Schematic presentation of the selection strategy.

a) The phage library, containing billions of different phages, was amplified from the bacterial stock. ScFv are fused to minor coat protein III and as such displayed on the phage surface. b) In a subtraction step, the phage library was incubated with secretome from the healthy control tissue. Binders to common proteins were removed in this way. c) Phages that did not bind to the control secretome were incubated with the atherosclerotic secretome in this panning step. d) Unbound phages were washed off, and e) bound phages were eluted and used to infect a suitable E.coli strain (E.coli TG1). f) Bacteria infected with the selected and eluted phages were plated on large agar plates. g) To further enrich for phages that specifically bind to the atherosclerotic secretome, the selection round was repeated. h) Single colonies were induced to produce monoclonal phages. i) Monoclonal phages were analysed for their reactivity with atherosclerotic versus control secretomes in ELISA. In total, six different selections were performed. For each separate selection the control and atherosclerotic secretomes from one individual patient were used. In order not to loose diversity, only two subtractive panning rounds were performed for each selection. To analyse whether enrichment of atherosclerotic secretome-specific binders had taken place, polyclonal phage pools (as obtained in step a) after each subtractive panning round (and of the unselected library as a control) were analysed in ELISA for reactivity with atherosclerotic and control secretomes.