GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies

Glutathione S-transferase (GST) fusion proteins are used frequently for inv estigating protein-protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives dur ing selection of phage-displayed single-chain antibody fragments (scFvs) sp ecific for three domains of the movement protein (NS,) of tomato spotted wi lt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzy me-linked immunosorbent assay (ELISA) was compared with capture phage ELISA . Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, usi ng capture phage ELISA, all 106 selected clones were identified as false po sitives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody f ragments and it is essential to use capture phage ELISA, instead of the ind irect phage ELISA used commonly to exclude false positives in characterizat ion of selected clones with GST fusion proteins. (C) 2001 Elsevier Science B.V. All rights reserved,