Fig 1: Singleplex (hGH coating) (A) and duplex (both hGH and COMP coating) (B) detection of different hGH concentrations (50 nM, 25 nM, 5 nM) in 96 well plate vs microvolumetric format. In 96-well format, signal was detected from the bottom of the plate (80 μL volume) and in microvolumes (signal detected on a microvolume glass slide (2 µL/sample). Blank – every protocol step followed, excluding coating of the bottom of the well with analytes (non-specific signal). P-values indicate statistical significance (*P ≤ 0.05). Error bars represent the standard deviation of the average value (96-well plate N=2; microvolumes N=3).
Fig 2: Optimization of COMP analyte detection on the microslide. Non-diluted and diluted 1:1 (v/v) with PBS 10 mM NaOH and 0.5% SDS buffer on a glass microslide. At concentrations 50 nM, 25 nM, and 5 nM COMP was analyzed using 17C10 anti-COMP-biotinylated Ab and streptavidin-coated QD655. MFI – median fluorescence intensity; Blank – sample without COMP protein coating, followed by all of the other protocol steps. P-values indicate statistical significance (***P ≤ 0.001). Error bars represent the standard deviation of the average value (N=3).
Fig 3: Scheme of the classical QLISA detection method. COMP (cartilage oligomeric matrix protein) and hGH (human growth hormone) proteins are attached to the bottom of the plate, incubated with biotinylated detection antibodies, and then conjugated with streptavidin-coated QDs, followed by the direct registration of fluorescence signal from the bottom of the plate.
Fig 4: Different COMP concentration (50 nM to 3.125 nM) detection in 96-well plate vs microvolumetric format. 17C10 anti-COMP-biotinylated Ab and streptavidin-coated QD655 were used. Purple bars indicate the signal detected from the bottom of the plate, excitation 400 nm, emission 655 nm, 80 μL volume. Blue bars indicate the signal detected on a microvolume glass slide (2 µL/sample), excitation 400 nm, emission 655 nm. Blank – every protocol step followed, excluding coating the bottom of the well with COMP (non-specific signal). P-values indicate statistical significance (****P < 0.0001). Error bars represent the standard deviation of the average value (96-well plate N=2; microvolumes N=3).
Fig 5: Singleplex (COMP coating) (A) and duplex (both COMP and hGH coating) (B) detection of different COMP concentrations (50 nM, 25 nM, 5 nM) in 96-well plate vs microvolumetric format. In 96-well format, signal was detected from the bottom of the plate (80 μL volume) and in microvolumes (signal detected on a microvolume glass slide (2 µL/sample). Blank – every protocol step followed, excluding coating of the bottom of the well with analytes (non-specific signal). P-values indicate statistical significance (*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001). Error bars represent the standard deviation of the average value (96-well plate N=2; microvolumes N=3).
Supplier Page from BioVendor Laboratory Medicine, Inc. for Cartilage Oligomeric Matrix Protein Human HEK293