A second filter membrane (B) was coated with the antigen of interest (represented by pyramids), laid on a solid medium capable of inducing the expression of scFvs, and in contact with membrane A. antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant (3,4) and displayed on phages (5) opened new avenues for antibody engineering and for the isolation of useful binding specificities. The display of antibody repertoires on the surface of bacteriophages, together with stringent selection protocols, allow to by-pass hybridoma technology and to isolate specific monoclonal antibody fragments against Rifabutin virtually any antigen, both self and foreign (6). It is generally recognised that good quality antibody phage libraries are reliable sources of useful antibodies when panned against purified antigen (6C10) and even complex antigen mixtures (11). However, it has been noticed that multiple rounds of biopanning may give rise to immunodominance, with the loss of diverse binding specificities and of valuable antibody clones (12). Furthermore, a complex patent situation has limited the availability of commercial antibody phage libraries for academic laboratories (13). Filter screening techniques could in principle represent a valuable alternative to phage display. Clones (typically Rifabutin <106) are simultaneously assayed for their ability to generate the binding specificities of interest, thereby preventing the Rifabutin loss of binders that may occur during biopanning. Gherardi (14) described a filter-screening methodology for the identification of the few clones secreting an antibody of given antigen specificity, out of several thousand hybridoma clones. This screening methodology was improved by Skerra and colleagues (15,16), who developed a two-membrane system for detection of antigen binding by antibody Fab fragments secreted by bacterial colonies. In brief, antibody-expressing Rabbit polyclonal to PELI1 bacterial colonies were grown on a porous master filter. Secreted Fab fragments were captured on a second membrane, which was later probed for antigen binding. Clones expressing binding antibodies could be identified on the master filter and regrown. However, this interesting screening methodology was only tested in model screening experiments with two antibody clones, and it was not clear whether it could work with libraries containing billions of antibody clones. In this article we investigated whether the two-membrane screening methodology allows the isolation of good quality monoclonal antibodies from a large (>7 108 clones) synthetic antibody repertoire expressed in TG1 strain) harbouring phagemid DNA. Bacteria were grown in liquid medium as described (7) and, once in the exponential phase of growth, 108 bacteria were spread on a 20 cm diameter PVDF filter membrane Durapore (type GVWP; Millipore, Bedford, MA). This master filter was placed on a 20 Rifabutin cm diameter Petri dish containing TYE agar (1 l TYE agar contains 15 g agar, 10?g bactotryptone, 5 g yeast extract), 100 g/ml ampicillin, 1% glucose and incubated at 37C for 8 h. After incubation, a lawn of completely confluent bacterial colonies was visible on the filter. A second (capture) membrane was prepared as follows. A 20?cm diameter PVDF membrane (Immobilon-P; Millipore) was pre-wet and coated with the capture antigen, the recombinant protein 7B89 (29) and a fibronectin fragment containing the ED-B domain. Preliminary screening experiments had shown that the small recombinant ED-B domain of fibronectin alone did not bind to the membrane. The filter coating was achieved by incubation of the membrane for 6 h at 37C in phosphate-buffered saline (PBS: 50 mM phosphate, pH 7.4, 100 mM NaCl) containing 150 g/ml 7B89. The filter was then blocked in 5% milk/PBS (MPBS) for 2 h at 37C, washed Rifabutin four times in PBS 0.2% (v/v) Tween-20 (PBST) and soaked in 2?TY medium containing 100 g/ml ampicillin and 1 mM isopropyl–d-thiogalactopyranoside (IPTG). The capture membrane was then placed onto a TYE agar plate containing 100 g/ml ampicillin and 1 mM IPTG and covered in turn with the Durapore membrane, with the bacterial colonies on top. The resulting sandwich was incubated at room temperature for 16 h. The presence of IPTG in the medium and the absence of glucose allow for the Lac-promoter regulated expression of.