Analytical and Bioanalytical Chemistry

, Volume 408, Issue 29, pp 8325–8332 | Cite as

Quantification of antibody coupled to magnetic particles by targeted mass spectrometry

Research Paper


Quantifying the amount of antibody on magnetic particles is a fundamental, but often overlooked step in the development of magnetic separation-based immunoaffinity enrichment procedures. In this work, a targeted mass spectrometry (MS)-based method was developed to directly measure the amount of antibody covalently bound to magnetic particles. Isotope-dilution liquid chromatography-tandem MS (ID-LC-MS/MS) has been extensively employed as a gold-standard method for protein quantification. Here, we demonstrate the utility of this methodology for evaluating different antibody coupling processes to magnetic particles of different dimensions. Synthesized magnetic nanoparticles and pre-functionalized microparticles activated with glutaraldehyde or epoxy surface groups were used as solid supports for antibody conjugation. The key steps in this quantitative approach involved an antibody-magnetic particle coupling process, a wash step to remove unreacted antibody, followed by an enzymatic digestion step (in situ with the magnetic particles) to release tryptic antibody peptides. Our results demonstrate that nanoparticles more efficiently bind antibody when compared to microparticles, which was expected due to the larger surface area per unit mass of the nanoparticles compared to the same mass of microparticles. This quantitative method is shown to be capable of accurately and directly measuring antibody bound to magnetic particles and is independent of the conjugation method or type of magnetic particle.

Graphical Abstract

Schematic illustration of the isotope-dilution mass spectrometry-based workflow to directly measure antibody bound to magnetic particles (MP)


Bioanalytical methods Immunoaffinity enrichment Mass spectrometry Nanoparticles/Nanotechnology 

Supplementary material

216_2016_9948_MOESM1_ESM.pdf (399 kb)
ESM 1(PDF 398 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2016

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of MarylandCollege ParkUSA
  2. 2.Biomolecular Measurement DivisionNational Institute of Standards and TechnologyGaithersburgUSA

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