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!

Our hybridoma development service content

Achieve your unique project goals by choosing the market’s most robust and flexible solution for therapeutic and diagnostic antibody development. For therapeutic applications, our hybridoma development process is tailored to maximize antibody affinity towards clinically relevant targets. Most of these targets are membrane-bound receptors with complex native conformations requiring precise antigen design and the use of adequate hybridoma screening approaches. In contrast, for diagnostic applications, our hybridoma development process takes into account the antibody’s final application. All diagnostic antibodies generated in our hybridoma platform are optimized and validated in the application of your choice considering assay format and conditions, sample composition, and processing protocol. In this way, we ensure all antibodies keep the highest level of performance.

Antigen design

  • Definition of the most relevant immunization strategy. Antigen design: peptide synthesis, gene synthesis & protein production in 2 systems or DNA Immunization.


  • Immunization of 5 mice with our optimized proprietary protocol.

Cell fusion

  • Collection of the splenocytes from 2 mice for 2 fusions with a myeloma cell line.

Hybridoma selection ans screening (Polyclonal stage)

  • Hybrid cells selection (HAT selection) Culture supernatant screening vs. target antigen (ELISA screening).

Isolation and selection of the best monoclones

  • Isolation of monoclones by limiting dilution. Expansion and screening of the monoclones by ELISA or in target application.

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.”

We congratulate the team of Dr. rer. nat. Ubelhart for their impressive achievements which lead to a publication in PNAS and we are proud that the antibodies generated by ProteoGenix contributed to their success!

“I requested ProteoGenix’s services for the generation of an anti-peptide monoclonal antibody to be used in my research. I required the antibody primarily for western blotting and immunoprecipitation experiments. Although the peptide antigen proved challenging in terms of immunogenicity, ProteoGenix was able to propose a range of customised strategies to overcome production issues encountered and finally succeeded in generating not only one but three antibodies of interest, thus offering me extra options in terms of final choice and use. During the whole project, Proteogenix strived to ensure maximal quality and to adapt to my needs, and my Account Manager always provided prompt and helpful services and solutions. I can highly commend both the level of expertise and the service I received.”

“We contacted ProteoGenix for a project concerning the detection of a phytopathogen. ProteoGenix helped us to the production of a specific antigen and the development of a monoclonal antibody to detect it by ELISA test and western blot. We appreciated the contact, the reactivity and professionalism of ProteoGenix for the good running of the project. We are satisfied with the services of the company.”

“I contacted ProteoGenix regarding the generation of monoclonal antibodies for the detection of a chemokine. ProteoGenix generated for us multiple monoclonal antibodies to detect the chemokine by ELISA, western blot and to test these antibodies in functional assays. They provided an excellent service, with regular updates, quick response to our queries and an excellent delivery of the product. I recommend their service for their efficacy and professionalism.”

“I highly recommend ProteoGenix for their fast and efficient services, providing top-quality antibodies suitable for diverse applications. Their exceptional communication, quick turnaround time, and reasonable pricing make them an excellent choice even for non-standard projects.”

“We contacted ProteoGenix to develop repertoires of specific antibodies against three antigens sharing high homology. As determined by ELISA, ProteoGenix developed several specific antibodies that exhibited no cross-reactivity with the other antigens. We appreciated their proactive approach to increase our chances of success (e.g., performing fusions at their own risks). We are satisfied with the services of the company.”

We needed to develop two monoclonal antibodies for Western-Blot, from antigen synthesis to hybridoma cell line and purified antibody delivery. ProteoGenix met all our expectations with efficient communication, satisfying deadlines, and successful production of specific and functional antibodies. For future antibody production, ProteoGenix will remain our preferred partner.

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.

Sample tested Positive control Mouse 3 Mouse 4
Mice serum after 3rd injection 90% 42% 29%
Mice serum after 4th injection 90% 76% 47%

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.

Clone ID 65 66 67 68 69 70 71
OD value 1.254 1.615 1.306 1.412 1.224 1.198 1.334
Clone ID 72 73 74 75 76 77 78
OD value 1.251 1.011 1.394 1.225 1.636 0.153 1.647
Clone ID 79 80 81 82 83 85 86
OD value 1.07 1.223 1.267 1.847 1.395 1.282 1.783
Clone ID 86 87 88 89 Negative Negative
OD value 1.036 0.983 1.173 1.116 0.088 1.731

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

Clone ID 65 66 67 68 69 70 71
FC result / % 37.48 1.27 20.27 62.50 1.18 11.40 1.38
Clone ID 72 73 74 75 76 77 78
FC result / % 75.09 1.01 6.50 56.76 73.16 1.18 1.09
Clone ID 79 80 81 82 83 84 85
FC result / % 1.07 1.05 1.49 6.75 71.92 1.18 1.02
Clone ID 86 87 88 Positive
FC result / % 1.04 1.01 7.36 80.73

11 positive clones were obtained after fusion in FC. These clones were subcloned for further antibody production.

Similar ELISA and FC analyses were performed on mouse 3. 14 positive clones were obtained after FC. Results are available in the complete report.

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.


  • 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.

ProteoGenix’s Hybridoma platform (FAQ)

What is a Hybridoma ?

Definition of hybridomas and their importance in antibody discovery

Hybridomas are immortal antibody-secreting cell lines first developed by scientists Georges Kohler and Cesar Milstein in 1975. Conventionally, these cell lines are generated by fusing short-lived spleen cells (plasma cells) from pre-immunized hosts with a compatible myeloma partner (malignant plasma cells).

This delicate process of cell fusion, often induced by electric pulses (electrofusion) or polyethylene glycol (PEG), renders these highly productive cell lines amenable to cryopreservation for the long-lasting production of high-quality monoclonal antibodies.

What are the major sources of hybridomas?

Hybridomas have played and continue to play a key role in antibody discovery for multiple applications. They allow capturing highly mature antibodies with the highest stability, affinity, and specificity towards specific targets.
These hybrid cell lines embody the possibility of indefinite production of monoclonal antibodies in vitro. Many of the highly valuable hybridoma cell lines are currently stored in vast cell banks allowing their widespread distribution and ultimately supporting research and diagnostic efforts.
The major sources of hybridomas are rodent species such as mice, rats, and hamsters. In these cases, monohybridoma production (fusion of a healthy plasma cell with malignant myelomas from the same species) is the most common route for achieving cell immortalization. However, some heterohybridomas (cell fusion between partners from different species) such as mouse-rat and rat-mouse are known to be as efficient as their monohybridoma counterparts.
Heterohybridomas have often been used to generate immortal antibody-secreting cell lines for alternative species such as chicken, rabbit, or human. However, most of these heterologous cell lines have proven to be genetically unstable and the process of clonal selection often leads to the loss of antibody-encoding genes.

How to grow hybridomas in vitro?

There are several methods for growing hybridomas in vitro. Up to recently, the ascites production method was the most common process. However, this method requires animal use and it often yields stocks with trace amounts of unspecific antibodies and other animal-derived contaminants.
Our recommendation is to grow hybridomas in suspension cultures. Most hybridoma cell lines can be grown under standard laboratory conditions and we believe it to be a more humane method for antibody production. Our recommendation is also in tune with the EU’s latest report discouraging animal use in antibody production when viable alternatives exist (EURL ECVAM Recommendation on Non-Animal-Derived Antibodies, issued in May 2020).
With this recommendation in mind, hybridomas can be grown in suspension in the presence of serum (e.g. 8-10% FCS – fetal calf serum or similar reagents) supplemented with glutamine (known to stimulate antibody production) and antibiotics (to reduce bacterial contamination). However, sera also contain trace amounts of unspecific antibodies and other animal-derived contaminants. To overcome this issue and produce stocks with higher levels of purity, hybridomas can be acclimatized to serum-free and chemically-defined medium.

Major uses of hybridoma cell lines

After proper screening and monoclone selection, mouse hybridomas are able to secrete large quantities of IgG monoclonal antibodies. They can then be conjugated either to enzymes or fluorescent labels for the detection or capture of specific antigens.
This versatility is especially useful for diagnostics and research, where an antibody’s specificity and sensitivity may help to unravel the hidden causes of certain conditions or aid in the early-stage detection of many diseases. But in the long-term, hybridomas may suffer from instability resulting from mutations or chromosome loss that impact their long-term productivity.
To overcome this limitation, hybridoma cell lines can be sequenced so that the antibody-encoding genes may be easily adapted to recombinant expression in more stable mammalian systems.

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.

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.

How are monoclonal antibodies produced by Hybridomas?

How does B cell activation influence the performance of hybridomas?

Antibodies are the key players in the humoral immune response. Their production in naïve B cells (or B lymphocytes) is triggered by the presence of foreign molecules (antigens). The activation of these naïve cells typically proceeds via:
• Direct activation by an antigen (e.g. polymers of bacterial origin) tends to generate a short-lived immune response and antibodies with lower affinity and specificity
• T cell-dependent activation tends to generate a strong and long-lasting immune response and antibodies with high affinity and specificity towards a specific target
Ideally, hybridomas should be produced from mature B cells (plasma cells) activated in a T cell-dependent way. This response takes longer to develop and it is often dictated by the dosage and frequency of the immunizations, as well as, by the quality and stability of the immunogen.
In a nutshell, the development of a strong immune response is paramount for the production of highly productive hybridomas. But this process requires the linked recognition of the same antigen particle by both B and T cells; the former is the precursor of antibody-secreting plasma cells while the latter catalyzes the process of B cell activation.

How does T cell-dependent activation occur?

The frequent exposure to an antigen allows the host to produce a strong and highly specific response and to develop immunological memory. This process starts when professional antigen-presenting cells (APC) capture and degrade the injected antigens into epitope-containing peptides. These epitopes subsequently complex with class II MHC molecules (major histocompatibility complex) and become exposed on the surface of professional APC (typically dendritic cells).
These cells then migrate to the lymph nodes through the vast network of lymph vessels. At each node, the APCs carrying the antigen-MHC complexes are free to interact with naïve T cells possessing compatible T cell receptors (TCRs). Subsequently, this interaction activates the naïve T cells which, in turn, differentiate into armed effector T cells.
This process of differentiation generates three distinct classes of effector T cells, the most important of which is the CD4 TH2 class that are ultimately responsible for activating B cells. But not all B cells can be activated through this process, only the ones that have previously internalized a compatible antigen through their membrane Ig, processed it into the corresponding peptide fragments, complex them with class II MHC, and carry the complex exposed on their surfaces.
Once the TH2 cells are further activated by the linked recognition of an antigen, the production and secretion of cytokines take place. In turn, these serve as chemical signals that trigger antibody affinity maturation and production in plasma cells or generate immunological memory in memory B cells.
At this point, the plasma cells lose their membrane receptors and start secreting pentameric IgM molecules, providing the first line of humoral defense against pathogens. Subsequently, further stimulation by specialized T cells leads to class or isotype switching allowing the production of different antibody classes including IgG, IgA, or IgE.

What applications best benefit from the use of the hybridoma-generated antibodies?

Considering most hybridomas are sourced from mice or other rodents, hybridoma-generated antibodies are typically murine IgG molecules. Murine antibodies have a low level of homology with human antibodies, for this reason, the prolonged use of these molecules may elicit the development of the Human Anti-Mouse Antibody (HAMA) response, leading to a faster clearance from the organism, lower therapeutic efficiencies, and, on occasion, adverse allergic reactions.
For this reason, murine antibodies need to be humanized before they can be considered suitable for therapeutic applications. Despite the longer turnaround time of this approach in comparison to in vitro methods, humanized antibodies from mice hybridomas continue to be one of the most successful biotherapeutics.
In contrast, murine antibodies are extremely suited for diagnostic applications. The vast majority of diagnostic antibodies are murine in origin due to the cost efficiency of the mouse hybridoma-technology and the possibility of using hybridomas for the native production of small quantities of monoclonal antibodies (sufficient for most diagnostic applications).

Can a single hybridoma-generated antibody serve all types of diagnostic applications?

It is very challenging to develop an antibody able to perform well across many different applications such as flow cytometry, ELISA, Western Blot, Immunohistochemistry, etc. The reason for this is that these platforms differ in terms of assay conditions and sample preparation. For instance, WB applications require antibodies targeting linear epitopes (peptides) since all proteins from a given sample are denatured before antibody binding. In contrast, flow cytometry works primarily with liquid samples where the antigen is expected to maintain its native conformation, and thus, the antibody needs to be able to target exposed regions.
The sample composition also influences the performance of a given antibody. For instance, some tissues or biological fluids may be rich in components that share some conserved regions with your antigen of interest. For this reason, some specific samples may promote off-target binding, resulting in high background noise and often leading to inconclusive or false results.
For this reason, all antibodies used for diagnostics should be properly validated in the specific assay format, conditions, samples type, and employing the desired sample preparation protocol.

What are the 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.