Development & validation of an ELISA for quantification of soluble IFN-β receptor Research Article
Table 3. Selectivity assessment of sIFNAR2 assay in human serum. Sample Dilution
Low spike
Observed (ng/ml)
S1
1/2 1/4 1/8 1/10
S2
1/2 1/4 1/8 1/10
S3
1/2 1/4 1/8 1/10
S4
1/2 1/4 1/8 1/10
S5
1/2 1/4 1/8 1/10
92.46 49.51 25.18 20.21
122.54 67.00 32.03 25.01
60.00 31.01 15.22 12.36
144.77 65.30 38.83 30.82
146.74 71.90 45.08 35.22
Expected (ng/ml)
46.23 23.12 18.49
61.27 30.63 24.51
30.00 15.00 12.00
72.38 36.19 28.95
73.37 36.68 29.35
Accuracy (obs/exp × 100)
107.09 108.91 109.29
109.35 104.54 102.05
103.36 101.49 103
90.22 107.30 106.43
98.00 122.87 120.00
Observed (ng/ml)
123.11 64.09 30.99 23.54
132.03 71.56 40.26 25.32
86.41 42.62 20.23 18.12
227.93 105.18 55.79 54.15
266.48 120.10 68.57 56.90
High spike
Expected (ng/ml)
61.55 30.78 24.62
66.02 33.01 26.41
43.21 21.60 17.28
113.96 56.98 45.59
133.24 66.62 53.30
Accuracy (obs/exp × 100)
104.11 100.68 95.61
92.25 121.98 95.89
98.65 93.65 104.85
92.29 97.90 118.78
90.14 102.92 106.75
Selectivity assessment of sIFNAR2 assay in human serum. 8 ng/ml (low spike) and 125.0 ng/ml (high spike) of recombinant sIFNAR2 was spiked in five serum samples. Accuracy was calculated using the sIFNAR2 concentration determined at dilution 1:2 as a reference value.
Discussion sIFNAR2 is found in body fluids such as peripheral blood and urine [14]; although its function is not fully defined, it can bind type I IFN [15] and appears to modulate the bioactivity of endogenous and systemi- cally administered IFN-β [16,17]. Despite the evidence that sIFNAR2 is a potential regulator of type I IFN responses [17], the role of sIFNAR2 in MS and in other immunological diseases is unknown. Although biomarkers have become an important
tool for optimizing the benefit/risk ratio of therapeu- tics, validated methods must be used in biomarker studies to improve the quality of the data and their application in clinical practice. Although previous studies have evaluated serum sIFNAR2 levels in diseases such as hepatitis C [23], AIDS [24] and neo- plasms [25] proposing sIFNAR2 as a biomarker, none of these reports describe the detection method explic- itly and no validated assays for sIFNAR2 measure- ment in serum have been reported. Here we describe the development, validation and implementation of a sensitive ELISA for quantification of sIFNAR2 in human serum. Since no commercial sIFNAR2 peptide was avail- able, we synthesized a recombinant
sIFNAR2 in- future science group
house. The first step in the development was to clone, express and purify the recombinant sIFNAR2 using a prokaryotic system. Recombinant sIFNAR2 was iden- tified by western blot with the same antibodies used in the ELISA and then by MS (MALDI-TOF) and pep- tide mass fingerprinting, which confirmed the identity of the recombinant protein. Throughout the validation process, we used a single
batch of protein with a purity >95% to generate the standards. The other reagents used in the assay were commercially available. Incubation times, reagent concentrations and buffers
used in the ELISA were optimized prior to validation to confirm the suitability and reliability of assay per- formance for measurement of sIFNAR2. The method was fully validated in human serum, although addi- tional validation would be necessary for measurement of sIFNAR2 in matrices such as plasma or CSF. The assay was shown to be accurate and precise over
a dynamic range from 3.9 to 250 ng/ml, and sensitivity in serum samples was 7.8 ng/ml. Experiments of linearity with serum samples were
performed to demonstrate proportionality between the endogenous form of the biomarker and the reference standard, showing that the relationship between sIF-
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