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Abstract
A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b.
Highlights
Membrane proteins are extremely attractive as targets for research, diagnostic and therapeutic applications
Each of the target membrane proteins, including their trans-membrane domains, were cloned in-frame with Green Fluorescent Protein (GFP), such that the target protein is displayed on the extracellular surface of transfected cells and the GFP is intracellular, as a transmembrane fusion protein (Fig. 1a)
To validate the surface expression, Chinese Hamster Ovary (CHO) cells transfected with CD83-GFP were probed with an antibody (3C12) to the extracellular domain of CD8315
Summary
Membrane proteins are extremely attractive as targets for research, diagnostic and therapeutic applications. To avoid mouse-derived sequence entirely, mAbs may be isolated using transgenic, humanized mice[6] (followed by hybridoma technology), or using antibody library display technologies[7]. The former technology is very expensive and hindered by intellectual property protection, while display technologies are relatively cheap and available to research laboratories. The technology was later extended to allow cloning into simpler phagemid vectors and the creation of libraries of antibody fragments (scFv or Fab), cloned from human blood and displayed on phage[9,10] These libraries were screened against immobilized target proteins to isolate antibodies of defined specificity, in a process known as “biopanning”[11]. A solution to this problem is to biopan libraries using whole cells as the antigen source, thereby maintaining a native conformation of the protein
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