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Antibody/Hybridoma Sequencing

Antibody sequencing/hybridoma sequencing allows to determine the variable antibody gene sequences quickly and efficiently. This opens new possibilities in the field of recombinant antibody technologies and permanently establishes antibody identity. Due to our commitment to provide our customers with a complete service package, we offer hybridoma sequencing as part of monoclonal antibody development projects. Optional we accept external hybridoma clones. For sequencing, we use the Sanger method, which delivers highly precise results, allowing for the identification of even small variances that might otherwise be missed.

Development Strategy

Each project is conducted in close cooperation with the customer and consists of different work phases, each of which can be adapted according to the project’s specific aims. 

 

Phase 1 - RNA Isolation from Antibody Expressing Cells

For hybridoma clones stored at BioGenes, the clones will be recultivated and tested in an ELISA for activity. If the hybridoma sequencing is part of the “Take Care” or antibody production package, this work is already included. For foreign hybridoma clones, you have two options: you can either send us fresh cell pellets with 1x106 hybridoma cells (frozen, including one back-up vial), or you can send us two cryo vials of the same lot. Total RNA from 1x106 hybridoma cells will be extracted.

Phase 2 - cDNA Synthesis and VH/VL Amplification

After extraction, the total RNA will be reverse transcribed. Subsequently, the heavy and light chain variable antibody fragments (VH and VL) are amplified via RACE. The advantage of using RACE instead of normal RT-PCR is that we are independent of (forward) primer sets and can also amplify completely unknown sequences without having to design/order new primers.

Phase 3 - Cloning

The resulting product is then cloned into a PCR-cloning-vector.

Phase 4 - Sequencing

Following this, several bacterial clones containing inserts of the correct size are submitted to sequencing, providing the VH and VL consensus sequences.

Phase 5 - Analysis and Sequencing Report

Good quality sequences are aligned to get the VH and VL consensus sequences. Finally, the consensus sequences are verified using the IMGT online database.

 

Benefits

  • A broad range of applications: Thanks to our experience from more than 30 years in antibody services, we can sequence antibodies from various species (mouse, rat, rabbit, human…) and from different subtypes (IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgE, IgA…).
  • A reliable process: Between 4 and 6 positive clones will be selected for sequencing and invalid sequences will be excluded after analysis.
  • Flexibility: We can sequence full-length antibodies or heavy and light variable regions.
  • Full Service Provider: We take care from every step from hybridoma generation to its sequencing.

FAQs

What are the advantages of sequencing your antibody?

  1. Security: Ensuring your antibody can always be produced recombinantly, even if the hybridoma line is lost.
  2. Chimerization/Humanization: Facilitating the humanization process for therapeutic antibody projects once the VH and VL sequences are identified.
  3. Flexibility: Allowing the conversion of your antibody to any species, isotype, or format. 
  4. Intellectual Property: Enabling you to patent the unique variable domain sequences of your antibody.

What are the advantages of Sanger vs NGS?

In terms of accuracy, the Sanger method remains the Gold Standard method for single nucleotide variants and small variations (insertions/deletions). It is cost-effective when it comes to low target numbers (1-20 targets), and remains a quite easily apprehensible method, less reliant on computer tools and analysis than NGS. In some cases, it can also allow the reading of sequences up to 1000 bps.