Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein

Abstract

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.

Keywords: RPS4Y1; diagnosis; fetal cells; hemophilia; male cells; male marker; monoclonal antibody; ribosomal protein.

Figure 1

Analysis of RPS4X and RPS4Y1 RT-PCR products. (A) Agarose gel electrophoresis showing RPS4X and RPS4Y1 cDNA bands amplified from male (Y) and female (X) peripheral blood mononuclear cells (PBMCs), chorionic villi, male HepG2 and female HEK293 cells. The bands of the GeneRuler 50 bp marker (M) are indicated on the left. Size of RPS4X and RPS4Y1 bands are also reported in bold. (B) Bar graph showing the relative quantitation (RQ) of RPS4Y1 transcript in male PBMCs, chorionic villi and HepG2 cells.

Figure 2

Alignment of RPS4X and RPS4Y1 amino acid sequences. Amino acid difference between the two proteins are indicated by asterisks (∗). Regions selected as antigens for mice immunization are boxed.

Figure 3

Y3 antigen specificity of monoclonal antibodies. The bar graph showed the optical density (OD; y-axis) from the ELISA assay (one out of three) performed using Y3 (black bar) or X3 (white bar) capture peptides and 2 μg/mL of antiRPS4Y1 antibodies #1, #2, #3, #4 or mouse serum (x-axis).

Figure 4

mRPS4Y1 antibody binding to the male RPS4Y1 protein. Representative results of SDS-PAGE and Western blotting (one out of three) performed on cell lysates of male HepG2 (Y) and female HEK293 (X) cells are showed. AntiRPS4Y1 antibodies were tested individually (#1, #2, #3, #4) and in combination (mix). AntiRPS46 and anti-tubulin antibodies were used as loading controls. The bands of the Precision Plus Protein marker (M) are indicated on the left.

Figure 5

mRPS4Y1#3 antibody binding to the native RPS4Y1 protein. Representative results of immunoprecipitation experiments (one out of three) of RPS4Y1-antibody#3 complex performed with magnetic beads coupled to protein G (G) are showed. Immunoprecipitated proteins (IP) and supernatants (–) were loaded and analyzed by SDS-PAGE and Western blotting. The arrow indicated the RPS4Y1 band.

Figure 6

mRPS4Y1#3 antibody specificity for male cells. (A) Representative spinning disk confocal microscopy images of mRPS4Y1 antibody #3 staining (red fluorescence) performed over night at 4 °C (ON) or at room temperature for 3 h (3h) on HepG2 and HEK293 cells (lower and upper panels, respectively). (B,C) Results of the digital imaging analysis of mRPS4Y1 antibody #3 staining reported as the percentage of positively stained cells relative to total cells (B) and the mean fluorescence intensity per field of view (mfi/FOV) (C). (D) Representative spinning disk confocal microscopy at 100× magnification followed by PFS deconvolution via Richardson Lucy algorithm for evaluation of co-localization signals of RPS4Y1 (red fluorescence) and endoplasmic reticulum (ER)-marker calnexin (green fluorescence) on HepG2 cells. Highlighted zoomed-in area in the right upper image showed one single HepG2 cell from the FOV in the left bigger image. Co-localization analysis of signal intensities for each pixel in the FOV is shown in the zoomed in right lower image. Co-localizing pixels with signals from both RPS4Y1 and calnexin are in grey. (E) Dot-plot showing the mean Pearson correlation index per FOV resulting from pixel by pixel digital analysis of mRPS4Y1 antibody #3 and ER-marker fluorescent signals performed on 50 FOVs from best-focus deconvolved Z plan at 100× magnification, with a mean number of 30 cells/FOV.