BMC Cancer 2004, 4:78 Table 1: Lambda display libraries list. http://www.biomedcentral.com/1471-2407/4/78 Library name Point of fusion with gpD Source of cDNA Library complexity T5 T6 T7 T9 T11 N-terminus C-terminus N-terminus N-terminus N-terminus MCF-7 + MDA-MB-468 MCF-7 + MDA-MB-469 Human breast carcinoma (patient B81) Human breast carcinoma (patient B84) Human testis 1.7 × 107 3.4 × 107 1.5 × 106 2.3 × 107 1.3 × 107 PAGE. DNA smear, corresponding to 300–1000 base-pair fragments, was cut and eluted from gel according to stand- ard procedure [13]. After digestion with SpeI and NotI enzymes, in order to remove the biotinylated extremities and uncut fragments, a 20-minute incubation with streptavidin M-280 Dyna- beads (DYNAL, Norway) was performed. After additional filtration on Microcon-100 the insert was cloned in λKM8 or λKM10 vectors. The vector was digested with SpeI, NotI enzymes and dephosphorylated. For each library 5 ligation mixtures, each one containing 0.5 µg of vector and about 3 ng of insert, were performed. After overnight incubation at 4°C the ligation mixtures were packaged in vitro by lambda packaging extract (Stratagene, La Jolla, CA). BB4 cells were infected by lambda and plated in top-agar on 100 (15 cm) NZY plates. After overnight incubation phages were eluted from the plates with SM buffer, purified, PEG/NaCl pre- cipitated [13] and stored at -80°C in SM buffer, 7% DMSO. Affinity selection Two µl of human serum were preincubated with 10 µl of BB4 bacterial extract and 10 µl of UV-killed lambda phage in 1 ml of blocking buffer (3% BSA, 1X PBS, 10 mM MgSO4, 1% Triton) for 30 minutes at 37°C under gentle agitation. 1010 pfu of lambda library were then added to the preincubated mixture for a further incubation of 1 hr. Magnetic beads (100 µl), linked to Protein A (Dynabeads Protein-A, Dynal, Norway) were washed twice with the blocking solution. Mixture of library with serum was incu- bated with the beads for 10 min at RT under agitation. The beads were washed 10 times with 1 ml of washing solu- tion (1X PBS, 1% Triton, 10 mM MgSO4). The bound phages were recovered by infection of 600 µl BB4 cells added directly to the beads. After a 20-minute incubation 10 ml of molten NZY-top agar (48°C) was added to the mixture of beads with infected cells and immediately poured onto NZY plates (15 cm). Next day the phage par- ticles were harvested by incubation of the plates under agi- tation with 15 ml of SM buffer for 4 hours at 4°C. The phage particles were purified by PEG/NaCl precipitation and stored in 1/10 of initial volume of SM with 0.05% NaN3 at 4°C. Analysis of gene expression by PCR Five hundred ng of poly(A)+ RNA from breast carcinomas or normal tissue were used to synthesize full-length cDNA by SMART cDNA library construction kit (Clontech, Palo Alto, CA). For maximum sensitivity specific primers for the different genes were designed to amplify sequences located near the 3' end of gene's transcript. Twenty-five cycles of PCR were performed from 1 µl of each cDNA template, normalized through PCR amplification of the β- actin gene. Results Construction of the libraries Lambda libraries were constructed by directional cloning of randomly primed cDNA from human breast carcinoma cell lines MCF-7 and MDA-MB-468, from human breast carcinomas or from human testis into the phage display vector λKM8 to generate fusions with the N-terminus of gpD (see list of libraries in Table 1). Only library T6 was built like C-terminal fusions with protein D by cloning cDNA into λKM10 vector. λKM8 and λKM10 are deriva- tives of λKM4 vector [6] obtained by introducing a flexible GS-linker between the displayed protein and gpD (Fig. 1). The insert size in the majority of the clones in the libraries ranged from 100 to 400 bp (Fig. 2). Only a tiny fraction of out-of-frame clones of this length do not contain stop codons, and are therefore displayed in the libraries con- structed as N-terminus fusions, thus greatly reducing the probability of the selection of mimotopes. Selection of tumor-associated antigens The scheme of TAA identification is shown in Figure 3. Typically, one or two rounds of biopanning, performed according to the selection protocol described in Materials and Methods, were sufficient to obtain 2–50% of positive clones in the following immunoscreening procedure. Then, the identified phage clones were tested with a panel Page 4 of 10 (page number not for citation purposes)