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Development & validation of an ELISA for quantification of soluble IFN-β receptor Research Article


Std conc. (ng/ml)


250 125 62.5 31.25 15.62 7.81 3.90


Table 1. Example of data from calibration curve obtained during assay validation. Optical density


Values Mean 2.604 2.540 1.479 1.589 0.823 0.831 0.470 0.478 0.298 0.298 0.209 0.198 0.156 0.159


2.572 1.534 0.827 0.474 0.298 0.203 0.157 SD 0.045 0.078 0.006 0.006 0 0.008 0.002


%CV 1.8


5.1 0.7 1.2 0.1 3.9 1.3


Values 254.26 245.16 120.63 131.74 60.58 61.24 30.95 31.64 16.61 16.56 8.88 7.92 4.02 4.29


Back-calculated concentration (ng/ml) Accuracy (obs/exp) ×100 Mean


SD %CV 249.710 6.435 2.6 126.185 7.856 6.2 60.910 31.295 16.585 8.400 4.195 0.467 0.8 0.488 1.6 0.035 0.2 0.679 8.1 0.195 4.6 99.88 100.95 97.46 100.16 106.20 107.55 106.49


Standard curve was based on six serial dilutions of recombinant sIFNAR2 with a concentration range of 3.9 to 250 ng/ml. %CV: Coefficient of variation expressed as percentage; Obs/exp: Observed/expected; Std conc: Standard concentration; SD: Standard deviation.


the range of 91.04 to 111% and imprecision (CV) within the range of 4.24 to 8.32% (Table 4).


Intra-assay accuracy & imprecision of the serum samples (QC) Five serum samples were analyzed in duplicate four- times in a single run to evaluate ELISA repeatability. Nominal values of the samples were determined previ- ously and were used as reference values for intra-assay accuracy and imprecision assessments. The intra-assay accuracy of the sIFNAR2 determination was within the range of 80.42 to 107.03%; intra-assay imprecision with the back-calculated concentration ranged from 2.50 to 5.16% (Table 4).


Reproducibility Inter-assay accuracy & imprecision of the standards Interassay imprecision was determined with three concentrations of the standard on five different days, performed by at least two different analysts. Interas- say accuracy was calculated for each point with the back-calculated concentration and ranged from 86.54 to 112.69%. Interassay imprecision was 0.24% for the highest concentration, 4% for medium concentration and 10.1% for the lowest concentration (Table 5).


Interassay accuracy & imprecision of the serum samples (QC) Five serum samples were assayed in five different exper- iments to evaluate the interassay variability. As before,


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nominal values of the samples were determined previ- ously and used as reference values for interassay accu- racy and imprecision assessments. Interassay accuracy of the sIFNAR2 determination ranged from 80.43 to 115.05%, and imprecision with the back-calculated concentration from 3.9 to 16.6% (Table 5).


Interference To test analytic interference from complex forma- tion between sIFNAR2 and IFN-β, five different sera were preincubated with each of the five IFN-β con- centrations (Betaferon 400, 800, 1200, 1600, 2000 IU) and without IFN-β. No analytical interference was observed when recombinant IFN-β was added up to 2000 IU, since the accuracy ranged from 96.2 to 100.3%, using the sIFNAR2 concentration without IFN-β as the reference value.


sIFNAR2 stability in serum sIFNAR2 stability in serum samples was analyzed with the same samples tested for interassay impreci- sion, after four cycles of freezing (-20°C for at least 8 weeks) and thawing (at RT for at least 3 h). Recovery was assessed against the concentration measured at day 1 and ranged from 80.2 to 97.3% for the four samples. Only the sample with the lowest sIFNAR2 concentra- tion showed a percentage of recovery less than 80%. The results showed that endogenous sIFNAR2 con- centrations in human serum were stable for at least 8 weeks at -20°C and after four freeze–thaw cycles.


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