Fully in vitro selection of recombinant antibodies

Abstract

Antibodies are essential for the identification and characterization of proteins. In the current postgenomic era the need for highly specific antibodies has further increased not only for research applications but also because they represent one of the most promising therapeutic options, especially in the field of cancer treatment. One appealing approach for rapid and inexpensive antibody generation is the use of phage display. This technique allows for a fast and animal-free selection of highly functional alternatives to classical antibodies. However, one strong limitation of this recombinant approach has been the difficulty in producing and purifying antigens. These steps have to be adjusted for each new target, are time consuming and sometimes present an insurmountable obstacle. Here we report the development of new antibody selection approach where antigens are produced through in vitro translation and are used directly and without the need for purification. With this approach we were able to rapidly select recombinant antibodies directed against GFP and the mammalian protein tsg101, respectively. We believe that our method greatly facilitates antigen preparation and thus may broaden the use of the recombinant approach for antibody generation, especially since the technique could in the future be adapted to a high-throughput technology, thus further accelerating antibody selection.

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

“…Reagents PMA (used at 150 ng/ml from a 1-mg/ml stock in DMSO), taxol (paclitaxel, used at 10 μ M from a 10-mM stock in ethanol), nocodazole (used at 0.5 μ M from a 10-mM stock in DMSO), latrunculin A (used at 0.1 μ M from a 500-μ M stock in ethanol), and complement C5-defi cient serum were obtained from Sigma-Aldrich. The following antibodies were used: anti -CLIP-170 (clone F-3; Santa Cruz Biotechnology, Inc.); #2221 serum (a gift from N. Galjart, Erasmus University, Rotterdam, Netherlands); anti-EB3 serum (a gift from A. Akhmanova, Erasmus University); anti -␣ -tubulin (DM1A; Sigma-Aldrich); anti-myc (Roche); anti-mDia1, anti-EB1, anti-CLIP-115, and anti-clathrin heavy chain (BD); anti -p16-Arc (Synaptic Systems GmbH); anti-GST (home made); anti-tubulin (clone F2C; Nizak et al, 2003 ;Moutel et al, 2009 ); purifi ed rabbit anti-SRBC (IGN Biochemicals); purifi ed rabbit anti-SRBC IgM (Accurate); AMCA-, Cy2-, Cy3-, or Cy5-labeled F(ab ' ) 2 anti-mouse, -human, and -rabbit (Jackson Immuno-Research Laboratories); HRP-labeled anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Alexa 350/488 -coupled phalloidins were obtained from Invitrogen.…”

A new anti-mesothelin antibody targets selectively the membrane-associated form

“…25 Phage display libraries have also been reported as a viable alternative to hybridoma technology for development of antibodies with desired antigen-binding characteristics. 26 Indeed, occluded epitopes on the cell surface may be difficult to access by full-size immunoglobulin G (IgG) antibodies produced by hybridoma technology. These epitopes can be targeted by fragments of antibodies such as antibody-binding fragments (Fabs) and single-chain variable fragments (scFvs).…”