ELISA microplate Research Article


actual binding surface area being used. The volume capacity/well for the plates was 200 μl. When a plate was incubated with 150 μl of capture protein solution, it could be reasonably assumed that all the well inner surface area was coated. When only 50 μl of serum samples was applied into a coated well, approximately only a fourth of the coated area was effectively used for the binding, which makes the binding be reduced to a fourth. For protein-bound magnetic beads, linear capture


was observed with analyte concentrations from 10 to 1000 ng/ml in 200 μl of serum samples (data were not shown). Sample dilution was also attempted briefly. However, the dilution prevented the assay from achiev- ing the required LLOQ of 10 ng/ml and therefore was not pursued further.


Sensitivity, linearity, selectivity, precision & accuracy The performance of the microplates and magnetic beads was characterized by the sensitivity,


A


50 40 30 20 10 0


C


100 120 140 160


20 40 60 80


0 0


24 68 Time (min)


7.24


selectivity, precision and accuracy in the quantita- tion of a protein therapeutic, ASP2409, under the optimized conditions as discussed above. Sensitivity was expressed as the assay’s LLOQ


(reproducible response with signal/noise ratio greater than five). Linearity was examined by using ASP2409/ IS peak area ratios of eight calibration standards and applying a weighted (1/concentration2


) least-squares linearity,


linear regression algorithm through Sciex Analyst 1.5.2 software. The results are summarized in Table 2, and chromatograms for the LLOQ and ULOQ stan- dards are presented in Figure 2 for microplates and magnetic beads. The experiments for magnetic beads were performed much earlier than for microplates. This resulted in the retention time shift because of the column history. Compared with magnetic beads, GBO microplates provided a comparable linear range and lower LLOQ. NIA microplates provided a wider linear range and lower LLOQ as well. The data from MA microplates are quite interesting. Although this plate had relatively less capture capability compared


B 4.36 1.49 2.48 4.17


02 46 8 Time (min)


4.21 D 5.15 5.36 0.81 3.033.22 7.55 8.74


10 20 30 40 50 60 70


0 0


24 68 Time (min)


Figure 3. Typical multiple reaction monitoring chromatograms. (A&C): analyte channel; (B&D): IS channel of blank human serum extracted using an unmodified plate (A&B) or magnetic beads (C&D).


future science group www.future-science.com 315 5.27 6.62 7.39 8.53 8.86


50 40 30 20 10 0


7.95


6.38 5.67


2.70 2.84 4.03 5.36


02


46 8 Time (min)


7.40 6.35


8.018.97 8.92


8.11


7.13 7.43


1.85 0.13 3.38 5.195.45


8.65 9.86


Intensity (cps)


Intensity (cps)


Intensity (cps)


Intensity (cps)


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