Journal: International Journal of Molecular Sciences

Article Title: The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

doi: 10.3390/ijms25116121

Figure Lengend Snippet: SSPN epitopes are used for antibody development. ( A ) Schematic diagram representing the predicted membrane topology of human SSPN protein and the corresponding epitopes of commercially available antibodies (in gray). ( B ) Summary of commercially available antibodies, their epitopes, applications, and limitations. ( C ) Schematic diagrams representing the predicted membrane topology of mouse (mSSPN) and human (hSSPN) SSPN proteins and the corresponding epitopes of newly developed antibodies (in gray). hSSPN has 243 amino acids, while mSSPN has 216, with the difference mostly being due to the longer N-terminus of hSSPN. ( D ) Comparison of the amino acid residues of hSSPN and mSSPN in the epitope regions used to immunize rabbits and mice. The amino acid sequence is shown in a single-letter code. Non-matching amino acids between hSSPN and mSSPN are labeled by gray in the amino acid sequence of mSSPN.

Article Snippet: The initial screening was performed by ABclonal with an ELISA with biotinylated synthetic peptides corresponding to the C-terminus and LEL fragment of hSSPN.

Techniques: Membrane, Comparison, Sequencing, Labeling

Journal: International Journal of Molecular Sciences

Article Title: The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

doi: 10.3390/ijms25116121

Figure Lengend Snippet: Testing rabbit immune response to mSSPN with the ELISA and immunoblotting. Panels ( A , D , G , J , M ): mSSPN-specific rabbit sera titration curves with the ELISA. The blood was collected seven days after the second (bleed I) and third (bleed II) booster doses. Serial dilutions of each rabbit serum were added to the ELISA plates coated with SUMO-tagged mSSPN N-terminus (MGRKPSPRAQELPEEEARTCCGCRF, U02487). HRP-conjugated goat anti-rabbit IgG was used to detect the mSSPN-specific rabbit IgG. TMB was used as a substrate for the HRP. The enzymatic reaction was stopped by 2 M of sulfuric acid, and the plates were read at 450 nm. Panels ( B , C , E , F , H , I , K , L , N , O ): Immunoblotting of bleeds I and II from each rabbit. Rabbit 8870—panels ( B , C ); rabbit 8871—panels ( E , F ); rabbit 8872—panels ( H , I ); rabbit 8873—panels ( K , L ); and rabbit 8874—panels ( N , O ). Immunoblotting of bleeds I and II with total muscle lysates from mice overexpressing mouse SSPN (mSSPN-TG, arrow) (lane 1), human SSPN (hSSPN-TG) (lane 2), wild type (lane 3), mdx (lane 4), and SSPN-null (lane 5). m-SSPN-TG, wild type, and mdx lysates were used for the evaluation of the specific immune response to mouse SSPN. h-SSPN-TG lysate was used for detection of possible cross-reactivity between the SSPN of mouse and human origins. The SSPN-null lysate was used to exclude rabbits generating a non-specific response. mSSPN was detected at 25 kDa (arrow) by immunoblotting. The skeletal muscle was solubilized using an RIPA buffer, and the protein samples (20 μg of total protein per line for all lines, except mSSPN-TG (lane 1) where 2 μg of total protein per line was loaded) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were probed with crude non-purified rabbit sera diluted 1:150 (bleed I) or 1:600 (bleed II) in the blocking buffer. Arrows indicate the mSSPN. The ~27 kDa bands presenting in all muscle lysates (panels ( C , E , F , I , L , N , O )) were not SSPN-related and may represent secondary antibody cross-reactivity with endogenous antibody light chains in the skeletal muscle protein lysates.

Article Snippet: The initial screening was performed by ABclonal with an ELISA with biotinylated synthetic peptides corresponding to the C-terminus and LEL fragment of hSSPN.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Titration, SDS Page, Purification, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

doi: 10.3390/ijms25116121

Figure Lengend Snippet: Validation of rabbit polyclonal antibodies to mouse SSPN N-terminus. ( A ) Validation of purified rabbit polyclonal antibodies with an ELISA. Terminal bleeds from rabbits #8870, #8871, #8872, and #8873 were purified by affinity chromatography. The titer of purified antibodies was estimated by the ELISA with SUMO-tagged mSSPN (MGRKPSPRAQELPEEEARTCCGCRF, U02487). HRP-conjugated goat anti-rabbit IgG was used to detect the mSSPN-specific rabbit IgG. TMB was used as a substrate for the HRP. The enzymatic reaction was stopped by 2 M of sulfuric acid, and the plates were read at 450 nm. ( B ) Immunoblotting of the total muscle lysates from the mSSPN-TG (lane 1), hSSPN-TG (lane 2), WT (lane 3), mdx (lane 4), and SSPN-null (lane 5) mice with purified rabbit polyclonal antibodies #8870. There was 20 μg of total protein per line for all lines, except mSSPN-TG where 2 μg of total protein per line was loaded. The arrow indicates the mSSPN. Film exposure time, 15 s. ( C ) Immunofluorescence images of the transverse cross-sections of the quadriceps muscles of the WT and SSPN-null mice and the human control (hamstring) stained with purified rabbit polyclonal antibodies #8870. The acquisition time for all muscle cross-sections was 0.8 s. Scale bar, 50 μm.

Article Snippet: The initial screening was performed by ABclonal with an ELISA with biotinylated synthetic peptides corresponding to the C-terminus and LEL fragment of hSSPN.

Techniques: Biomarker Discovery, Purification, Enzyme-linked Immunosorbent Assay, Affinity Chromatography, Western Blot, Immunofluorescence, Muscles, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

doi: 10.3390/ijms25116121

Figure Lengend Snippet: Validation of rabbit monoclonal antibodies to mouse SSPN N-terminus. ( A ) Immunoblotting of total muscle lysates from mSSPN-TG (lane 1), hSSPN-TG (lane 2), WT (lane 3), mdx (lane 4), and SSPN-null (lane 5) with newly developed rabbit monoclonal antibody 10B8. There was 20 μg of total protein per line for all lines, except mSSPN-TG where 2 μg of total protein per line was loaded. The arrow indicates the mSSPN. Film exposure time, 15 s. ( B ) Immunofluorescence images of transverse cross-sections of the quadriceps of WT and SSPN-null mice, WT hearts, and the human control hamstring stained with newly developed rabbit monoclonal antibody 10B8 and commercially available mouse monoclonal antibody E2 for comparison. The acquisition time for E2 was 1 s, and for 10B8, it was 0.8 s. Scale bar, 50 μm.

Article Snippet: The initial screening was performed by ABclonal with an ELISA with biotinylated synthetic peptides corresponding to the C-terminus and LEL fragment of hSSPN.

Techniques: Biomarker Discovery, Bioprocessing, Western Blot, Immunofluorescence, Control, Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

doi: 10.3390/ijms25116121

Figure Lengend Snippet: Reactivity of mouse sera to C-terminus and LEL fragment of human SSPN in an indirect ELISA. hSSPN-specific mouse IgG titration curves. Blood was collected seven days after the second (bleed I) booster dose. Serial dilutions of each mouse serum were added to the ELISA plates coated with a biotinylated peptide of hSSPN C-terminus (HRYQVFYVGVRICSLTASEGPQQKI, AF016028) ( A ) or with a biotinylated peptide of hSSPN LEL fragment (PSSEPLSRTFVYRDVTDCTS) ( B ). HRP-conjugated goat anti-mouse IgG was used to detect hSSPN-specific mouse IgG. TMB was used as a substrate for the HRP. The enzymatic reaction was stopped by 2 M of sulfuric acid, and the plates were read at 450 nm.

Article Snippet: The initial screening was performed by ABclonal with an ELISA with biotinylated synthetic peptides corresponding to the C-terminus and LEL fragment of hSSPN.

Techniques: Indirect ELISA, Titration, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

doi: 10.3390/ijms25116121

Figure Lengend Snippet: Validation of final mouse monoclonal antibodies to C-terminus and LEL fragment of human SSPN by IFA. Immunofluorescence images of transverse cross-sections of human healthy control, human Becker muscular dystrophy (BMD) skeletal muscles, and mouse WT and mouse SSPN-null quadriceps stained with newly developed mouse monoclonal antibodies to hSSPN C-terminus (clones 289-17, 289-18), hSSPN LEL fragment (clones 290-4, 290-11), and commercially available mouse monoclonal antibody E2 for comparison. Newly developed mouse monoclonal antibodies to the hSSPN C-terminus are human-specific and do not cross-react with mouse SSPN. Clone 290-04, developed to the hSSPN LEL fragment, recognizes both mSSPN and hSSPN, and clone 290-11 is mouse-specific. All three human-specific clones shown in the figure recognize hSSPN in muscles from the BMD patient more efficiently than E2. The acquisition time for all antibodies was 1 s. Scale bar, 50 μm.

Article Snippet: The initial screening was performed by ABclonal with an ELISA with biotinylated synthetic peptides corresponding to the C-terminus and LEL fragment of hSSPN.

Techniques: Biomarker Discovery, Bioprocessing, Immunofluorescence, Control, Muscles, Staining, Clone Assay, Comparison