The study protocol was approved by the Central South University’s Institutional Review Board, Changsha, Hunan, China and written informed consent was obtained from each subject. Demographic and clinical information was obtained, and a blood sample was collected from each subject. All the subjects were recruited from the clinics at Xiangya Second Hospital, Changsha, Hunan between March 2012 and November 2012. The diagnosis of HCC was made based on guidelines from the Chinese Society of Hepatology, the Chinese Society of Infectious Diseases and the Chinese Medical Association (11–13). A 4 ml blood sample obtained from each subject prior to the initiation of HCC treatment was centrifuged and the serum aliquoted and stored at −80˚C until testing (n=30; age, 54.30±10.14 years; HBV-positive). Control subjects were enrolled from staff with no liver disease or risk factors for viral hepatitis, and who were in normal physiological condition [n=30; age, 49.87±9.23 years; normal control (NC)]. Balb/c mice were maintained according to institutional animal care and use committee (IACUC-BIDMC) protocols.

The recombinant plasmid pET21a(+)-TRX-Golph2, which was constructed in the laboratory, was transformed into Escherichia coli Rosetta (ATCC, Manassas, VA, USA) and optimal expression of recombinant proteins was achieved through controlling the concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) and growth conditions. After induction, bacteria were harvested and centrifuged at 4˚C, 4,449 × g for 10 min, and the pellet was resuspended in 50 mmol/l sodium phosphate with 0.3 mol/l NaCl (pH 8.0). The resuspended cells were then lysed by sonication and centrifuged at 4°C, 10,012 × g for 10 min. The expression form of fusion protein TRX-Golph2 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel).

The recombinant protein TRX-Golph2 was further purified by immobilized metal affinity chromatography (MagneHis™ Protein Purification System; Promega, Madison, WI, USA) under native conditions. The purified protein was dialyzed in 1X phosphate buffered saline (PBS) at 4°C overnight and condensed to a high concentration.

The concentrated recombinant protein was separated by SDS-PAGE on a 12% polyacrylamide gel and then transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA) by electroblotting. Fat-free milk (5%) was used to block the membrane at 37°C for 2 h. The membrane was then incubated with anti-His tag antibody (1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight in a 4°C refrigerator. The membrane was then washed with PBS-Tween-20 (PBST) three times and incubated with the horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (1:1,000, Sigma, St. Louis, MO, USA) in a 37°C shaker for 1 h. After washing three times with PBST, the membrane was visualized with an enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Inc., Rockford, IL, USA).

Eight-week-old Balb/c mice were immunized three times with purified TRX-Golph2 fusion protein at days 0, 21 and 42 with 100, 50 and 50 μg, respectively, by peritoneal injection. Complete and incomplete Freund’s adjuvant (1:1, Sigma) were used to emulsify the protein in the first and other immunizations, respectively. Ten days after the third immunization, mice were bled from the caudal vein and the serum titer was detected by indirect ELISA. Three days after the last booster immunization (100 μg), the spleen of the mouse with the highest titer was removed under sterile conditions to prepare a splenic lymphocyte suspension for cell fusion.

Mouse myeloma cell line SP2/0 was cultured in 10% fetal bovine serum (Hyclone, Tauranga, New Zealand) and RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA) in a 37°C, 5% CO₂ incubator. The feeder cells, obtained from the peritoneal cavity of a normal BALB/c mouse by peritoneal injection of RPMI-1640 medium, were plated in 96-well plates and cultured in a 37°C, 5% CO₂ incubator for 1 day prior to fusion. Splenic lymphocytes (1×10⁸) were fused with SP2/0 cells (1×10⁷) in 50% polyethylene glycol 1500 (PEG 1500, Sigma-Aldrich, St. Louis, MO, USA). The hybridoma cells were cultured in 96-well plates containing feeder cells and screened by hypoxanthine-aminopterin-thymidine (HAT; Sigma-Aldrich) and cultured in a 37°C, 5% CO₂ incubator. Two weeks later, indirect ELISA using purified TRX-Golph2 protein and HepG2 cells as coating proteins, respectively, was performed to detect the positive clones and then screened clones were recloned by limiting dilution in hypoxanthine-thymidine (HT; Sigma-Aldrich) medium for a further 2 weeks.

By observing the 96-well plates, we selected a single clone to perform the indirect ELISA using purified TRX-Golph2 protein and HepG2 cells as coating proteins, respectively. The optical density (OD) was read at a wavelength of 405 nm (microplate reader, Tianshi, Beijing, China) and considered positive when the ratio of OD test to OD control was greater than 3 times. The wells that were positive for both purified TRX-Golph2 protein and HepG2 cells were considered as positive wells and then recloned 3–5 times by limiting dilution until their antibody secretion was 100%. Finally, the positive clones in the 96-well plates were transferred to 24-well plates in 10% fetal bovine serum RPMI-1640 medium. The strain of hybridoma cells that secreted specific mAb was established.

Balb/c mice were injected with incomplete Freund’s adjuvant (0.5 ml) by peritoneal injection. Three days later, the mice were peritoneally injected with 5×10⁶ hybridoma cells diluted by D-Hank’s. Ten days later, ascitic fluid was collected and centrifuged at 278 × g for 5 min to remove the cellular deposition. Anti-Golph2 mAb in supernatant was diluted into different gradients (1:100; 1:200; 1:400; 1:800; 1:1,600; 1:3,200; 1:6,400; 1:12,800; 1:25,600; 1:51,200) and added into 96-well plates coated with TRX-Golph2 antigen, respectively. Indirect ELISA was performed to detect the titer of anti-Golph2 mAb. The OD was read at 405 nm.

The subclass of anti-Golph2 mAb was determined using the SBA Clonotyping System (SouthernBiotech, Birmingham, AL, USA) according to the manufacturer’s instructions. The anti-Golph2 mAb (3 ml) was added with equivalent precooled ammonium sulfate overnight in a 4°C refrigerator. They were then centrifuged at 4°C, 1,738 × g for 15 min. The supernatant was decanted, solubilized by adding PBS (0.02 M, 3 ml), and then filtered by Sephacryl S-300 HR gel filtration (Pharmacia, USA). PBS containing 0.02% NaN₃ was used to wash the column and collected components. The positive components confirmed by indirect ELISA were dialyzed in PBS at 4°C overnight and condensed to a high concentration. The purity was identified by SDS-PAGE (12%).

A 96-well immunoplate was coated with the TRX-Golph2 fusion protein (4 μg/ml) at 4°C overnight, and then was blocked with 2% fetal bovine serum at 37°C for 2 h. Serial dilutions of the purified mAb were incubated at 37°C for 2 h. The plate was rinsed and incubated with the HRP-conjugated goat anti-mouse antibody (1:4,000 dilution) at 37°C for 1 h. After washing, TMB (3,3′,5,5′-tetramethylbenzidine) substrate was used for color development. The OD value was measured at 405 nm in order to determine the relative affinity.

Total proteins extracted from HepG2 cells were separated by SDS-PAGE on a 12% polyacrylamide gel and then transferred onto a nitrocellulose membrane (Millipore) by electroblotting. Fat-free milk (5%) was used to block the membrane at 37°C for 2 h. The membrane was then incubated with purified anti-Golph2 mAb (1:2,000) overnight in a 4°C refrigerator. The membrane was then washed with PBS-Tween-20 (PBST) three times and incubated with the HRP-conjugated goat anti-mouse secondary antibody (1:1,000) in a 37°C shaker for 1 h. After washing three times with PBST, the membrane was visualized with an ECL system (Pierce Biotechnology, Inc.).

Appropriate amounts of cells were cultured on microslides overnight. When the cells were adherent, slides were fixed with 4% paraformaldehyde for 15 min. PBST (0.5%) was used to wash the slides three times. Endogenous peroxidase activity was blocked by 3% H₂O₂/methanol and non-specific binding was blocked with 2% BSA-PBS. Diluted anti-Golph2 mAb (50 μl; 1:50) was added and incubated overnight in a 4°C refrigerator. After three 0.5% PBST washes, the appropriate (1:200) HRP-conjugated goat anti-mouse secondary antibody was added and incubated at room temperature for 1 h. Slides were washed with 0.5% PBST three times. Diaminobenzidine (DAB) was added and allowed to react for 1 min. The reaction was stopped by adding water and hematoxylin was added to counterstain for 2 min. Slides were dehydrated with alcohol and mounted with neutral gummi.

Modified sodium periodate oxidation was used for labeling anti-Golph2 mAb (8A7B4) with high purity. The result and the optimal working concentration of labeled anti-Golph2 mAb with HRP were analyzed by direct ELISA.

A 96-well immunoplate was coated with purified anti-Golph2 mAb (5C6D5, 20 μg/ml) at 4°C overnight and then blocked with 2% BSA-PBS at 4°C overnight. After washing plates with PBST three times, serum dilutions (1:2, 1:4, 1:8), strong positive (50 ng/ml antigen), weak positive (3 ng/ml antigen) and negative, were incubated at 37°C for 2 h, respectively. The plates were washed three times and enzyme-labeled anti-Golph2 mAb (1:300, 1:400, 1:500, 1:600) was added and the plates were incubated at 37°C for 1 h. The plates were washed again and colored with 100 μl 2,2-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Boehringer Mannheim GmbH, Germany) for 25 min at 37°C. The OD was read at a wavelength of 405 nm. A chessboard titration method was designed to examine the optimal working concentration of HRP-labeled mAb and the dilution ratio of serum.

The expression level of Golph2 in serum dilutions was analyzed by s-ELISA.

All of the values which presented as the mean of three paralleled individual experiments were entered into SPSS 13.0 software. The mean and standard deviation (SD) of the OD of individual groups were calculated. The expression of Golph2 in serum was described using box-and-whisker plots. The difference between groups was analyzed using Student t-tests. P<0.05 was considered to indicate a statistically significant result.

The recombinant fusion protein was expressed under the optimizing prokaryotic expression conditions of 1 mM IPTG, 30°C, 2 × g, for 10 h and purified by immobilized metal affinity chromatography under native conditions. The expressed and purified protein were identified by 12% SDS-PAGE and subsequently stained with Coomassie Brilliant Blue R250 (Fig. 1). The molecular weight of TRX-Golph2 protein was ~73 kDa. The protein was then further identified by western blot analysis using anti-His tag antibody (Fig. 2).

Through the procedures of immunization, fusion and clone selection, five hybridoma cell lines (5C6D5, 5B7F5, 7F5F3, 8A7B4, 8C9E8) that stably secreted anti-Golph2 mAb were obtained. The identity, subclass and titer are shown in Table I.

The relative affinities of mAbs were defined by the antibody concentration at which the OD value reached half the maximal signal at the plateau stage of antigen-antibody binding. The results revealed that the order of relative affinity of the five selected mAbs was 8A7B4>5C6D5>5B7F5>8C9E8>7F5F3 (Fig. 3).

The anti-Golph2 mAb was purified by Sephacryl S-300 HR gel filtration. After dialyzing and condensing, the purity was identified by SDS-PAGE (12%) (Fig. 4).

Immunocytochemistry (HepG2) revealed that the membrane of the Golgi was positively stained (Fig. 5A and B). By western blot analysis, anti-Golph2 mAb (8A7B4) was shown to react specifically with Golph2 protein (45kDa) in accordance with the theoretical value (Fig. 5C).

The anti-Golph2 mAb (8A7B4) was labeled with HRP by modified sodium periodate oxidation. By direct ELISA, the working concentration of labeled mAb was shown to be 1:500.

One serum sample from a known patient with overexpression of Golph2 in serum was used for the determination of the dilution ratio of serum. The optimal working concentration of HRP-labeled mAb and the dilution ratio of serum were fixed on 1:500 and 1:2, respectively, by the chessboard titration method (Table II).

The levels of antigen in the samples were detected by s-ELISA using HRP-labeled anti-Golph2 mAb (Fig. 6). The median serum level of Golph2 in the HCC group significantly exceeded that of the healthy controls (P<0.05). As shown in Fig. 6, the average serum content of Golph2 in HCC patients is >100 μg/l, consistent with other studies using s-ELISA (8).