Figure 4.

Detection of vesicles and vesicle-associated antigens via immuno-SPR. (a) Fluorescence microscopy analysis of the binding of fluorescent s-MVs (at a concentration of 0.35 µg/µl) to anti-E. coli antibody in the test channel and to the BSA-treated surface in the reference channel following 20 min of s-MV binding and 20 min PBS rinse. The measurements indicated represent the size of the PDMS master. (b) Overlaid SPR sensorgrams showing concentration-dependent binding of OMVs to immobilized anti-E. coli antibodies. For each binding experiment, 200 µl of OMV-containing samples (diluted to the concentrations indicated) was introduced to the test or reference channel for 20 min, followed by a 20 min PBS rinse. The SPR signal was recorded as wavelength shift (nm) versus time and plotted as a “sensorgram”. All binding experiments were performed at 25°C ± 1°C with a flowrate of 10 µl/min. Each vesicle sample was assayed in triplicate and the standard error was determined to be less than 5%. (c) Vesicle standard curve generated using the SPR immunosensor. The steady-state SPR signal change was calculated by subtracting the average SPR signal during the PBS wash step following OMV binding from the average SPR signal collected during the initial PBS wash step prior to OMV addition. The equation y = 0.92ln(x) + 3.63 with an R² value of 0.95 describes the fit of a straight line through the logarithm of the data and was determined using SigmaPlot. The results are the average of the values calculated for the SPR signal change from three independent binding measurements with error bars showing ± standard error. It is noteworthy that the lower detection limit for this system was determined to be 0.01 µg/µl (10% of the compensated SPR wavelength shift in the standard curve) and that for vesicle concentrations ≥0.18 µg/µl, SPR wavelength shifts >2.5 nm were recorded, which is ~10× greater than the baseline signal. (d) Representative sensorgrams for antibody binding to s-MV surface-displayed GFP in test and reference channels. Channels were prepared identically so that fluorescent s-MVs were captured in both channels. The change in SPR signal over time was measured following addition of 1 µg/µl anti-GFP (black line) or 1 µg/µl anti-his6× (gray line) monoclonal antibody to surface-captured s-MVs. Antibody binding proceeded for 20 min followed by a 10 min PBS rinse. Each antibody was assayed in triplicate and the standard error was determined to be less than 5%.