Rabbits were injected intradermally at multiple sites with 1 mg oPRL emulsified in Freund’s complete adjuvant and received similar booster injections of oPRL in incomplete Freund’s adjuvant at two-week intervals. The rabbits were bled weekly after the first booster injection and 1 week after receiving the last injections. After four immunisations, immunized rabbits were shown to generate high-titre sera against oPRL, as determined by enzyme-linked immunosorbent assays (ELISA). Immunoglobulin G (IgG) fragments were purified from each rabbit by ammonium sulphate precipitation followed by protein-A affinity chromatography.

Female BALB/c mice (6 to 8 weeks old) were purchased from the Peking university laboratory animal centre. The mice were immunized intraperitoneally with 0.2 mg of rabbit anti-oPRL-F(ab′)₂ emulsified in Freund’s complete adjuvant and received similar booster injections of anti-oPRL-F(ab′)₂ emulsified in incomplete Freund’s adjuvant at two-week intervals. The animals were immunized four times. Three days after final immunization, splenocytes from mice immunized with anti-oPRL-F(ab′)₂ were harvested and fused with sp2/0 myeloma cells at a 5:1 ratio using 50% (w/v) polyethylene glycol (PEG) 1500. After fusion, the cells were distributed in 96-well plates at 1×10⁵ cells per well in the hypoxanthine/aminopterin/thymidine (HAT) selection medium. The culture supernatants of hybridomas selected on HAT medium were tested by ELISA to determine the presence of antibodies against anti-oPRL-F(ab′)₂. Antibody-secreting hybridomas were subcloned four times via limiting dilution, and a number of monoclonal anti-oPRL-F(ab′)2 antibody-secreting cell lines were selected for expansion into mass culture and production of ascetic fluids.

To test the rabbit anti-oPRL polyclonal antibodies, 96-well plates were coated with 0.1 μg per well of oPRL in PBS (PH 7.2) overnight at 4°C. The plates were subsequently washed four times with PBS in 0.05% Tween 20 (PBST), blocked with 2.5% non-fat powder in PBS and incubated with Rabbit antisera diluted to their optimum concentration in PBST. The plates were incubated for 1.5 h at 37°C, washed and reincubated with the HRP-conjugated goat anti-rabbit IgG. After 1 h, the plates were washed and incubated with the tetramethylbenzidine (TMB) substrate. The results were read at 450 nm in an automatic ELISA plate reader (Multiskan FC, Thermo Fisher Scientific Inc., Waltham, MA, USA).

To detect the monoclonal anti-idiotypic antibodies to PRL, 96-well plates were coated with purified anti-PRLR F(ab′)₂ fragments or with F(ab′)₂ fragments of control antibodies overnight at 4°C. The plates were then washed four times with PBS in 0.05% Tween 20 (PBST), blocked with 2.5% non-fat powdered in PBS and incubated with the culture supernatants from the hybridomas, and the wells were incubated at 37°C for 1 h. After washing, goat anti-mouse IgG-FC HRP secondary antibody was added, and after 1 h, the plates were washed and incubated with TMB substrate, and the results were read at 450 nm in an automatic ELISA plate reader (Multiskan FC, Thermo Fisher Scientific Inc., USA).

CHO-PRLR cells were trypsinised and detached from the plate surface and adjusted to a density of 1×10⁵ cells/mL. The cells were then incubated with 100 μL in either a negative control medium or in one of the positive hybridoma supernatants for 1 h at 4°C. After three washes with PBST, cells were fixed and stained with FITC-labelled goat-anti-mouse IgG. After staining, the cells were incubated with FITC-labelled goat anti-mouse IgG for 1 h at 4°C in the dark. After incubation, the cells were washed and resuspended in 0.5 mL FACS buffer. All flow cytometric analysis was conducted on FACS Calibur cytometer (Becton Dickenson, New York, NJ, USA). The data were analysed using the Cell Quest software.

Competitive ELISAs were performed according to method of Lan et al. (2015). For the competition studies, ELISA plates were coated with 0.5 μg of F(ab′)₂ fragments of Mab2s or control antibody overnight. The next morning, the plates were washed and blocked as described above. Then, a constant amount of rabbit anti-PRL antibody (Ab1, 1 mg/mL) was mixed with different dilutions of PRL and incubated for 1 h at 37°C prior to application of the blocked ELISA plate. The plates were washed again, and goat anti-mouse IgG (Fc fragment-specific)-HRP secondary antibody was added to the wells for 1 h at 37°C. After incubation for 1 h, the plates were washed and incubated with TMB substrate; results were read at 450 nm in an automatic ELISA plate reader (Multiskan FC, Thermo Fisher Scientific Inc., USA).

In addition, a similar competitive ELISA was performed. The ELISA plates were coated with 0.5 μg F(ab′)2 fragments of rabbit anti-PRLR antibodies overnight. Then, a constant amount of Mab2s was mixed with different dilutions of PRL and incubated for 1 h at 37°C prior to application to the blocked ELISA plate. The subsequent treatments were performed as described as above.

CHO-PRLR cells were cultured in serum-free F-12K medium containing 1% bovine serum albumin for 12 h. The cells were subsequently washed and incubated with 125I-PRL (5,000 CPM/well) together with increasing concentrations of unlabelled PRL and Mab2s or isotype-matched control antibody for 2 h. After the incubation, the cells were washed and detached from the plate. The cells were then counted in a gamma counter. The non-specific binding was determined in the presence of 5 μg unlabelled PRL.

CHO-PRLR and Nb2 cells were cultured in six-well plates as described as above. The cells were cultured in serum-free medium for 10 h prior to the experimental treatments. For the dose-dependent experiments, serum-starved CHO-PRLR and Nb2 cells were stimulated with the different concentration of B7 for the indicated time. For the time-course experiment, serum-starved CHO-PRLR and Nb2 cells were stimulated with the indicated concentration of B7 for different times. Unless otherwise noted, all treatments were performed at 37°C. At the end of the treatment, cells were solubilized in lysis buffer on ice for 30 min before centrifugation at 12,000×g for 10 min. The resulting supernatants were collected and concentrated by ultrafiltration. Samples were lysed directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 5 min, and the protein concentration was determined by BCA Protein Assay according to manufacturer’s instructions. Samples (60 μg protein per lane) were subjected to SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were washed and blocked with 5% non-fat milk for nonspecific binding for 1 h at 37°C. After three washes, the membranes were incubated with the primary antibodies (anti-phospho-JAK2 anti-phospho-STAT5) according to the manufacturer’s protocols. Following three washes, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (secondary antibody) for 1 h at 37°C. After three final washes, the blots were then developed using the ECL Plus chemiluminescent detection system according to the manufacturer’s instructions. After ECL detection, the membranes were stripped of primary and secondary antibodies using the Restore Western Blot Stripping Buffer according to the manufacturer’s instructions, and the blots were subsequently probed with antibodies against total JAK2 and total STAT5.

The [³H]thymidine incorporation assay was performed according to the method of (Clevenger et al., 1994). Briefly, Nb2 or Ba/F3-PRLR cells were grown and maintained in RPMI-1640 medium containing 10% FBS, 50 U/mL penicillin, and 50 μg/mL of streptomycin for 24 h. After washing with PBS, the cell suspensions were then prepared. The cell suspensions were added to the wells of 96-well round-bottomed tissue culture plates in a volume of 100 μL (1×10⁶ cells/mL), and the cells were stimulated with the indicated concentrations of PRL, Mab2s or control antibody in a final volume of 0.2 mL for 24 h at 37°C. Ligand-stimulated Nb2 or Ba/F3-PRLR cells were labelled for 4 h with 0.5 μCi of [³H]thymidine. Next, [³H]thymidine incorporation was determined by liquid scintillation counting.

The data are presented as the mean values±standard error.

Employing the hybridoma technique, a panel of anti-idiotypic monoclonal antibodies to PRL was obtained. To determine whether Mab2s that can bind to PRLR on CHO-PRLR cells, flow cytometry analysis was performed, and nine mab2s exhibited strong positive signals. Among these, one Mab2 (designed B7) was selected for further characterization as described as below. B7 was classified as an IgG1 isotype using a commercial mouse mAb isotyping kit.

Competitive ELISA was performed to assess whether B7 could mimic a PRL epitope (namely possesses an internal image nature, Ab2β). The results indicated that PRL could inhibit the interaction between the anti-PRL antibodies and anti-idiotypic monoclonal antibody B7. As illustrated in Figure 1, PRL inhibited polyclonal anti-PRL binding to B7 in a dose-dependent manner, and control antibody showed no effects. Moreover, PRL could also compete with B7 for binding to polyclonal anti-PRL antibodies in a dose-dependent manner (Figure 2). These results indicate that B7 and PRL share a common epitope, which also suggest that B7 possesses an internal image nature (Ab2β).

To demonstrate further whether B7 could specifically bind to PRLR on CHO-PRLR, we performed a competitive receptor-binding assay. As shown in Figure 3, unlabelled PRL displaced ¹²⁵I-PRL from the cells as expected. B7 also displaced ¹²⁵I-PRL in a dose-dependent manner; however, the control antibody elicited no effects. These results demonstrated that B7 specifically binds to PRLR expressed on target cells.

The abovementioned experiments demonstrated that B7 could specifically bind to PRLR. However, it is unclear whether B7 can trigger PRLR-induced intracellular signalling. Therefore, the western-blot was performed to check whether the agonistic effect of B7 exerted on CHO transfected with PRLR or not could mimic the signal transduction of PRL. As shown in Figure 4, PRL strongly induced phosphorylation of JAK2-STAT5 in CHO-PRLR but not parent CHO cells. Similar to PRL, B7 also activates the JAK2-STAT5 pathway in CHO-PRLR but not parent CHO cells. However, the control antibody shows no effect.

Next, the kinetics of JAK2-STAT5 activation by B2 or PRL was determined by western-blot analysis in CHO-PRLR cells, and representative results are shown in Figure 5A and 5B, respectively. The dose–response of JAK2 activation by either PRL or B7 was similar to each other. PRL or B7 treatment resulted in the rapid phosphorylation of JAK2, and phosphorylation was detectable at 3 nM. The maximal activation in response to PRL or B7 was observed between 10 and 20 nM and subsequently began to decline. In addition, the time courses of JAK2 and STAT5 activation by PRL or B7 were also similar in CHO-PRLR cells. For each stimulator, JAK2 and STAT5 activation was observed as early as 5 min, maximised at 15 to 45 min, and declined dramatically thereafter.

To confirm the agonistic effect of B7 further, we also evaluated JAK2-STAT5 signalling induced by B7 in Nb2 cells. As shown in Figure 6A and 6B, the dose–response of Jak2 activation by either PRL or B7 was similar to each other. PRL or B7 treatment resulted in the rapid phosphorylation of JAK2, and phosphorylation was detectable at 3 nM. The maximal activation in response to PRL or B7 was observed between 10 to 20 nM, and subsequently began to decline. In addition, the time courses of JAK2 and STAT5 activation by PRL or B7 were also similar in CHO-PRLR cells. For each stimulator, JAK2 and STAT5 activation was observed as early as 5 min, maximised at 15 to 45 min, and declined dramatically thereafter.

The [³H]-thymidine incorporation assay was performed to further assess agonistic activity of B7. As shown in Figure 7A and 7B, both B7 and PRL promoted Nb2 and Ba/F3-PRLR cell proliferation in a dose-dependent manner. The maximal proliferation effect of PRL was shown at 1 to 10 nM (Nb2) and 1 to 10 nM (Ba/F3-PRLR), and the maximal proliferation effect of B7 was shown at 1 to 5 nM (Nb2) and 1 to 10 nM (Ba/F3-PRLR), and at higher concentrations, the cell proliferation levels was observed to decline slightly. However, the control antibody exhibits no effect when compared with B7.