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


METHODS
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.


RESULTS
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 5alpha-tetrahydrocortisol (5alpha-THF) glucuronide.


CONCLUSIONS
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 5alpha-THF, found to be one of the causes of inter-kit assay discrepancies.
Horie
Hitoshi
H
Mitsubishi Kagaku Bio-Clinical Laboratories, INC., 3-30-1 Shimura, Itabashi-ku, and Department of Clinical Laboratories, Keio University Hospital, Tokyo, Japan. h-horie@nm.mbcl.co.jp
Kidowaki
Takeshi
T
Koyama
Yuhei
Y
Endo
Tatsuji
T
Homma
Keiko
K
Kambegawa
Akira
A
Aoki
Norihiko
N
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006854:Hydrocortisone / Q000276:immunology / Q000652:urine*; D008297:Male; D047748:Pituitary ACTH Hypersecretion / Q000652:urine; D011863:Radioimmunoassay / Q000379:methods*; D011933:Reagent Kits, Diagnostic
D011933:Reagent Kits, Diagnostic; D006854:Hydrocortisone