ELISA microplate Research Article E


2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4


0.0 0 F


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24 68 Time (min)


2 4 6 Time (min) 6.50 H


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Figure 2. Multiple reaction monitoring chromatograms at LLOQ for (A) unmodified plate (5 ng/ml), (B) maleic anhydride-activated plate (2 ng/ml), (C) Nunc®


Immobilizer™ Amino Plate (5 ng/ml), (D) magnetic beads


(10 ng/ml), and at ULOQ for (E) unmodified plate plate (850 ng/ml), (F) maleic anhydride-activated plate (100 ng/ml), (G) Nunc®


The study used 30 μl of the beads solution, which is roughly 0.9 mg of the beads. Therefore, 5 μg of the capture protein was used in the study without further optimization.


Capture capability of protein-immobilized surfaces The capture capability of the immobilized protein on the surface greatly affects assay sensitivity and linear range. A series of reference standard samples prepared in human serum with ASP2409 concentrations from 5 to 1000 ng/ml were used to evaluate these features. The relationship between the response and the


analyte concentration for the protein-adsorbed GBO plate is shown in Figure 1A. In Figure 1A and the fol- lowings, the concentration was expressed in the initial serum. Using 200 μl of serum samples, the response increased linearly up to 400 ng/ml. The capture capacity seemed to be reached because the response plateaued at higher concentrations. This was further confirmed by using 100 μl of serum sample diluted


future science group Immobilizer™ Amino Plate (1000 ng/ml) and (H) magnetic beads (1000 ng/ml) (cont.).


with 100 μl of PBS. As shown in Figure 1A, under this condition the linear response was extended up to 850 ng/ml, which is proportional to the dilution factor. The capture capacity could be estimated as 85 ng of ASP2409. Similar observations for the protein-immobilized


MA plate are presented in Figure 1B. The response versus concentration was linear up to 50 ng/ml. The linear response increased proportionally with a twofold or fourfold dilution of the serum. The capture capacity was roughly 10 ng of ASP2409. The protein-bound MA plate had significantly lower


capture efficiency compared with the protein-adsorbed GBO plate, for example, 10 versus 85 ng/ml. This could be interpreted as follows. Although the saturated con- centrations for surface binding for the two plates were


Key term


Capture capability: Ability of the immobilized ligand on the surface of a plate well to capture the target from the sample solution.


8 6.51 G


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6.39 6.51


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Intensity (cps)


Intensity (cps)


Intensity (cps)


Intensity (cps)


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