Why are abnormal binding protein concentrations an issue for immunoassays?

Immunoassay detection relies on noncovalent binding interactions between the receptor and the target analyte. These interactions are equilibrium reactions, and the results that are obtained are snapshots of one moment in the equilibrium process of very complex sample mixtures with potentially high inter-individual variability.

Most antibodies used as receptors in immunoassays have binding affinities (Ka) ranging from 108–1010 L/mol. For many analytes, this level of binding selectivity is excellent. For small molecule hormones that are transported by specific binding globulins, however, this level of binding selectivity may not be adequate, especially when concentrations of binding globulins are abnormal.

This is because specific binding proteins such as thyroxine-, corticosteroid-, and sex hormone-binding globulins (TBG, CBG, SHBG) and vitamin D binding protein (DBP) also have Kas for their target hormones (e.g., thyroxine, cortisol, estradiol, testosterone, and vitamin D) ranging from 108–1010 L/mol (JBMR Plus 2020; doi: 10.1002/jbm4.10418).

Which assays are affected by abnormal concentrations of binding proteins?

In general, there is consensus that total hormone levels can fluctuate due to abnormally high or low binding protein concentrations. However, a high or low concentration of total hormone may not necessarily mean that there is an excess or a deficiency of biologically active, or free hormone, that is available to the tissue. For this reason, free hormone levels for analytes such as cortisol, thyroxine, triiodothyronine, testosterone, and vitamin D are also important to measure.

Unfortunately, researchers have shown that the measurement of free hormone by immunoassay, particularly free thyroxine, is affected by both nonspecific (albumin) and specific binding protein concentrations (TBG) in comparison with a reference liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which does not exhibit a bias due to binding protein concentrations (Clin Chem 2011; doi: 10.1373/clinchem.2010.154088).

There are many occasions when the measurement of total hormone can be used to confirm a diagnosis or monitor a patient’s treatment. Immunoassay measurements of total hormones are considered less susceptible to binding protein concentrations, because they typically include additional reagents such as acid to denature binding proteins, or ligands with higher binding affinities to displace the native target hormone.

However, total hormone measurements by immunoassay also still can be affected by abnormal concentrations of binding globulins. For example, one study found immunoassay measurements of total cortisol to be falsely low compared with LC-MS/MS measurements in patients with abnormally high concentrations of CBG (Clin Endocrinol (Oxf) 2013; doi: 10.1111/cen.12039).

Which major patient populations are affected by abnormal binding protein concentrations?

The following patient populations have conditions or treatments that can lead to abnormal binding protein concentrations: patients who are pregnant, patients receiving estrogen treatment or taking oral contraceptives, patients with liver diseases, and patients with compromised renal function.

What can be done to ensure accurate laboratory results for these patients?

There are several strategies that both clinical and laboratory teams can take to ensure accurate measurements for patients with abnormal levels of binding proteins. Initially, if laboratory measurements do not agree with the clinical presentation of the patient, the analyte-specific binding globulin can be measured to rule out bias due to abnormal concentrations of binding proteins. If possible, the sample can be analyzed by the gold standard method, LC-MS/MS, which removes binding proteins by precipitation or, in the case of free hormone measurements, physically separates the bound from the free fraction via equilibrium dialysis or ultracentrifugation.

If measurement by LC-MS/MS is not feasible, the clinical and laboratory teams can collaborate to define appropriate reference intervals for specific patient populations.

Anastasia Gant Kanegusuku, PhD, is a clinical chemistry fellow at the University of Chicago. Email: [email protected]