Hybridoma Sequencing Service

Background Description Service Highlight Published Data FAQ Resources

With years of experience in the field of antibody development, Creative Biolabs has established a high-accuracy sequencing platform to provide top quality antibody sequence determination services from hybridoma cell lines. Creative Biolabs’ hybridoma sequencing service is highly versatile and is applicable for both IgG and IgM types of monoclonal antibodies produced by mouse and rat hybridoma cell lines. We are dedicated to providing first-class customized service to contribute to the success of your project.

The Value of Antibody Sequence

The protein sequence of an antibody is one of its most important attributes that enables various downstream applications and developments:

Patent application: the uniqueness in the amino acid sequence of an antibody, especially that from the variable region, is the key factor that dictates the novelty of the antibody and plays a crucial role in the patent application to ensure the exclusive rights in downstream antibody developments.
Antibody recombinant expression: antibody sequence serves as a blueprint for vector construction and recombinant expression in various systems.
Antibody downstream development: the original antibody sequence provides a template for various downstream engineering platforms such as humanization and affinity maturation.
Other applications: the antibody sequence is also useful in hybridoma cell line validation, antibody format alteration, and to serve as a precaution in case of the loss of hybridoma cell line due to contamination.

Hybridoma Sequencing

Hybridoma sequencing refers to the process of obtaining sequence information regarding the cDNA encoding the variable heavy (VH) and variable light (VL) domains of the antibody produced by your hybridoma cell line. Before sequencing, total mRNA of the hybridoma cells is extracted followed by PCR amplification of antibody variable regions (VH and VL) as well as non-variable flanking constant region sequences. The antibody sequence information is derived from the amplified PCR products by a sequencer. With antibody variable region protein sequence, different antibody formats can be designed by grafting the variable region onto various framework sequence templates. Antibodies generated by this method can be produced via recombinant expression in a mammalian expression system. Alternatively, the full-length sequence can also be derived from hybridoma sequencing to enable antibody production via recombinant expression and to serve as an insurance policy to secure the source of the antibody in case of losing the hybridoma cell line due to contamination or other reasons.

Fig 1. Schematic representation of the approach for high-throughput sequencing of the Ig sequence repertoire. (Georgiou, G., 2014)

Hybridoma sequencing service at Creative Biolabs

Full-length antibody sequencing service

In this service, the 5'RACE method is applied to amplify, clone and sequence the complete sequences of both heavy and light chains of an antibody, which are subsequently cloned into two cloning vectors for downstream sequence manipulation and expression vector constructions.

V-region sequencing service

In this service, only the sequences of the heavy and light chains at the variable region of an antibody are amplified by specially designed degenerated primers and sequenced. The resulted variable region can be grafted into any framework to yield an Ig isoform that is most suitable for the client’s project.

Recombinant antibody production

With the antibody sequence, scientists at Creative Biolabs provide services to convert the sequences into various forms of antibodies, including multi-isoform full-length antibodies and fragment antibodies such as Fab or scFv. A detailed project report, as well as vectors containing the antibody constructs, are delivered to the client at the end of the project.

Service Features

The sequencing service is applicable for hybridoma cell lines from most rodent species and antibody isotypes.
Highly versatile sequencing services to reveal both variable region and full-length antibody protein sequences.
One-stop service from mRNA extraction to sequence determination as well as antibody format conversion.
Short turn-around time: the projects are usually finished within 3 weeks.

Creative Biolabs also offers high-throughput hybridoma platform and other hybridoma-related services to facilitate your antibody discovery projects. Please feel free to contact us for more information.

Other optional antibody analysis services:

Published Data

The Development of Humanized Antibodies for CCHFV by Mouse Hybridoma Approach with Subsequent Sequencing

Fig. 2 Model of the EG5 VH and VL domains. (Stuart D. Dowall, 2023)

Humanized antibodies to the Crimean-Congo hemorrhagic virus (CCHFV) are essential for the development and standardization of serological tests. The researchers generated humanized monoclonal antibodies against CCHFV through two schemes and compared the two methods. The first is the traditional mouse hybridoma method, followed by sequencing and humanization of the antibody, and the second is a non-animal alternative using the human combinatorial antibody library (HuCAL). The results of the experiment and comparison showed that humanization after hybridoma in mice could produce antibodies with higher affinity. The sequencing data obtained can be used to produce recombinant antibodies in subsequent production and research processes, thus reducing the use of animals for this application in the future. Finally, the researchers provided information about the development of a humanized standardized control, which could be an important positive control component for serological testing of CCHFV.

References

Georgiou, G., (2014). "The promise and challenge of high-throughput sequencing of the antibody repertoire." Nature biotechnology, 32(2), 158-168.
Dowall, Stuart D., et al. "Development of humanised antibodies for Crimean-Congo Haemorrhagic fever virus: Comparison of hybridoma-based versus phage library techniques." Journal of Immunological Methods 512 (2023): 113405.

FAQ

What is hybridoma sequencing?
Hybridoma sequencing is a molecular technique used to determine the specific genetic sequence of the variable regions of both heavy and light chains in monoclonal antibodies produced by hybridoma cells. This method is essential for characterizing and replicating monoclonal antibodies for research and therapeutic purposes.
Why is hybridoma sequencing important?
Hybridoma sequencing is crucial for ensuring the specificity and efficacy of monoclonal antibodies. By determining the exact sequence of the antibodies, researchers can reproduce them reliably, study their properties in detail, and modify them for improved performance in diagnostic or therapeutic applications.
What are the steps involved in hybridoma sequencing?
The typical steps in hybridoma sequencing include: 1) Isolating RNA from hybridoma cells, 2) Converting RNA into cDNA using reverse transcription, 3) Amplifying the antibody gene segments using PCR, and 4) Sequencing the PCR products to identify the variable regions of the antibody genes. This process allows researchers to obtain the precise sequence data necessary for further analysis and application.
Can hybridoma sequencing be used for any type of antibody?
Hybridoma sequencing can be applied to any type of antibody produced by hybridoma cells. It is a versatile technique that can be used to sequence both monoclonal and polyclonal antibodies, although it is primarily used for monoclonal antibodies due to the nature of hybridoma technology, which produces a single type of antibody molecule.
How accurate is hybridoma sequencing?
Hybridoma sequencing is highly accurate when performed with modern sequencing technologies. It provides precise information about the variable regions of antibody genes, ensuring detailed and reliable characterization of monoclonal antibodies. The accuracy of the sequencing results depends on the quality of the sample preparation, the efficiency of the RNA extraction and PCR amplification, and the resolution of the sequencing platform used.
What challenges might one encounter during hybridoma sequencing?
Challenges in hybridoma sequencing can include inadequate RNA quality or quantity, which can compromise the cDNA synthesis; PCR amplification biases or errors, particularly in GC-rich or complex regions; and sequencing artifacts or errors. Overcoming these challenges often requires optimization of sample preparation protocols, careful design of PCR primers, and the use of high-fidelity enzymes and reliable sequencing platforms.
How long does hybridoma sequencing typically take?
The duration of hybridoma sequencing can vary depending on the specific protocols and equipment used, but it typically takes between a few days to a week. This time frame includes RNA extraction, cDNA synthesis, PCR amplification, and the actual sequencing process. Additional time may be required for data analysis and interpretation of the sequencing results.
Can hybridoma sequencing be automated?
Several aspects of hybridoma sequencing, such as PCR amplification and sequencing, can be automated to increase throughput and reduce human error. Automation is particularly useful in high-throughput environments where large numbers of monoclonal antibodies need to be sequenced. Automated systems can help streamline the workflow, from sample preparation to data analysis, enhancing the efficiency and reproducibility of the sequencing process.