Deutscher et al. BMC Cancer 2010, 10:519 http://www.biomedcentral.com/1471-2407/10/519 the presence of TF and Tn antigen. The results of this preliminary study demonstrated significant differences in the expression of TF and Tn between NAF from a breast with cancer compared to asymptomatic women [5]. Based on the encouraging findings, we instituted a multicenter prospective clinical trial in women with a breast lesion requiring diagnostic biopsy to exclude cancer. Methods Patients and Sample Collection After receiving Institutional Review Board approval from the three institutions listed below, informed consent was obtained from all subjects prior to enrollment. 166 sub- jects were prospectively enrolled and samples collected in Grand Forks, ND at the University of North Dakota, in Columbia, MO at the Ellis Fischel Cancer Center and in London, UK at the Royal Marsden Cancer Center. Participants were enrolled from September 2005 to Feb- ruary 2008. The time between sample collection and analysis ranged from 0.7 to 25.9 months, with a median of 7.3 months. A subject was classified as postmenopau- sal if at least one year had passed without a menstrual period or she had undergone bilateral oophorectomy prior to enrollment. Women who had undergone hyster- ectomy without bilateral oophorectomy were considered postmenopausal if they were over 50 years old. If follicle stimulating hormone (FSH) levels were available, a level of 34 mIU/mL or greater was used to classify women as postmenopausal. There were four women in this cate- gory. All ND samples were collected prior to surgical biopsy. All comparisons of TF and Tn concentrations with disease were based on the histopathologic findings in the clinical report. ND samples ND (1-10 μL) samples were obtained from the breast with a lesion prior to surgery. Lesions included women with 1) PND; 2) a suspicious lesion identified on ima- ging, be it mammogram, ultrasound or breast magnetic resonance imaging; and/or 3) a palpable lesion that was not a simple cyst. Samples were collected as described previously [6]. Briefly, after informed consent was obtained, ND fluid was aspirated using a breast pump (NAF) or collected after the participant massaged her breast (PND). Samples were collected into capillary tubes and stored at -80°C until use. Assessment of TF and Tn ND samples from women with and without breast can- cer were analyzed in blinded fashion. All assays were run in triplicate. It was initially thought that an indirect immunoassay would be more sensitive than a direct assay to small changes in TF and Tn concentration, so the 42 initially collected ND samples were analyzed Page 2 of 7 using an indirect sandwich immunoassay. The indirect method entailed capturing TF and Tn antigen molecules in NAF on an antibody coated surface. The surface was interrogated with a biotinylated TF or Tn ligand. Ligand binding was determined by a streptavidin-alkaline phos- phate color reaction. TF and Tn concentrations were inversely proportional to the reporter signal. This assay proved unreliable, so a direct assay was developed and proved more reliable. Herein we present results for the 124 ND samples that were analyzed using a direct immunoassay. Reagents All reagents were purchased from Sigma (Sigma Chemi- cal, St. Louis, MO) unless specifically noted. The anti- TF antibody A78-G/A7 was purchased from Glycotope (Berlin, Germany) and the BRIC-111 anti-Tn antibody was purchased from Serotec (Oxford, UK). Both antibo- dies were raised against desialylated red blood cells of the appropriate blood group. The TF Ab is specific for both anomeric forms of the disaccharide, including related structures on the glycolipids and shows no cross-reactivity with sialylated glycophorin [7]. The Tn antibody is specific for the alpha O-linked N-acetyl-D- galactosamine. The alpha-O linkage appears to be important. Biotinylation of antibodies and lectins was performed according to published procedures [7]. TF and Tn Direct Immunoassay Each NAF sample was assayed in triplicate on the same ELISA plate for TF or Tn and the mean values were reported to the statistician. Anti-TF and anti-Tn monoclo- nal antibodies (200 ng/well) in a 0.1 M sodium carbonate pH 9.6 buffer were applied to Nunc-immunomax 96-well plates for 4 h at 37°C. The plates were washed with Tris- buffered saline pH 8.0 containing 0.5% Tween-20 (TTBS) and blocked with 2% bovine serum albumin (BSA) in TTBS for 2 h. One-hundred microliters of diluted ND samples (1:50-1:500) in Tris-buffered saline pH 8.0 (TBS) were added to the wells. Control wells contained various dilutions of asialofetuin (ASF) or asialo-ovine submaxillary mucin (OSM) in TBS (0 ng - 2 ng/well). ASF contains TF antigen while AOSM contains exposed Tn antigen. The ND, ASF and OSM samples were incubated at 25°C for 2 h followed by a TTBS wash. Biotinylated secondary detec- tion proteins, anti-TF antibody (0.625 μg/ml) or Tn bind- ing Vicia villosa lectin (1 μg/mL) in 100 μL TBS, were added to the wells and incubated for 1 hr. The unbound secondary detection proteins were removed by two washes with TTBS, while the bound biotinylated secondary detec- tion proteins were bound with a streptavidin-horse radish peroxidase conjugate (SA-HRP) (100 μL/well of a 1 μg/mL stock) for 1 hour at 25°C. Unbound SA-HRP was removed by two TTBS washes and the wells were developed with 100 μL/well of the chromogenic substrate 2, 2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic) (Sigma) detected at