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Abstract Number: 27

Top-Down Proteomics Coupled with Antibody Sequencing from Single B Cells Reveals a Monoclonal Anti-Sm Clone Present in the Serum of an SLE Patient over Three Years

Zhe Wang1, Jennifer Muther2, Judith A. James3, Si Wu1 and Kenneth Smith2, 1Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 2Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, 3University of Oklahoma Health Sciences Center, Oklahoma City, OK

Meeting: 2018 ACR/ARHP Annual Meeting

Keywords: autoantibodies and proteomics, B cells, SLE

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Session Information

Date: Sunday, October 21, 2018

Session Title: B Cell Biology and Targets in Autoimmune and Inflammatory Disease Poster

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose:

Proteomics is becoming an increasingly powerful tool to study autoantibodies. Current bottom-up approaches are yielding a plethora of knowledge about public clonotypes and clonal turnover. However, reports using top-down proteomics are scarce and the advantages of not using digestions to increase the complexity of already complex mixtures are enormous. De novo top-down sequencing approaches, however, are extremely difficult. To overcome these limitations, we have coupled antibody sequencing from single B cells with targeted top-down MS for analysis and quantification of antibodies in autoimmune patient serum.

Methods:

Fully human recombinant monoclonal antibodies were generated from single B cells. Antibody secreting cells (ASCs), sorted 7 days after vaccination of an SLE patient, have specificities to the vaccine antigen, but SLE patients also have persistent ASCs with auto-specificities which we have detected using this methodology. After identifying monoclonal specificities of interest, we then applied top-down proteomics to search for this antibody clone of known heavy and light chain sequences in longitudinal serum samples from the same donor. The detected ion intensities of known masses were used for quantitation. This allows us to directly link the presence of a specific antibody-producing B cell with a specific monoclonal antibody in the polyclonal serum antibody pool.

Results:

An anti-Sm monoclonal was characterized from an SLE donor with high (>mg/ml) levels of anti-Sm. This particular antibody was isolated from an ASC following vaccination with the influenza vaccine. Although this antibody does not bind to influenza virus, it binds to both SmB’ (1.5 nM affinity) and SmD (2.6 nM affinity). The serum of the donor was also carefully analyzed over a three-year period and this patient’s antibody response to Sm affinity matured, increasing in overall affinity in each year. In order to determine whether this antibody was present in the serum during these three years we purified IgG from the donor’s serum using protein A, and applied the polyclonal IgG to an Ultra High-pressure Liquid Chromatography-High Resolution Mass Spectrometry system for top-down proteomics analysis. We determined that the antibody is present in the serum in the year the ASC was isolated, as well as the years before and after.

Conclusion: Our results demonstrate the ability of top-down proteomics, coupled with single cell sequencing of monoclonal antibodies, to perform targeted analysis of specific serum autoantibody clones over time. In this particular SLE patient, we characterized an anti-Sm monoclonal antibody from a single B cell and determined that this antibody is present in the serum over a period of at least three years. Although clonal turnover may produce new predominant clones over a period of months, this technique shows that particular antibody clones are still present for extended periods of time (years). Overall, using single cell sequencing to enable “targeted” top-down proteomics, is a potent method for analyzing single monoclonal antibody specificities in the serum.


Disclosure: Z. Wang, None; J. Muther, None; J. A. James, None; S. Wu, None; K. Smith, None.

To cite this abstract in AMA style:

Wang Z, Muther J, James JA, Wu S, Smith K. Top-Down Proteomics Coupled with Antibody Sequencing from Single B Cells Reveals a Monoclonal Anti-Sm Clone Present in the Serum of an SLE Patient over Three Years [abstract]. Arthritis Rheumatol. 2018; 70 (suppl 9). https://acrabstracts.org/abstract/top-down-proteomics-coupled-with-antibody-sequencing-from-single-b-cells-reveals-a-monoclonal-anti-sm-clone-present-in-the-serum-of-an-sle-patient-over-three-years/. Accessed March 23, 2023.
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