Antibody Sequencing Services

Creative Biolabs has established a solid platform for DNA sequencing of monoclonal antibodies produced by mouse or 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 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. scFv sequencing by PCR amplification and subcloning of the variable domains;

        4. Full antibody sequencing by subcloning both variable and constant 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 order to make sure the cloned cDNA fragments are derived from the antigen-binding antibody, we usually express the sequenced scFv domain and validate its authenticity in an ELISA assay. In extreme cases, a small phage display scFv library is constructed for each monoclonal antibody, and then the right scFv clones are selected against the original antigen. This important point is widely forgotten by our peers in the field.

We have extensive experience in cloning and sequencing both IgG and IgM types of monoclonal antibodies derived from both mouse and rat hybridoma clones.