Review
The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry

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Abstract

Immunoassays have made it possible to measure dozens of individual proteins and other analytes in human samples for help in establishing the diagnosis and prognosis of disease. In too many cases the results of those measurements are misleading and can lead to unnecessary treatment or missed opportunities for therapeutic interventions. These cases stem from problems inherent to immunoassays performed with human samples, which include a lack of concordance across platforms, autoantibodies, anti-reagent antibodies, and the high-dose hook effect. Tandem mass spectrometry may represent a detection method capable of alleviating many of the flaws inherent to immunoassays. We review our understanding of the problems associated with immunoassays on human specimens and describe methodologies using tandem mass spectrometry that could solve some of those problems. We also provide a critical discussion of the potential pitfalls of novel mass spectrometric approaches in the clinical laboratory.

Section snippets

Immunoassays are imperfect

Ever since the description of the first immunoassay in the late 1950s (Yalow and Berson, 1960), the use of antibodies to measure the concentration of proteins and small molecules in clinical samples has been changing the face of medicine. The platform for immunoassays has evolved from its initial conception as a competitive radioimmunoassay to enzyme linked immunosorbent assays on plastic surfaces to sandwich liquid phase chemiluminescent immunometric assays using paramagnetic beads on

Could mass spectrometry be the answer?

In the 1980s, scientists at Yale described robust methods for ionizing intact molecules and introducing them into mass analyzers (Whitehouse et al., 1985). These ionization strategies combined with gas-phase fragmentation permitted the de novo identification of peptides and proteins from complex mixtures, thus birthing the field of proteomics. Later advances in electronics and architecture have made modern mass analyzers capable of directly detecting attomoles of analyte. With this level of

Potential pitfalls of LC-MS/MS in the clinical quantification of proteins

However exciting mass spectrometric assays for protein quantification might seem, they will undoubtedly have significant problems of their own. In this section we will highlight recognized issues with mass spectrometric assays, but we must keep in mind that unanticipated problems will also likely surface over time. Indeed, the abovementioned problems with immunoassays were only elucidated after years of clinical use.

Conclusions

Over the past five decades, we have learned that immunoassays can be used to help diagnose human disease. Sadly, we have also learned that immunoassays can hurt patients. It is important for clinicians to remember how immunoassays can give inaccurate results in many clinical situations. It is also important for the community as a whole to look for solutions, which may reside in novel detection technologies like tandem mass spectrometry. And as with all novel technologies, we must be prepared

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