Research Article Órpez-Zafra, Pavía, Pinto-Medel et al.
Table 2. Parallelism of sIFNAR2 assay. Dilution
Sample 1
1/2 1/4 1/8 1/10
Sample 2
1/2 1/4 1/8 1/10
Sample 3
1/2 1/4 1/8 1/10
Sample 4
1/2 1/4 1/8 1/10
Sample 5
1/2 1/4 1/8 1/10
OD values
0.845 0.536 0.360 0.302
0.995 0.665 0.451 0.384
0.916 0.578 0.376 0.344
0.781 0.504 0.341 0.308
0.825 0.570 0.391 0.335
Mean adjusted concentration (ng/ml)
110.10 119.32 128.13 116.14
155.60 160.55 184.97 178.97
122.17 132.47 138.36 148.30
99.36 109.07 116.98 120.62
106.85 129.89 137.46 139.11
Accuracy (obs/exp × 100)
108.37 116.38 105.49
103.18 118.88 115.01
108.43 113.26 121.39
109.77 117.73 121.39
121.57 128.65 130.20
Parallelism assessment of sIFNAR2 assay in human serum. The accuracy was calculated using the sIFNAR2 concentration determined at dilution 1:2 as a reference value.
Distribution of sIFNAR2 in serum of MS patients & healthy controls The validated sIFNAR2 ELISA was used to deter- mine sIFNAR2 concentration in serum samples from 137 untreated MS patients and 88 HC. There were not significant differences in gender distribution among patients and controls. sIFNAR2 levels in MS patients (median [IR]: 71.67 [39.67–128.18 ng/ml]) were sig- nificantly lower than those in HC (median [IR]: 134.3 [76.10–179.21 ng/ml]; p <0.00001). A slight overlap
0.000 0.200 0.400 0.600 0.800 1.000 1.200
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
was observed over the distribution ranges in the two subject groups (Figure 3). We tested our recombinant sIFNAR2 protein with
a commercial kit (VeryKine Human IFN Alpha/Beta Receptor 2 ELISA kit; cat. number 41,385) and the standard protein provided in the kit was included in the ELISA we developed. The distribution pattern of sIFNAR2 in serum of MS patients and HC as determined by the commercial kit was similar to that obtained with our ELISA (not shown).
100 120 140 160 180 200
40 60 80
20 0
1/2 1/4 1/8 Serum dilution 1/10 1/2 1/4 1/8 Serum dilution
Figure 2. Parallelism assessment of sIFNAR2 assay in human serum. The data for five samples were plotted as absorbance versus 1/dilution factor (A) and dilution-adjusted concentration versus 1/dilution factor (B).
2874 Bioanalysis (2015) 7(22) future science group
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
1/10
Absorbance
Adjusted concentration
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