Polyclonal Antibody Sequencing Methods
Polyclonal Antibody Sequencing Methods
Polyclonal antibody sequencing is a powerful tool for understanding the humoral immune response.1 Two primary approaches exist: Proteogenomic Polyclonal Antibody Sequencing and De Novo Polyclonal Antibody Sequencing. This guide will differentiate these methods, highlighting their workflows, applications, benefits, and limitations.
Proteogenomic polyclonal antibody sequencing can identify lower abundance serum clones, but abundant clones may be missed if no corroborating B cell is detected.
De novo polyclonal antibodies recover the most abundant antibodies in the sample, but potentially miss rare clones.
Proteogenomic Polyclonal Antibody Sequencing Methods
Overview of a typical proteogenomic polyclonal antibody sequencing method. Starting from a host animal or patient, serum is collected and enriched for antigen-binding antibodies using affinity chromatography. The enriched antibody is analyzed by tandem-mass spectrometry (Top track of diagram). From the same host, a B cell source is collected, then the antibody transcripts are captured, amplified, and sequenced (Bottom track of diagram). Software is needed to integrate the mass spectrometry data and next-generation sequencing data, and to identify the set of best candidate monoclonal antibodies.
The proteogenomic polyclonal antibody sequencing method has been successfully used as a method for antibody discovery in rabbits [1,2], llamas [3], and humans [4,5,6]. This method forms the basis for our own serum-focused antibody discovery platform, Alicanto. In the proteogenomic approach, the B cell repertoire forms an in silico ‘library’ of candidate antibodies, and the serum antibody measurements are used to select which candidates are present in antigen-specific polyclonal antibody. Serum clones must have a corroborating B cell sequence in order to be identified.
Key considerations when planning a proteogenomic polyclonal antibody sequencing project are the source of the B cells and what B-cell processing will be performed. As to the source of B cells, our proteogenomic studies in rabbit [2] showed that the spleen has the highest corroboration with serum antibodies of any of the B cell sources collected.
A second consideration is what B cell population to target. B cells derived from peripheral blood mononuclear cells are predominantly naive and non-antigen exposed. Memory B cells contain the immune memory of past exposures, however, depending on the recency of the challenge, these may yield the highest corroboration with serum. Antibody-secreting cells, such as plasmablasts, have good corroboration with serum, but are rare in the blood. They also don’t have B-cell receptors on their cell surface, making them difficult to sort based on antigen-reactivity.
Major B cell subsets that are found in common B cell sources. The majority of B cells in blood are naive, and have never been exposed to an antigen. The serum antibodies are produced by antibody secreting cells, like plasmablasts and plasma cells, that are rare in blood. Memory B cells are a major component of B cells in blood, and capture a broad history of past exposures.
A third consideration is how to sequence the B cells. Single cell sequencing methods are available from a growing number of suppliers, with kits available for B cell sequencing from common species. The benefit of single cell sequencing is the retention of heavy and light chain pairs, with the limitation of throughput. An alternate approach is to perform bulk repertoire sequencing, which can be ideal for capturing cell populations where the cells are rare, but the transcripts are abundant (e.g. plasmablasts). Deep bulk repertoire sequencing results in large repertoires, however, heavy and light chain pairs are lost.
Key benefits of proteogenomic polyclonal antibody sequencing:
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High sequence confidence since clone is observed in both B cells and serum.
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High sensitivity, since complete peptide coverage of the serum clone is not required.
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Lower sample requirements (~25ug of target specific polyclonal antibody for Alicanto) compared to de novo polyclonal antibody sequencing.
Limitations:
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Requires B cells collected contemporaneously with serum antibodies.
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Different B cell subsets may corroborate more or less with serum antibodies.
De Novo Polyclonal Antibody Sequencing Methods
Overview of a de novo polyclonal antibody sequencing workflow. Serum or plasma is collected from the host, typically an immunized animal or patient. Antigen-binding antibodies are isolated from the input using affinity chromatography, and analyzed by tandem mass spectrometry. Specialized software is needed to recover full-length antibody sequences from the mass spectrometry data.
De novo polyclonal antibody sequencing methods enable the identification of individual antibodies in serum or plasma, without the need for a B cell source. These methods are ideal for:
- converting existing polyclonal antibodies to monoclonal antibodies to enable more reproducible production and function,
- analyzing biorepository samples where cell viability may be low,
- analyzing non-blood biofluids where B cells are rare (e.g. cerebral spinal fluid),
- cataloging the dominant clones in a patient sample [7]
Typically, de novo polyclonal antibody sequencing methods require more extensive mass spectrometry data generation in order to capture sequences from the entire length of the antibody. We use this approach for our own polyclonal antibody sequencing platform, Griffin. You can listen to a brief overview of our Griffin method in a recent talk. The higher sample depth required for de novo sequencing as compared to proteogenomic methods means that only the highest abundance clones can be sequenced.
Key benefits of de novo polyclonal antibody sequencing:
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Captures the most abundant antibodies in the sample, which often have the highest impact on the activity and may be most relevant to the application.
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Enables sequencing from samples without any B cells – enabling samples/use-cases that elude proteogenomics.
Limitations:
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Higher sample requirement as compared to proteogenomic antibody sequencing
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May miss out on lower abundance antibodies in the sample.
Summary of polyclonal antibody sequencing methods
References
[1] Cheung, Wan Cheung, et al. “A proteomics approach for the identification and cloning of monoclonal antibodies from serum.” Nature biotechnology 30.5 (2012): 447-452.
[2] Bonissone, Stefano R., et al. “Serum proteomics expands on identifying more high-affinity antibodies in immunized rabbits than deep b-cell repertoire sequencing alone.” BioRxiv (2019): 833871.
[3] Fridy, Peter C., et al. “A new generation of nanobody research tools using improved mass spectrometry-based discovery methods.” Journal of Biological Chemistry 300.9 (2024): 107623.
[4] Lavinder, Jason J., et al. “Identification and characterization of the constituent human serum antibodies elicited by vaccination.” Proceedings of the National Academy of Sciences 111.6 (2014): 2259-2264.
[5] Gilchuk, Pavlo, et al. “Proteo-genomic analysis identifies two major sites of vulnerability on ebolavirus glycoprotein for neutralizing antibodies in convalescent human plasma.” Frontiers in immunology 12 (2021): 706757.
[6] Patel, Anand, et al. “Serum proteomics reveals high-affinity and convergent antibodies by tracking SARS-CoV-2 hybrid immunity to emerging variants of concern.” Frontiers in Immunology 16 (2025): 1509888.
[7] Guthals, Adrian, et al. “De novo MS/MS sequencing of native human antibodies.” Journal of proteome research 16.1 (2017): 45-54.