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Antibody mRNA Sequencing

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Creative Biolabs has established a solid platform for DNA sequencing of both IgG and IgM types of monoclonal antibodies produced by mouse and rat hybridoma cell lines. The sequences of a monoclonal antibody are important for patent protection and therapeutic approval.

Starting from a hybridoma cell line, we offer the following services:

  1. Validation of the hybridoma cell line in terms of antibody production, antibody isotyping and antigen-binding specificity;
  2. RNA extraction and reverse transcription;
  3. Full length IgG or Fab sequencing by PCR amplification and subcloning of the variable domains.

Antibody mRNA Sequencing 

Of note, hybridoma cells may contain pseudogenes and mRNAs encoding non-functional antibody chains, which might be accidentally amplified by the primer pairs intended to clone the target antibody sequences. Therefore, in a total RNA (as well as cDNA) pool of the hybridoma cells, there are other antibody-like sequences (e.g. other IgG) that can be amplified along with the sequences of the target antibody. In fact, PCR amplifications intended to clone coding sequences (cDNAs) of the mouse/rat monoclonal antibody will inevitably amplify some other antibody-like sequences. This is the key challenge in sequencing the cDNAs of a particular monoclonal antibody. In extreme cases, a small phage display scFv/Fab library is constructed for each monoclonal antibody, and then the right scFv/Fab clones are selected against the original antigen.

Note that, it is even harder to clone the right light chain.

This important point is widely forgotten by our peers in the field. In order to make sure the cloned cDNA fragments are derived from the antigen-binding antibody, we usually express the sequenced scFv/Fab and validate its authenticity in an ELISA assay. Frequently, we even express the sequenced VL and VH in full IgG format to make sure the binding specificity and affinity is unchanged in comparison with the parental antibody. We will make our best effort to produce the conjugate you request. Optimal labeling must be determined empirically; and it is technically impossible to guarantee the yield and biological function of the final products. We refund payments for the failed services. Whatever labels you want to add to your protein, just let us know; in most cases we can accommodate your request.

Another feature of our service is that we always keep the original FR1 sequence since we do not design degenerate PCR primers matching this region.

For antibodies intended for therapeutic use, we also sequence the N-terminals of the heavy chain and the light chain using protein sequencing methods to confirm the cDNA sequences cloned from hybridoma cell lines.




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