Academy of Diagnostics & Laboratory Medicine - Scientific Short

Is it time for antibodies and mass spectrometers to dissociate?

Frederick G. Strathmann

The following post was written to update a previous Short. Please see the past Short on this topic here.

Almost 10 years have passed since I asked the question – Should I be embarrassed that my mass spec assay uses an antibody? Since then, many of my colleagues have provide me numerous examples as to why I should be embarrassed in general. However, during this same period the number of immunoaffinity enrichment methods and use of antibody-mediate sample prep approaches continues to increase which indicates the combination of antibody-mediated sample preparation with mass spectrometric detection is a workflow that is here to stay. This time around, we’ll look at a few well-known and a few still evolving clinical applications using antibodies to augment mass spectrometric methods.

Thyroglobulin (Tg) is a well-known example where use of mass spectrometry has resolved problems with antibody-based detection methods caused by the presence of autoantibodies. In 2013, Kushnir et al. published a method using rabbit polyclonal anti-Tg antiserum, protein precipitation, tryptic digestion, and use of an antibody specific to the Tg tryptic peptide used for analysis(1). In 2016 a multi-center study was published as a first step in harmonizing clinically validated LC-MS/MS thyroglobulin assays across four performing laboratories – all of which used an antibody-mediate enrichment strategy(2). Numerous other clinical assays have been developed and published using a similar antibody-based enrichment strategy and the sheer number of targets available through patented technologies such as SISCAPA® (even COVID-19 reagents) demonstrate how deeply entrenched this approach is for mass spectrometry.

Enrichment of peptides using an antibody is a powerful approach but by no means the only application using antibodies in mass spectrometry. A review by Delaunay et al. from 2020 highlights the use of immunosorbents to selectively extract various types of toxins from small mycotoxins to large proteins(3). Antibodies immobilized on a solid sorbent are becoming more widely available and can be found packed into conventional solid-phase extraction cartridges or dispersed directly into the sample. Immobilizing different antibodies on the same sorbent combines the sensitivity and multiplexing capability of immunoaffinity enrichment with the resolving power of a mass spectrometer. Going one step further is the new yet also old version of an antibody – aptamers. Oligosorbents or aptamer affinity columns offer several advantages as selective extraction tools: they can be prepared for targets that do not induce an immune response, are substantially easier to produce with less lot-to-lot variation, are typically highly stable, and allow faster regeneration times between samples.

It seems inevitable that the future of mass spectrometry will involve antibodies or nearest neighbors in some form or another. But if you have not yet found your way to clinical mass spectrometry and are feeling left out - all is not lost. Yamashita et al. in 2021 published a promising comparison of a fully automated chemiluminescence enzyme immunoassay against an immunoprecipitation mass spectrometric method to detect β-amyloid in plasma samples(4). The ratio of Aβ42 to Aβ40 in plasma has shown promise in comparison to amyloid -β positron-emission tomography as a predictor of brain amyloid-β-positive status associated with Alzheimer’s disease. The automated method used the High Sensitivity Chemiluminescence Enzyme Immunoassay (HILIC™) platform with a biotinylated capture antibody immobilized on magnetic beads and an alkaline phosphatase-conjugated detection antibody. The automated assay used 30 µL of sample and 17 minutes of analysis time in comparison to 250 µL and a several hour workflow using immunoaffinity enrichment and LC-MS/MS. Future studies are still needed but this demonstrates an encouraging continuum from the cutting edge work using mass spectrometry-based methods to the eventual use of a platform amenable to high throughput and lower complexity testing for widespread availability.

Perhaps in another 10 years the next question to ask will be should I be embarrassed that my mass spec assay does not use 3D printed reagents or consumables(5) or incorporate artificial intelligence(6)?

  1. Kushnir MM, Rockwood AL, Roberts WL, Abraham D, Hoofnagle AN, Meikle AW. Measurement of thyroglobulin by liquid chromatography–tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clinical Chemistry 2013;59:982-90.
  2. Netzel BC, Grant RP, Hoofnagle AN, Rockwood AL, Shuford CM, Grebe SK. First steps toward harmonization of lc-ms/ms thyroglobulin assays. Clin Chem 2016;62:297-9.
  3. Delaunay N, Combès A, Pichon V. Immunoaffinity extraction and alternative approaches for the analysis of toxins in environmental, food or biological matrices. Toxins (Basel) 2020;12.
  4. Yamashita K, Watanabe S, Ishiki K, Miura M, Irino Y, Kubo T, et al. Fully automated chemiluminescence enzyme immunoassays showing high correlation with immunoprecipitation mass spectrometry assays for β-amyloid (1-40) and (1-42) in plasma samples. Biochem Biophys Res Commun 2021;576:22-6.
  5. Grajewski M, Hermann M, Oleschuk RD, Verpoorte E, Salentijn GI. Leveraging 3d printing to enhance mass spectrometry: A review. Anal Chim Acta 2021;1166:338332.
  6. Xiao Q, Zhang F, Xu L, Yue L, Kon OL, Zhu Y, Guo T. High-throughput proteomics and ai for cancer biomarker discovery. Adv Drug Deliv Rev 2021;176:113844.

 

Academy of Diagnostics & Laboratory Medicine Designation

Fellows of the Academy use the designation of FADLM. This designation is equivalent to FACB and FAACC, the previous designations used by fellows of the National Academy of Clinical Biochemistry and AACC Academy. Those groups were rebranded as Academy of Diagnostics & Laboratory Medicine in 2023.