Research Article Yang, Kernstock, Simmons & Alak
similar, approximately 3 μg, it is not known how much capture protein was actually immobilized on the sur- faces. It could be less for the MA plate, because only active maleic anhydride groups were available for the binding. On the other hand, assuming a similar amount of capture protein was immobilized on the two surfaces, the chemical binding on the MA plate surface might make the conformation of the capture protein more rigid or provide fewer binding sites and therefore limit the accessibility for the target. Based on the product information, MA plates preferentially immobilize pep- tides rather than proteins. Further exploration into this low capture efficiency was not attempted in this study. The results for protein-immobilized NIA plates are
presented in Figure 1C. These plates had linear binding for 200 μl of serum samples up to 500 ng/ml, or 1000 ng/ml when diluted twofold with PBS. These results implied this plate has a capture capacity of roughly 100 ng of ASP2409. Compared with the protein-immobilized MA plate,
the protein-immobilized NIA plate had significantly higher capture capability. This may result from its unique coating feature. In contrast to the MA coating where the active maleic anhydride groups are directly attached to the surface and could limit the access of large molecules, the coupling groups on NIA plates are introduced through an ethylene glycol spacer by
QC level Platform LLOQ
GBO plate MA plate NIA plate
LQC
Nominal conc. (ng/ml)
5.00 2.00 5.00
Magnetic beads 10.00 GBO plate MA plate NIA plate
15.00 6.00
15.00 MQC
Magnetic beads 30.00 GBO plate MA plate NIA plate
HQC
150.00 40.00
150.00
Magnetic beads 160.00 GBO plate MA plate NIA plate
800.00 80.00
800.00 Magnetic beads 800.00 Accuracy (%RE)
Intra-day (6 replicates, 3 days)
6.4 – 12.3 −7.7 – 8.8 6.4 – 10.8 −13.9 – 9.6 4.4 – 10.8 0.7 – 9.3 5.1 – 10.1
−8.0 – (−2.1) −19.1 – 6.9 6.3 – 16.3 −0.8 – 8.2
−17.2 – (−2.4) −15.0 – (−3.1) −19.5 – (−12.8) −19.1 – 0.7
−13.1 – (−3.5) HQC: High quality control; LQC: Low quality control; MQC: Mid quality control.
Inter-day (n = 18)
8.3 1.6 8.7
−2.5 7.3 5.0 7.2
−4.7 −4.0 10.2 4.6
−10.6 −7.3
−16.1 −7.1 −8.2
5.9 – 10.9 3.8 – 14.5 7.5 – 14.4 16.7 – 24.1 5.9 – 10.7 5.8 – 10.1 5.8 – 10.4 14.8 – 18.9 6.8 – 11.2 5.9 – 18.6 6.8 – 18.6 7.2 – 12.2 5.7 – 11.6 8.2 – 17.2 5.7 –17.2 9.3 – 18.2
a photochemical method. The density of the coupling groups on the surface and especially the spacer design were optimized for protein coupling [9]. Sample dilution is typically an optimization factor
in LBA to achieve the maximal signal-to-noise ratio and quantitation range. Figure 1 shows that in most cases a twofold dilution gave relatively higher responses than nondiluted samples at the same final concentra- tion level. To understand this, one needs to convert the initial concentration expressed in Figure 1 to the final concentration. This observation suggests that the proper sample dilution could not only extend response linearity, but also increase capture efficiency due to the reduction of the matrix complexity resulted from sample dilution. It was also noticed in Figure 1C that when dilution
was not employed, 50 and 200 μl of serum sample saturated the binding surface at the same concentra- tion level (the two curves level off at 480 ng/ml), and the signals (bound analyte accordingly) from 50 μl of serum sample were lower than those from 200 μl of the sample. Considering the absolute amount of the ana- lyte in 50 μl of serum samples is only a fourth of that in 200 μl of the sample, if the binding surface was satu- rated by applying 200 μl of the serum samples, 50 μl of the sample should not have saturated the surface. The reason for this ‘conflict’ could be attributed to the
Table 3. Intra- and inter-day accuracy and precision of ELISA plates and magnetic beads for quantitation of protein therapeutic ASP2409.
Precision (%CV)
Intra-day (6 replicates, 3 days)
Inter-day (n = 18)
8.1 9.2
10.4 21.7 9.0 8.4 8.2
16.5 8.8
10.1 10.5 11.5 7.7
13.1 10.6 13.9
314
Bioanalysis (2015) 7(3)
future science group
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