All experiments in the present study were approved by the Northeast Agricultural University Provincial Experimental Animal Management Committee (Harbin, China) and were performed in accordance with the guidelines of this committee.

Freund's adjuvant (complete), incomplete Freund's adjuvant, bovine serum albumin (BSA) and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (A21010) was purchased from Abbikine, Inc. (Redlands, CA, USA). SAB Clonotyping System-HRP (5300–05) was purchased from SouthernBiotech (Birmingham, AL, USA). Fluorescein isothiocyanate (FITC) Antibody Labeling Kit (53027) was purchased from Thermo Fisher Scientific, Inc., (Waltham, MA, USA). Glucose Assay Kit (0105102), which utilzizes the GOD-PAP method was purchased from Sichuan Maccura Biotechnology Co., Ltd (Chengdu, China). Other reagent grade chemicals were purchased from Sigma-Aldrich (Merck Millipore). DNA maker₂₀₀₀, λEcoT₁₄ DNA Marker, and prestained protein MW Marker were purchased from Fermentas (Thermo Fisher Scientific, Inc.). All polymerase chain reaction (PCR) primers (Tables I and II) were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.).

Sp2/0 lymphocytes, 3T3-L1 adipocytes and DH5α Escherichia coli were lab stocks. DH5α (MLCC3002) was purchased from Miaolingbio Bioscience & Technology Co., Ltd., (Wuhan, China; −80°C). Sp2/0 (CC-Y2093), which were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% CO₂, and 3T3-L1 adipocytes (CC-Y2002), which were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS at 37°C, 5% CO₂, were both purchased from Enzyme Research Biotechnology Co., Ltd., (Beijing, China). RPMI-1640 (CM0302) was purchased from You Kang Biotechnology Co., Ltd., (Beijing, China). DMEM (PM150310) was purchased from Procell (Wuhan, China).

Whole hFGF-21 protein was expressed and purified during previous studies conducted in our laboratory (2).

Six female and six male BALB/c mice (age, 6–8 weeks old; weight, 11–13 g) were purchased from Harbin Veterinary Research Institute (Harbin, China), and housed in starter batteries with access to water and commercial feed.

BALB/c mice (female, n=3) were immunized with 100 µg hFGF-21 (as 400 ml of 1:1 hFGF-21: Freund's adjuvant), after 2-weeks of feeding, followed by second immunization with 100 µg hFGF-21 (400 ml of 1:1 hFGF-21: incomplete Freund's adjuvant), third immunization was the same as the second immunization and was performed 2-weeks later. Prior to hybridoma production, the mice received a booster immunization of 100 µg hFGF-21 in phosphate-buffered saline (PBS; pH 7.5), and separated eyeball blood samples as positive serum. BALB/c mice (female, n=3) under the same rearing conditions were used to obtain negative serum by separating eyeball blood sampling. The establishment methods of hybridoma were performed according to previously described methods (15).

Indirect ELISA was performed to screen for individual clones secreting hFGF-21 mAb. Prior to cell plating, the 96-well plates were coated with 20 µg/ml hFGF-21 (100 µl) and incubated at 4°C overnight. Then, plates were washed three times with washing buffer (0.05% Tween-20 in PBS), blocked with 5% skimmed milk in PBS for 2 h at 37°C, followed by washing (as previously described). Hybridomas cultures (100 µl) as primary antibodies (positive serum as positive control, negative serum as negative control) were added to the wells and incubated for 1 h at 37°C. Following washing (as described above), plates were incubated with HRP-conjugated goat anti-mouse secondary antibody (1:15,000; 100 µl) for 1 h at 37°C, followed by washing (as previously described). The chromogenic reagent TMB (100 µl) was added to the wells and following 20 min the reaction was stopped with 2 mol/l H₂SO₄ (50 µl), and the plates spectroscopically analyzed at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). hFGF-21-positive clones were then subcloned and rescreened (as previously described).

For large-scale production of the hFGF-21 mAb, a single highly positive subclone hybridoma cells (2D8) were injected into the peritoneal cavity of BALB/c mice (male, n=3) and allowed to proliferate prior to collection of fluid from ascites, the Sp2/0 cells were injected into the peritoneal cavity of BALB/c mice (male, n=3) and allowed to proliferate prior to collection of fluid from ascites as negative control. The fluid was centrifuged (12,000 × g, 10 min; 4°C) and the supernatant purified using a Protein A-Sepharose (GalaxyBio, Beijing, China). The concentration of purified mAb was measured by a spectrophotometer, and it was used as the source of the 2D8 hFGF-21 mAb used in the present study. The mAb was further analyzed using SDS-PAGE (resolving gel, 15%; stacking gel, 5%; 1 h at 200 V; 10 µl sample; 10 ng). The class and subclass of the mAb was determined using the mouse mAb isotyping kit.

With the exception of to the purified mAbs (gradient dilution from 250×2⁰-250×2¹³; 100 µl) replaced with hybridomas cultures (100 µl) as primary antibodies, (purified Sp2/0 ascites replaced hybridomas cultures as negative control), the other operating methods were the same as indirect ELISA, as previously described. Each sample was tested in triplicate and the results presented as the mean ± standard deviation. The maximum dilution ratio of the positive well value and negative control greater than or equal to 2.1 is the titer of the mAb.

The hFGF-21 protein and negative control (BSA) were used analyzed by western blot. Once the quality of hFGF-21 and BSA were consistent, samples (10 ng) were loaded onto a 12% gel, subjected to SDS-PAGE and blotted on to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk in tris-buffered saline with Tween-20 (TBST; 10 mmol/l pH 7.5 Tris-HCl; 150 mmol/l NaCl) at 4°C overnight. Following washing three times for 15 min in TBST, the membrane was incubated with the mAb (1:500 dilution) for 1 h at room temperature. Following washing (as described above), the membrane was incubated with HRP-conjugated goat anti-mouse secondary antibody (1:7,500 dilution) at room temperature for 1 h. The membrane was then washed again (as described above) and blots were visualized by Super Signal West Femto Maximum Sensitivity Substrate (34095; Thermo Fisher Scientific, Inc.) and SmartChem™ II image analysis system (SG2011060; Sagecreation, China).

Indirect ELISA was performed to determine the cross-reactivity between hFGF-21 and mouse FGF-21 (mFGF-21) produced in our laboratory (16). In the ELISA, 20 µg/ml of hFGF-21, mFGF-21 or BSA (negative control) were used as the coating antigen and 2.5 µg/ml of the mAb (100 µl) was used as the primary antibody. All other steps of the ELISA were as previously described. All assays were performed in triplicate and the results presented as the mean ± standard deviation.

Anchored periplasmic expression (APEx) bacterial display vectors containing the new lipoprotein A (NlpA) sequence (CDQSSS) plus 6 amino acids, obtained via previous studies published by our laboratory (17–19). hFGF-21 sequence was obtained from our previous study (2).

For the first round of screening, the hFGF-21 polypeptide sequence was divided into 2 segments that included polymerase chain reaction upper sequence (PU: 1–91 amino acids upstream of hFGF-21) and polymerase chain reaction downstream sequence (PD: 92–181 amino acids downstream of hFGF-21), and then cloned (primers described in Table I) into the APEx. The cloning vector was constructed from the secretory expression vector pET27b(+) as previously described (20). The vectors formed (APEx-PU and APEx-PD) were analyzed using the restriction enzyme Sfi I, and 1% gel analysis was performed to determine the size of PU and PD.

Vectors were transformed into DH5α E. coli, which were cultured in LB media (Amp 100 µg/ml; 37°C for 4 h). When the optical density (OD) reading of the culture at 600 nm (OD₆₀₀₎ reached between 0.3 and 0.4, 0.25 mmol/l of isopropyl-β-D-thiogalactoside (IPTG) was added. The E. coli were grown at 37°C for 4 h to induce the expression of PU or PD. Then, bacterial cells (1 ml) were collected, centriguged (12,000 × g; 5 min; 4°C), washed twice with PBS and resuspended in 350 µl of ice-cold suspension solution (0.75 mol/l sucrose, 0.1 mol/l Tris-HCl pH 8.0) supplemented with 10 mg/ml lysozyme (35 µl; Shanghai Yi Sheng Biotechnology Co., Ltd., Shanghai, China). Cells were treated with 1 mmol ice-cold EDTA (700 µl) and 0.5 mol MgCl₂ (50 µl), and centrifuged again as previously described. Following this, cell pellets were centrifuged as previously described, resuspended in 90 µl PBS, and incubated with 2 mg/ml FITC-labeled anti-hFGF-21 mAb (4 µl) and 10 µl 1% BSA at 4°C for 1 h. Then, the cells were washed three times with 0.5% PBS-Tween and resuspended in 500 µl of PBS for fluorescence-activated cell sorting (FACS) analysis at 488 nm. The negative control was treated the same, except that it was not incubated with the FITC-labeled mAb.

For the second round of screening, the PD was divided into 5 overlapping segments, which included PD₁ (amino acids 92–121 of hFGF-21), PD₂ (amino acids 122–151 of hFGF-21), PD₃ (amino acids 152–181 of hFGF-21), PD₄ (amino acids 107–136 of hFGF-21) and PD₅ (amino acids 137–166 of hFGF-21). The 5 segments were cloned and cloned into the APEx bacterial display vector to produce APEx-PD₁, APEx-PD₂, APEx-PD₃, APEx-PD₄ and APEx-PD₅. The steps described in the first screening were then performed.

3T3-L1 adipocytes (1×10⁶ per ml) were seeded into 96-well plates and incubated at 37°C in a 5% CO₂, and 95% humidified atmosphere overnight. Then, the cells were starved for 12 h in serum-free DMEM, followed by stimulation with hFGF-21 (1,000 nmol/l) and anti-hFGF-21 mAb (0.0, 0.075, 0.15, 0.3, 0.6 or 1.2 µg/ml) for 24 h. Controls were not stimulated.

Glucose uptake of 3T3-L1 adipocytes was measured following stimulation using the glucose uptake assay kit, according to the manufacturer's instructions. Absorbance was recorded at 490 nm and the glucose consumption rate was calculated. Each sample was tested in triplicate and the results presented as the mean ± standard deviation.

Secretory hybridoma cells were cloned, followed by recloning three times to increase monoclonality of the produced immunoglobulins (Igs). One stable hybridoma, designated 2D8 was obtained. The concentration of the mAbs produced following purification was 2.3 mg/ml. As expected, the heavy chain of the mAbs was 50 ku, and light chain was 25 ku (Fig. 1A). The isotype of the mAb was determined to be IgG2b. The titer of the mAb was 1:1.024×10⁶ (Fig. 1B). Western blot analysis revealed that hFGF-21 could be detected by the mAb, whereas the negative control (BSA) had no reaction with the mAb, indicating good specificity (Fig. 1C). Cross-reactivity analysis identified that compared with the negative control (BSA), the mAb reacted significantly with mFGF-21 and hFGF-21 (P<0.01; Fig. 1D), which have ~80% amino acid sequence homology (16). The reaction with hFGF-21 reaction was stronger compared with the mFGF-21 reaction (Fig. 1D), which is consistent with previous published data (16).

Clones of different hFGF-21 segments showed that PU and PD were both 273 base pairs (bp) in length (Fig. 2A), and that the 5 different segments of PD were all 90 bp (Fig. 2B). The results of restriction enzyme analysis identified that each segment was successfully cloned into the APEx bacterial display vector (Fig. 2C and D). The first round of FACS (Fig. 3A-C) showed that, compared with the negative control (Fig. 3A), the mAb could not bind to the PU segment (Fig. 3B), whereas it could strongly bind to the PD segment (Fig. 3C), which suggests that the epitope the mAbs recognizes is in the PD segment. The second round of FACS (Fig. 3D-I) determined that, compared with negative control (Fig. 3D), the mAb could not bind to PD₂, PD₃ or PD₅ (Fig. 3F, G and I, respectively), whereas it could strongly bind to PD₁ and PD₄ (Fig. 3E and H). This revealed that the epitope that the mAb binds to was in the region overlapping the PD₁ and PD₄ segments, a specific fifteen amino acid sequence (YQSEAHGLPLHLPGN).

In the glucose uptake assay a control group (without hFGF-21 and the mAb) and 6 groups stimulated with hFGF-21 (1,000 nmol/l) and different concentrations of the mAb were tested. Results of the assays determined that, compared with the control, the mAb (0.0 µg/ml) and hFGF-21 group had significantly increased glucose uptake (P<0.01; Fig. 4A), which is consistent with the results of a previous study (1). The mAb (0.075 µg/ml) and hFGF-21 group also had a significantly increased glucose uptake compared with the control (P<0.05; Fig. 4A). However, the mAb (0.15, 0.3, 0.6 or 1.2 µg/ml) and hFGF-21 groups did not have increased glucose uptake compared with the mAb(0) group (Fig. 4A), which indicates that the mAb reduces hFGF-21-dependent glucose uptake in vitro.

GLUT1 mRNA expression was quantified in a control group (without hFGF-21 and the mAb) and in 6 groups stimulated with hFGF-21 (1,000 nmol/l) and different concentrations of the mAb. The results of qPCR analysis identified that, compared with the control, the mAb (0.0 µg/ml) and hFGF-21 group had significantly increased GLUT1 mRNA expression (P<0.01; Fig. 4B), which is consistent with the results of a previous study (1). The mAb (0.075 µg/ml) and hFGF-21 group also had significantly increased GLUT1 mRNA expression (P<0.05; Fig. 4B). However, mAb (0.15, 0.3, 0.6 or 1.2 µg/ml) and hFGF-21 groups did not have increased GLUT1 mRNA expression (Fig. 4B). This indicates that the mAb reduces hFGF-21-dependent GLUT1 mRNA expression in vitro compared with the Ab(0) group.