Figure 5

Immunoassay for C-reactive protein in whole blood. Capture antibodies against CRP and insulin (control) were covalently immobilized on an activated glass slide. Whole blood spiked with CRP to give a concentration of 0, 5, 10, 20, 40 or 80 mg/liter above the endogenous level was hydrodynamically guided over both capture antibodies for 2.0 s. The lane was then washed, and Cy3-labeled secondary antibody was applied to the lane. After all lanes were completed, the entire slide was scanned in a fluorescence scanner. (A): Segments of this scan (each 68 × 470 pixel), showing a fluorescence signal from the area with anti-CRP capture antibody, but not from the area with anti-insulin capture antibody (control). The width of the lanes increased with increasing concentration of CRP, possibly due to diffusion of the antigen. Scale bar, 100 μm. (B): The mean fluorescence intensities of the 470 columns of pixels of the upper segment in (A) plotted against pixel column number. (C): The fluorescence signal from the area with anti-CRP capture antibody increased with increasing CRP concentration. The relationship between CRP concentration and area under the mean fluorescence intensity curve in (B) was linear (● R² = 0.97), whereas the relationship between CRP concentration and peak in the mean fluorescence intensity curve in (B) was better described by a model of saturation (○ MFI_peak = K_a + μ × [C_CRP / (C_CRP + K_s)]).