Abstract
Antigen Used for Immunization
Antigen: (C-terminus) sequence of human Ghrelin receptor (GHS-R)
The human GHS-R amino acid sequence was carefully analyzed and a 23-mer synthetic peptide, corresponding to the C-terminal intracellular portion of human GHS-R, was selected.
Amino acidic sequence: SQRKLSTLKDESSRAWTESSINT
Method of Immunization
CD2F1mice (Charles River), 7–12 weeks old, were immunized subcutaneously four times at 2 weeks intervals with 50 μg/mouse of antigen emulsified in the same volume (100 μL/mouse) of Incomplete Freund's Adjuvant (Sigma-Aldrich, St. Louis, MO). Blood from the mice was collected for sera 5 days after the previous immunization. Mice were housed in appropriate animal care facilities and handled according to international guidelines for experiments with animals.
Parental Cell Line Used for Fusion
P3 × 63Ag8.653 mouse myeloma cells (from Biological Bank; Istituto Nazionale per la Ricerca sul Cancro IST, Genova, Italy).
Selection and Cloning Procedure
Cells were cultured at 37°C in Roswell Park Memorial Institute (RPMI) 1640 medium that was supplemented with 10% fetal calf serum, L-glutamine, penicillin, streptomycin, and hypoxanthine-aminopterin-thymidine 100, 0.4, and 16 μM, respectively (Sigma-Aldrich) to select hybrid cells, in a humidified atmosphere in the presence of 5% carbon dioxide and 95% air. Supernatants from the growing hybridomas were screened with an enzyme-linked immunosorbent assay (ELISA). Positive wells were subcloned by limiting dilution and tested by indirect ELISA.
Heavy and Light Chains of Immunoglobulin
IgG1; Light chains K.
Specificity
ELISA was used to evaluate the specificity of monoclonal antibodies (mAb) 5C9H2 to bind the amino acidic sequence SQRKLSTLKDESSRAWTESSINT, corresponding to the C-terminal portion of human GHS-R.
For this assay, 96-well plates (Immobilizer-Nunc; Thermo Scientific, Waltham, MA) were coated with 1 μg/well of antigen diluted in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The plates were washed three times with PBS +0.05% Tween-20 (PBST; Sigma-Aldrich) and blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) in PBS at 37°C for 1 hour. Supernatants (50 μL) from hybridoma cultures, immune serum, or purified antibody diluted in PBS +0.5% BSA were added and incubated at 37°C for 2 hours. The plates were washed three times with PBS +0.2% Tween-20 (Sigma-Aldrich) before incubation with secondary goat antimouse IgG antibody, that was conjugated to alkaline phosphatase (Millipore, Bedford, MA), at 37°C for 1 hour. After three washes with PBS +0.2% Tween-20, the plates were incubated with p-nitrophenyl phosphate (Sigma-Aldrich) that was diluted to 1 mg/mL in diethanolamine substrate buffer (Thermo Scientific) at 37°C for 30 minutes. The optical density at 405 nm was detected on a Victor3 plate reader (Perkin Elmer, Waltham, MA). Isotypes of the mAb were determined by using the Mouse Mono-Ab-ID kit (Invitrogen, Carlsbad, CA) through the capture assay according to the manufacturer's instructions.
Specific binding of mAb 5D9H2 was confirmed by immunoprecipitation analysis using 22RV1 cells, a human prostate cell line described as positive for GHS-R.(1) Analysis was also performed on lysates of rat hippocampus, where GHS-R is expressed at high levels.(2,3)
For these experiments, protein extracts of cells or tissue were lysed with lysis buffer containing 20 mM Na2HPO4 (pH 7.2), 150 mM NaCl, 2 mM EGTA, 25 mM NaF, 1 mM Na3VO4, protease inhibition cocktail (Sigma-Aldrich), 1% Triton X-100, and 0.5% saponine (Sigma-Aldrich). Lysates were clarified by centrifugation at 15,900 × g at 4°C for 30 minutes. The bicinchoninic acid assay (BCA) reagent kit (Thermo Scientific-Pierce) was used to determine protein concentrations. Clarified lysates were incubated with 10 μg/mL mAb 5D9H2 or anti-GHS-R commercial polyclonal antibody (LSBio; LS-C346069) (2 μg) at 4°C overnight, then bound to protein A/G agarose (Sigma-Aldrich) at 4°C for 2 hours. The agarose was washed three times with lysis buffer, and proteins were released from the agarose by boiling in NuPage LDS Sample buffer (Invitrogen) for 5 minutes. The proteins were then subjected to sodium dodecil sulphate polyacrilamide gel electrophoresis (SDS-PAGE) and transferred to 0.45 μm nitrocellulose membranes (Invitrogen) at 30 Volts for 110 minutes at 4°C. After washing three times with PBS +0.1% Tween and twice with distilled water, the blots were incubated overnight at 4°C with anti-GHS-R purified commercial polyclonal (LSBio) or monoclonal antibody that was specific for GHS-R diluted in blocking solution (PBS +0.1% Tween +5% Skim milk). The membranes were washed three times with PBS +0.1% Tween and incubated with goat anti-rabbit Ig that was conjugated to horseradish peroxidase (BIO-RAD) at room temperature for 45 minutes. After three washes with PBS +0.1% Tween and twice with distilled water, the membranes were incubated with the Protein Detection System (Genespin s.r.l.). The immunoblotting analysis of the immunocomplexes detected a 48-kDa band corresponding to the predicted size of GHS-R.
The specificity of mAb 5C9H2 was also demonstrated by silencing GHS-R RNA in 22RV1 cells. In this case, we used a quantitative immunofluorescence assay (In-Cell Western assay). mAb 5C9H2 was able to recognize GHS-R in 22RV1 cells, whereas we observed a significant reduction of signal on siRNA GHS-R transfected cells, confirming the specificity of the binding.
For in-cell western assay, cells were cultured overnight in 96-well plates in complete medium culture, then fixed with 3.7% formaldehyde for 20 minutes, washed with PBS, and finally permeabilized with Triton X-100 (0.1% in PBS) for 15 minutes. Cells were blocked with Odyssey blocking buffer (LI-COR Bioscience) for 90 minutes at room temperature. After incubation in a humid chamber overnight at 4°C with mAb 5D9H2 diluted in Odyssey blocking buffer, cells were washed with PBS +0.1% Tween-20 and incubated for 1 hour at room temperature with LI-COR IRDye 800 labeled secondary antibodies (1:800) and with CellTag 700 (1:500) for cell number normalization, diluted in Odyssey blocking buffer. CellTag700 is a nonspecific cell stain that accumulates in both the nucleus and cytoplasm. Finally, cells were washed five times with PBS +0.1% Tween-20 before the scan of the plate by the Odyssey Infrared Imaging System (LI-COR Bioscience).
Immunofluorescence assay also confirmed the mAbs ability to stain hippocampal neurons. Primary neurons at different stages of development (4, 9, and 18 days in vitro) were fixed in 4% paraformaldehyde and 4% sucrose at room temperature, for 10 minutes. Primary and secondary antibodies were applied in gelatin diphosphate buffer (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl) for 2 hours at room temperature. The confocal images were acquired with a Leica SPE confocal microscope, by using a Nikon (Tokyo, Japan) 40x objective with a sequential-acquisition setting at a resolution of 102461024 pixels. Each image was a z-series projection taken at 0.8 mm deep intervals. The following antibodies were used: Polyclonal antibodies against VGlut-1 (1:1000 dilution) were from Synaptic System (Gottingen), and secondary antibody Alexa 546 was from Life Technologies.
Specific Antigen Identified
GHS-R1a isoform of GHS-R. Species: Human, rat and mouse.
The mAb detects the C-terminal portion of GHS-R that is expressed only in the 1a isoform of GHS-R.