FAQS – HYBRIDOMA

Hybridomas can be defined as a hybrid cell line able to produce antibodies indefinitely under standard laboratory conditions. Hybridoma-generated antibodies are characterized by their higher affinities, stabilities, and specificities in comparison to other antibody discovery methods.

To learn more about hybridomas, read the complete article: What is a hybridoma?

The fusion of plasma cells (actively secreting high-affinity antibodies) with compatible myeloma partners (malignant plasma cells typically from the same host) produces a hybridoma cell line. This procedure can be mediated by chemicals or physical signals (i.e. electrical signals) and it leads to the immortalization of these highly-productive and antibody-secreting cell lines.

To learn more about how hybridomas are produced, read the complete article: How are hybridoma cells produced?

The fusion of plasma cells with their corresponding myeloma partners is not 100% effective. Even in optimal conditions and under the most potent stimuli, cell fusion still generates a mixture of unfused and fused cells that need to be separated.

Plasma cells are easily eliminated due to their short lifespans but eliminating unfused myelomas is more complex. To improve the efficiency of the selection process, myeloma partners used for fusion lack HGPRT, a key enzyme of the nucleotide salvage pathway. The mixture is then grown in HAT medium (hypoxanthine-guanine-phosphoribosyltransferase) where only cells with HGPRT enzyme (hybridomas that have inherited HGPRT from plasma cells) can survive.

On an initial stage, hybridomas can be screened with ELISA (enzyme-linked immunosorbent assay) which is suitable for fast screening of multiple samples. However, ELISA is not always the most suitable method for all applications. For instance, therapeutic applications designed to target membrane-bound receptors may be best evaluated using alternative methods such as flow cytometry that can predict interactions with membrane proteins more accurately than ELISA. In contrast, for diagnostic applications, it is important to screen for antibody activity in the target application (e.g. ELISA, Flow Cytometry, Western Blot, Immunohistochemistry, etc.) because an antibody’s binding affinity will vary according to the sample type and specific assay conditions.

After a positive well is identified during initial ELISA screening, hybridomas are transferred to larger volumes (i.e. 24-well plates) in preparation for the subcloning procedure. Subcloning of hybridomas is typically done via the limiting dilution method to ensure the isolation of stable monoclones. The method consists in diluting hybridoma cultures and dispersing them into 96-well plates to achieve monoclonality (one cell per well). To reduce the risks of producing mixed hybridoma populations, limiting dilution should be performed at least two times.

Successful hybridoma generation depends, first and foremost, on the host’s unique immune response. For this reason, antigens used for immunization need to elicit a strong humoral response or, in other words, they need to be immunogenic. Not all antigenic substances (proteins, small molecules, lipids, carbohydrates, peptides) are immunogenic. This ability depends on the homology shared between the organism of origin of the antigen and the host organism used for hybridoma generation. The more distant these two organisms are, the better the chances the host will mount an efficient immune response against the desired target.

The most commonly used antigens include cells, proteins, peptides, and DNA. Proteins and peptides are the most popular choices since they allow more precise targeting. By using proteins, antibodies generated in hybridomas can be forced to target specific regions of a pathogen or protein complex. Moreover, by using peptides, hybridomas can be designed to target a specific epitope or a strict number of different epitopes. But since most peptides aren’t sufficiently immunogenic, they are often conjugated with carriers (adjuvants) to ensure the host organism can recognize them as foreign.

DNA immunization and cell immunization (recombinant cells expressing the antigen on their surface) are the preferred approaches when working with unstable or complex antigens such as G protein-coupled receptors (GPCRs) and other membrane-bound proteins. DNA immunization can also be used when the antigen presents specific post-translational modifications that play a decisive role in the activity of the antibody.

In sum, choosing the right antigen depends not only on its native structure and origin but also on the immune system of the host used for hybridoma generation.

Hybridomas are the product of immortalized plasma cells that have previously undergone a process of activation
(by an antigen) and affinity maturation (by recombination). For this reason, these cell lines represent the most
advanced stage of B cell development. These hybridomas produce monoclonal antibodies (i.e. IgG or IgA in mice hybridoma)
with high affinity and secrete them to the culture medium. Provided that the growth medium is devoid of serum
(contains trace amounts of unspecific antibodies), the antibodies of interest can be easily extracted by straightforward purification using resins with immobilized protein A, G, or L.

To learn more about monoclonal antibody production, read the complete article:
How are monoclonal antibodies produced by hybridomas?

The hybridoma technology was first developed in the 1970s by scientists George Köhler and Cesar Milstein working at MRC Laboratory of Molecular Biology in Cambridge. The benefits and limitations of the hybridoma technology for antibody discovery are well established. These hybrid cell lines are prized for their ability to generate antibodies with high affinity, stability, and specificity in the most cost-effective way. In contrast, the development of hybridomas and their corresponding myeloma fusion partners are time-intensive in comparison to in vitro antibody generation technique.

Hybridomas are obtained from fully mature plasma cells (effector B cells). These cells often result from
a complex process known as linked recognition. Antigens are what stimulates antibody production,
but the actual process requires the collaboration between several cell types including antigen resenting cells (APC, necessary to digest complex foreign molecules), T cells (activated by APC), and B cells (activated by T cells).
When B and T cells recognize the same antigen (linked recognition), T cells release chemical signals
that drive the process of antibody affinity maturation and class switching (sift from IgM to IgG/IgA/IgE production)
which ultimately leads to the production of high affinity monoclonal antibodies.

The hybridoma technology was initially developed in mice. Due to the genetic proximity, this technology was
easily adapted to other rodents such as rats or guinea pigs which can typically render stable hybridomas when
mice myeloma partners are used for cell fusion

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).

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.

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.

We have diversified our antigen design and immunization strategies to ensure you always get the best possible
antibody for your desired application. Antigen design and production are done in-house and we are currently
able to use peptides, proteins, DNA, and other small molecules (haptens) for immunization.
Customer-provided antigens are also accepted.

Currently, our generation platform is fully optimized for the production of high-quality mice hybridomas.

We have several protocols in place that allows us to elicit an optimal immune response according to the nature of the antigen. For instance, protein immunization is performed via a 51 to 65 days protocol, which means 4 to 5 injections are done until optimal response. In DNA-based immunization campaigns, the necessary time to generate a strong immune response depends on the complexity of the antigen (27-34 days). Finally, for peptide-based immunization it may take up to 79 days (a little over 2 months) to generate high antibody titers.

At ProteoGenix, we know that antigen design can make or break your hybridoma production projects.
For this reason, all projects include a preliminary stage of antigen design and evaluation where
our team analyses your unique project needs considering the format of your final application.
This detailed analysis allows us to provide the best solution according to your goals.

Depending on the nature of the antigen, we can then develop optimized protocols to produce the strongest
immune response and to adapt our hybridoma screening procedures to ensure you receive the most relevant
antibodies for your target application.

During early development, we can also provide you the parental clone supernatants so you
and your team can test them in your samples and assay conditions.
By participating in the early selection process, we ensure that you receive the most biologically relevant
monoclones at the end of the project.

Project deliverables are 100% customizable to your unique needs. But as a general rule, we typically include:

• Parental clone supernatants
• Best hybridomasprovided as 4 frozen cell vials + 2 mg purified antibodies
• A small quantity of antigen

Hybridoma development at our facilities comes with a double guarantee that allows you to minimize the risks,
secure your investment, and establish our strong commitment to delivering the highest quality product.

Our guarantee works under the principle of double testing. All antibodies are tested by us and by you during the early
(parental clone supernatants) and later stages (monoclone supernatants) of development in:

• Your chosen diagnostic application(i.e. ELISA, Western Blot, Flow Cytometry, Immunochemistry/
  Immunofluorescence, etc.) or using antigens in their native conformations (important for therapeutic applications)
• Your unique specifications(i.e. in your samples and respecting the affinity and sensitivity you expect)

You only pay the full price if you are satisfied with the performance of your custom antibody with your samples
in your target application.

Antibody productivity yields of hybridoma cell cultures vary between 40 mg/l in medium with serum and 20 mg/l
in serum and animal free medium. Our animal/serum free process ensures you receive antibody stocks with
the highest level of purity (>95%) and very low levels of contamination, making these antibodies compatible
even with in vivo applications.