HYBRIDOMA DEVELOPMENT SERVICES

Owing to a success rate of over 98% on more than 500 monoclonal antibody production projects, ProteoGenix is able to offer the strongest guarantees on the market for hybridoma development. We are sharing this success with you by only requesting payment once your application is successful and has been handed over to you. Being able to offer this high level of guarantee makes us particularly proud!

Hybridoma development testimonials

“We collaborated on a project for generating a monoclonal antibody that recognizes a one-amino-acid mutation in a cancer-associated cell-surface protein. Although the project was technically challenging, we succeeded in generating a hybridoma clone that produced the desired antibody. During the course of the project, we found additional clones that recognized adjacent epitopes and ProteoGenix proceeded to perform additional subclonings in order to ensure the delivery of clones that work perfectly. In summary, ProteoGenix always strived to ensure maximal product quality and customer satisfaction, and the account Manager provided prompt and competent support at all times.”

Case study: Monoclonal antibody production for the detection of a human cell membrane protein

Aim Of The Project

A customer requested the production of a monoclonal antibody for the detection of a human cell membrane protein. Design of the antigen was particularly challenging as the protein was known to be difficult to express in mammalian cells.

Application guaranteed: Flow cytometry

Antigen Design And Production

• A protein fragment estimated as the possible extra-cellular domain, and possible to express in mammalian cells, was designed.
• The corresponding gene was designed and optimized for mammalian cell expression.
• Mammalian cells were transfected with a plasmid expressing the target protein coupled to GFP.

Protein expression was performed both in HEK293 and in CHO cell lines. Final production was performed in HEK.

Production method: protein secreted in culture supernatant Yield: 0.43mg/L Quantity produced: 0.86mg A final QC was performed by SDS-PAGE.

Results for the other mice are available in the complete report.
Mouse 3 demonstrated the best immunogenic reactivity against the antigen in FC.
As our project includes 2 fusions, both mice 3 and 4 were selected to perform fusion.

24 positive clones were selected in ELISA.
Determination of the best clones for FC is done directly in the target application.

Antibody Production

30 clones were selected by our customer for further production. Supernatant samples were tested in ELISA and FC after 2 subcloning steps. Results are provided in the complete report.

Conclusion

• We succeeded in producing a difficult-to-express protein in sufficient quantities to develop monoclonal antibodies. Several expression tests, with several mammalian cells strains, were performed to reach this goal.
• The immunization protocol in mice allowed us to develop good antibodies against the antigen.
• After 2 fusions, 30 hybridomas/clones detecting the antigen in flow cytometry were produced.
• 30 clones and 30 purified antibodies were delivered to our customer.

Please click on the button below to obtain the complete report.

Would you like to know more about our hybridoma generation services? Please take a look at another anti-peptide case study.

What are the advantages of hybridoma development for antibody generation?

Hybridoma technology revolutionized the field of biotechnology by allowing the unlimited and reproducible production of monoclonal antibodies in standard laboratory conditions. These hybrid cell lines are readily stored and distributed among different laboratories allowing the fast progression of scientific research.

Beyond these advantages, hybridoma cell cultures can be used for the native production of high-quality antibodies in serum-free or animal-free medium (with a low amount of contaminants) on a small scale, readily reducing the costs of antibody production in the early stages of development.

Today, this technology is still a reference method for high sensitivity antibody generation. Due to their enhanced biophysical properties, hybridoma-generated antibodies are also considered ideal for diagnostic applications. The most successful of these include ELISA (enzyme-linked immunosorbent assay) and flow cytometry in which conjugated immunoglobulins (with enzymatic or fluorescent tags, respectively) are used to capture, detect, or quantify antigens of interest in complex samples.

Therapeutic antibody development in hybridomas is also one of the most successful approaches used in drug discovery. This technology is complementary to phage display for therapeutic antibody generation. Unlike phage display, hybridomas preserve the natural pairing between heavy and light chains ensuring the resulting antibodies have the best stabilities.

However, hybridomas typically produce murine antibodies known to cause adverse allergic reactions in patients after prolonged use. Engineering techniques such as antibody humanization can be used to overcome this issue by grafting murine antigen-binding residues into human immunoglobulin frameworks. This approach to therapeutic antibody generation remains highly valuable because it allows capturing the unique immune response of different organisms to create a diversity of therapies for the treatment of complex diseases.

For additional resources, access our Frequently Asked Questions (FAQs) page focused on the hybridoma technology.

How to ensure hybridomas produce highly specific antibodies?

The production of high-quality hybridomas starts by choosing the most adequate immunization strategy for each project.

Most clinically relevant targets are membrane-bound proteins which are hard to express in recombinant systems and quickly lose their native conformations when expressed in a soluble form. In these cases, immunization can be best achieved using DNA to stimulate the in situ production of the target antigen. This strategy ensures the antigen keeps its native conformation and that the antibodies produced in vivo target only the epitopes which are exposed on the protein’s surface.

However, most projects focused on diagnostic or therapeutic antibody development generate the best results when recombinantly expressed proteins or synthetic peptides are used as immunogens. In some cases, the diagnostic application may be carried out in denaturing conditions demanding the use of antibodies tailored to recognize linear epitopes (peptides). In other cases, the target protein is soluble and easy to express in vitro making it the best immunogen to trigger antibody production in the host.

Alternative uses for hybridoma-generated monoclonal antibodies

Although most antibodies target conventional antigens such as proteins or peptides, some applications require the recognition of non-conventional and often synthetic targets. These antibodies are collectively called small molecule antibodies or anti-hapten antibodies.

They can bind, with high affinity, a wide range of compounds like pesticides, antibiotics, toxins, lipids, etc. This specificity makes them extremely useful for a variety of applications including food and environmental monitoring technologies, invaluable to quickly detect pollution hotspots or contaminated food sources that endanger human health.

From a therapeutic point-of-view, small molecule antibodies serve either to deliver drugs to specific tissues or cell types (i.e. in bispecific conformations) or prolong the half-life of small drugs in patients’ plasma thus improving their therapeutic efficacy.

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