A total of 5 BALB/с female mice (6–8 weeks old, weighing 20–22 g; The Jackson Laboratory, Bar Harbor, ME, USA) were used in all the experiments. The animals were maintained in the animal facility at the Northwestern University (Chicago, IL, USA). All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Northwestern University.

The LNCaP human prostate cancer cell line, C3H10T1/2 (10T1/2; mouse embryonic fibroblasts) and the SP-2/0 mouse myeloma cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); 293T cells, MCF7 breast carcinoma cells, HeLa cervical carcinoma cells, Caco2 colon carcinoma cells and T98G human glioblastoma cells were obtained from the Culture Collection of the Institute of Cytology RAS (St. Petersburg, Russia). The Saos2 human osteosarcoma cells were kindly provided by Dr K. Helin (European Institute of Oncology, Milan, Italy). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FCS) and gentamicin (50 μg/ml) in a CO₂ incubator under conditions of 5% CO₂ and 100% humidity.

The BALB/c female mice were immunized by intraperitoneal injections of 50 μg of affinity purified recombinant AMACR in 150 μl of sterile phosphate-buffered saline (PBS). As an adjuvant to the injection solution, 50 μg of the oligonucleotide 5′-TCC-ATG-ACG-TTC-CTG-ACG-TT-3′ were added (28). The injections were repeated 5 times at intervals of 2 weeks. At 7 days after the fourth injection, the animals were bled from the retro-orbital sinus for the detection in their serum the antibodies against AMACR by immunoblotting and enzyme-linked immunosorbent assay (ELISA) The bleeding was performed under anesthesia [xylazine 10 mg/kg + ketamine 80 mg/kg administered intraperitoneally (i.p.)].

These procedures were carried out as previously described (29). The SP-2/0 myeloma cell line was passaged 1 day prior to fusion to yield 1×10⁷ cells the following day. The cells were centrifuged for 5 min at, 200 × g at room temperature to remove FCS, resuspended in 10 ml of DMEM/F12 without FCS and counted with 0.2% Trypan blue. Immune mouse spleen was removed under anesthesia as mentioned above followed by 5–7 min with euthanasia and cervical dislocation. The spleen was removed under sterile conditions and perfused with DMEM/F12 medium without FCS using a 21G needle with a 10 ml syringe; the splenocytes were washed twice in 10 ml of serum-free medium. For counting, 50 μl of the cell suspension were mixed with 450 μl of 1% acetic acid and then with 500 μl of 0.2% Trypan blue in PBS. Subsequently, 7×10⁷ splenocytes and 1×10⁷ myeloma cells were mixed in a 50-ml tube, washed once by centrifugation as described above, the supernatant was removed and the pellet was resuspended by tapping the tube. The cells were placed in a water bath at 37°C, mixed with 1 ml of warm 50% polyethylene glycol 1500 (Roche Diagnostics, Indianapolis, IN, USA) for 1 min with constant stirring of the slurry. Immediately thereafter, the cells were supplemented with 1 ml of serum-free medium and then for 3–4 min with 3 ml of serum-free medium followed by the addition of 20 ml of serum-free medium and 20 ml of medium with 15% FCS. The cells were incubated for 30 min at 37°C, pelleted at 200 × g resuspended in 42 ml of DMEM/F12 with 15% FCS and 10% BM-Condimed H1 (Roche Diagnostics), 1X HAT (Roche Diagnostics), and cultured at 5 × 96-well plates of 100 μl per well. After 7 days, the growth medium was exchanged with HAT-free medium. Within 3 days, the hybridomas were screened for antibody production by ELISA. The cells in the wells with positive reaction were cloned by inoculating 100 cells in 96-well plates and the supernatants were screened for antibody production. Cloning was continued until all clones did not exhibit a positive reaction.

Antibody isotyping was performed using the mouse hybridoma subisotyping kit purchased from Calbiochem (La Jolla, CA, USA) according to the manufacturer's recommendations. Brifly, separate wells of a 96-well ELISA plate were sequentially filled with the goat anti-mouse IgGs, blocking solution, an the hybridoma supernatants were diluted 10-fold, isotyping antisera were added, and the wells were then visualized by TMB reagent and read at OD450 nm.

Phage peptide sequences specifically recognized by in-house made murine (clones 6H9 and 2A5) and the commercial rabbit monoclonal (clone 13H4; Zeta Corp., Arcadia, CA, USA) antibody against AMACR (mimotopes), were determined using a commercial phage peptide library Ph.D.-7C7 (BioLabs) based on combinatorial plurality of random 7-mer peptides fused to the pIII coat protein of M13 phage (30). In this library, the randomize peptide sequence is flanked by a pair of cysteine residues. Under non-reducing conditions, the cysteines form a disulfide crosslink, resulting in the phage display of cyclized peptides. The library consists of 1.2×10⁹ sequences amplified to produce ~200 copies of each sequence in 10 μl of the phage library.

The selection of phage particles containing mimotopes (biopanning), was performed by incubating the antibodies coupled to protein A/G immobilized on agarose beads (Pierce, Rockford, IL, USA). The E. coli ER2738 cells (included in the Ph.D.-7C7 BioLabs kit) were grown in 5 ml of LB medium on a shaker at 37°C until early logarithmic phase (OD600 to 0.01–0.05) for titration and at the same time in 20 ml of LB medium for the amplification of phage particles. Subsequently, 50 μl of 50% protein A-agarose was resuspended in 1.5 ml microtube in 1 ml of TBST [10 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.1% Tween-20] and precipitated. The precipitate was washed in TBST by 3-fold centrifugation for 5 min at 2,000 × g. Protein A-agarose was resuspended in 1 ml of blocking buffer [0.1 M NaHCO₃ (pH 8.6), 5 mg/ml bovine serum albumin (BSA), 0.02% NaN₃], incubated for 60 min at 4°C followed by stirring. Subsequently, 10 μl of the phage library in suspension (2×10¹¹ Pfu) and 300 ng of anti-AMACR antibodies were mixed in 200 μl of TBST, incubated for 20 min at room temperature, transferred to a 1.5 ml tube containing the affinity sorbent, incubated for 15 min at room temperature with irregular stirring and centrifuged for 5 min at 2,000 × g at room temperature. The supernatant was removed and the sorbent was washed 10 times in 1 ml of TBST and precipitated by centrifugation as described above. The phage particles bound to protein A-agarose were eluted for 10 min at room temperature in 1 ml of 0.2 M glycine-HCl (pH 2.2) containing 1 mg/ml BSA. The eluate was centrifuged for 1 min at 13,000 × g, the supernatant was transferred to a new microtube, neutralized by adding of 150 μl of 1 M Tris-HCl (pH 9.1) and titrated on plates containing LB agar with isopropyl-β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). The eluted phage particles were amplified in 20 ml of ER2738 cell culture in the logarithmic growth phase and incubated at 37°C for 4–5 h on a shaker. The culture of the bacterial cells was centrifuged the following day for 10 min at 13,000 × g and 4°C, the supernatant was transferred to new tube, and centrifugation was repeated; 80% of the top layer of supernatant was transferred to new tube, followed by the addition of a 1/6 volume of 20% polyethylene glycol (PEG) 8000, containing 2.5 M NaCl; the solution was left for 2 h at 4°C, the precipitate was spun down for 15 min at 13,000 × g and 4°C, the pellet was resuspended in 1 ml of TBS [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.2% NaN₃], centrifuged for 1 min at 13,000 × g, and the supernatant was transferred to a new tube and titrated on plates with LB/IPTG/X-Gal using the predicted titer levels of 10⁻⁸–10⁻¹¹ Pfu. To do that, 200 μl of bacterial cultures in microtubes were supplemented with 10 μl of phage particles in each dilution for 1–5 min, the infected cells were transferred to tubes containing 3 ml of warmed up to 45°C agarose, mixed and applied as the upper layer on the cup agar plates containing IPTG/X-Gal; the plates were cooled for 5 min, inverted and incubated overnight at 37°C. The following day, the numbers of blue colonies were counted and the phage titer was determined using as a baseline titer of 2×10¹¹ Pfu. During the course of second round of phage concentration, we used protein G-instead of protein A-agarose and the Tween-20 concentration in TBST was increased up to 0.5%. During third round of phage selection, we used protein A-agarose and 0.5% Tween-20 for washing. Finally, the phage particles from 10 different colonies were transferred and amplified in separate tubes using overnight culture of the ER2738 cells for 4–5 h at 37°С. The bacterial cultures were then transferred to microtubes, centrifuged for 30 sec at 13,000 × g at +4°С, 500 μl of upper layer of the supernatant was transferred into new tube, supplemented with 200 μl of PEG/NaCl, and the tube content was mixed by inverting and left at room temperature for 10 min, centrifuged for 10 min at 13,000 × g; the supernatant was removed, the pellet was resuspended in 100 μl of the buffer containing iodide chloride [10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 4M ICl], supplemented with 250 μl of ethanol for 10 min at room temperature and spun down for 10 min; the supernatant was removed, the pellet was washed with 70% ethanol, dried and diluted in 30 μl of deionized water. The phage DNA was sequenced using dye-labeled dideoxynucleotides in the Sequencing Facility of the Northwestern University (Chicago, IL, USA).

This was performed using the program Pepitope available online (http://pepitope.tau.ac.il) (26), the algorithm PepSurf (27) and the crystal structure of MRC, PDB 1×74 (1).

In total, 100 μl of recombinant AMACR or BSA as a negative control (10 μg/ml) in 0.05 M sodium carbonate buffer (pH 9.6) were added into 1 well of a 96-well plate for ELISA for 3 h at room temperature or overnight at +4°C. Unbound material was removed by washing 3 times with PBST buffer (PBS containing 0.05% Tween-20). The unbound sites were saturated with the addition of 3% BSA in PBST for 1 h. The plate was washed 3 times with PBST and 100 μl of hybridoma supernatant, and diluted mouse immune serum or BSA were added into 1 well for 1 h at room temperature. The plate was then washed as described above and 100 μl of anti-mouse-HRP antibody was loaded into each well for 1 h at room temperature. To visualize peroxidase staining, 100 μl of tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, St. Louis, MO, USA) containing 0.01% hydrogen peroxide for 5–30 min were added into each well. TMB was prepared from the 1 mg/ml stock solution diluted 1:100 by 0.1 M sodium acetate buffer. The reaction was terminated by the addition of 50 μl of 10% phosphoric acid and the absorbance was read at 450 nm using a Dynex MRC TC Revelation microplate reader (Dynex Technologies, Inc., Chantilly, VA, USA).

Intracellular proteins were stained as follows: coverslips with spread cells grown in culture or attached to the glass as a result of close contact with sections of various mouse organs (kidneys, liver, heart, bladder and thymus, which were obtained under the conditions described above in the section entitled 'Animal care') were placed in 35-mm plates, washed with PBS for 5 min, fixed with 4% paraformaldehyde for 15 min, and then with 70% ethanol overnight at 4°C, treated with 0.2% Triton X-100 for 10 min and washed with PBS twice for 5 min. Unspecific binding was blocked by 3% BSA with 0.1% Tween-20 for 1 h. The cells were incubated with primary antibodies (rabbit monoclonal anti-AMACR, cat. no. 13H4; Zeta Corp., Sierra Madre, CA, USA; mouse monoclonal anti-AMACR, cat. no. ab63340; Abcam, Cambridge, UK; in-house made mouse monoclonal antibodies against AMACR 6H9 and 2A5; antibody dilutions were 1:50–1:200) in blocking solution for 1 h at room temperature, washed 3 times with PBS for 5 min and incubated with labeled species-specific antibodies [Cy3 goat anti-mouse IgG (H+L), cat. no. A10521; and Cy5 goat anti-rabbit IgG (H+L), cat. no. A10523, both from Molecular Probes, Eugene, OR, USA], washed 3 times with PBS for 5 min and mounted in Anti-Fade (Bio-Rad Laboratories, Hercules, CA, USA) containing DAPI for nuclei staining. Images were recorded on Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) or Olympus FV3000 (Olympus, Tokyo, Japan) confocal scanning microscopes using lasers at a 405, 488 and 561 nm wavelength.

Protein electrophoresis and immunoblotting were performed as previously described (31). Electrophoresis was run on 8% SDS-PAGE on mini or large vertical protein electrophoresis chambers (Bio-Rad Laboratories). The probes for SDS-PAGE were prepared from human prostate tissues and cells of established cell lines. The prostate tissues were obtained from 5 male cancer patients, 60–70 years of age, subjected to radical prostatectomy in accordance with the procedure approved by the Ethics Committee of the Northwestern University Review Board (IRB) protocol. All patients provided written informed consent. The prostate tissue probes included 5 samples of tumors and 4 samples of normal tissues adjacent to the tumors obtained from the same patients.

Tissue samples of approximately 10 mg were placed in tubes with 100 μl of lysis buffer containing 1% SDS, 50 mM TrisCl (pH 6.8), 100 mM β-mercaptoethanole, protease and phosphatase inhibitor cocktails (Sigma-Aldrich); treated with Cole-Parmer 130 Watt Ultrasonic Processor (Cole-Parmer, Vernon Hills, IL, USA) with 70% amplitude 6 times for 10 sec, incubated for 0°C for 20 min, and centrifuged at 13, 000 × g for 15 min at +4°C.

The cells grown on culture plates were washed twice with PBS and removed with a plastic scrapper, sedimented by centrifugation as described above and lysed with periodic resuspending on ice for 30 min in 3 volumes (relative to the volume of the dense cellular pellet) of buffer solution composed of 25 mM Tris-HCl (pH 7.4), 250 mM NaCl, 0.25% NP-40, protease and phosphatase inhibitor cocktails in 1:100 dilution. Cellular extracts were centrifuged at 13,000 x g for 15 min at 4°C. Supernatants were applied for further experiments. The probes were equilibrated for protein content determined by Bradford reagent (Bio-Rad Protein Assay kit II, cat. no. 5000002; Bio-Rad Laboratories), transferred to micro-tubes with an equal volume of loading buffer [5% SDS, 20% glycerol, 200 mM dithiotreitol, 120 mM Tris-HCl (pH 6.8), 0.002% bromophenol blue] and boiled for 5 min in a water bath. Probes in the volume of 25 μl containing 30–90 μg of total protein were loaded on one line of polyacrylamide gel. Electrophoretically separated proteins were transferred onto a PVDF membrane by semi-dry electrotransfer. The proteins on the membrane were revealed using specific antibody against AMACR described above and secondary Peroxidase AffiniPure goat anti-mouse IgG (H+L) (cat. no. 115–035–003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and anti-rabbit IgG, HRP-linked antibody, cat. no. 7074S; Cell Signaling Technology, Danvers, MA, USA) and visualized with ECL™ Western Blotting Detection Reagent (cat. no. RPN2209; Sigma-Aldrich).

To evaluate the ability of the anti-AMACR antibody to immunoprecipitate (IP)-specific antigen, we first prepared the affinity matrix. Two 1.5 ml Eppendorf microtubes were loaded with 50 μl of 50% protein G-sepharose (Invitrogen, Carlsbad, CA, USA), washed 5 times with 0.5 ml of PBS and supplemented with 1.25 ml of the serum-free hybridoma supernatant containing anti-AMACR antibody 6H9 or with 2 μl of the immune serum from an immunized with AMACR mouse in 1.25 ml of PBS. The supernatant was prepared using ex-cell 610-HSF hybridoma serum-free medium (SAFC Biosciences, Lenexa, KS, USA). The microtubes were rotated for 1.5 h at room temperature to couple the antibody to protein G-sepharose and washed 5 times with the IP buffer containing 25 mM TrisCl (pH 7.5), 50 mM NaCl, 0.2% SDS, 1% NP-40, protease and phosphatase inhibitor cocktails in 1:100 dilution. The prepared affinity matrix was loaded with extracts from mouse liver containing 0.5–2 mg of total protein. The tubes with IP were rotated overnight at 4°C to couple the AMACR protein from the cell extracts to protein G-sepharose, the affinity matrix was washed 5 times with the IP buffer and twice with the same buffer without detergents. After a final washing, the tubes containing the immune complexes were loaded on 25 μl of SDS-PAGE loading buffer, boiled for 5 min and centrifuged for 5 min at 13,000 x g. The supernatants were loaded into 10% polyacrylamide gel to separate proteins under reducing conditions. The separated proteins were transferred from gel onto a PVDF membrane and visualized with ECL as described above.

The 6H9 and 2A5 hybridoma supernatants were prepared by growing the cells for 15 days in Ex-Cell 610-HSF hybridoma serum-free medium in CELLine 1000 flasks (Integra Biosciences, Hudson, NH, USA). The supernatants were spun down for 30 min at 2, 000 × g, filtered through a 0.45-μm filter and evaluated for protein concentration and antibody activity by ELISA and immunoblotting. For affinity purification, the supernatants were loaded into a 1 ml protein G-column (HiTrap Protein G HP; GE Healthcare Life Sciences, Pittsburgh, PA, USA) equilibrated with 3 column volumes of binding buffer (0.02 M sodium phosphate, pH 7.0). The column was washed with 10 column volumes of the binding buffer to remove impurities and unbound material. The bound antibody was eluted with 1 ml of elution buffer (0.1 M glycine-HCl, pH 2.7) into a microtube containing 100 μl of neutralization buffer (1 M Tris-HCl, pH 9.0). To exchange the elution buffer, 200 μl of the sample was loaded on a Zeba Spin 0.5 ml desalting column (Thermo Fisher Scientific, Waltham, MA, USA). Prior to desalting, the column was washed 3 times by centrifugation with 300 μl of PBS for 1 min at 1,500 × g. Subsequently, 200 μl of the eluate was applied on the top of the resin and supplemented with 15 μl of the stacker. The sample recovery was performed by spinning at 1,500 × g for 2 min, and the protein concentration was estimated by Bradford assay.

Antibody delivery into cells was performed using PULSin reagent (Polyplus-transfection Inc., New York, NY, USA). Subsequently, 6 wells of a 24-well plate were loaded with cover glasses (one 8-mm glass into each well) and seeded with 2×10⁵ exponentially growing HeLa or T98G cells in DMEM containing 10% FCS. The following day, when the cell saturation density was 70–80%, the cells were washed twice with PBS and the growth medium in wells was exchanged to 900 μl of DME serum-free medium. Three master mixes were prepared: i) 250 μl of 20 mM HEPES buffer (pH 7.4) and 2 μg of the non-specific 2A5 antibody; ii) 250 μl of 20 mM HEPES buffer (pH 7.4) and 2 μg of the specific 6H9 antibody against AMACR; iii) 250 μl of 20 mM HEPES buffer (pH 7.4) and 2 μg of the specific 63340 antibody against AMACR purchased from Abcam (Cambridge, UK). The mixes were filtered and supplemented with 5 μl of PULSin reagent (Polyplus-transfection Inc.) for 15 min at room temperature. Each mixture was then divided in two and added to the wells with the HeLa or T98G cells. Following 4 h of incubation at 37°C, the growth medium was exchanged with fresh one containing 10% FCS and the cells were kept in a CO₂ incubator overnight. The following day, the adherent cells on coverglasses were stained with anti-mouse antibody conjugated with Cy3 dye (Invitrogen). In the case of effective proteofection (≥80% exhibited positive immunofluorescence with specific anti-AMACR antibody), the cells were trypsinized, washed, counted and seeded into 15 wells of a 96-well plate at a density of 2×10⁴ cells per well in 100 μl of culture medium. For growth curves, the number of cells was evaluated in triplicate each day for the following 5 days using counting chambers.

The proliferation rate was estimated using the Cell Proliferation kit I MTT (Sigma-Aldrich) according to the manufacturer's instructions. The cells treated with the antibodies were seeded at a concentration of 5×10³ cells/well in 100 μl culture medium in 9 wells of a 96-well plate and incubated for 24 h. After the incubation period, 10 μl of MTT labeling reagent (Cell Proliferation kit, REF 11465007001; Roche Diagnostics, GmbH, Mannheim, Germany) were added to each well and the cells were placed into a CO₂ incubator for 4 h. Subsequently, 100 μl of the solubilization solution were added to each well and the plate was incubated overnight in a CO₂ incubator followed by measuring the optical density (OD) at 570 nm using an ELISA reader (Fluorofot; Probanauchpribor LLC, St. Petersburg, Russia).

The HeLa or T98G cells were labeled with CellLight Peroxisome-GFP, BacMam 2.0 reagent (Molecular Probes, Eugene OR, USA) as follows: The cells asynchronously growing on 60-mm cell culture with 70% density were supplemented with 10 μl of CellLight reagent (10 particles per cell) for 18 h. Co-localization was evaluated the following day after staining the cells with mouse monoclonal antibodies to AMACR (ab63340; Abcam, in-house made 6H9) and anti-mouse antibody coupled to Cy3 as described above. For the study of peroxisome co-localization with AMACR detected by the antibody delivered into live cells, the cells with labeled peroxisomes were transferred into the wells of a 24-well plate containing coverglasses and proteofected the following day as described above. Immunofluorescence was visualized using an Olympus FV3000 fluorescence confocal microscope (Olympus) with 405, 488 and 561 nm lasers.

Statistical analysis was performed using Microsoft Excel 2010 software. Each experiment was repeated at least 3 times.

The immunization of mice with recombinant human AMACR and adjuvant oligonucleotide comprising the CpG residues caused a rapid and strong immune response. In 1 week after the fourth intraperitoneal injection of the antigen, the antibodies which recognized AMACR were clearly detected in the mouse serum diluted 1:4,000 were by ELISA and immunoblotting (Fig. 1A). In the result of the fusion of the immune splenocytes and myeloma cells followed by HAT selection and screening by ELISA, we selected 20 hybridomas producing antibodies which showed the signal exceeding 1.0 standard unit at 450 nm (>50% of the full scale value). In total, 5 of the 20 hybridomas produced antibodies of the IgG1 isotype, 4 of the IgG2b isotype and 11 of the IgM isotype. The antibodies secreted by 4 different hybridomas bound the 1A AMACR isoform in both extracts from the mouse kidney (37 kDa) and human prostate cell line LNCaP (42 kDa), as shown by immunoblotting; the remaining 16 antibodies did not bind any protein or interacted with several proteins The blots in Fig. 1A shows that 3C7, 6D6, 6H9 antibodies bound a 42 kDa protein, 3G5, 6B9 do not recognize any protein, while 2A5 and 4B7 recognize several non-specific proteins in immunoblotting. For the following experiments, we selected 6H9 antibody which a specifically recognized 1A isoform of AMACR and 2A5 non-specific antibody as a negative control for 6H9.

We found that in-house made antibody which bound AMACR in extracts of LNCaP human prostate cells (Fig. 1A), also recognized this antigen by immunoblotting in various mouse tissues. The highest levels of AMACR expression were detected in the kidney, liver and heart; however, in the bladder, brain, spleen and thymus, the antibody bound small amounts of the protein (Fig. 1B). Human HeLa cells exhibited the high levels of AMACR production similar to mouse liver and kidney tissues; however, human AMACR migrated as a 42 kDa band, while all mouse tissues contained the 37 kDa AMACR isoform (Figs. 1C and 2B). In immunoprecipitation assay, the antibody to AMACR similar to the immune serum of mouse-donor splenocytes used for hybridoma production bound minimal amounts of AMACR from mouse liver in contrast to the high levels of this protein detected by this antibody in immunoblotting (Fig. 1D). These results were confirmed by immunofluorescence staining of the mouse tissues performed with the 6H9 antibody (Fig. 2A). The cell lines of human origin, similar to the mouse tissues, exhibited a distinct AMACR expression dependent on their tissue origin. AMACR was highly expressed in HeLa cervical carcinoma and Caco2 colon carcinoma cells, in contrast to MCF7 breast carcinoma, 293T and Saos2 osteosarcoma cells (Fig. 2B). To compare the specific activity of the in-house made 6H9 and commercial antibody, 13H4, we performed a site-by-site immunoblot comparison of AMACR detection in probes from cancer or normal prostate tissue from patients with prostate cancer. This experiment revealed that the in-house made antibody, 6H9, possessed a higher antigen sensitivity and detected AMACR in all 5 probes from the cancer tissue of the patients with prostate cancer compared with 3 from 5 probes detected by the commercial antibody, 13H4 (Fig. 1E).

The antibodies, 6H9, 2A5 and 13H4, demonstrated a high and reproducible signal in ELISA; however, they yielded different images of antigen binding in immunoblotting. The 6H9 and 13H4 antibodies recognized the 42 kDa 1A human AMACR isoform, while the 2A5 antibody bound several proteins with different molecular mass (MM) (Fig. 1A). Given the differences in the characteristics of the antibody/antigen interaction in immunoblotting, we used the 2A5 antibody as a negative control. Sequencing of the phage DNA from 10 different clones obtained by biopanning and subsequent translation of the phage DNA revealed that the 6H9 and 13H4 antibodies bound the same AA sequence in 7 and 5 out of 10 cases, respectively. By contrast, the 2A5 antibody recognized 10 different mimotopes in biopanning (Table I).

Mapping of 10 phage mimotopes selected with the 6H9 antibody revealed 3 clusters on the the surface of MRC, the AA sequences, in which most closely corresponded to those in the phage polypeptides. In accordance with the program, the most possible conformational epitope (probability is 89.112 conventional units) recognized by this antibody included AA G113, W114, R120, Q122, Q123, A124, G125, Y130, S132, L133, N134, V189 and W200 of MRC. This epitope was homologous to the phage mimotopes LKWGVHW and LKYGQHR, identified, respectively, at 7 and 1 of the 10 phage clones (Table I). To find the epitope recognized by the 13H4 antibody, we used sequences of 9 phage mimotopes (mimotope KSLPXHS contains an unspecified residue and was not included in the analysis). In total, 3 clusters of AA were found for this antibody among which the greatest homology (46.293 conventional units) showed the sequence which included W114, P119, R120, H126, I128, N129, Y130, S132, L133, N134, G135 and W200 of MRC. This epitope was homologous to polypeptides WWWSLQP and HKSLGTW, identified, respectively, in 5 and 1 from 9 phage clones (Table I). Antibody 2A5 interacted with 10 different mimotopes, from which the sequences FPTFPNQ, TPSNKHT, NNILPMT, PWGFPYK revealed the greatest degree of homology (probability is 37.089 conventional units) to the epitope cluster of MRC comprising N309, E310, P311, H312, I313, R316, N317, W318, F319, Y320, G325, W326, Q327, P328 and M329 (Fig. 3).

All AA forming the epitopes recognized by the 6H9 and 13H4 antibodies were located in the region of MRC, composed of helices α5 and α8, and unstructured sequence between helices α5 and α6, helix α5 and sheet β5 (Fig. 4). The vast number of the AA forming these epitopes, 24 out of 27, were located between helices α5 and α6, helix α5 and sheet β5, whereas in the region of helix α8, there were mapped only 3 AA (Table I). The epitopes recognized by the 6H9 and 13H4 antibodies indicated high levels of conservatism, 71/69%, 50/38% and 71/77% of their AA were identical, respectively, in MRC, human or mouse AMACR and conservative. The epitopes detected by the 6H9 and 13H4 antibodies in human or mouse AMACR were virtually identical (Table I).

The epitopes recognized by the 2A5 antibody, which was used as a negative control localized in the helix α13 and sheets 10–11 of MRC, that represent a different region of the AMACR fold compared with the region between helices α5 and α8, in which the epitopes of the 6H9 and 13H4 antibodies were localized (Fig. 3). Of note, despite the high levels of identity of phage mimotopes and MRC epitopes recognized by the 2A5 antibody (64% identity), these sequences revealed low levels of conservatism (36%). When compared with homologous sequences in mouse and human AMACR the levels of their identity and conservatism were, respectively, 28 and 36% (Table I) (the percentage identity scores were obtained by dividing the number of identical AA by the total number of AA in all mimotopes).

To examine the growth-associated activity of the 6H9 antibody, we first tried to deliver the antibody into cells using a liposome-based PULSin reagent (Polyplus-transfection). For these experiments, we exploited HeLa human cervical adenocarcinoma cells, which produce high levels of AMACR (Figs. 1C and 2B) and exhibit a high sensitivity in antibody delivery with PULSin reagent (32). AMACR catalyzes the oxidation of fatty acids and is localized in peroxisomes and mitochondria. The 6H9 antibody similar, to the commercially available 63340 antibody (Abcam), detected abundant amounts of AMACR in HeLa cells (Fig. 5A). In HeLa cells treated with paraformaldehyde, AMACR was mostly detected outside peroxisomes, observed in both preparations stained with 6H9 or 63340 antibodies (Fig. 5B). The distribution pattern of specific 6H9 and 63340 antibodies proteofected into HeLa cells was similar and differed from that of non-specific 2A5 antibody. While specific antibodies were distributed over the entire cytoplasm compartment preferentially in the form of small particles, non-specific antibody forms large cytoplasmic clusters, often connected in chains near or around the nuclei (Fig. 5C). The levels of co-localization with peroxisomes of different antibodies against AMACR proteofected into HeLa cells depended on the type of antibody used. While non-specific 2A5 antibody did not exhibit any co-localization (Fig. 5D), 6H9 antibody, low level and 63340 antibody, exhibited a greater level of co-localization visible due to the change in the color of peroxisomes from green to yellow (Fig. 5D). The growth rate of the HeLa and T98G cells proteofected with antibody 6H9 or non-specific antibody 2A5 was similar when determined using growth curves or MTT assay (Fig. 6).