The present study was approved by the Institutional Animal Care and Use Committee of Tianjin First Central Hospital (Tianjin, China) and conformed to the Guide for the Use and Care of Animals published by the National Institutes of Health (NIH Publication number 85–23, revised 1996) (11). In total, eight female BALB/c mice (age, 8 weeks; weight, 18–20 g) were obtained from the Academy of Military Medical Science [Beijing, China; certificate no. SCXK (JUN) 2007–004]. Mice were housed at a temperature of 22–24°C, relative humidity of 45–60%, a 12-h light/dark cycle, with free access to food and water. Mouse myeloma cells (Sp2/0) and a human hepatoblastoma cell line (HepG2) were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Medical Sciences, Shanghai, China). Hepatoblastoma is a rare disease; however, it is the most common type of primary hepatic malignancy encountered in infants and young children (12). SP2/0 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). HepG2 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

The template for GPC3 was created in accordance with the sequence from GenBank (accession no. NM_001164617; http://www.ncbi.nlm.nih.gov/genbank/); the primer sequences for amplification of the GPC3 gene were as follows: Forward, 5′-ACGGGATCCGCCAAGAACTACACCAATGC-3′ with the BamHI restriction site underlined; and reverse, 5′-ATTCTCGAGTTACTCCACCACACCTGCCATACA-3′ with the XhoI restriction site underlined (Sangon Biotech Co., Ltd., Shanghai, China). The sequence was inserted into pDNOR223-GPC3 (IMMUSOFT, Seattle, WA, USA). The primers were synthesized by Takara Bio, Inc. (Tokyo, Japan). The polymerase chain reaction (PCR) amplification conditions were the following: In total, 30 cycles of 98°C for 10 sec, followed by annealing at 55°C for 15 sec, extension at 72°C for 30 sec using an ABI 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR product was analyzed by electrophoresis on a 1.0% agarose gel. Following staining with ethidium bromide solution (10 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), gel images were obtained.

PCR fragments were digested using BamHI and XhoI restriction enzymes (10 U/µl; Takara Bio, Inc.) for 1 h at 37°C and then cloned into the BamHI- and XhoI-digested pET16b vector ecoding a His-tag (Novagen; Merck KGaA); successful cloning was confirmed by sequencing (Sangon Biotech Co., Ltd.). The recombinant plasmid was transfected into E. coli BL21 (DE3, Novagen; Merck KGaA) with the soluble fusion protein GPC3 expressed following induction with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Takara Bio, Inc.), and non-IPTG cells were used as control. The cultures were induced for 4 h at 37°C. Affinity chromatography was used to purify the expressed fusion protein via a His-tag. The target fusion protein was expressed in inclusion body; thus, the bacterial cells were lysed using 4 ml lysis buffer containing 50 mM Tris-Hcl (pH 8.0), 300 mM NaCl and 8 M urea. Lysed cells were subsequently mixed with 1 ml of a solution provided in the kit containing the Nickel chelating resin (GE Healthcare, Chicago, IL, USA). The binding buffer contained 50 mmol/l Na₂HPO_4, 300 mM NaCl, 6 M guanidine at pH 6.0–8.0 and was used as the mobile phase at a flow rate of ~1 ml/min at 4°C. The protein was eluted using the elution buffer containing 50 mmol/l Na₂HPO₄, 300 mM NaCl and 250 mM imidazole at pH 8.0. The identity of the purified fusion protein was confirmed by SDS-PAGE and western blot analysis (13). The primary antibody used was rabbit anti-His-tag antibody (cat. no. ABIN398410; 1:5,000; antibodies-online GmbH, Aachen, Germany), and the secondary antibody was goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase antibody (cat. no. ABIN398323; 1:10,000; antibodies-online GmbH). The concentration of the purified protein was determined using a Bradford protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China).

Recombinant GPC3 protein (20 µg) was deposited onto nitrocellulose (NC) membranes (Sigma-Aldrich; Merck KGaA) in a drop-wise manner using a micro-syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland). BALB/c mice were anesthetized with via isoflurane inhalation (1.0–2.0%, induction; 2.0–2.5% maintenance; Lunan Pharmaceutical Co., Ltd., Linyi, China) usinga VMR Small Animal Anesthesia Machine System (Midmark Corporation, Orchard Park, NY, USA). An NC membrane was embedded into the spleen via a 1-cm surgical incision on the left side and the embedding procedure was repeated 2 weeks later. On day 21 following immunization, blood samples were obtained from the tail vein of mice. The serum was collected by centrifugation at 6,000 × g and 4°C for 20 min. An indirect ELISA was applied to assay serum antibody titers against GPC3 (13). The fusion protein (10 µg/ml) was used to coat microtiter plates, plates were incubated with dilutions of mice immunized serum. Subsequently, goat anti-mouse IgG-horseradish peroxidase (cat. no. sc-2005; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added. The final injection was administered intraperitoneally with 100 µg of fusion protein antigen purified following chromatography and after 3 days, mice were sacrificed prior to harvesting of the spleens. Splenocytes from the immunized mice were collected and subsequently fused with pre-prepared SP2/0 cells with the standard methodology (14). In brief, the cells were washed twice with RPMI-1640 medium and pre-warmed 50% polyethylene glycol 4000 (Sigma-Aldrich; Merck KGaA), and then added to the cell pellet slowly with continuous agitation. Hypoxanthine-aminopterin-thymidine selection media (Sigma-Aldrich; Merck KGaA) was used to select for fused cells under 37°C in a humidified environment with 5% CO₂. The non-fused dead cells were removed by replacing the medium. The fused cells were seeded with RPMI-1640 medium into 96-well plates; the hybridoma culture supernatants were then initially screened by an indirect ELISA (15). Positive hybridomas, which secreted antibodies against the recombinant GPC3 protein, were cloned at least four times by a limiting dilution assay in Hypoxanthine-thymidine selection media at 37°C in a humidified environment with 5% CO₂. Single positive hybridoma clones were expanded 4 days at 37°C in 75-cm² culture flasks and cryopreserved in liquid nitrogen [Air Products and Chemicals (Tianjin) Co., Ltd., Tianjin, China].

Selected positive clones of 5×10⁶ hybridoma cells were injected into the peritoneum of three sterile female BALB/c mice (age, 8 weeks; weight, 18–20 g) (The Academy of Military Medical Science) primed with pristane (cat. no. P2870; Sigma-Aldrich; Merck KGaA). Mice were housed at a temperature of 22–24°C, relative humidity of 45–60%, with a 12-h light/dark cycle. After 10 days, ascitic fluid (3–5 ml) was first drained from the peritoneum for when the body weight of mice increased to 20–30%; ascites collection was conducted for a total of three times. Multiple harvests were conducted two days apart; the second and third harvests were performed two days apart. Providing mice manifested signs of pain or were due for final ascites harvest, mice were euthanized by cervical dislocation prior to ascites collection. Subsequently, mAbs against GPC3 were purified using G-Sepharose 4B (Sigma-Aldrich; Merck KGaA), the affinity purification column (size, 5×0.7 cm) was filled with a sepharose cross-linked with protein G. In total, 10 ml ascitic fluid sample was clarified using centrifugation at a speed of 10,000 × g for 10 min at 4°C prior to column-based purification. The supernatant at pH 7.0 was loaded through the protein G-agarose column at a flow rate of 1 ml/min at 4°C following by washing the column using 5 volumes of washing buffer containg 20 mmol/l Na₂HPO₄, 150 mmol/l NaCl and 2 mmol/l EDTA at pH 7.0 at the same flow rate and temperature. The antibodies were eluted using the elution buffer containing 100 mM glycine at pH 2.7. The immunoglobulin-containing fraction was identified by measuring its absorbance at 280 nm.

The mAb isotypes were determined using a mouse mAb isotyping kit (cat. no. 501240; Santa Cruz Biotechnology, Inc.) according to the manufacturer's protocols.

The chromosome number of the hybridomas was determined in accordance with the International System for Chromosome Nomenclature 2013 (16). Cell division was inhibited in metaphase by adding 200 µg/ml colcemid (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 18 h. The chromosome number was then assessed using an Ni-U fluorescence microscope (Nikon Corporation, Tokyo, Japan; magnification, 100×).

For western blotting, HepG2 cells were collected and lysed in radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.). In total, 50 µg protein was loaded in each lane of the separation gel following protein concentration determination (Bradford's assay). Proteins were separated by 12% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (Merck KGaA) with Mini PROTEAN (Bio-Rad Laboratories, Inc., Hercules, CA). Following three washes with Tris-buffered saline with 0.1% Tween 20, the membrane was incubated with purified mAb (1:1,000) at 37°C for 2 h. Followed by washing as aforementioned, then goat anti-mouse IgG-horseradish peroxidase (cat no. sc-2005; 1:5,000; Santa Cruz Biotechnology, Inc.) was used as a secondary antibody and incubated at room temperature for 1 h. The membranes were washed again and blots were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Inc.). All experiments were repeated three times.

HepG2 cells were plated onto a glass slide and cultured overnight at 4°C; following washing with PBS, cells were fixed with ice-cold 4% paraformaldehyde for 1 h and were blocked with bovine serum albumin (Beyotime Institute of Biotechnology) for 1 h at 37°C. Following three washes with PBS, cells were incubated with mAb in a humidified chamber overnight at 4°C followed by washing as aforementioned. A fluorescent-labeled goat anti-mouse IgG (cat. no. sc-2010; 1:5,000; Santa Cruz Biotechnology, Inc.) was applied as the secondary antibody and cells were incubated at room temperature for 1 h. Immunofluorescence was analyzed in 10 randomly selected fields of view using a Ni-U fluorescence microscope (magnification, ×400).

The PCR product (~510 bp) was analyzed by 1% agarose electrophoresis (Fig. 1). Subsequently, the recombinant plasmid was digested by incubation with two restriction endonucleases, BamHI and XhoI. Gel electrophoresis revealed that the cleavage of BamHI and XhoI had generated the target fragment, which was determined to have the expected size of 500 bp (Fig. 2). The fragment obtained from endonuclease cleavage was verified by sequencing (data not shown); the results were consistent with the sequence of GPC3 (Genbank, accession no. NM_001164617). These findings indicated the successful construction of the expression vector.

The plasmid pET16b-GPC3 was transfected into E. coli BL21 (DE3). Protein expression was induced by adding IPTG; a dense protein band with a molecular weight of ~19 kDa was detected in IPTG-induced cells (Fig. 3A). The location of the band corresponded to the theoretical molecular weight calculated for the recombinant GPC3 protein. Purification of the recombinant GPC3 protein by affinity chromatography via a His-tag was conducted with a purity of ≤95%. Following purification, there appeared to be only one clear protein band with a molecular weight of ~19 kDa (Fig. 3B). This result was further confirmed by western blotting (Fig. 3C).

Following immunization, splenocytes were fused with Sp2/0 to establish hybridomas; these were cultured in 96-well culture microtitration plates. Hybridoma cells secreting antibodies were screened by an indirect ELISA and sub-cloned three times in order to guarantee the monoclonal behavior of the IgGs. Finally, we obtained three positive hybridoma cell clones against recombinant GPC3 following cell fusion and sub-cloning.

A stable hybridoma clone designated as 2F3 and the corresponding antibody titers were determined to be 1:10⁵ in ascites. Furthermore, 2F3 was identified as being an IgG1/κ isotype. In addition, the median chromosome number of 2F3 was 98 (Fig. 4).

Western blot analysis demonstrated that mAb 2F3 yielded a positive signal with the total cell extracts of HepG2 cells at a molecular weight of ~70 kDa (Fig. 5).

HepG2 cells were observed via fluorescence microscopy following staining with mAb 2F3 (Fig. 6). The results indicated that mAb 2F3 could react specifically with the native GPC3 protein in liver carcinoma cells.