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Hybridoma Sequencing Pitfalls

Top 4 pitfalls to avoid when sequencing hybridomas:  

  1. Selecting the right sequencing technology (Sanger vs Next-Generation Sequencing): Choosing an inappropriate technology for your project can be costly and time-consuming.
  • Both technologies can detect monoclonal antibodies but Sanger sequencing is not as sensitive as NGS to adequately detect polyclonal antibodies. NGS provides greater sequencing depth and would be able to capture polyclonal antibodies from a hybridoma sample.
  1. Impurity of cells/hybridomas: Diluting and culturing hybridomas as monoclonal is not always accurate, so a sample can potentially have more than one hybridoma.
  • Not all hybridomas produce monoclonal antibodies. In addition, a sample could have contamination of other organisms like bacteria and or yeast [1].
  • Abterra Bio uses NGS for hybridoma sequencing and our software can assess clonality of your hybridomas. We will give you access to all the relevant antibodies.
  1. Mutations introduced by PCR: Mutations can be introduced during the amplification step of the cDNA especially with the use of degenerate primers.
  • Abterra Bio uses 5’RACE strategy to prevent the introduction of mutations at the primer annealing sites of the variable region.
  1. Inaccurate sequences: No sequencer calls nucleotides without some errors.
  • To achieve high accuracy, our proprietary process involves deeply sequencing the full variable region of each chain for every sample complemented by software that corrects substitution errors typically found in ILMN Miseq data. Read more about our publication on error correction.

 

References:

  1. National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Monoclonal Antibody Production. Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies. (NCBI)

 

Antibody Resources

Antibodies as Tools*

Antibody Production*

Antibody Reference Guide (downloadable)

Hybridoma Resources

Hybridoma Technology

Hybridoma Sequencing

Sequencing Pitfalls

Case Studies (Downloadable)

Reptor

Alicanto

Valens

*Stay tuned for these upcoming topics!

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