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Antibody mRNA Sequencing

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Abstract or Description:

Starting from a hybridoma cell line:

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.

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 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.  Optimal labeling must be determined empirically; and it is technically impossible to guarantee the yield and biological function of the final products.

Another feature of it is that the original FR1 sequence wil be kept since we do not design degenerate PCR primers matching this region.
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Posted By: Candy Ton on 5/15/2015 6:15:26 AM
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