This article provides an alternative technique for creating high-affinity binders against small molecules, difficult proteins, and toxins.
An in vitro-derived recombinant monoclonal antibody produced against a complex protein — Diphtheria toxin.
Human monoclonal IgG1 to Diphtheria toxin (ab209329): As a result of toxic effects, it is hard to create antibodies using an in vivo method against toxins, therefore Abcam has isolated an antibody using the in vitro phage display library.
As the scientific community pursues studying complex biological systems, there exists a bigger requirement for antibodies against difficult targets such as nucleotides, toxins, and membrane-bound proteins. Regrettably, it is not always feasible to use an in vivo model. Being aware of this unmet demand, Abcam can now develop such antibodies using AxioMx’s in vitro phage display technology and library to screen, validate, and produce recombinant monoclonal antibodies.
Through having both in vivo and in vitro procedures, Abcam is currently positioned to deliver high-quality antibodies to all targets, particularly difficult ones.
Recombinant Monoclonal Antibody Development Using an In Vitro Approach
The in vitro method is based upon a large library of bacteriophage particles (>1010 clones), each containing the genetic information and the exclusive phenotypic binding function of a single antibody clone. Abcam’s libraries are M13 phagemid libraries with diversity limited to the most variable positions in the complementary determining regions (CDRs) of natural antibody sequences. This technique restricts the amount of parental clones in the library to less than 1%. Screen procedures are listed below:
- Target peptides or proteins are trapped on an ELISA plate which is used to screen the phage display library
- Plates are washed to eliminate non-specific binders
- The specific phage display binders are later eluted and transduced into bacterial cells for amplification
- Two extra rounds of panning/screening (steps 1 and 2) are processed to make sure that each individual clone is pure
- Target specificity is established through binding assays, such as an ELISA
- After the clone is isolated, the antibody gene sequence is designed into a mammalian or bacterial expression vector for mass manufacture of the recombinant monoclonal antibody and then validated in applicable assays such as flow cytometry, immunofluorescence, western blotting, and immunohistochemistry
References for AxioMx’s Phage Display Recombinant Antibody Technology
- pMINERVA: A donor-acceptor system for the in vivo recombineering of scFv into IgG molecules. J Immunol Methods. 2016 Apr; 431:22-30, Batonick M. et al.
- Generating Recombinant Antibodies to Membrane Proteins through Phage Display, Antibodies 2016, 5(2), Huang R. et al.
- AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins. J Immunol Methods. 2013 Aug 30; 394(1-2):55-61. Holland EG. et al.
- Use of micro-emulsion technology for the directed evolution of antibodies. Methods. 2012 Sep; 58(1):28-33. Buhr DL, et al.
Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.
Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.
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