Journal: Scientific Reports

Article Title: Targeting of specific neuronal types in the non-human primate brain by using a murine CD25-specific recombinant immunotoxin

doi: 10.1038/s41598-026-39662-6

Figure Lengend Snippet: Production of mCD25-specific recombinant monoclonal antibodies. ( a ) Strategy for screening rabbit monoclonal antibodies that specifically react to mCD25 by ISAAC technology. A rabbit was immunized with purified mCD25/His-tag protein, and IgG + cells were isolated from peripheral blood lymphocytes, arrayed on the microarray chip, and cultured to trap secreted IgG. The chip was blocked with hCD25/His-tag protein, and then labelled with biotinylated mCD25/His-tag protein, followed by addition of Cy3-conjugated streptavidin. The cells producing mCD25-specific antibodies were visualized under a fluorescence microscope and collected from individual wells. The mRNA samples were extracted from single cell populations and subjected to reverse transcription (RT), and cDNA fragments encoding V H and V L regions were amplified by PCR, which were then cloned into the expression vectors containing rabbit immunoglobulin Y and K chains. The recombinant antibodies were produced in cultured cells and secreted into the medium. ( b ) Binding property of an mCD25-specific recombinant antibody to target proteins. Microplates were coated with either mCD25/His-tag or hCD25/His-tag, and then various concentrations of RMAb-52 were added to the wells. The binding of antibody was detected using a secondary antibody conjugated to alkaline phosphatase. ( c ) Competitive binding of antigen for the mCD25-specific recombinant antibody. RMAb-52 solution was incubated with various concentrations of mCD25/His-tag. Microplates were coated with mCD25/His-tag, and then the incubated mixture at equilibrium was added to the wells. The binding of free antibody was detected using a secondary antibody conjugated to alkaline phosphatase. The K D value was determined by using Scatchard plots. Data are expressed as mean ± SEM of four independent experiments. Individual data are overlaid in ( c ).

Article Snippet: The chip was incubated with 10 μg/mL hCD25/His-tag (Sino Biological Inc., Cat#10165-H08H) for 30 min as a blocking step, and then with 10 μg/mL biotinylated mCD25/His-tag for 30 min, followed by addition of Cy3-conjugated streptavidin (Sigma-Aldrich, Cat#S6402) for 30 min.

Techniques: Recombinant, Bioprocessing, Purification, Isolation, Microarray, Cell Culture, Fluorescence, Microscopy, Single Cell, Reverse Transcription, Amplification, Clone Assay, Expressing, Produced, Binding Assay, Incubation

Journal: Scientific Reports

Article Title: Targeting of specific neuronal types in the non-human primate brain by using a murine CD25-specific recombinant immunotoxin

doi: 10.1038/s41598-026-39662-6

Figure Lengend Snippet: Properties of anti-mCD25-PE38 ITX. ( a ) Binding property of the recombinant ITX for target proteins. Microplates were coated with either mCD25/His-tag or hCD25/His-tag, and then various concentrations of ITX protein were added to the wells. The binding of ITX was detected using a PE38-specific monoclonal antibody and a mouse IgG-specific secondary antibody conjugated to horseradish peroxidase. ( b ) Competitive binding of antigen for the recombinant ITX. ITX solution was incubated with various concentrations of mCD25/His-tag protein. Microplates were coated with mCD25/His-tag, and then the incubated mixture at equilibrium was added to the wells. The binding of free ITX was detected using a PE38-specific monoclonal antibody and a mouse IgG-specific secondary antibody conjugated to horseradish peroxidase. The K D value was determined using Scatchard plots. ( c ) Cytotoxic activity of the recombinant ITX toward HT-2 and EL-4 cells expressing mCD25 and hCD25, respectively. Cells were incubated with various concentrations of the ITX. Viable cell number was evaluated by a cell viability assay using WST reagent. Relative ratios to the average of control cell number without the ITX were calculated. Data are expressed as mean ± SEM of four independent experiments. Individual data are overlaid in ( b ).

Article Snippet: The chip was incubated with 10 μg/mL hCD25/His-tag (Sino Biological Inc., Cat#10165-H08H) for 30 min as a blocking step, and then with 10 μg/mL biotinylated mCD25/His-tag for 30 min, followed by addition of Cy3-conjugated streptavidin (Sigma-Aldrich, Cat#S6402) for 30 min.

Techniques: Binding Assay, Recombinant, Incubation, Activity Assay, Expressing, Viability Assay, Control