Hybridoma development services

Monoclonal Antibody form

    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 solutions for hybridoma development

    Therapeutic applications

    Hybridoma production for therapeutic applications

    Developing therapeutic antibodies?

    Our process of hybridoma production for therapeutic applications is tailored to maximize antibody functionality, epitope specificity, and therapeutic efficiency. We ensure your antibodies will recognize your clinical targets even if they have complex native conformations.

    Diagnostic applications

    Hybridoma generation for diagnostic applications

    Developing diagnostic antibodies?

    Our process of hybridoma generation for diagnostic applications includes a broad offer of application guaranteed packages ensuring your antibodies keep their high affinity, specificity, and stability in your unique assay format and conditions.

    Why choose ProteoGenix for your hybridoma development ?

    Guaranteed hybridoma development
    Strongest guarantees

    Test purified antibodies and only pay if you are satisfied! The best guarantees on the market!

    Successful hybridoma generation
    High success rate

    More than 300 monoclonal antibody generation and over 98% success rate!

    Animal immunization for antibody generation
    Immunization approach diversity

    Protein, peptide, DNA immunization… We carry out all types of immunization strategies!

    aaalac hybridoma production
    AAALAC accreditation

    ProteoGenix applies the highest ethical standards and is committed to the ethical use of animals science.

    Application guaranteed antibody
    Screening in target application

    Developing a diagnostic tool? Flow cytometry, Sandwich ELISA, IHC, IF, IP, WB… Your antibody is guaranteed in the application of your choice!

    Therapeutic antibody expert
    Therapeutic antibody expert

    Looking for therapeutic antibody development? Reformatting (bispecific, ADC), humanization, affinity maturation… Use our wide range of services and go straight to the clinic!

    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.

    1
    Antigen design for hybridoma generation
    • Antigen design

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

    2
    Immunization fro hybridoma development
    • Immunization

    Immunization of 5 mice with our optimized proprietary protocol

    3
    cell fusion for hybridoma generation
    • Cell fusion

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

    4
    Hybridoma selection
    • Hybridoma selection and screening (polyclonal stage)

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

    5
    Hybridoma cells expansion and selection
    • 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.”

    Satisfied monoclonal antibody customer

    Dr. rer. nat. Rudolf Übelhart, Project Leader, Ulm Institute of Immunology, Germany

    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.”

    Dr. David Pryce, Lecturer in Biomedical Sciences (Immunology), Bangor University, UK

    “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.”

    Université de Lille

    Dr. Karine Lecointe, Researcher, Lille University, France

    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.

    ANIMAL IMMUNIZATION AND SCREENING

     

    5 mice were immunized with 4 injections containing the protein antigen. More information about the protocol is available in the complete report.

    FC results of the mice serum tested against the immunogen:

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

    SUPERNATANT SAMPLES AFTER FUSION (MOUSE 4): TEST IN ELISA

     

    Confirming screening after 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 84 85
    OD value 1.07 1.223 1.267 1.847 1.395 1.282 1.783
    Clone ID 86 87 88 89 Negative Positive
    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.

     

    SUPERNATANT SAMPLES AFTER FUSION (MOUSE 4): TEST IN FC

     

    Details about the protocol are provided in the complete report.

    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.

     

    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.