How Mouse Monoclonal Antibodies Are Made: A Step-by-Step Overview
Monoclonal antibodies have become indispensable tools in biomedical research, diagnostics, and therapeutic development. Among the various production methods, the use of mouse models remains a gold standard for generating high-affinity, antigen-specific monoclonal antibodies. In this article, we walk through how monoclonal antibodies are made, focusing specifically on the well-established process of mouse monoclonal antibody development using hybridoma technology.
Why Mouse Monoclonal Antibodies?
Mouse monoclonal antibodies offer unmatched specificity and uniformity. Because they recognize a single epitope, they provide reliable and reproducible results across assays, making them ideal for ELISA, Western blot, immunohistochemistry, and flow cytometry applications.
They are also widely used as templates for therapeutic antibody development. Many FDA-approved biologics began as mouse monoclonals and were later humanized or chimerized using molecular engineering techniques, highlighting a continuing trend in therapeutic development (Nelson et al., 2010). and were later humanized or chimerized using molecular engineering techniques. Their predictability and stability make them especially valuable in quality-controlled environments like clinical diagnostics.
The foundational technique for producing these antibodies—hybridoma technology—has remained largely unchanged since its introduction by Köhler and Milstein in 1975 (Köhler & Milstein, 1975). Despite advancements in recombinant technologies, hybridomas remain a cost-effective, scalable, and dependable solution.
Step-by-Step: How Monoclonal Antibodies Are Made Using Mouse Models
Step 1: Antigen Preparation
The first step involves preparing the antigen that the antibody will target. This may include full-length proteins, peptides, or post-translationally modified molecules. For increased immunogenicity, the antigen may be conjugated to a carrier protein such as KLH.
The choice of antigen format is critical. Peptides must be carefully selected for surface exposure and biological relevance, while full-length proteins offer more conformational epitopes but may be harder to express and purify. Green Mountain Antibodies offers assistance with epitope prediction and antigen design.
Step 2: Immunization
Mice are immunized over a period of weeks with the antigen using adjuvants to enhance the immune response. Antibody titers are monitored through periodic serum collection and ELISA testing to confirm successful immune activation.
The immunization schedule can be tailored based on the desired immune response, host strain, and downstream assay. Typically, multiple booster injections are administered to ensure a robust pool of high-affinity B-cells.
Step 3: Cell Fusion
B-cells from the immunized mouse spleen are harvested and fused with immortal myeloma cells using a fusion agent like polyethylene glycol (PEG) or electrofusion. This produces hybridomas—cells that combine antibody production with long-term proliferation.
The choice of myeloma fusion partner (e.g. NS1, SP2/0, P3X63Ag8.653) affects fusion efficiency, antibody yield, and growth kinetics. Following fusion, cells are plated in 96-well plates for selective outgrowth.
Step 4: Selection and Screening
The hybridoma cells are cultured in selective HA medium, which allows only fused cells to survive. Supernatants are screened via ELISA, flow cytometry, Western Blot, or immunocytochemistry to identify clones producing the desired antibody.
Additional screening steps may include specificity tests (e.g., cross-reactivity checks), titer quantification, and functional assays. This phase is iterative and can take several weeks depending on project requirements.
Step 5: Cloning
Positive hybridomas are cloned (typically by limiting dilution) to ensure monoclonality. Each clone is tested again for specificity, affinity, and isotype. This guarantees that each antibody batch originates from a single B-cell lineage.
Subsequent isotyping identifies the heavy and light chain subclasses, which is essential for downstream assay compatibility and therapeutic development.
Step 6: Expansion and Cryopreservation
Once a desirable hybridoma is confirmed, it is expanded for antibody production in a flask or bioreactor cultures. The clone is also cryopreserved in liquid nitrogen for long-term storage and reproducibility.
Large-scale expansion allows gram-level antibody production, depending on the required application. Cell banking ensures that identical batches can be produced at any time in the future.
Step 7: Antibody Purification
Antibodies are harvested from hybridoma supernatants and purified using protein A/G affinity chromatography or antigen-specific purification methods.
Step 8: Antibody Sequencing
Sequencing of the hybridoma-derived antibody is performed to determine the variable region sequences of both the heavy and light chains. This step is essential for intellectual property documentation, future recombinant production, and humanization workflows.
Step 9: Recombinant Expression
Once the antibody sequences are confirmed, they can be cloned into expression vectors and transfected into mammalian cell lines such as HEK293 or CHO cells. Recombinant expression enables scalable, serum-free, and animal-free production of the same monoclonal antibody, preserving its specificity while allowing for higher purity and consistency.
Step 10: Stability Testing
The final recombinant antibody undergoes stability testing under different storage and assay conditions to ensure it maintains binding activity, solubility, and structural integrity over time. This step is crucial for diagnostic and therapeutic applications where long-term reagent stability is required.
The final product undergoes quality control to assess purity (via SDS-PAGE or SEC), concentration, and endotoxin levels. Additional steps, such as buffer exchange and sterilization, prepare the antibody for application in sensitive assays or in vivo studies.
Additional Considerations in Monoclonal Production
- Isotype Switching: In some cases, isotype switching may be engineered to match desired effector functions or detection reagent compatibility.
- Subcloning for Stability: Clones may require additional rounds of subcloning if antibody production decreases over time.
- Cross-application Validation: Antibodies can be validated across Western blot, ELISA, IHC, and flow cytometry to ensure broad utility.
Applications of Mouse Monoclonal Antibodies
- Diagnostics: Used in rapid tests, ELISA kits, and immunoassays for infectious diseases, biomarkers, and hormones.
- Therapeutics: Serve as precursors for humanized or chimeric antibodies used in cancer immunotherapy, autoimmune disorders, and more.
- Research: Critical for protein detection, localization, and quantification in academic and industrial laboratories.
Many mouse monoclonal antibodies developed through hybridoma technology have become industry standards for reference reagents in clinical assays and are widely cited in peer-reviewed publications, prompting calls for reproducibility and standardization across research labs (Bradbury & Pluckthun, 2015). have become industry standards for reference reagents in clinical assays and are widely cited in peer-reviewed publications.
References
Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–497. Link
Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov. 2010;9(10):767–774. Link
Bradbury ARM, Pluckthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518(7537):27–29. Link
Learn More About Our Antibody Development Process
At Green Mountain Antibodies, we offer expert-driven monoclonal antibody development services using proven hybridoma techniques. From antigen design to final purification, our U.S.-based team supports every stage of the process with transparency, scientific rigor, and a commitment to quality.
Learn more about our antibody development process or contact us to get started.
