Detection of low-abundance monoclonal antibodies in a population of polyclonal antibodies is critical for various applications including diagnostics, vaccine development and immunological studies. Next-generation protein sequencing has revolutionized how researchers approach protein analysis.
The latest advances in next-generation protein sequencing overcome the limitations of previous sequencing technologies including high costs, complexity, and time-intensive workflows, by providing a first-of-its-kind benchtop solution to simplify rapid and efficient analysis of complex protein populations.
This application note demonstrates the successful identification of two low-abundance monoclonal antibodies using next-generation protein sequencing.
Download this application note to learn about:
- The latest advances in next-generation protein sequencing technology
- The advantages of next-generation protein sequencing compared to other analysis methods
- Accurately detecting specific antibodies in complex polyclonal populations
APP NOTE
For Research Use Only. Not for use in diagnostic procedures.
Identifying Monoclonal
Antibodies with Quantum-Si’s
Next-Generation
Protein Sequencing Technology
SUMMARY
Identifying low-abundance monoclonal antibodies in a population
of polyclonal antibodies can provide critical insights for diverse
applications such as understanding disease mechanisms, disease
diagnostics, vaccine development, therapeutic antibody discovery, and serological surveillance. Existing methodologies, despite
their utility, come with significant disadvantages including high
costs, complex procedures, and time-intensive workflows, which
often limit their broad application. In this application note, we
present a method utilizing Quantum-Si’s next-generation protein
sequencing technology on the Platinum™ instrument to successfully identify two low-abundance monoclonal antibodies in a
population of polyclonal antibodies.
INTRODUCTION
The detection of low-abundance monoclonal antibodies from a
diverse population of polyclonal antibodies is critical in various
research and clinical settings. In disease diagnostics, particularly
in cancers and autoimmune diseases, the presence of monoclonal
antibodies can serve as valuable biomarkers for early detection or
monitoring disease progression.1–3 They also play a significant role
in immunology research, where understanding the diversity and
specificity of an immune response to an antigen can offer insights
into disease mechanisms. Additionally, in the field of vaccine development and therapeutic antibody discovery, researchers need
to isolate specific monoclonal antibodies that exhibit high affinity
for target antigens, even if present in low amounts. Serological
Q-SI TECHNOLOGY
Quantum-Si’s benchtop Platinum™ instrument enables protein sequencing
from biological samples in a simple
user-friendly workflow. Our technology utilizes dye-tagged N-terminal
amino acid recognizers and semiconductor chip technology to detect the
binding characteristics and binding
order of N-terminal amino acids,
resulting in unique kinetic signatures
that can be used to differentiate and
identify amino acid residues and
PTMs. A more detailed overview of
the workflow and technology can be
found in our Science Paper.
For Research Use Only. Not for use in diagnostic procedures.
2
surveillance also relies on detecting such antibodies to track infectious disease exposure in populations over time.4 Similarly, the
identification of low-abundance monoclonal antibodies can lead
to the development of novel antibody-drug conjugates (ADCs),
where an antibody selectively targets and delivers a drug to tumor
cells, sparing normal cells.5
Several methods exist to identify low-abundance monoclonal
antibodies in a population of polyclonal antibodies. ELISA and
Western blots are commonly used as an easy way to screen for
antibodies; however, these assays might not exhibit the required
sensitivity to detect very low-abundance antibodies and often
demand larger sample volumes. Similarly, flow cytometry and
fluorescence-activated cell sorting (FACS) have the potential for
high sensitivity but demand sophisticated and expensive equipment and entail a technically challenging process of deriving
monoclonal antibodies from sorted B cells. Mass spectrometry
(MS), while offering a detailed view of the antibody repertoire, is
complex, time-consuming, and requires high levels of expertise
to interpret data, which in turn requires more time and resources.
Finally, phage display, while advantageous for high-throughput
screening, is labor-intensive and requires specialized molecular
biology techniques.
Quantum-Si’s Platinum next-generation protein sequencing
platform is a compact, affordable benchtop instrument that
effectively addresses many challenges of traditional antibody
identification techniques. Its user-friendly interface eliminates
the need for specialized expertise to operate or interpret data,
making it a practical choice for a wide range of labs. Moreover, it
offers a fast and efficient way to identify low-abundance monoclonal antibodies, accelerating the pace of research and clinical
diagnostics without the time-consuming processes associated
with many traditional methods. To demonstrate the capabilities of
Quantum-Si’s technology to identify low-abundance monoclonal
antibodies, we sequenced the Fab fragment of two monoclonal
antibodies individually and successfully identified the Fab fragments in a mixed population of polyclonal antibodies.
Identifying Monoclonal Antibodies with Quantum-Si’s Next-Generation Protein Sequencing Technology
REFERENCES
1. M. J. Monroy-Iglesias, S. Crescioli, K. Beckmann, N. Le, S. N.
Karagiannis, M. van Hemelrijck,
A. Santaolalla. Antibodies as biomarkers for cancer risk: a systematic review. Clin. Exp. Immunol.
209 (1), 46–63 (2022).
2. M. A. M. van Delft, T. W. J. Huizinga. An overview of autoantibodies in rheumatoid arthritis. J.
Autoimmun. 110, 102392 (2020).
3. P. D. Burbelo , S. M. Gordon,
M. Waldman, J. D. Edison, D. J.
Little, R. S. Stitt, W. T. Bailey, J.
B. Hughes, S. W. Olson. Autoantibodies are present before
the clinical diagnosis of systemic sclerosis. PloS One 14 (3),
e0214202 (2019).
4. C. Y.-P. Lee, R. T. P. Lin, L. Renia, L.
F. P. Ng. Serological Approaches
for COVID-19: Epidemiologic
Perspective on Surveillance and
Control. Front. Immunol. 11, 879
(2020).
5. M. S. Castelli, P. McGonigle, P.
J. Hornby. The pharmacology
and therapeutic applications of
monoclonal antibodies. Pharmacol. Res. Perspect. 7 (6), e00535
(2019).
6. Reed BD et al. Real-time dynamic single-molecule protein
sequencing on an integrated
semiconductor device. Science.
2022 Oct 14;378(6616):186-192.
For Research Use Only. Not for use in diagnostic procedures.
Identifying Monoclonal Antibodies with Quantum-Si’s Next-Generation Protein Sequencing Technology 3
METHODOLOGY & WORKFLOW
The Fab fragments of two monoclonal antibodies (mAb) were
isolated, enriched, and individually sequenced on Platinum using
our previously established library preparation and real-time sequencing procedure.6 Subsequently, the mAbs were mixed into a
population of polyclonal antibodies (pAb) at the mAb abundance
levels of 12.5% and 2.5% before sequencing on the Platinum.
The total antibody concentrations at the start of the library preparation process were 5–10 µM. In brief, the process involves digesting proteins into peptide fragments and conjugating the peptides
to macromolecular linkers at the C-terminus. The peptides are
then immobilized on Quantum-Si’s semiconductor chip at a loading concentration of 7 nM, exposing the N-termini for sequencing.
Dye-tagged recognizers bind on and off to N-terminal amino acids
(NAAs), creating pulsing patterns with distinct kinetic properties.
Sequential removal of individual NAAs is achieved using aminopeptidases present in the solution, allowing for the detection of
subsequent amino acids. Fluorescence lifetime, intensity, and
binding kinetics of each NAA binding event are collected and
processed using Cloud-based software to determine the peptide
sequence of the Fab fragment and the corresponding antibody.
RESULTS & DISCUSSION
THE MONOCLONAL ANTIBODIES ARE SUCCESSFULLY IDENTIFIED INDIVIDUALLY
We first sought to demonstrate the capability of the Platinum in
sequencing the monoclonal antibodies by analyzing their unique
peptide sequences. We sequenced each antibody on the Platinum instrument using five NAA recognizers with specificity for
11 NAAs (F, Y, W, L, I, V, R, A, S, N, and Q)—and analyzed data to
identify recognition segments (RSs), determine the mean pulse
duration (PD) of each RS, and characterize the kinetic signature of
the peptides of interest. For antibody A, we focused on a unique
peptide sequence of TTLFLQMNSLRAEDTAVYYCAK, located in
the heavy chain of its variable region. Similarly, antibody B has
a unique peptide sequence of NSLYLQMNSLRAEDTAVYYCAREGNSGYEPLDYWGQGTLVTVSSASTK in its variable region’s heavy
chain. The successful identification of these unique peptides indicates the accurate sequencing of the corresponding antibodies.
For Research Use Only. Not for use in diagnostic procedures.
Identifying Monoclonal Antibodies with Quantum-Si’s Next-Generation Protein Sequencing Technology 4
The two peptides were detected in the sequencing data and displayed characteristic pulsing patterns (Figure 1). The unique peptide of antibody A was sequenced up to at least the tenth amino
acid (L), while the unique peptide of antibody B was sequenced
up to at least the eighth amino acid (N), as demonstrated by the
representative traces. The kinetic properties of these peptides
were demonstrated through the mean PD values for each recognition segment (RS). These results demonstrate the effectiveness
of Quantum-Si Platinum in accurately sequencing monoclonal antibodies, enabling the identification of unique peptide sequences
specific to each antibody.
FIGURE 1
The Sequencing Data of Unique Peptides From Two Monoclonal Antibodies. Each
residue in the sequence is colored by the corresponding recognizer. Pulse data
is collected over a 10-hour period of sequencing, with each recognition segment (RS) colored by the corresponding recognizer. The coverage for each RS is
indicated by the height of color in the box, and the corresponding pulse duration
distribution.
For Research Use Only. Not for use in diagnostic procedures.
Identifying Monoclonal Antibodies with Quantum-Si’s Next-Generation Protein Sequencing Technology 5
THE MONOCLONAL ANTIBODIES ARE SUCCESSFULLY IDENTIFIED AT LOW ABUNDANCE IN A POPULATION OF POLYCLONAL
ANTIBODIES
Next, we sought to demonstrate the capability of the Platinum
in sequencing the monoclonal antibodies at low abundance in
a population of polyclonal antibodies by identifying their unique
peptide sequences. Antibodies A and B were mixed with polyclonal antibodies at mAb:pAb ratios of 1:7 and 1:39, which correspond to mAb abundance levels of 12.5% and 2.5%, respectively.
Similar to individual sequencing results above, the two unique
peptides of antibodies A and B were readily detected in the sequencing data. The unique peptide of antibody A was sequenced
up to at least the tenth amino acid (L), while the unique peptide
of antibody B was sequenced up to at least the eighth amino acid
(N). These results demonstrate the effectiveness of the Platinum’s
ability to sequence and identify unique peptides of monoclonal
antibodies.
CONCLUSION
In this application note, we demonstrated the successful identification of individual and low-abundance monoclonal antibodies in
a population of polyclonal antibodies using the Platinum protein
sequencing platform. The results underscore the platform’s
potential to accelerate research involving monoclonal antibody
detection and characterization. By overcoming traditional challenges of cost, complexity, and time consumption, Quantum-Si’s
technology can be an effective tool for advancing immunological
research, disease diagnostics, and therapeutic development,
potentially opening new opportunities for precision medicine.
For Research Use Only. Not for use in diagnostic procedures.
6
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