Research Article Yang, Kernstock, Simmons & Alak
Table 2. Quantitative features of ELISA plates and magnetic beads for protein therapeutic ASP2409. Platform
Cost (US$/96-well plate)†
GBO plate NIA plate MA plate
0.5 2
30 Magnetic beads 250 Calibration curve
Serum sample volume (μl)
y = 0.00411x + 0.00425 100 y = 0.00412x + 0.000976 100 y = 0.00161x + 0.00302 100 y = 0.00398x + 0.00542 200
†The cost was calculated based on the current market price and the assay setting. GBO: Unmodified; MA: Maleic anhydride-activated; NIA: Nunc®
immobilization capacity for CD86 protein could be roughly estimated as 4.8 μg/well. Based on this esti- mate, immobilization capacity for the three plates was experimentally investigated with the protein varying from 0.5 to 20 μg/well using the optimal immobiliza- tion conditions established above and 200 μl of 100 μg/ml ASP2409 in human serum as a probe. Although the absolute responses to ASP2409 were varied among
A
100 80 60 40 20 0
6.51 Immobilizer™ Amino.
MA, NIA and GBO plates, they all showed a similar trend in immobilization capacity where the responses reached the plateau roughly between 3 to 10 μg of the capture protein, CD86. Therefore, 3 μg of CD86 was used in the study. According to the product information, 1 mg of
Tosylactivated beads typically binds 1–10 μg of tar- get protein, but varies from application to application.
C
100 120
6.25
02 4 3.21
B
100 80 60 40 20
1.59 0 0
24 68 Time (min)
4.35 0 68 Time (min) 6.49 D
100 150 200
7.22 5.24 3.1 4.578 0
02 46 8 Time (min)
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®
Immobilizer™ Amino Plate (1000 ng/ml) and (H) magnetic beads (1000 ng/ml). 312 Bioanalysis (2015) 7(3) future science group 8.50 8.86 50 0 2 4 6 Time (min) 6.39 8 8.00 8.52
80 60 40 20
6.51
Linear range (ng/ml)
5.00–850.00 5.00–1000.00 2.00–100.00
10.00–1000.00 r
0.9939 0.9945 0.9927 0.9934
7.16 8.87 0.861.70 3.23 4.77 5.24
4.22 5.16 6.27 7.08 8.81
Intensity (cps)
Intensity (cps)
Intensity (cps)
Intensity (cps)
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