Hybridoma Sequencing Service

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:

1. 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.
2. Antibody recombinant expression: antibody sequence serves as a blueprint for vector construction and recombinant expression in various systems.
3. Antibody downstream development: the original antibody sequence provides a template for various downstream engineering platforms such as humanization and affinity maturation.
4. 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.

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.