kambegawa, Akira, Aoki, Norihiko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2007
• Subjects: Adult; Chromatography, High Pressure Liquid; Cross Reactions; Cushing's disease; Gas Chromatography-Mass Spectrometry; HPLC; Humans; Hydrocortisone - immunology; Hydrocortisone - urine; Immunoassay; Male; Pituitary ACTH Hypersecretion - urine; Radioimmunoassay - methods; Reagent Kits, Diagnostic; Urinary free cortisol; Immunoassay; GCMS; FPIA; 5α-THF; 5β-DHF; 5α-THE; Urinary free cortisol; 6β-OHF; UFC; 6β-OHE; Cushing's disease; 5β-THE; 5β-THF; HPLC
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2006.10.018

In immunoassay kits for determination of urinary free cortisol (UFC) concentrations, the results vary markedly from kit to kit, so we compared in this study the reaction specificity among 4 commercially available immunoassay kits to determine the applicability of these assays in routine determination of UFC concentrations. Using 4 commercially available kits, cross-reaction was investigated. In addition, urine samples were fractionated by HPLC to investigate endogenous immunoreactive cortisol responses. HPLC fractions were subjected to gas chromatography-mass spectrometry (GCMS) to identify substances causing inter-kit assay discrepancies. Among the 4 kits, cortisol Kit “TFB” (Immunotech; IOT-RIA method) showed the lowest cross-reaction (2.5%) for prednisolone. Furthermore, on HPLC, 87.8% of the reaction of the entire fraction was seen in the fractions corresponding to the elution position of standard cortisol with the IOT-RIA method; this was the highest percentage among the 4 kits. GCMS revealed that the substance that showed a cross-reaction with the other 3 kits was 5α-tetrahydrocortisol (5α-THF) glucuronide. The IOT-RIA method was found to be the most specific for UFC. The other 3 commercially available kits showed cross-reaction with a conjugate of 5α-THF, found to be one of the causes of inter-kit assay discrepancies.