Mass Spectrometric Immunoassay

Abstract

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus.

An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

“…The iMALDI technique belongs to a general category of techniques which couples affinity capture with direct mass spectrometric analysis of target proteins (Experimental section and Figure 1) [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]. In iMALDI, anti-peptide antibodies are immobilized on sepharose beads for affinity capture, rather than on the surface of a plate, thus eliminating the need for special surface chemistry.…”

“…Unlike conventional affinity capture/elution/MALDI techniques [33,34], the "stable isotope standards with capture by anti-peptide antibodies" (SISCAPA) approach [35], or on-target elution followed by removal of affinity beads as was done by Li [50], in iMALDI the affinity beads are placed directly on the MALDI target and analyzed without prior elution of the bound analytes [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49], which greatly reduces the sample loss and therefore increases the detection sensitivity. In iMALDI, after immuno-adsorption, the antibody beads are arranged in a microarray/spot format on the MALDI-target plate.…”

Glycosylation status of vitamin D binding protein in cancer patients

“…Since its inception in 1995 by R.W. Nelson et al, 21 it has been applied to study a variety of proteins and peptides, [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] and has served as the foundation for the nascent field of Population Proteomics. [37][38][39][40][41][42] Roughly, MSIA can be envisioned as ultra high resolution, semiquantitative Western blotting in which protein variants are thoroughly resolved and their molecular masses determined to within less than 2 Da.…”

Population Proteomics

“…In its simplest form, surface-immobilized ligands are utilized to affinity retrieve a protein of interest from a biological sample after which the protein (with or without the affinity ligand) is introduced in a mass spectrometer. One of the first affinity MS methods was developed in this laboratory and termed mass spectrometric immunoassay (MSIA) 1 (41,50). The approach combines targeted protein affinity extraction with rigorous characterization using MALDI-TOF mass spectrometry (Fig.…”