CHO-K1 cells (ATCC) were grown in RPMI (Sigma) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma). The TurboFect reagent (Thermo Sci.) was used for transient and stable transfections. The procedure was carried out in accordance with the manufacturer’s protocol. Early passages of CHO-K1 cells were stably transfected (1.5 mg DNA) with the plasmid pcDNA3.1(+) encoding the human 5-HT_1AR or the human D₂R (UMR cDNA Resource Centre) separately or cotransfected with both vectors. Stable cell lines expressing D₂R and/or 5-HT_1AR were obtained after the addition of the selection antibiotic, G418 (Sigma), at a final concentration of 0.75 mg/ml. Cells resistant to the antibiotic and stably expressing investigated receptors were analysed by RT-PCR (data not shown). Forty-eight hours before the selection experiment, stable cell lines were transiently transfected with 0.5 mg DNA (per 10 mm plate area) encoding the desired receptors. Additionally, the HEK-293 cell line (ATCC) was used. The cells were grown in MEM (Sigma) supplemented with 1% L-glutamine and 10% heat-inactivated FBS (Sigma).

All cells were cultured at 37°C inside a humidified incubator in an atmosphere of 5% CO₂.

The human antibody scFv phagemid library Tomlinson I+J (Geneservice) was used. The library J was amplified and titrated according to the manufacturer’s protocols using E. coli TG1 cells. Biopanning was conducted against the D₂-5-HT_1A heteromer using CHO-K1 cells expressing D₂-5-HT_1A heteromers(CHO-positive cells; CHO+) and CHO-K1 cells expressing D₂R mixed with CHO-K1 cells expressing 5-HT_1AR (1:1) (CHO-negative cells; CHO-). Five rounds of positive selection followed by negative preselection and finally 2 negative selections were performed. Preselection was performed as follows: amplified phages were blocked for 2 hr at RT (or overnight at 4°C) in 3% MPBS and then added to culture medium with CHO- cells grown on 150 mm plates (20x, 95% confluence) and incubated for 2 hr at 0°C. Then, the temperature was changed to 37°C for 20 min. The CHO- cells were centrifuged (10 min at 1000 rpm), and the supernatant containing unbound phages was added to the medium with CHO+ cells grown on 150 mm plates (10x, 95% confluence). From that moment, the positive selection began and extended for 2 hr at 0°C. In the next step, the cells were washed 4 times with cold PBS. Between washings, RPMI medium was added, and the cells were incubated on ice for 10 min. Similar to preselection, for the next 20 min, the temperature of incubation was changed to 37°C. In the next step, the cells were washed 4 times using elution buffer (100 mM glycine, 150 mM NaCl, pH 2.8). The number of washes increased (twice each time) with subsequent rounds of selection. Finally, CHO+ cells were harvested from the plates and resuspended in PBS containing trypsin (1 mg/ml) for approximately 15 min (until cell lysis). Then, the obtained suspension was centrifuged (10 min, 1000 rpm, 4°C), and the supernatant containing the desired phages after titration and amplification was used for another round of biopanning. The biopanning rounds were repeated until the results of polyclonal phages ELISA related to output phages reached a plateau or started to decline.

The efficiency of the biopanning process was determined using polyclonal phage ELISA. A total of 50 ml of amplificated phages (obtained after each round of selection) was added to each well of the 96-well V-microtiter plate (Sarstedt) and incubated with 50 ml of 4% MPBS for 2 hr at 37°C. Then, 1.5 x10⁵ CHO+ cells (resuspended in 50 ml of RPMI with 5% FBS) expressing D₂-5-HT_1A heteromers (positive probe) were added to the wells with blocked phages and incubated on ice for 1 hr. The negative probe consisted of CHO- cells, i.e., cells expressing a single type of receptor mixed at the 1:1 ratio. The washing step was conducted 3 times at 4°C using 200 ml cold PBS. After each round, the cells were centrifuged (300 x g, 10 min, 4°C), and the supernatant was rejected. Bound phages were detected using horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibodies (GE Healthcare). Briefly, after washing, the cells were incubated with the antibody resuspended in a 1:5000 ratio in 3% MPBS for 30 min on ice and then washed 4 times as described above. Finally, 100 ml of TMB substrate (GE Healthcare) was added to each well. The reaction was stopped with 1 M HCl (100 ml per well), and the absorbance was measured at 450 nm. Experiments were performed in triplicate.

Based on the results of polyclonal phage ELISA, phage clones from rounds characterized by the highest affinity against CHO+ cells were randomly selected for monoclonal phage ELISA experiments. Individual colonies were inoculated into 96-well plates containing 100 ml 2xTYAG (2xTY (Bioshop) with 100 μg/ml ampicillin (Sigma) and 1% glucose (Bioshop)) medium per well and cultured overnight at 37°C (250 rpm). Then, 5 ml of the culture (from each well) was added to fresh 200 ml 2xTYAG medium and cultured with shaking (250 rpm) at 37°C for 2 hr. After that time, 10⁹ helper phages were added to the wells and incubated for 1 hr and at 37°C with shaking at 250 rpm. The plates were then centrifuged (1800 x g, 10 min), the supernatants were aspirated off, and bacterial pellets were resuspended in 200 ml 2xTYAKG (2xTY containing 100 μg/ml ampicillin, 50 mg/ml kanamycin (Sigma) and 1% glucose) medium and incubated overnight at 30°C (250 rpm). Finally, the plates were centrifuged (1800 x g, 10 min), and 50 ml of supernatants (containing monoclonal phages) was used in the phage ELISA as described above (2.4).

To obtain soluble scFv expressed in E. coli HB2151, a single colony was used to inoculate 5 ml of 2xTY medium containing 100 μg/ml ampicilin, and the cultures were grown overnight at 37°C. A total of 2.5 ml of the overnight culture was used to inoculate 25 ml, and then this culture was used to start a 1 L culture at 37°C. The expression was induced in the host bacteria by adding IPTG to the final concentration of 1 mM when the OD600 reached 0.9; the incubation continued for 16 h at 28°C, and then the cells were harvested by centrifugation (4000 x g, 15 min, 4°C). The cells were placed on ice until scFv purification.

ScFvs are secreted into the periplasmic space located between the outer and inner membrane of E. coli. The pellet of bacterial cells was resuspended in buffer consisting of 50 mM Tris-HCl (Bioshop), pH 8.0; 20% sucrose (Bioshop); 1 mM EDTA (Bioshop); and 1x Protease Inhibitor Cocktail (Sigma) using 20 ml buffer/1 L overnight culture). The cells were incubated on ice for 15 min with gentle agitation, and then the cell suspension was centrifuged at 30000 x g for 30 min at 4°C. The supernatant is the osmotic shock fluid containing periplasmic proteins and was divided into two equal parts: one was dialyzed extensively against Protein L Binding Buffer (100 NaH₂PO₄, pH 7.2; 150 mM NaCl) (Bioshop). The dialysis was carried out at 4°C overnight before continuing with the purification. The supernatants were centrifuged (30000 x g, 30 min, 4°C) after dialysis. ScFvs were purified at room temperature.

The extract of E. coli scFvs was loaded onto a column filled with 5 ml of Protein L Agarose equilibrated with Protein L Binding Buffer at a flow rate of 1 ml/min. After washing with 10 column volumes of Binding Buffer, the proteins were eluted from the column with Elution Buffer that contained 0.1 M glycine, pH 2.5 (at flow rate 1 ml/min). Eluted fractions were immediately adjusted to physiologic pH by adding 100 μl of the Neutralization Buffer (1 M Tris, pH 8.0) to 1 ml of eluate. Fractions containing scFvs (as judged by SDS-PAGE and Coomassie Brilliant Blue staining) were pooled and dialyzed against 1x PBS at 4°C overnight. The concentration of protein was calculated via Bradford assay. The purified fractions were stored at -80°C.

Samples for analysis were mixed with 2x Electrophoresis Buffer containing 50 mM Tris-HCl, pH 6.8; 10% glycerol; 2% SDS; 0.1% bromophenol blue and 5% β-mercaptoethanol and heated for 5 min at 95°C. SDS-PAGE was performed according to Laemmli, 1970 12 with a 4% stacking gel and a 12% resolving gel (Mini-Protean 3, Bio-Rad). Gels were stained by Coomassie Brilliant Blue.

The specificity of purified scFvs was determined by ELISA and flow cytometry analysis. In the case of the ELISA experiments, 100 ml of purified antibodies at different concentrations, resuspended in 4% BSA, were added to wells of a 96-V-well plate and incubated for 1.5 h at 37°C. Then, 1.5 x105 CHO+ cells as well as CHO- cells (resuspended in 50 ml of RPMI with 5% FBS) were added to the suitable well with blocked antibodies and incubated on ice for 1 hr. The washing step was conducted 3 times at 4°C using 200 ml cold PBS. After each round, the cells were centrifuged (300 x g, 10 min, 4°C), and the supernatant was aspirated off. Then, the pellets were resuspended in 50 ml protein L conjugated with HRP (protein L was resuspended in 0.5% BSA/PBS, 1:2000 ratio) and incubated for 30 min on ice. After washing (carried out 4 times as described above), the reactions were developed by TMB substrate. Experiments were performed in triplicate.

Moreover, similar experiments were conducted with HEK-293 cells. HEK-293+ represents cells stably expressing D₂R and 5-HT_1AR, HEK-293- represents a mixture of HEK-293 cells expressing individual forms of the receptors.

Before experiments using flow cytometry analysis, purified antibodies were conjugated with fluorescent dye. The idea of the tests was the same as described above in the case of ELISA. The same types of cells were used. The experiments were conducted to confirm the results from ELISA.