Exploring Single B-Cell Technologies for Antibody Discovery

What Are Single B Cells?

B lymphocytes, commonly known as B cells, are a type of white blood cell produced in the bone marrow. They are part of the immune system and play a crucial role in adaptive immunity – the part of our immune system that learns to recognize pathogens, cancer cells, and other foreign substances inside our body. They do this by producing Y-shaped proteins called antibodies that bind foreign molecules (antigens), which help neutralize and eliminate the target.

B cells are incredibly diverse. Our body contains millions of distinct B cells, each with a unique receptor located on the B cell’s surface called a B cell receptor. Understanding how B cells make antibodies and harnessing custom antibodies is crucial for vaccine development, diagnostics, and therapeutic antibody production. Researchers use single B cell technologies to isolate and study individual B cells, leading to the discovery of potent monoclonal antibodies.

They continue to be a focal point of scientific investigation, especially due to their pivotal role in the onset of autoimmune disorders and cancers when their development or functionality is impaired.

When Were B Cells First Discovered?

The first clear functional indication of the existence of cells that we now know as B cells came in 1890. It was the discovery by Emil von Behring and Shibasaburo Kitasato of the importance of circulating antitoxins in immunity to diphtheria and tetanus.

In the 1960s, Max Cooper elucidated the role of B cells by conducting experiments on irradiated chickens. He observed that surgical removal of the Bursa of Fabricius, the primary site of B-cell development in birds analogous to the bone marrow in mammals, led to a complete cessation of antibody production in these animals. This pivotal research established the significance of B cells in antibody generation.

Contrary to common belief, the designation “B” in B cells actually originates from the term “bursa,” rather than “bone marrow,” as it is commonly assumed. Cooper’s findings underscored the distinct differentiation pathways for B and T cells within the adaptive immune system, marking a significant milestone in immunology research.

What Are the Two Main Types of B Cells?

The two main types of B cells are plasma cells and memory cells. While plasma cells specialize in producing antibodies to fight current infections, memory cells prepare your immune system for future encounters. Memory B cells are formed after the initial encounter of a B cell with its specific antigen. 

Following activation and proliferation, some of the naive B cells differentiate into memory B cells while other naïve B cells differentiate into plasma cells. Memory B cells are long-lived cells that persist in the body after the primary immune response has resolved. They provide immunological memory and can rapidly respond to subsequent encounters with the same antigen by proliferating and differentiating into short-lived antibody-producing plasma cells.

Single B-cells and Monoclonal Antibodies 

Antibodies play a crucial role in our immune system, helping identify and neutralize foreign substances in the body. Over the years, the development of monoclonal antibodies (mAbs) has revolutionized the field of diagnostics and therapeutics, providing targeted treatments for various diseases, including cancer, autoimmune disorders, and infectious diseases.  Monoclonal antibodies comprise four polypeptide chains, two identical heavy chains, and two identical light chains.

Antibody discovery methods, such as hybridoma technology and phage display, have been and still are instrumental in generating mAbs. However, as with any method, they have their pros and cons, with limitations including time-consuming workflows, low throughput, and the potential loss of rare antigen-specific antibodies, as is the case with hybridoma technology. Single-B cell technologies emerged to overcome these challenges.

The Rise of Single B Cell Antibody Technologies

This shift towards single B cells has marked a significant advancement over traditional methods of hybridoma cell line production, which involve isolating, immortalizing, and culturing B cell cultures or bulk populations in vitro. This method is inferior to the antibody production method known as single B cell isolation for one key reason. 

You must convert B cells with a finite life span and make them live forever by fusing them with a myeloma (cancer) cell line. This process is inefficient, resulting in the death of many B cells and potentially the loss of high-quality antibodies of interest. Single B cell antibody technologies overcome this limitation. Instead of fusing isolated B cells with myeloma cells, the unique VH and VL antibody gene regions are collected from the individual B cells. 

Therefore, single B cell technologies more efficiently capture the entire B cell compartment, ensuring that the B cells that produce rare and potentially valuable antibodies are captured. offer an advantage in terms of a more efficient and streamlined approach to antibody discovery, relying on directly amplifying genes encoding the VH and VL regions from individual human B cells.

By isolating and analyzing individual B cells from diverse sources, researchers can uncover valuable information about antigen specificity and immune memory and identify high-quality antibodies with specific antigenic selectivity from humans and immunized animals (antigen-specific single B cells).

This access to individual B cells facilitated the identification of rare and potent antibodies with therapeutic potential tailored to target specific diseases or conditions, supercharging the development of novel biologics for various disease treatments.

Key Advantages of Single B-Cell Methods for Antibody Discovery

Each antibody discovery methodology has its own set of benefits. Here’s where single B-cell technologies excel:

• Precision and High Specificity: By analyzing single cells, researchers can directly identify and isolate antigen-specific antibodies without the need for hybridoma fusion. This approach ensures that the complete antibody repertoire is captured, improving the likelihood of identifying potent antibodies that bind rare antigens or antigens with low immunogenicity.
• Enhanced Efficiency: Single B-cell screening allows for the parallel analysis of multiple individual B cells, significantly increasing throughput and expediting the antibody discovery process. The ability to analyze large cell populations enhances the identification of rare antigen-specific B cells that may be missed using traditional methods.
•  Versatility in Antibody Engineering: The availability of antibody sequences from single B cells facilitates the engineering of antibodies with desired properties such as increased affinity or altered effector functions. Researchers can manipulate the heavy chain and light chain sequences independently to optimize antibody characteristics.

The Workflow of Single B-Cell Antibody Discovery

Single B-cell antibody discovery involves isolating and characterizing monoclonal antibodies (mAbs) from individual B cells. Here’s an overview of the process:

1. Isolation: Single B cells are isolated from a heterogeneous population of cells in the blood and the spleen using advanced techniques such as fluorescence-activated cell sorting (FACS) or microfluidic sorting to isolate any antibody-secreting cells.
2. Single B Cell Sorting and Screening: Isolated single B cells are individually screened for the presence of antigen-specific antibodies. They can undergo direct lysis for gene amplification or in vitro expansion, and the resulting cell culture supernatants can be examined using ELISA or FACS assays.
3. Antibody Sequencing: The antibody-coding sequences of antigen-specific B cells are retrieved.
   Miniaturization of reaction volumes aids in efficient sequencing.
4. Clonal Selection: Antigen-specific clones are identified and selected based on predetermined selection criteria such as binding affinity and specificity.
5. Characterization: Antibody clones are further characterized to assess their functionality and specificity.

When to Opt for Single B Cell Methods?

You should consider single b-cell technologies for monoclonal antibody discovery when:

• The objective is to procure antibodies with native characteristics and minimal alteration. This approach ensures that the obtained antibodies retain their natural specificity without extensive manipulation.
• Working with limited sample sources or targeting rare B cells. By isolating individual B cells, researchers can maximize the chances of obtaining valuable antibodies even from scarce samples.
• Early assessment of cross-reactivity or functional activities (such as ligand blocking) is crucial. By analyzing individual B cells, researchers can swiftly identify antibodies with desired characteristics, facilitating early decision-making in the research process.

As with any method, single b-cell techniques also come with certain disadvantages:

• The development and implementation of single B-cell technologies can be technically complex.
• Although single B-cell technologies reduce off-target binding, it is not completely eliminated. Some mAbs may still exhibit unintended interactions.
• Developing and maintaining single B-cell systems can be resource-intensive. Research costs may be higher compared to other approaches.

Key Single B Cell Screening Technologies & Methods

What are some of the techniques that rely on B-cells for antibody discovery and generation?  

Flow Cytometry-Based Sorting of B-Cells

A pivotal technique enabling the identification and isolation of individual B cells based on specific markers. This method allows for precise cell selection and is instrumental in capturing rare B cells present in a heterogeneous population.

By using flow cytometry, researchers can isolate single B cells with desired characteristics, such as antigen specificity or expression levels of certain receptors, facilitating downstream analysis and antibody discovery processes.

Microfluidics

These methods offer the identification of individual antigen-specific B cells in a high-throughput manner and single-cell manipulation within a short time frame. Through microfluidic devices, individual B cells can be encapsulated in tiny droplets, allowing for parallel processing of multiple cells simultaneously.

This technology minimizes cross-contamination and enhances the efficiency of isolating and analyzing single cells. Moreover, microfluidics enables the screening of a large number of cells in a short time, accelerating the antibody discovery workflow and enhancing the robustness of results.

BCR Sequencing and Antibody Cloning

By sequencing the B cell receptor (BCR) of individual B cells, researchers can identify the genetic information encoding the antibodies. This information is pivotal for antibody discovery, as it allows for the reconstruction and production of monoclonal antibodies from specific B cells.

Through antibody cloning, researchers can further characterize and validate the function of antibodies, paving the way for the development of novel therapeutic antibodies and diagnostic tools.

Applications of Single B Cell Technologies

Single B cell technologies have revolutionized antibody discovery and development with their diverse applications. 

• Targeted Therapeutics: Monoclonal antibodies derived from single B-cell discovery hold immense potential for the development of personalized therapies targeting specific antigens implicated in various diseases, including autoimmune diseases and cancer. Checkpoint inhibitors and bispecific antibodies are examples of such successful applications.
• Monoclonal Antibody Treatment: Rapid identification of antigen-specific mAbs also enables the development of monoclonal antibody treatments against infectious agents. The ability to isolate potent antibodies from single B cells allows for the design of more effective and targeted treatments against infectious diseases. 
• Disease Research & Diagnostics: The precise identification of antigen-specific antibodies enables the development of diagnostic assays for early disease detection and monitoring. Single B-cell antibody discovery can also facilitate the discovery of novel biomarkers for disease prognosis and treatment response assessment.
• Advancements in Biotechnology: Integration of single B cell antibody discovery with cutting-edge technologies such as phage display and single-cell RNA sequencing will further expand its applications in biotechnology and drug development. The use of mammalian cells for antibody production ensures the generation of antibodies with native post-translational modifications, enhancing their therapeutic efficacy.

The future studies of antibody discovery with single B cell technologies hold immense promise in revolutionizing how we combat diseases. By isolating individual B cells and unraveling the antigen specificity encoded within them, advancements in antibody discovery are on the horizon, paving the way for tailored therapeutic solutions for various ailments.

Thinking of Using the Single B-Cell Approach for Monoclonal Antibody Discovery?

Single B-cell technologies for antibody discovery represent a paradigm shift in antibody engineering and therapeutics. Their ability to precisely identify and isolate antigen-specific antibodies from individual B cells offers many advantages in terms of specificity and efficiency.

The integration of high-throughput technologies and advanced analytical tools further enhances the capabilities of single B-cell approaches, making them indispensable in the quest for innovative solutions.

If you’re seeking to create a monoclonal antibody while preserving its inherent characteristics and optimizing its development potential,  ProteoGenix’s single B-cell sorting service offers antibodies with exceptional affinity, maintaining natural VH and VL pairing and maximizing B-cell diversity.

With our high-throughput single B-cell antibody technology platform, we ensure prompt delivery of a minimum of three antibody sequences within a maximum of two months, from animal immunization to antibody screening. Take advantage of our services – get in touch today to accelerate your antibody discovery journey and achieve unparalleled results.