Generation and Identification of Monoclonal Antibodies Against FNIII Domain D of Human Tenascin-C

Cell culture

Murine myeloma cell line Sp2/0 and human osteosarcoma cell line MG-63 were incubated in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 100 mL/L fetal calf serum (FCS, Gibco-Invitrogen, San Diego, CA). Hybridomas were incubated in RPMI 1640 supplemented with 20 mL/L FCS.

Molecular cloning of FNIII D domain cDNA

A total of 10⁶ MG-63 cells were solubilized in RNA extraction solution (TRIzol, Invitrogen). Total RNA was isolated using chloroform, and precipitated with isopropanol. The resulting 5 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 specific for human FNIII D of TN-C and β-actin were synthesized by Shanghai Sangon Biological Engineering and Technology Service (Shanghai, China). Primers were as follows: sense, 5′-gaagccgaaccggaagttga-3′ (20 nt at position 4851–4870 relative to the start codon of human tenascin-C, NM_002160) and antisense, 5′-tgttgttgctatagcactg-3′ (19 nt at position 5123-5105) for FNIII D domain of TN-C; 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; 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.

Expression of FNIII D-GST fusion protein in Escherichia coli

The cDNA of FNIII-D was subcloned with pyrobest polymerase (Takara, China) and inserted into a pGEX-4T-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The corresponding protein was expressed in E. coli as glutathione S-transferase (GST) fusion protein induced by isopropyl-b-D-thiogalactopyranoside (IPTG, Sigma) and purified with glutathione-sepharose.

Preparation of hybridomas secreting MAb to FNIII D of TN-C

Female Balb/c mice (4 weeks old) were immunized with 40 mg of purified FNII D-GST fusion protein in complete Freund's adjuvant (Sigma) by subcutaneous (sc) injection. Subsequently, immunizations were carried out twice with 40 mg of antigen in incomplete Freund's adjuvant (Sigma) by sc and intraperitoneal (ip) injection, respectively, at 3-week 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 mg of FNII D-GST fusion protein by ip injection 3 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, Darmstadt, Germany).⁽⁹⁾ Positive hybrids were selected by indirect ELISA and subcloned three times using the limiting dilution method. MAbs were produced either from supernatants of the hybridoma culture or from ascitic fluid of Balb/c mice intraperitoneally injected with hybridoma. Immunoglobulin (Ig) isotypes were identified by using an isotyping kit (Pierce, Rockford, IL).

ELISA

ELISA was performed on 96-well plates (Nunc, Nagel Inc., Roskilde, Denmark), which were coated with 5 mg/L FNIII D-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 incubation at 37°C for 1 h and three more washes, the substrate 2,2’-azino-bis (3-ethylbenzo thiasoline-6-sulfonic acid [ABTS], Sigma) was added to each well and incubated for 15 min; then the absorbance at 410 nm (A410 nm) was detected with a microplate reader.

Western blot analysis

Samples containing 50 μg of purified recombinant protein were separated on a polyacrylamide gel with Tris-glycine-SDS running buffer and were subsequently transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) for 1 h. Membranes were blocked with 5% milk in TBS-Tween-20 and incubated with the primary antibody overnight. Membranes were washed and incubated with a secondary peroxidase linked antibody, and the reactive bands were detected by enhanced chemiluminescence (ECL) according to the manufacturer's protocol (Amersham Biosciences).

Immunohistochemistry

Formalin-fixed and paraffin-embedded malignant human osteosarcoma tissue sections were maintained in our department. 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 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 6.0) for 10 min in a microwave oven. After blocking 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 the manufacturer's manual. In brief, tissue sections were stained with MAb against FNIII D of TN-C with a concentration of 50 mg/L for 1 h at room temperature and washed three times; after that 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 coloring.

FNIII D domain of TN-C cDNA cloning and fusion protein expression

FNIII D domain of TN-C cDNA was successfully cloned from human osteosarcoma cell line MG-63 (Fig. 2), and the sequence was identical to the data in GenBank. The optimal condition inducing the soluble FNIII D-GST fusion protein to the greatest extent was at 30°C for 8 h with 80 nmol/L IPTG. The SDS-PAGE results showed the FNIII D-GST fusion protein had a molecular weight (MW) of ∼38 kDa (Fig. 3).

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FIG. 2.

Representative RT-PCR results of FNIII D domain of TN-C from osteosarcoma cells. Lane 1, FNIII D cDNA; lane 2, marker of DL2000.

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FIG. 3.

SDS-PAGE and Western blot of FNIII D-GST fusion protein. Lane 1, bacteria culture with IPTG induction; lane 2, bacteria culture without IPTG induction; lane 3, Western blot of MAb 3A8; lane 4, Western blot of MAb 4E2.

Characterization of MAbs against FNIII D domain of TN-C

Altogether two hybridoma clones secreting MAbs to FNIII D were obtained by the routine hybridoma technology and designated as 3A8 and 4E2, respectively. The clone name, isotype, and ascitic titers are listed in Table 1.

Both of the MAbs could be used for Western blot (Fig. 3) and immunohistochemical staining (Fig. 4) on tissue sections. In the Western blot assay, the MAbs stained the protein bands corresponding FNIII D. In the IHC results, TN-C was found distributed within the matrix around the malignant tissues.

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FIG. 4.

Tenascin-C staining with anti-FNIII D domain of TN-C MAb on osteosarcoma tissues (× 400). (A) Control MAb; (B) MAb 3A8; (C) MAb 4E2.