SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays

Abstract

This study presents a novel method based on size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) for the characterisation and quantification of immunoassays employing lanthanide-labelled antibodies. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD=3.7 ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid high-resolution chromatographic method with separation times under 7 minutes of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided new insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153 Eu labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156 Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated only 1.9% from the immunochemical approach employing 153 Eu labelled polyclonal antibodies.

Immunoassays based on inductively coupled plasma mass spectrometry detection: So far so good, so what?

“…DTPA, DOTA, MeCAT®, etc.) and a reactive group which binds covalently to functional groups present in the antibody [24,25,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47]. As regards transition metals, they have mostly been used as NPs [30][31][32]34,.…”

The Immuno-Mass Spectrometry Chemical Microscope

“…Accurate characterization of nanoparticle dimensions using electron microscopy and REE content by ICP-MS should be used to determine the ideal nanoparticle reporter what gives adequate sensitivity while also limiting potential steric hindrance that reduces total antigen-antibody binding. Chromatography with ICP-MS detection and quantification by isotope dilution can determine the number of metal atoms bound to a single antibody using the sulfur content of the immunoglobulin as an analytical proxy [7].…”

Matching sensitivity to abundance: high resolution immuno-mass spectrometry imaging of lanthanide labels and endogenous elements in the murine brain

“…Although IHC-based methods have improved dramatically over the past decades [9][10][11] , multiplexed visualisation of low abundant proteins is limited due to problems such as photo-bleaching 12 , co-localisation of proteins, and matrix or background autofluorescence 13,14 . Emerging novel methods that combine IHC and mass spectrometry have considerable potential to address the spatiotemporal and quantitative challenges presented by the measurement of proteins in biological systems [15][16][17][18][19] . IHC was first integrated into the workflow of mass imaging via laser ablation -inductively coupled plasma -mass spectrometry (LA-ICP-MS) by Zhang et al for the investigation of thyroid stimulating hormone 20 .…”

“…IHC was first integrated into the workflow of mass imaging via laser ablation -inductively coupled plasma -mass spectrometry (LA-ICP-MS) by Zhang et al for the investigation of thyroid stimulating hormone 20 . Since then, various labelling protocols that tag one or several metals to antibodies and different protocols to characterise and validate labelling processes have been developed 14,19,21 . Up to 32 different proteins may be labelled with different metal tags and detected with high sensitivity and subcellular lateral resolution (1 µm) 22 .…”