BMC Cancer 2004, 4:78 http://www.biomedcentral.com/1471-2407/4/78 IFnisgeurrtele2ngth distribution Insert length distribution. Forty-eight random clones from T5 library were amplified by PCR using a couple of primers on the sides of insert. Size of inserts was calculated according to their electrophoretic mobility in 3% agarose gel. of positive and negative human sera by picking the clones in arrayed order on the bacterial lawn, blotting onto nitro- cellulose membrane and probing with a number of differ- ent sera as previously described [6]. The nucleotide sequences of 21 clones that exhibited specific or preferen- tial reactivity with sera from breast tumor patients as com- pared to sera from healthy donors were identified, and their nucleotide sequences were determined (Table 2 [see Additional file 1]). Serological analysis of tumor antigens Phage lysates were prepared from all the selected clones as previously described [6] and tested in ELISA first with a collection of negative, and subsequently, with positive sera (Table 2 [see Additional file 1]). All the antigens tested reacted exclusively or preferentially with sera from breast cancer patients. Eight of the antigens reacted only with the patient serum used in the corresponding selec- tion. Five antigens had cancer-related profile of reactivity, P < 0.05 (T6-2, T6-7, T7-1, T9-21 and T9-27). The other antigens either reacted with a low percentage of cancer sera, or the total panel of the tested sera was too small to offer any clear conclusion. Sequence analysis of selected cDNA clones Twenty-one positive clones were found to encode frag- ments from 18 different gene products, such as 4 clones (T5-9, T9-21, T9-27, T11-7) showing homology to differ- ent regions of the same reverse transcriptase gene (Figure 4). Most of the clones correspond to known gene products in the correct orientation and reading frame, with the exception of clone T5-18 encoding myc oncogen in an alternative frame. Several of these known gene products, SFcigheumree 3of selection strategy leading to TAA identification Scheme of selection strategy leading to TAA identifi- cation. A phage-displayed tumor cDNA library is preincu- bated with patient serum. TAA-specific antibodies bind to antigens exposed on the phage surface. Abs-phage complex is captured by protein A-coated solid support (ELISA plate or dynebeads). Non-bound phage are washed away. Bound phage are eluted by infection of added bacteria and amplified. Positive clones are isolated by immunoscreening procedure and then picked in ordered array on a bacterial lawn, trans- ferred to nitrocellulose membrane and probed with different positive and negative sera. such as reverse transcriptase homolog (clones T5-9, T9-21, T9-27, T11-7), protein kinase C-binding protein (T6-1), trap ankyrin repeat (T11-3), heat shock protein apg-2 (T11-13), have been previously identified by SEREX [9,14-17]. Eight of the sequences listed in Table 2 [see Additional file 1] encode for proteins with unknown functions. Cancer-specific expression of selected tumor antigens Expression patterns for several of the selected genes were analyzed by semi-quantitative PCR from SMART cDNA Page 5 of 10 (page number not for citation purposes)