57 Isolation of single-chain variable fragment (scFv) antibodies against synthetic peptide fragments of human cathepsin S

Abstract

This transaction reports on the on-going attempt to isolate anti-peptide scFv antibodies from an scFv phage-display library capable of detecting the native protein from which the peptide sequence was derived.The display of antibody fragments on the surface of filamentous bacteriophage by fusion to the minor coat protein (plll) and selection of phage with antigen, has provided a powerful method of generating antibodies of pre-defined specificity without the need for immunization (for review seeHaving successhlly generated antibodies against human cathepsin S utilizing homology-based molecular modelling of cathepsin S, epitope prediction and multiple antigenic peptide synthesis followed by polyclonal antibody production in rabbits [2], it was wondered whether a linear version of the peptides would enable the isolation of scFv antibodies from a phage display library capable of cross-reacting with native cathepsin S (Table 1) Conc. of antigen Washing (Pglml) protocol 50 1 OxMPBS, 1 OxPBS 50 20xMPBS, 20xPBS 25 I O W B S , IOxPBS 25 20xMF'BS, 20xPBS 10 20xMPBS, 20xPBSFollowing each stage of biopanning a polyclonal phage mixture of the eluted phage prepared by precipitation using polyethylene glycol [5] was utilized in a dot-blotting experiment to determine binding to antigen. Essentially, antigen (100-300 ng, in water) was dotted onto nitrocellulose strips and allowed to dry. Following blocking of non-specific binding sites on the strips using 3% MPBS the blot was challenged with the polyclonal phage solution (in PBS) overnight with shaking at room temperature. Following extensive washing with PBS, a monoclonal anti-MI3 horseradish peroxidase conjugated antibody (Pierce) was used to detect bound phage (according to manufacturer's protocols).Atter five rounds of panning, polyclonal phage solution was seen to detect antigen (100 ng) well above background staining (by visual inspection).Polyclonal phage isolated from the fifth biopanning were reinfected into Ikherichin coli TGI cells and plated onto large bioassay dishes using appropriate selective media. Ninety individual colonies were selected at random from these plates and propagated to produce monoclonal phage solutions. Each of these phage solutions were utilized in a dot-blotting experiment essentially as above with the difference that a 96well dot-blot apparatus was used (Biorad, according to manufacturer's instructions) and I00 ng antigen was used. From the ninety clones, at least 23 were seen to recognize the antigen (by visual inspection) above background levels.Fourteen positive and three negative clones were chosen for characterization utilizing DNA sequencing of the CDR3 region.Analysis of the DNA sequences and assessment as to whether any of the clones isolated will cross-react with native cathepsin S and/or cathepsins B and L is in progress.This approach if successful will allow the generation of antibodies to proteins of biological interest, but of limited supply, if the primary sequence of the protein is known and possible antigenic regions can be predic...