ATIC NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH Public Access Author Manuscript J Immunol Methods. Author manuscript; available in PMC 2010 August 15. OF HEALTH Published in final edited form as: J Immunol Methods. 2009 August 15; 347(1-2): 3-11. doi:10.1016/j.jim.2009.06.003. The Fundamental Flaws of Immunoassays and Potential Solutions Using Tandem Mass Spectrometry Andrew N. Hoofnagle" and Mark H. Wener Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 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. Keywords Immunoassays; tandem mass spectrometry; autoantibody interference; hook effect; heterophile anti- reagent antibodies; standardization 1. Immunoassays Are Imperfect Ever since the description of the first immunoassay in the late 1950's (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 automated instruments (Bock, 2000) to microfluidic point of care testing 'lab on chip' immunoassays (Kartalov et al., 2008). The march of progressive technologies continues onward. Unfortunately, even after the many years we have spent optimizing antibody reagents for use in immunoassays, we frequently neglect to focus on the many limitations inherent to immunoassays as they behave with actual human samples in the clinical laboratory, which can be harmful to patients (Table 1). This review highlights how normal human biology and assay design can cause properly functioning C 2009 Elsevier B.V. All rights reserved. *Corresponding author: Andrew N. Hoofnagle, MD PhD, Dept of Lab Medicine, Box 357110, 1959 NE Pacific St, Room NW120, Seattle, WA 98195-7110, Fax: (206) 598-6189, Phone: (206) 598-6131, Email: E-mail: ahoof@u.washington.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all