Lee C, Goeger D E
Department of Clinical Laboratory Sciences, School of Allied Health Sciences, University of Texas Medical Branch, Galveston 77555-1028, USA.
Clin Biochem. 1998 Jun;31(4):229-33. doi: 10.1016/s0009-9120(98)00025-3.
To study the cross-reactivity of 6 beta-hydroxycortisol (6 beta-OHF) with anticortisol antibodies and subsequent interference with urinary free cortisol (UFC) quantitation in commercial immunoassay kits. A solid-phase-extraction (SPE) technique was evaluated for removal of 6 beta-OHF from the specimen.
Interference by 6 beta-OHF was studied in three enzyme immunoassay and three radioimmunoassay kits. Interference was assessed by the multiple regression equation derived from a 4 x 4 matrix composed of four levels of cortisol and four levels of 6 beta-OHF. Sep-Pak Plus C18 cartridges were used to remove 6 beta-OHF from the specimens, and optimum conditions for fractionating 6 beta-OHF from cortisol were determined by eluting the cartridge with a stepwise increment in methanol concentration. HPLC was used to monitor cortisol and 6 beta-OHF in the sample and cartridge eluates.
Cross-reactivity of 6 beta-OHF with anticortisol antibodies was variable and depended on the cortisol kit used with a range from 0.1 to 10%. Multiple regression analysis indicated that 6 beta-OHF interfered positively with the cortisol assay regardless of cortisol concentrations. Extraction of UFC by methylene chloride as recommended by some immunoassay kits yielded a significant constant error to the UFC results which was nearly proportional to the added levels of 6 beta-OHF. SPE of samples completely removed 6 beta-OHF from the specimen when a 40% methanol elution step was included. Consequently, specimens processed in this manner did not show interference in UFC measurements even in samples spiked with 2.38 mumol/L of 6 beta-OHF.
Elevated urinary 6 beta-OHF may be a significant source of interference in UFC immunoassays. SPE of samples prior to analysis would be a simple and inexpensive means for removing 6 beta-OHF from urine specimens, therefore, increasing accuracy and precision in UFC measurements by immunoassay.
研究6β-羟基皮质醇（6β-OHF）与抗皮质醇抗体的交叉反应性，以及随后在商用免疫分析试剂盒中对尿游离皮质醇（UFC）定量的干扰。评估了一种固相萃取（SPE）技术从标本中去除6β-OHF的效果。
在三种酶免疫分析试剂盒和三种放射免疫分析试剂盒中研究了6β-OHF的干扰情况。干扰通过由四个皮质醇水平和四个6β-OHF水平组成的4×4矩阵得出的多元回归方程进行评估。使用Sep-Pak Plus C18柱从标本中去除6β-OHF，并通过逐步增加甲醇浓度洗脱柱来确定从皮质醇中分离6β-OHF的最佳条件。采用高效液相色谱法监测样品和柱洗脱液中的皮质醇和6β-OHF。
6β-OHF与抗皮质醇抗体的交叉反应性各不相同，取决于所使用的皮质醇试剂盒，范围为0.1%至10%。多元回归分析表明，无论皮质醇浓度如何，6β-OHF均对皮质醇测定产生正向干扰。一些免疫分析试剂盒推荐的用二氯甲烷萃取UFC会给UFC结果带来显著的恒定误差，该误差几乎与添加的6β-OHF水平成正比。当包含40%甲醇洗脱步骤时，样品的SPE能完全从标本中去除6β-OHF。因此，即使在添加了2.38μmol/L 6β-OHF的样品中，以这种方式处理的标本在UFC测量中也未显示出干扰。
尿中6β-OHF升高可能是UFC免疫分析中一个重要的干扰来源。分析前对样品进行SPE将是一种从尿标本中去除6β-OHF的简单且廉价的方法，从而提高免疫分析UFC测量的准确性和精密度。
