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

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.

Figure 1. Endogenous antibody interference in sandwich immunoassays

A modern sandwich immunoassay for thyroglobulin is pictured on the left. A streptavidin coated paramagnetic bead (large sphere) binds biotin-labeled (small spheres) capture antibody (white), which forms a sandwich with analyte (gray globule) and enzyme-labeled (sunburst) reporter antibody (dotted). Sandwiches bound to beads are separated using a magnet, unbound reporter antibody is washed away, and enzyme is detected using one of a variety of automated methodologies. Unfortunately, in a high percentage of patients, the epitopes needed for sandwich formation are sterically protected by anti-analyte antibodies (black) as pictured in the middle. In other patients, non-specific anti-reagent antibodies (gray) are able to bridge the gap between capture and reporter antibodies in the absence of analyte as picture on the right.

Figure 2. HPLC-tandem mass spectrometry

Complex mixtures of peptides are first separated by HPLC based on hydrophobicity. If a UV detector were placed in-line after the HPLC column, peptides would be detected as they elute from the column. In this example, four peptides elute simultaneously (e.g. at the black arrow in the UV trace). In the first quadrupole (Q1), the mass-to-charge ratio (m/z) of the precursor peptide is selected. Here, two peptides of identical m/z make it to the second quadrupole (Collision), where they are fragmented by collision induced dissociation. A single fragment m/z is selected in the third quadrupole (Q3) and any ions that reach the detector have the correct precursor m/z and fragment ion m/z. Specificity in the HPLC-tandem mass spectrometric experiment thus arises from the retention time on the HPLC column and the precursor-fragment ion pair, which is also called a transition. In a multiple reaction monitoring experiment, multiple transitions are monitored.

Figure 3. Immunoaffinity peptide enrichment and isotope dilution tandem mass spectrometry

All proteins in the sample are digested to peptides with trypsin, which digests analyte (gray globule) and any interfering endogenous immunoglobulins (black). Stable isotope labeled peptide is added after digestion and the peptides are incubated with beads coated in polyclonal anti-peptide antibody. Unbound peptides are washed away and bound peptides are eluted and then analyzed using HPLC-tandem mass spectrometry.