HYBRIDOMA GENERATION FOR DIAGNOSTIC APPLICATIONS

Start creating next-generation antibody-based diagnostics using our risk-free approach to hybridoma production. Our process of hybridoma production for diagnostic applications guarantees your antibodies deliver the best performances independently of sample types or assay conditions. With ten different development packages and swift access to downstream services such as antibody conjugation, custom ELISA development, and recombinant production, we strive to ensure even your most challenging projects reach the market in as little time as possible.

Why the hybridoma technology generates
the best antibodies for clinical research and diagnostics?

Antibody-based diagnostics are among the most successful technologies for the accurate and timely detection of low-abundance disease markers, toxins, or other hazardous components in complex samples. These tests have evolved into multiple formats in response to the growing demand for accurate, fast, and sensitive tools to support the flourishing field of personalized and targeted medicine.

With this purpose in mind, several immunoassays have been developed. Most of these assays employ labeled antibodies generated through the hybridoma technology. This preference towards hybridoma-generated reagents is justified by their inherently higher affinities, stabilities, and specificities.

Moreover, among all reagents used in diagnostics and medical imaging, antibodies are the only reagents with sufficient sensitivity to recognize post-translational modifications (PTM). In this way, the development of anti-PTM antibodies can be used to detect specific disease-causing modifications as early as possible.

Several studies show that detecting these modifications during the early stages of the disease can dramatically change the prognosis, making antibodies the ideal reagents for high-throughput diagnostic applications.

The unique advantages of using hybridoma
generation for diagnostic applications

Hybridoma production is the dominant technology for diagnostic applications. These hybrid cell lines represent a robust and mature approach with a proven track record for the cost-effective generation of high-quality antibodies.

Hybridoma-generated antibodies are also known for their higher stability, which makes them ideal when testing the presence of specific markers even in harsh and complex environments. Moreover, in contrast to other diagnostic tools, antibodies have the unique advantages of allowing spatial, temporal, and accurate quantitative measurements

The process of antibody development via hybridomas is well-established and straightforward. Besides, it confers a unique advantage over in vitro production processes: highly efficient in vivo affinity maturation which saves time by forgoing the need to further engineer these reagents prior to use.

Depending on the final application and the nature of the biomarker, these antibody-secreting cell lines can be generated using integral proteins, peptides, or DNA for efficient immunization. The latter is also considered one of the most efficient approaches for generating antibodies targeting membrane-bound or unstable biomarkers.

In what areas can antibody-based
diagnostic tools be used?

The low cost and stability of these biomolecules have supported the development of antibody-based bioassays and diagnostic imaging techniques in several areas including:

• Cancer and autoimmune conditions – both conditions present a complex disease landscape driven by genetic and epigenetic modifications. In both types of conditions, timely and accurate diagnosis is crucial to either eradicate the cancerous cells and tissues or arrest the progression of aggressive autoimmune responses. Antibodies, due to their high specificity and sensitivity, are essential for the early detection of these conditions and, consequently, for improving the disease prognostics
• Infectious diseases – antibodies can give precise information regarding the type of pathogen and the state of infection. Due to their low cost, stability, and flexibility in terms of parallelization and miniaturization, antibodies serve as the best tools for monitoring and controlling the onset of infectious disease outbreaks.
• Allergies – testing for IgE sensitization is the cornerstone approach for detecting allergic conditions. Like all other diseases, allergies can be accurately monitored in vivo or in vitro with the support of high-affinity antibodies.
• Food safety – the risk imposed by foodborne illnesses is often overlooked, but these conditions still inflict a heavy burden on our health care systems. Due to their flexibility, specificity, and stability, hybridoma-generated antibodies can be quickly adapted and used in food monitoring tests, thus reducing the unpredictable effects of these silent diseases.

Conventional formats of antibody-based
assays for diagnostic

The validation of antibodies intended for diagnostic applications involves testing them in the right assay conditions and with the proper sample types as well as developing the proper positive and negative controls to minimize off-target binding and maximize the detection signal.

For this very reason, all our customers receive a purified sample of the antibody generated by our hybridoma technology so they can test it with their samples in specific assay conditions. Our double validation procedure (in-house and by the client) ensures the diagnostic antibody is fine-tuned for the best possible performance under real conditions.

Most of the immunoassays currently in use can be designed for direct detection (using a single antibody for binding and detecting the antigen) or indirect detection (using the primary antibody for antigen binding and a secondary antibody for detection and signal amplification). Moreover, the detection antibodies can be labeled with a wide array of different substrates including chromogenic, fluorogenic, or chemiluminescent.

Among the different immunoassay formats, ELISA and flow cytometry have established themselves as the best approaches for high-throughput screening and early detection of particularly complex and aggressive diseases.

In all cases, the validation of hybridoma-produced antibodies remains vital. Thus, the development of these reagents should take into account the:

1. Cell types or tissues that will be used in each specific assay
2. Assay conditions
3. Reporter systems and multiplexing parameters (how many targets will be detected in a single assay)

Currently, the most widely used immunoassays for diagnostics include:

Immunohistochemistry (IHC) And Immunocytochemistry (ICC)

IHC and ICC are antibody-based methods for staining specific cells (ICC) or tissues (IHC) samples. Together they represent a powerful microscopy-based technique providing a clear visual and spatial output at the tissue or cellular level of specific markers and how they correlate with different cell types, cellular compartments, or biological states.

Antibodies used in IHC or ICC assays are labeled with chromogenic reagents such as horseradish peroxidase (HRP) which then require a chemical substrate to produce a color change, making the samples easy to visualize under a light microscope. These assays are also amenable to multiplexing (the use of several enzyme reporter labels to produce different colors for different antigens).

Conventional IHC and ICC assays often use formalin-fixed paraffin-embedded (FFPE) tissues that preserve the histological morphology but can mask important epitopes. It is possible to partially revert the chemical crosslinking in FFPE tissues by using antigen retrieval protocols. However, these protocols need to be optimized for each specific tissue and antigen.

Alternatively, frozen tissues can be used to minimize this issue. But, in both cases, the conformation of target epitopes is expected to change during sample treatment, which makes antibody validation in the specific cell or tissue type vital to ensure the success of IHC/ICC experiments.

Western Blot (WB)

WC is a widely used method to separate and detect proteins. It involves transferring (also known as blotting) proteins, previously separated by electrophoresis, from a polyacrylamide gel to a nitrocellulose membrane for visualization. The membrane is then blocked and marker-specific antibodies (labeled with chromogenic, fluorogenic, or chemiluminescent substrates) are added for protein imaging.

This immunoassay is typically performed in denaturing conditions meaning that the secondary and tertiary structures of the desired marker are lost. The development of antibodies for these applications needs to take this loss of native structure into account. More importantly, it remains vital to validate antibodies for WB in denaturing conditions to ensure they detect only the marker of interest.

Despite its enhanced sensitivity, WB assays remain complex and labor-intensive. For this reason, nowadays they are mostly used to confirm the results obtained by other techniques in preclinical and clinical diagnostics.

Immunoprecipitation (IP)

IP is a popular technique to capture and concentrate proteins from complex mixtures. It allows the enrichment of specific markers, which is particularly useful when dealing with low-abundance proteins.

Besides allowing the study of a protein outside its original environment, IP can be used to study the interaction of the target protein with other molecules (proteins, DNA, RNA, cells, etc.), as these complexes tend to co-precipitate. Additional reagents may be used to stabilize these complexes prior to precipitation, thus enhancing the usefulness of the assay for the study of important interactions in the context of disease.

This technique is often used in tandem with mass spectrometry providing a powerful method for measuring and identifying low-abundance proteins in complex samples. Thus, antibodies intended for IP use need to be carefully validated in the test conditions to ensure only the target protein or complex is efficiently recovered from specific samples.

Immunofluorescence (IF)

Fluorescent-labeled antibodies are the crucial reagents of IF methods, a specific type of immunostaining approach. In its essence, IF is similar to IHC/ICC, thus it can be used to detect and visualize a protein of interest on fixed (FFPE) or frozen tissues (immunohistofluorescence) or particular cell types (immunocytofluorescence).

The difference lies in how the samples are visualized. For instance, IHC/ICC assays rely on an enzymatic reporter and a substrate to produce a color change, while IF assays require only a fluorescent label and the proper lasers to produce a signal. In both cases, multiplexing (detecting several antigens) is possible provided that the fluorochromes or enzymatic reporters emit non-overlapping signals.

Developing antibodies for this approach can be especially challenging since these reagents need to be highly specific to the target and, at the same time, cause minimal background noise (minimal off-site binding). Moreover, when different antibody conjugates are used in the same assay, it is even more important to validate the panel or cocktail of the different antibodies to ensure the data generated by IF experiments are easily interpreted.

Flow Cytometry

These assays have been quickly gaining ground over classical immunoassays for the fast detection of disease markers in liquid samples (e.g. fluids, blood, plasma, etc.). Antibodies used in flow cytometry are typically used to detect low abundance markers (diagnostics) or determining the efficiency of specific treatments, substantially aiding the efforts of developing targeted or personalized therapeutic approaches.

These immunoassays can be performed with fixed or unfixed samples; moreover, they typically employ fluorescence-labeled antibodies in multiplexing conditions. Due to its enhanced sensitivity, off-target binding can become problematic in flow cytometry. For this reason, validation should foresee potential issues caused by different sample types, cell fixation protocols, and multiplexing conditions.

Elisa (enzyme-linked Immunosorbent Assay)

ELISA is a plate-based immunoassay for the detection of markers in complex samples. Several ELISA formats are currently employed in clinical diagnostics depending on the abundance of the target. Generally, ELISA antibodies are tagged with enzymatic labels (similar to IHC/ICC applications) and these assays can be designed for antigen detection or quantification.

Of all ELISA formats, sandwich ELISA has become the most useful and, consequently, the most challenging to develop. Sandwich ELISA employs two primary antibodies binding non-overlapping epitopes of a given antigen. Similar to all other immunoassays, the validation of antibodies for ELISA should consider the type of samples and detection conditions to be used.

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