Single B-cell screening provides a direct approach for identifying antibody sequences from individual antibody-producing B cells. By isolating individual cells and recovering their paired antibody sequences, the approach enables antibody candidates to be identified and subsequently produced for experimental characterization.
Biointron has established a single-B-cell antibody discovery platform, named AbDrop, based on microfluidic technology. The platform combines high-speed single B-cell sorting with Biointron’s rapid antibody expression capabilities, allowing identified sequences to move from screening into recombinant antibody production and experimental validation.
In this case study, the platform was applied to the discovery of antibodies targeting programmed cell death protein 1 (PD-1), an immune checkpoint receptor and established therapeutic target in oncology. The objective was to identify PD-1-specific antibodies while retaining sequence and epitope diversity among the resulting candidates.
The discovery workflow combines single B-cell screening with downstream recombinant antibody expression and characterization.
Using the microfluidic screening platform, individual B cells are rapidly screened while a large number of antibody sequences can be retained for subsequent analysis. Rather than advancing every sequence identified during screening, selected candidates can then be expressed and evaluated experimentally.
For the PD-1 project, the workflow consisted of:
Single B-cell screening → sequence recovery and candidate selection → antibody expression → PD-1 binding analysis → affinity characterization → sequence diversity analysis → epitope binning
From the hundreds of sequences retained during screening, 62 antibody candidates were selected for recombinant expression and further experimental validation.
The 62 selected antibody candidates were first evaluated for binding to PD-1 by ELISA.
Of these candidates, 59 of 62 showed positive binding to PD-1, corresponding to approximately 95% of the antibodies advanced for experimental characterization.
The ELISA concentration-response curves showed binding across the selected antibody panel, with a positive control included for comparison.
The 59 ELISA-positive antibodies were subsequently characterized by surface plasmon resonance (SPR) to evaluate binding affinity.
SPR analysis showed that most of the antibodies exhibited high-affinity binding to PD-1, with affinities reported predominantly within the low-nanomolar range. According to the study data, nearly all of the evaluated antibodies were within approximately the 0.1–1 nM range.
A nivolumab biosimilar was included as a positive control for the affinity analysis.
These results show that the single-B-cell screening workflow generated a substantial panel of PD-1-binding antibodies that could be carried forward into more detailed characterization rather than producing only a small number of initial leads.
Affinity alone was not the only objective of the screening campaign. Retaining antibody diversity was important because multiple antibodies against the same antigen can differ substantially in sequence and binding properties.
The complementarity-determining regions (CDRs) of the heavy and light chains from the 59 PD-1-binding antibodies were therefore analyzed to assess sequence diversity.
Phylogenetic analysis showed diversity across the antibody panel, indicating that the screening process recovered multiple distinct antibody sequences rather than a highly redundant population of closely related candidates.
This diversity provided a broader set of molecules for subsequent functional and epitope characterization.
The antibodies were further characterized by epitope binning to determine whether they recognized overlapping or distinct regions of PD-1.
The resulting interaction pattern showed that the antibody candidates could be primarily divided into four epitope groups.
B6924, a nivolumab biosimilar, and B2014, a pembrolizumab biosimilar, were included as reference controls. The analysis identified multiple antibodies with binding patterns distinct from these controls, demonstrating that the screening campaign generated antibodies recognizing different PD-1 epitopes.
This is particularly useful during early antibody discovery because it preserves multiple binding profiles for subsequent evaluation rather than narrowing the candidate pool solely on the basis of antigen binding or affinity.
The PD-1 campaign shows how combining single-B-cell screening with rapid recombinant antibody expression can generate a sizeable antibody panel for downstream characterization.
Starting from hundreds of retained sequences, 62 candidates were selected for expression and validation. Fifty-nine of the 62 selected antibodies bound PD-1 by ELISA, and subsequent SPR characterization identified high-affinity binders within the panel.
CDR analysis demonstrated sequence diversity, while epitope binning separated the antibodies into four major groups and identified candidates with binding profiles distinct from nivolumab and pembrolizumab reference antibodies.
Together, these measurements provided several dimensions for evaluating the candidate pool:
Antigen binding: 59 of 62 selected antibodies showed positive PD-1 binding by ELISA.
Affinity: SPR identified antibodies with binding affinities predominantly in the low-nanomolar range.
Sequence diversity: Heavy- and light-chain CDR analysis showed diversity among the 59 PD-1 binders.
Epitope diversity: Epitope binning identified four major groups, including candidates distinct from the reference antibodies.
For an antibody discovery campaign, this type of dataset allows candidates to be compared using multiple experimentally measured properties before deciding which molecules warrant further investigation.
A practical challenge in antibody discovery is moving from initial sequence identification to experimental characterization of the resulting molecules.
Biointron integrates its microfluidic single-B-cell screening platform with recombinant antibody expression, allowing selected sequences to proceed directly into antibody production and validation within the same discovery workflow.
In the PD-1 case study, this integration enabled dozens of candidates to be evaluated by binding, affinity, sequence diversity, and epitope characteristics following the initial single-B-cell screen. This provides one example of how this workflow can be used to generate and characterize a diverse antibody candidate pool.
Biointron’s single-B-cell antibody discovery platform combines microfluidic single B-cell screening with downstream antibody expression and characterization.
With hundreds of sequences during screening and integrating sequence selection with recombinant expression, the platform is designed to support the evaluation of multiple antibody candidates rather than relying on an individual lead identified at the screening stage.
In this PD-1 project, 62 candidates were selected for expression, 59 demonstrated PD-1 binding, and the resulting antibody panel contained both sequence and epitope diversity. The study demonstrates the platform’s ability to move from single-B-cell screening to experimental characterization of a substantial set of antibody candidates.
PD-1 is an inhibitory immune checkpoint receptor that regulates T-cell responses. Therapeutic antibodies targeting the PD-1 pathway are established in cancer immunotherapy, making PD-1 a relevant model target for antibody discovery and characterization.
Biointron’s AbDrop platform uses microfluidic technology for high-speed single B-cell screening and retains hundreds of antibody sequences for subsequent candidate selection. Selected sequences can then move into recombinant antibody expression and experimental validation.
Retaining a broader sequence pool allows candidates to be selected and compared beyond initial antigen recognition. In this study, the resulting antibodies were evaluated for binding, affinity, CDR sequence diversity, and epitope characteristics.
Of the 62 candidates selected for expression and validation, 59 showed positive PD-1 binding by ELISA. The resulting antibody panel contained high-affinity binders, demonstrated CDR sequence diversity, and could be separated primarily into four epitope groups.
Epitope binning helps determine whether antibodies have overlapping or different antigen-binding profiles. In this case study, binning separated the PD-1 antibodies into four major groups and identified candidates with epitope profiles distinct from the nivolumab and pembrolizumab reference antibodies.
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