Freshly excised colon cancer tissue was washed with 0.15 mol/L PBS and resuspended in 200 μL of lysis buffer (5 mL/L Triton X-100, 10 mol/L Tris-HCl, pH 8, 0.1 mmol/L EDTA, 0.32 mol/L sucrose, 3 mmol/L MgCl₂, 1 mmol/L PMSF (Sigma Chemical Co., St. Louis, MO), and 10 mg/L leupeptin (Sigma Chemical Co., St. Louis, MO). The tissue was ground using a mortar and pestle, and solubilized in RNA extraction solution (TRIzol, Invitrogen, San Diego, CA). Total RNA was isolated using chloroform, and precipitated with isopropanol. The resulting 1 μg of RNA was used as template for single-strand cDNA synthesis with 20 U avian myeloblastosis virus (AMV) reverse transcriptase (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. Primers for PCR amplification were synthesized by Shanghai Sangon Biological Engineering and Technology Service Co. Ltd. Primers were as follows: sense, 5’-CAGGAAGAATGTGTCTGTG-3’ and antisense, 5’-CCACGCACACACATTTGTAA-3’ for Ep-CAM; sense, 5’-GGCACCACACCTTCTACAATG-3’ and antisense, 5’-CAGGAAGGAAGGTTGGAAGAG-3’ for β-actin. PCR reaction mixture was subjected to pre-denaturation at 94 °C for 5 min followed by 31 amplification cycles each consisting of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 3 min, and then a final elongation cycle at 72 °C for 10 min was performed with thermo-DNA polymerase Pfu (Stratagene, La Jolla, CA). Products of PCR were electrophoresed on 10 g/L agarose gels and cloned into the pGEM-3Zf, which was digested with SmaI. cDNA clones were sequenced using automated DNA sequencing facilities from Shanghai Sangon Biological Engineering and Technology Service Co. Ltd.

Murine myeloma cell line Sp2/0, human colon carcinoma cell lines Colo205 and SW480 were incubated in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) containing 100 mL/L fetal calf serum (FCS, GIBCO-Invitrogen Co., San Diego, CA). Hybridomas were incubated in RPMI 1640 supplemented with 20 mL/L FCS.

The cDNA clone encoding the extracellular domain of Ep-CAM was subcloned with pyrobest polymerase (Takara, China) and inserted into a pGEX-4T-3 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The corresponding protein was expressed in Escherichia coli as glutathione S-transferase (GST) fusion protein induced by isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma Chemical Co., St. Louis, MO) and purified with glutathione-sepharose.

Female Balb/c mice (4 wk old) were immunized with 40 μg of purified Ep-CAM-GST fusion protein in complete Freund’s adjuvant (Sigma Chemical Co., St. Louis, MO) by subcutaneous (sc) injection. Subsequently, immunizations were carried out twice with 40 μg of antigen in incomplete Freund’s adjuvant (Sigma Chemical Co., St. Louis, MO) by sc and intraperitoneal (ip) injection respectively, at 3 wk intervals. Ten days after the third immunization, mice were bled from caudal vein and the serum titer was determined by ELISA. The immunized mice were boosted with 20 μg of Ep-CAM-GST fusion protein by ip injection three days before fusion. Splenocytes from immunized mice and Sp2/0 myeloma cells were fused according to the general procedure in the presence of polyethylene glycol (PEG, MW 4000, Merck, Germany)[9]. Positive hybrids were selected by indirect ELISA and subcloned 3 times using the limiting dilution method. MAbs were produced either from supernatants of the hybridoma culture or from ascites fluid of Balb/c mice intraperitoneally injected with hybridoma. Immunoglobulin (Ig) isotypes were identified by using an isotyping kit (Pierce, Rockford, IL).

ELISA was performed on 96-well plates (NUNC, Nagel Inc., Roskilde, Denmark), which were coated with 5 mg/L Ep-CAM-GST or GST respectively, in coating buffer (0.05 mol/L carbonate/bicarbonate buffer, pH 9.6) and incubated overnight at 4 °C. After washing three times with PBS/0.5 mL/L Tween-20, the supernatants of hybridoma culture or various dilutions of corresponding murine ascites were added and the plates were incubated for 1 h at 37 °C. The plates were washed three times and the working dilution (1:2000) of horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (DAKO, Glostrup, Denmark) was added. After incubated at 37 °C for 1 h and three more washes, the substrate 2,2’-azino-bis (3-ethylbenzo thiasoline-6-sulfonic acid) (ABTS, Sigma Chemical Co., St. Louis, MO) was added to each well and incubated for 15 min, and then the absorbance at 410 nm (A_{410 nm}) was detected on a micro-plate reader.

The reported Ep-CAM positive cell line Colo205 was incubated with anti-Ep-CAM MAbs or negative control anti-staphylococcal enterotoxin B (SEB) mAb (IgG1) at 4 °C for 30 min, respectively. After washing twice, the cells were suspended in Dulbecco phosphate-buffered saline (DPBS) containing a working dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG antibodies (Sigma) and incubated for 30 min at 4 °C. Cells were washed, fixed and then analyzed on a FACScan cytometer (ELITE ESP, Coulter Inc., Miami, FL).

Formalin-fixed and paraffin-embedded normal or malignant human colon tissue sections were obtained from the tissue bank of the Department of Pathology at the XJ Hospital (Xi’an, Shaanxi, China). All tissues were sampled from surgical specimens within 2 h of resection. Three-micrometer thick sections were mounted on poly-L-lysine-coated slides, followed by deparaffinization in xylene and dehydration in graded alcohol. The endogenous peroxidase activity was blocked by an incubation with 3 mL/L H₂O₂ in methanol for 30 min. Tissue sections were subjected to antigen retrieval by boiling in 0.01 mol/L sodium-citrate (pH 6) for 10 min in a microwave oven. After blocked with 15 mL/L normal goat serum for 1 h, immunohistochemistry assay was carried out with an Elivision plus staining kit (Maixub-bio, Fuzhou, China) according to its manual. In brief, tissue sections were stained with MAb against Ep-CAM with concentration of 50 μg/L for 1 h at room temperature and washed three times, after which an enhancer was added for 30 min, followed by three washes before HRP-conjugated goat anti-mouse was added and washed another three times. Finally, diaminobenzidine (DAB) was added for colouring. Human colon carcinoma tissue array prepared from seventy individual patients supplied by Cybrdi Corporation (Xi’an, China) was stained with the same procedure mentioned above.

Ep-CAM cDNA was successfully cloned from the malignant colon tissue (Figure 1) and its sequencing result was identical to the data in GenBank. The optimal condition inducing the soluble Ep-CAM-GST fusion protein to the greatest extent was at 37 °C for 7 h with 100 nmol/L IPTG. The SDS-PAGE results showed the Ep-CAM-GST fusion protein had a molecular weight (MW) of 53 ku (Figure 2).

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Figure 1 Representative RT-PCR results of Ep-CAM from colon malignant tissue. Lane 1: marker of DL2000; lane 2: Ep-CAM cDNA.

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Figure 2 SDS-PAGE of Ep-CAM-GST fusion protein. Lane Mr: low molecule weight protein marker; lane 1: bacteria culture without IPTG induction; lane 2: bacteria culture with IPTG induction for 0 h; lane 3: bacteria culture with IPTG induction for 2 h; lane 4: bacteria culture with IPTG induction for 7 h.

Four hybridoma clones secreting MAbs to Ep-CAM were obtained by the routine hybridoma technology and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively. The clone name, isotype, titers, ability of immunohistochemical staining, and reaction with membrane Ep-CAM in FCM are listed in Table 1. Among them, FMU-Ep4 could recognize the natural Ep-CAM on Colo205 and SW480 cells (Figure 3), and all of them could be used for immunohistochemical staining on tissue sections.

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Figure 3 Flow cytometric analysis of Ep-CAM distribution with MAb FMU-Ep4. A: Colo205 cell line; B: SW480 cell line.

Ep-CAM mAb FMU-Ep1 was selected for the immunohistochemistry staining. In the normal colon tissues, Ep-CAM antigen mainly distributed on the basolateral membrane and the region between the basolateral membrane of colon gland epithelia and the cytoplastic part near the nucli, but not all the junctions of cells, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues (Figure 4).

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Figure 4 Ep-CAM staining with anti-Ep-CAM mAb FMU-Ep1 on colon tissues (×400). A: normal colon tissue; B: malignant colon tissue.

Seventy-two tissue dots were collected from 70 individual patients with colon carcinoma tissue array, including adenocar-cinoma (grades I to III), papillary adenocarcinoma (grades I to III), signet-ring and squamous cell carcinomas as well as other non-tumor tissues such as fibrous, fatty and mucous membrane inflammation tissues, smooth muscle and B cell lymphoma. We found that Ep-CAM was expressed only on the epithelia-origined normal and malignant tissues. Among them, Ep-CAM was positive on cell surface and cytoplastic region of signet-ring carcinoma cells and only on the surface of squamous cells (Figure 5). In adenocarcinoma and papillary adenocarcinoma, the expression of Ep-CAM was increased from grades I to III.

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Figure 5 Representative Ep-CAM staining with anti-Ep-CAM MAb FMU-Ep1 on human colon carcinoma tissue array (×400). A: signet-ring carcinoma; B: squamous cell carcinoma.