Antibodies are innate products of the body’s adaptive immune system, produced in response to disease-causing pathogens called antigens. Their primary function is to attach to antigens via a lock-and-key mechanism, neutralizing the latter. However, while endogenous antibodies are literal lifesavers, their specificity limits their applications outside the body.
Thankfully, advances in biotechnology and recombinant protein production have led to the development of custom antibody techniques. Custom antibody production services facilitate antibody creation for projects where commercially available antibodies will not suffice. Below is an overview of custom antibody production, including the production steps, applications, and advantages of using custom antibody services.
Brief History of Antibodies
According to one historical account, the first mention of antibodies was in the 1890s in a publication by two pioneering immunologists. The two co-authored a paper showing that the transfer of blood serum from livestock immunized against diphtheria to livestock infected with the disease cured the latter.
The scientific community was already aware of elements in the body that prevent disease, thanks to the inoculation (vaccination) campaign against smallpox in the 1770s. However, the groundbreaking publication above not only identified antibodies as the agents behind successful inoculation but also revealed their potential as curative agents. Therefore, besides preventing disease, epidemiologists and other researchers could use antibodies to cure illnesses.
Other significant developments in antibody development include discovering the antibody structure and the discovery of B-cells as the specific lymphocytes that produce antibodies. Also, two researchers developed the clonal selection theory, highlighting how the B-cells produce an antibody before releasing it to fight an antigen, challenging the then-popular notion that the antigen functions as an antibody creation template.
However, the most groundbreaking development in the history of antibodies was the development of the first custom monoclonal antibody in 1975. Fast forward to 2023, and antibody production has gradually migrated from hybridoma technology to more advanced phage display systems. Moreover, custom antibody services utilize techniques like gene synthesis and peptide synthesis, protein production services, and antibody sequencing services to meet the growing demand in the monoclonal antibody custom services market.
Below is an overview of the steps in custom antibody production for monoclonal and polyclonal antibodies, including the pros and cons of monoclonal antibodies.
Step-by-step Custom Antibody Production
As stated earlier, antibodies have multiple applications beyond traditional vaccine preparation. The recombinant antibodies available on the market are typically monoclonal or polyclonal.
An antibody production company typically has commercially available polyclonal and monoclonal antibodies for standard applications. However, there are times when a unique antigen necessitates the production of unique antibodies that are not commercially available. This is where custom antibody services come in.
Below is an overview of the general custom antibody production process. Note that different antibody production service providers may charge different rates for additional steps and may include specialized support and follow-up services.
1. Custom Monoclonal Antibody Production
Monoclonal antibodies (mAbs) are the most widely used in many research applications. As previously stated, antibodies utilize a lock-and-key mechanism to bind to complementary target antigens.
mAbs' distinguishing factor of mAbs is that they originate from identical B-cells during production. Therefore, they are exclusively compatible with one specific antigen. They feature a single binding site that recognizes a specific antigen determinant or epitope on the antigen. Consequently, the primary advantage of mAbs is enhanced specificity.
Given the distinction between monoclonal and polyclonal antibodies, their production processes differ slightly, as highlighted below.
Peptide Design
Antibodies and antigens are natural proteins, and proteins consist of amino acids and peptides as their building blocks. Therefore, the first step in the process is to generate a peptide that matches the target antigen’s characteristics. This is achieved via antibody sequencing conducted by peptide synthesis service providers. Because peptides have unique features, the amino acid sequence must be carefully designed to meet the desired characteristics.
Antigen Synthesis/ Purification
The next step after peptide design is generating the target antigen from the designed peptides. Custom peptide synthesis companies are often one-stop shops offering end-to-end services, including custom protein synthesis under their protein expression services. Therefore, generating the custom antigen is straightforward.
However, there are certain cases where the entity seeking custom antibody production services already has the target antigen. In the case of an existing antigen, the peptide design and antigen synthesis steps are unnecessary. The first step is then antigen purification; an antigen must be at least 90% pure to function effectively in monoclonal antibody production.
Conjugation With a Carrier
The antigen produced during synthesis is often a hapten. Immunologists define haptens as molecules that are too small to trigger an immunogenic effect on their own.
Therefore, immunologists attach the hapten to a larger protein molecule derived via a recombinant protein production service to add molecular weight to the hapten, unlocking its immunogenic effects. However, the conjugation step is unnecessary if the synthesized antigen is large enough to trigger an immune response independently.
Immunization
Immunization entails injecting the purified or conjugated antigen into the host animal to stimulate spleen cells. Although multiple mammalian animal hosts can produce monoclonal antibodies, studies show that mice are the most popular for mAb production because their immune systems are well-characterized and they are cost-effective.
Second, mice readily produce the myeloma cells that fuse with the target B-cells to generate the hybridomas necessary for monoclonal antibody production. Hybridoma technology remains the primary method used to generate mAbs.
Cell Fusion
Cell fusion entails fusing the B-cells from the animal host with myeloma cells to form a hybridoma. A hybridoma is an immortal cell line that can be maintained indefinitely and constantly produces antibodies.
Hybridoma Selection
Hybridoma selection entails separating the B-cells that fused with the myeloma cells from those that did not fuse. This process is crucial to ensuring monoclonal antibody quality and quantity. It entails incubating the cell mixture derived from the host's spleen in a selection medium, typically hypoxanthine-aminopterin-thymidine (HAT), for up to 14 days.
Screening of the Retrieved Cells
Antibody screening is necessary to identify and confirm that the target antibody is present in the cell mixture. The hybridoma population may produce other antibodies rather than just the target antibody.
Cloning and Propagation
Cloning entails selecting a single line of hybridoma cells that produces the target antibodies. It ensures that the custom antibody service provider retrieves homogeneous antibodies, facilitating consistent quality across different mAb batches produced from the same cell line.
Propagation entails generating large quantities of the target monoclonal antibodies from the cloned hybridoma cell line. Cloned cell lines receive a clone number that facilitates tracking the mAbs back to the parent cell line.
Characterization and Storage
Monoclonal antibody characterization utilizes peptide mapping to establish the peptide sequence, primary structure, and the antibody's molecular mass. The process uses mass spectrometry techniques to confirm the expected target antibody sequence and antibody homogeneity.
After characterization, the service provider stores the target monoclonal antibodies in ideal conditions for shipment to the customer. Monoclonal antibody production using hybridoma technology can take several months, depending on the antibody's complexity and whether the process commences from peptide design or antigen purification.
2. Monoclonal Antibody Production Using Phage Display
In addition to the hybridoma technology described above, immunologists can utilize phage display to generate custom monoclonal antibodies. The phage display technique entails isolating the genes encoding an antibody and introducing them into a bacteriophage coat protein.
Consequently, the bacteriophage particles display the specific antibody fragments on their surface. After creating the bacteriophage display library, antibody production companies compile these sequences into a gene library.
Researchers then screen the library against the antigens of interest to identify the gene sequences that correspond with the target antigen. After identifying the target genes, the final step involves expressing the antibodies in a recombinant system, followed by characterization and storage.
3. Custom Polyclonal Antibody Production
Unlike monoclonal antibodies, which are generated from a singular B-cell line, polyclonal antibodies arise from different B-cell lines. Therefore, while they target a single corresponding antigen, they recognize different epitopes on that antigen. Polyclonal antibodies share some production steps with monoclonal antibodies but have specific, unique steps.
Peptide Design and Antigen Synthesis
The first two steps in custom polyclonal antibody production (peptide design and antigen synthesis) are identical to the initial stages of monoclonal antibody production. Therefore, please refer to the segment above for details on how each step works.
Immunization
Unlike custom monoclonal antibodies, custom polyclonal antibody production is effective in a wider range of animal hosts than just mice. Rabbits are the preferred hosts for custom polyclonal antibodies because their larger size facilitates higher serum collection per host with fewer injections.
Furthermore, rabbits are easier to inject, allowing for more humane serum collection practices. However, chickens are also favorable polyclonal antibody hosts because the antibodies accumulate in the egg yolks, facilitating non-invasive antibody retrieval.
Serum Collection
Serum collection entails retrieving the antiserum from the host animal after exposure to the antigen. The timeline for recovering the serum depends on how long it takes the specific animal host to produce an adequate immune response.
Titer Analysis
A titer analysis is a test to determine the concentration of the target custom polyclonal antibody present in the harvested antiserum. Researchers typically use enzyme-linked immunosorbent assays (ELISA kits) to perform these titer tests.
Antibody Purification
Antibody purification entails separating the target custom polyclonal antibody from a crude organic mixture. Antibody production companies utilize different techniques, including affinity chromatography and mass spectrometry, depending on the target antibody's characteristics, to avoid denaturing the protein.
Antibody Characterization
Custom polyclonal antibody characterization entails examining the purified antibody to establish its physical and chemical characteristics. It is a form of quality analysis and quality control to ensure that only the highest-quality custom antibodies make it to the order fulfillment batch.
Importance of Custom Antibody Production
Custom antibodies have numerous applications within and outside the biotech industry, as highlighted below.
1. Therapeutics
Non-commercially available monoclonal and polyclonal antibodies play a vital role in treating and managing various illnesses, including cancers and highly infectious diseases. Researchers use their antigen-binding activities to carry drugs directly to targeted areas. According to current 2025 industry reports, the FDA has approved over 130 monoclonal medications for use, while hundreds more await approval in the clinical research stage.
2. Diagnostics and Clinical Projects
Custom polyclonal antibodies and their affinity for multiple antigens play a significant role in antigen detection devices like ELISA kits. Researchers and biochemists use polyclonal antibodies for the biochemical analysis of biological samples. Additionally, they function as biorecognition elements coupled with labeling agents to facilitate diagnostic imaging.
3. Basic, Clinical, and Translational Research Applications
Recombinant custom antibodies play a significant role in research, facilitating concept development, actualization, and implementation. First, they are integral to basic research, which helps improve the understanding of antibody behavior and how other body systems work.
Second, they are pivotal to clinical research, helping to explore the effectiveness of the various antibody therapeutics and vaccines employed in disease management. Third, custom antibodies are also helpful in translational research, helping to apply findings from clinical research to real-life situations.
Advantages of Custom Antibody Production
Both antibody types highlighted above offer numerous benefits for research and other industry-specific applications. Below is an overview of these benefits:
- Custom monoclonal antibodies provide unparalleled homogeneity thanks to the cloning stage included in the process. This allows researchers to avoid fluctuating results that could derail or compromise a project. Moreover, clone number assignment allows researchers to order additional batches of the same homogenous antibodies for future projects.
- Fewer antigens are necessary to induce the antibody production process in both custom monoclonal and polyclonal services. Moreover, custom peptide design and recombinant antigen production eliminate the need for manual antigen characterization.
- Scaling up custom monoclonal antibody production is efficient thanks to the molecules' enhanced stability, which is facilitated by upstream modifications.
- Custom polyclonal antibodies are cost-effective and require a significantly shorter production period, thanks to fewer production steps.
- Custom polyclonal antibody production can be adapted to different hosts to suit specific project needs.
Conclusion
Custom antibody production plays a crucial role in medical advancement by developing novel monoclonal and polyclonal antibodies. However, you should consult your custom antibody service provider to ensure that the antibody type and production methods you choose are ideal for your project. Most custom antibody production companies offer free consultations.