Monoclonal Antibody Production

Hybridomas are immortal antibody-secreting cell lines developed by Georges Kohler and Cesar Milstein in 1975. Created by fusing short-lived plasma cells from pre-immunized hosts with myeloma cells, hybridomas offer long-lasting production of high-quality monoclonal antibodies.

Hybridomas offer natural advantages, such as preserving VH/VL pairing, efficient in vivo affinity maturation, and the ability to produce full-length IgG antibodies without additional sequencing or cloning. These features make hybridomas invaluable for antibody discovery in therapy, research, and diagnostics.

BioGenes employs mice of the strains BALB/cJRj, RjOrl:SWISS, and C57BL/6NRj, as well as rats of the strain RjHan:SD - Sprague Dawley for immunization. These strains possess a high repertoire of antibody genes. The genetic foundations of antibody production in humans and mice are biologically very similar compared to other animal species. Furthermore, the required fusion partners and techniques, which are patent-free, are exclusively based on the mouse system.

After proper screening and monoclonal selection, mouse hybridomas secrete large quantities of IgG monoclonal antibodies. These antibodies can be conjugated to enzymes or fluorescent labels for specific antigen detection or capture. The versatility of hybridomas is particularly beneficial for diagnostics and research, aiding in disease detection and unraveling underlying causes.

1. Proven Track Record: Hybridoma technology has a well-established track record and remains a proven method for generating monoclonal antibodies (mAbs). With over 20 years of experience, BioGenes has successfully completed over 1,000 mAb projects, demonstrating the effectiveness of this technology.
2. Specificity and Sensitivity: Hybridoma technology allows for the generation of highly specific and sensitive antibodies. This is crucial for applications in diagnostics and research where precise results are required.
3. In Vivo Affinity Maturation: BioGenes' immunization strategies are designed to harness the positive effects of in vivo affinity maturation of antibodies. This leads to the development of highly specific and sensitive immunoassay reagents.

Hybridomas may suffer from instability due to mutations or chromosome loss over time. To address this, hybridoma cell lines can be sequenced, enabling adaptation of antibody-encoding genes to recombinant expression systems for improved stability.

The hybridoma selection process involves separating a mixture of unfused and fused cells generated during the fusion of plasma cells with myeloma partners. Plasma cells, with short lifespans, are easily eliminated, while unfused myelomas are selectively removed by utilizing myeloma partners lacking HGPRT. The subsequent culture in HAT medium allows only cells with the HGPRT enzyme (hybridomas) to survive, ensuring the isolation of successfully fused cells for sustained antibody production.

Hybridomas are commonly screened using the Enzyme-Linked Immunosorbent Assay (ELISA). During ELISA screening, the supernatant from each hybridoma is tested for its ability to bind to a specific antigen. Positive hybridomas, which successfully produce antibodies targeting the antigen, are then selected for further characterization and development.

After identifying a positive well through initial ELISA screening, hybridomas are transferred to larger volumes (e.g., 24-well plates) in preparation for subcloning. This process, often conducted via the limiting dilution method, ensures the isolation of stable monoclones. By diluting hybridoma cultures and dispersing them into 96-well plates, monoclonality (one cell per well) is achieved. Performing limiting dilution at least two times helps reduce the risks of producing mixed hybridoma populations, ensuring the purity and clonality of selected hybridoma cell lines for consistent antibody production.

The key to successful hybridoma generation lies in the host's immune response, necessitating immunogenic antigens for effective immunization. The choice of antigens, such as proteins, peptides, or haptens, plays a critical role. Proteins and peptides, preferred for precise targeting, enable antibodies from hybridomas to focus on specific regions of pathogens or protein complexes. While peptides may lack sufficient immunogenicity, their conjugation with carriers (adjuvants) enhances recognition by the host organism. To obtain more information about our antigen requirements please click here.

Murine antibodies are well-suited for diagnostic applications. The cost efficiency of mouse hybridoma technology and the ability to produce small quantities of monoclonal antibodies through hybridomas make them suitable for diagnostic purposes. To make them suitable for therapeutic applications, murine antibodies often undergo humanization, a process that extends the turnaround time but is essential for minimizing adverse reactions and enhancing therapeutic efficiency.

Developing an antibody that performs effectively across diverse applications like flow cytometry, ELISA, Western Blot, Immunohistochemistry, etc., is highly challenging. The variation in assay conditions and sample preparation methods among these platforms poses a significant hurdle. For example, Western Blot applications require antibodies targeting linear epitopes (peptides) due to protein denaturation, whereas flow cytometry relies on maintaining the native conformation of antigens in liquid samples. Therefore, it is essential to validate antibodies for diagnostics in the specific assay format, under relevant conditions, with the appropriate sample type, and using the desired sample preparation protocol.

At BioGenes, we understand that the success of hybridoma production projects hinges on effective antigen design. Our process begins with a meticulous preliminary evaluation, where our team analyzes your project's unique requirements, considering the format of your intended application. Factors such as antigen immunogenicity, biological relevance, size (e.g., peptide), buffer conditions, potential cross-reactants, and antigen design are carefully assessed. This thorough analysis enables us to tailor the best solution to align with your project goals.

Moreover, we offer in the early development phase the option to provide you with parental clone supernatants. This allows you and your team to test them in your specific samples and assay conditions. 

BioGenes provides cryopreservation services for relevant hybridomas, offering convenient storage options for clients.

The client retains exclusive ownership of the generated clones, ensuring that the results of the project belong solely to them.