Use of Living Columns to Select Specific Phage Antibodies

Abstract

Here we demonstrate that it is possible to confront two recombinant microorganisms in order to select one using the other. We have shown that an epitope derived from p21ras expressed within the outer membrane protein, LamB, can be recognized both by the monoclonal antibody Y13-259, as well as the single chain Fv fragment derived from it. This specificity, which is maintained when the Y13-259 single chain Fv is expressed as a fusion protein with the phage fd gene 3 protein, has allowed us to use the living column of LamB-ras to purify Y13-259 phage from a background of non-binding phage, even at dilutions as high as 10 phage in 10(10) irrelevant phage.

Topological analysis of the human beta 2-adrenergic receptor expressed in Escherichia coli.

“…We then performed an ELISA (Fig. 6A) which detects the epitope directly on spheroplasts (26,27). This analysis showed that antibody 9E10 reacts very strongly with the tagged whole hup2AR, indicating that the epitope at the N terminus is exposed on the outside ofthe membrane.…”

Selection of antibodies for intracellular function using a two-hybridin vivosystem

“…25) into pVP16*. pscFvPM163-VP16* and pscFvPM163R4-VP16 were subcloned as NcoI-NotI fragments from pPM163 and pPM163R4 (12) (provided by P. Martineau, Institut Pasteur, Paris) into pCANTAB6 and subcloned again as SfiI-NotI fragments from pCANTAB6scFvPM163 and pCANTAB6scFvPM163R4, respectively, into pVP16*.…”

Intracellular single chain Fv antibody inhibits Ras activity in T‐cell antigen receptor stimulated Jurkat cells

“…The Y13.259-ScFv gene was subcloned from the secretory bacterial expression vector, p3JF4 [8], as a PstI-BamHI fragment and inserted into the leaderless ScFv expression vector, pJM1. This plasmid has been generated by deleting the pelB secretory leader sequence of p3JF4, thus yielding a leaderless 5' of the ScFv gene (corresponding to the amino acid sequence Met Gin Val Gin ).…”