Understanding the Role of Immunoaffinity-Based Mass Spectrometry Methods for Clinical Applications

Abstract

Both ligand binding assays (LBA)2 and mass spectrometry (MS) methods are widely used in routine clinical analysis. In recent years, an interesting trend has emerged which blends these 2 techniques to combine the selectivity of immunoaffinity (IA) extraction with the specificity of MS detection (IA-MS). The brief communication by Laha and coworkers in the current issue of Clinical Chemistry , entitled “Characterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to Multiplexed Liquid Chromatography–Tandem Mass Spectrometry,” is an excellent example of the power of this combined approach (1). These authors, who have had previous experience with multiplexed liquid chromatography–tandem MS (LC-MS/MS) vitamin D analysis (2), showed that the limited specificity of an anti–1α,25-dihydroxyvitamin D capture antibody could be used with LC-MS/MS to deliver a sensitive, highly specific assay for 5 vitamin D–related metabolites. Because of differing affinities of the capture antibody for the various metabolites and their wide range in concentrations (1000-fold), Laha et al. performed a detailed binding assessment by LC-MS/MS using synthetic standards to verify the suitability of their approach. Moreover, because 2 known interfering metabolites were shown not to bind to the antibody, the authors were able to use a shorter chromatographic run time. The report by Laha et al. highlights the growing awareness about the need for highly specific assays in clinical biomarker analysis. Historically, lipids have been measured by competitive LBA formats. However, accumulating evidence has raised concerns about the specificity of these approaches, and several assays have been replaced by more specific LC-MS/MS methods. Testosterone analysis is probably the most widely recognized example of this trend (3). Still, although LC/MS/MS is a well-established tool for clinical biomarker analysis, comparatively few IA-MS applications have appeared for small molecules (4), and the uptake of IA-MS utilization is uncertain. Interestingly, the situation is entirely different for …