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Mouse IgG & IgM cDNA Sequencing Service

LD Biopharma Inc has established a streamline platform for DNA sequencing of monoclonal antibodies (IgG or IgM) produced by mouse hybridoma cell lines (1), and production of single chain fragment (sFv) protein using E.coli derived protein-refolding technology. The sequences of a monoclonal antibody are important for inventor’s patent protection and therapeutic approval. Functional sFv may provide customer opportunities for construction of dual-functional fusion proteins or fusion target specific sFV to various functional domains, such as cell toxin protein, or GFP et al.


Starting from a hybridoma cells, we offer following services:

1. RNA extraction and reverse transcription (RT);

2. TA vector based cloning and DNA sequence of the H and L chain variable domains;

3. Production soluble / functional sFv recombinant protein using E.coli derived inclusion body refolding technology;


Of note, mouse hybridoma cells may contain pseudogenes and mRNAs encoding aberrant kappa chain, 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 monoclonal antibody will inevitably amplify some other antibody-like sequences. This is the key challenge in sequencing the IgG or IgM cDNAs of a particular monoclonal antibody light chain due to high-level expression of abberant kappa chain mRNA in Sp2 derived hybridoma cells. In order to make sure the cloned cDNA fragments are derived from the antigen-binding antibody, we usually treat hybridoma cells with unique retroviral based ribozyme or in vitro treatment for eliminating endogenous aberrant kappa chain or multiple clone DNA sequence for validate DNA sequence authenticity.


Members of our R&D team have extensive experience in cloning and sequencing both IgG and IgM types of mouse monoclonal antibodies since 1994 (1) .

Please contact us for initial project discussion by send in your email: info@ldbiopharma.com.


Reference:

1) Lingxun Duan & Roger J. Pomerantz. Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections. Nuclei Acids Research. 1994, vol.22, No.24. 5433- 5438.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332093/pdf/nar00048-0295.pdf