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Clinical Chemistry 51: 401-407, 2005. First published December 8, 2004; 10.1373/clinchem.2004.034264
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(Clinical Chemistry. 2005;51:401-407.)
© 2005 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Immunoassay for Sex Hormone-Binding Globulin in Undiluted Serum Is Influenced by High-Molecular-Mass Aggregates

Markus Thaler1, Jochen Metzger1, Anita Schreiegg1, Barbara Denk2, Andreas Gleixner2, Hagen Hauptmann3 and Peter B. Luppa1,a

1 Institute for Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar der Technischen Universität München, Munich, Germany.
2 Roche Diagnostics GmbH, Werk Penzberg, Penzberg, Germany.
3 Institute of Organic Chemistry, Universität Regensburg, Regensburg, Germany.

aAddress correspondence to this author at: Institute for Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Strasse 22, D-81675 Munich, Germany. Fax 49-89-4140-4875; e-mail luppa{at}klinchem.med.tum.de.

Background: The new Elecsys® chemiluminescence assay for measurement of homodimeric sex hormone-binding globulin (SHBG) was designed for use with undiluted serum, in contrast to other methods that require predilution. During assay development, unexpected calibration difficulties were observed that were attributable to particular biochemical properties of the highly concentrated SHBG in solution.

Methods: We used a surface plasmon resonance (SPR) biosensor, which enables biomolecular interaction analysis of SHBG, and size-exclusion chromatography for this investigation. The immunoassay was evaluated for imprecision, linearity, and suitability of the dilution medium, and the method was compared with an IRMA for SHBG.

Results: The SPR biosensor characterized the special protein properties of SHBG in various concentrations. Above 200 nmol/L there was a strong tendency toward formation of high-molecular-mass aggregates. This was also detectable by size-exclusion chromatography and could be reversed by simple dilution of the sample. On the basis of these results, the dynamic measuring range of the SHBG assay is restricted to 0.350–200 nmol/L. Assay evaluation on a 2010 analyzer revealed excellent precision (CV ≤2.5%). Mean recoveries were 84.2–98.8%. Intermethod comparison with an IRMA yielded a satisfactory concordance of the two assays with a Spearman correlation coefficient of 0.8807.

Conclusions: Aggregates of human SHBG may have a detrimental impact on the accurate measurement of the protein if measurements are performed with undiluted serum samples. Further work is needed to clarify whether these high-molecular-mass aggregates influence the free fraction of steroid hormones in vivo.






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