Journal: Biochemistry and Biophysics Reports

Article Title: New breast cancer marker BF-09 is overexpressed in tumor extracts and secreted in serum

doi: 10.1016/j.bbrep.2025.102097

Figure Lengend Snippet: BF-09 sandwich ELISA optimization The BF-09 sandwich ELISA uses a matched capture and detection mAb pair. BF-09 standard immunoassay protocol involves coating a 96-well immunoplate plate (Maxisorp) with capture mAb in PBS, blocking unoccupied sites, incubating with surrogate antigen, binding captured BF-09 antigen with biotinylated detecting mAb, amplifying detection with streptavidin linked horseradish peroxidase (HRP) and revealing colorimetric reaction with HRP substrate as measured at 450 nm by microplate reader. Serum-free cell culture medium (SFCM) from the breast cancer cell line MCF-7 was used as an antigen source and diluted in standard assay buffer. Antigen concentration is expressed as total protein concentration in the SFCM (μgE/ml). Panel A . Titration of capture and detection antibodies using different concentrations of capture (i.e. 0.5, 1, and 2 μg/ml) and detection antibody (i.e. 0.5, 1, 2, and 3 μg/ml) to achieve optimal signal/noise ratio. OD 450 nm values from wells with standard assay buffer only (blank” or noise) and wells with a fixed amount of MCF-7 SFCM secreted BF-09 antigen diluted in standard assay buffer (signal) are reported. Panel B . Influence of various blocking agents to achieve optimal signal/noise ratio, used in 16 (labeled 1–16 in x-axis) combinations of blocking buffer and detection antibody diluent (Ab Diluent). Buffer used to block the assay plate is indicated on the x-axis of the column chart. PF (1–4): protein free/PBS (Pierce); SB (5–8): SuperBlock (Pierce); CS (9–12): 1 % casein/PBS (Pierce); BS (13–16): 1 % BSA/PBS. The corresponding buffer used for Ab Diluent is indicated by a plus symbol (“+”) in the grid below the column chart. Optimal capture (2 μg/ml) and detect (1 μg/ml) antibody concentration was used. Panel C . Comparison of assay buffers B1 and B2 used as antigen diluent at various antigen concentrations. The column chart shows OD 450 nm signals from blank wells with assay diluent only (0μgE/ml total protein), and from wells with various amounts of MCF-7 SFCM containing secreted BF-09 antigen (1.9–17.1μgE/ml total protein) diluted in either B1 or B2 buffer. Optimal capture (2 μg/ml) and detection (1 μg/ml) antibody concentrations and optimal blocking agent (PF/PBS) were used. B1 is standard assay buffer containing: 1 % BSA in PBS-Tween 0.05 %; B2 is serum optimized assay buffer containing: 1 % BSA in PBS-Tween 0.05 %, 10 mM EDTA, 5 mM DTT, 10 μg/ml of purified mouse IgG and 10 μg/ml of purified bovine IgG.

Article Snippet: Nunc Maxisorp immunoplate wells (Thermo Fisher Scientific, Waltham, MA) were coated with 100 μl of capture BF-09 mAb in PBS, and incubated overnight at 4 °C.

Techniques: Sandwich ELISA, Blocking Assay, Binding Assay, Cell Culture, Concentration Assay, Protein Concentration, Titration, Labeling, Comparison, Purification