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Research Article Órpez-Zafra, Pavía, Pinto-Medel et al. Key terms


Recovery: The quantified closeness of an observed result to its theoretical true value. It is used as a measure of accuracy.


<20%. Less than 2% of samples were below the lower limit of quantification and were not included as they were outside the calibration range.


Validation of the sIFNAR2 assay in human serum The assay was validated following the Q2 (R1) Valida- tion of Analytical Procedures of the ICH Harmonised Tripartite Guideline [19] and the recommendations of Lee et al. [20] and Valentin et al. [21].


Statistical analysis The data were analyzed with SPSS 15.0 (SPSS, Chi- cago, IL, USA). Quantitative variables were reported as median and interquartile range. As a non-normal dis- tribution was established in the Kolmogorov–Smirnov test, nonparametric Mann–Whitney U test was used to compare sIFNAR2 concentration between MS patients and controls. A p < 0.05 was considered statistically significant.


Results Calibration curve The curve was based on six serial dilutions of recom- binant sIFNAR2 in blocking buffer and had a con- centration range from 3.9 to 250 ng/ml. Back-cal- culated concentration of the standard in the defined range met the acceptance criteria of mean accuracy within the range of 80–120% and imprecision less than 20% [21]. The standard recovery showed val- ues from 97.46 to 107.55%. The assay imprecision (CV) was <5.1% for OD values and <8.1% for the back-calculated values (Table 1 & Figure 1B).


Parallelism of sIFNAR2 assay Five serum samples were freshly diluted in sample diluent (blocking buffer) at 1:2, 1:4, 1:8 and 1:10 and evaluated to assess the parallelism of the assay. Accu- racy of IFNAR determination in each sample was within the range of 103.18 to 130% compared with values determined at the 1:2 dilution, which complies with acceptance criteria for accuracy within the range of 70–130% (Table 2 & Figure 2) [21]. This experiment demonstrated that the signal was


produced by the analyte of interest, showed propor- tionality between the endogenous sIFNAR2 form and the reference standard, and that there was no apparent effect of dilution of human serum from 1:2 to 1:10 on sIFNAR2 determination. The mini-


2872 Bioanalysis (2015) 7(22)


mum required dilution (MRD) to achieve acceptable accuracy and precision was thus 1:2.


Spike & recovery Five samples of human serum were spiked with 8 ng/ml (low spike) and 125 ng/ml (high spike) of recombinant human sIFNAR2 and accuracy was assessed at 1:2, 1:4, 1:8 and 1:10 dilutions in assay buffer. The spiking solution was <5% of the final vol- ume [22]. Accuracy was calculated using the sIFNAR2 concentration determined at dilution 1:2 as a reference value. The accuracy of measurement of spiked serum samples ranged from 90.14 to 122.87%; for each dilu- tion factor, the five samples thus met the acceptance criteria (Table 3). These data showed that the bio- logical matrix does not interfere with the quantitative determination of sIFNAR2 in human serum.


Assay dynamic range & limits of quantification/detection The LLOQ is the lowest calibration point for which the concentration can be back-calculated on the regression curve with 80–120% accuracy and a CV below 20%. The ULOQ is the upper calibration point that meets these criteria. The dynamic range thus extends from the LLOQs to ULOQs. In our assay, the dynamic range defined with recombinant sIFNAR2 ranged from 3.9 to 250 ng/ml. In serum samples, the LLOQ, and therefore the sensitivity, was 7.8 ng/ml given the 1:2 predilution of the samples before analysis. The LOD is the lowest concentration of a sub-


stance that can be distinguished from the absence of that substance, and its signal is therefore consid- erably higher than the background. The LOD was calculated from mean signal at background + three standard deviations. The LOD of the sIFNAR2 assay, calculated from ten assays, was 2.44 ng/ml.


Repeatability Intra-assay accuracy & imprecision of the standards Three points of the standard curve (250, 62.5 and 7.8 ng/ml) were included five-times in duplicate in a single assay to evaluate repeatability. Intra-assay accu- racy and imprecision were calculated for each stan- dard level as the accuracy and mean imprecision of the five measurements. Nominal values for each standard were determined as the mean of the five determina- tions and were used as reference values for intra-assay accuracy and imprecision assessments. The accuracy was determined with the back-calculated concentra- tion as the observed value and the nominal value as the expected value. Intra-assay accuracy was within


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