Research Article Órpez-Zafra, Pavía, Pinto-Medel et al. Key terms
Multiple sclerosis: A T-cell-mediated autoimmune disorder characterized by inflammation, demyelination and axonal damage of the central nervous system.
IFN-β: Cytokine with antiviral, antiproliferative and immunomodulatory effects that acts through interaction with its cell surface receptor (IFNAR), composed of two subunits, IFNAR1 and IFNAR2.
sIFNAR2: Soluble isoform of the IFNAR2 subunit, generated by alternative splicing of the RNA encoding IFNAR2 or cleavage of membrane receptor, with ability to modulate the biological activity of IFN-β.
Validation: Is the confirmation, via extensive laboratory work, that the performance characteristics of an assay are suitable and reliable for intended analytical use.
Accuracy: The accuracy describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte.
Imprecision: The random dispersion of a set of replicate measurements and/or values expressed quantitatively by statistic such as standard deviation or coefficient of variation.
In this study, we cloned, expressed and purified a
recombinant human sIFNAR2 protein, after which we developed and validated an enzyme-linked immuno- sorbent assay (ELISA) for its quantification in human serum. We applied the validated assay to quantify serum sIFNAR2 in MS patients and healthy controls.
Materials & methods Cloning & expression of recombinant soluble IFNAR2 sIFNAR2 was cloned in the prokaryotic system pEcoli- Cterm 6xHN Linear (Clontech), which bears an ampicillin resistance gene. The insert was synthesized by PCR using specific primers and separated by aga- rose gel electrophoresis based on size (924 base pairs). The specific band was purified with the QIAquick Gel Extraction kit (Qiagen) and ligated to the plasmid using the In-Fusion Dry-Down pellet kit (Clontech) following manufacturer’s instructions. Replicative bac- teria (MAX Efficiency DH5α Competent Cells [Invi- trogen]) were transformed with the plasmid, seeded in lysogeny broth (LB)-agar plates supplemented with 100 μg/ml ampicillin and incubated (37°C, over- night). Colony-forming units were isolated, seeded in LB supplemented with ampicillin and incubated over- night with agitation. The plasmid was purified with the PureYield Plasmid Miniprep System (Promega), the nucleotide sequence and correct reading frames were verified, and BL21(DE3) bacteria (Invitrogen) were transformed to express the recombinant protein sIFNAR2. The culture was grown in LB supplemented with ampicillin to an optical density (OD) of 0.8
2870 Bioanalysis (2015) 7(22)
(λ = 600 nm) and protein expression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside followed by incubation (37°C, 4 h, with agitation). Bacteria were harvested, resuspended in lysis buf- fer containing a protease inhibitor cocktail (Roche), incubated for (30 min, room temperature; RT) with constant shaking and sonicated. The suspension was centrifuged and the supernatant filtered. Recombinant sIFNAR2 was purified on high- -iminodiacetic acid resin columns
capacity Ni+2
and detected in western blot with anti-IFNAR2 human MaxPab (Abnova) (Figure 1A). Recombi- nant sIFNAR2 was also identified by MALDI-TOF (Supplementary Figure 1).
Standardization of a sandwich ELISA to detect sIFNAR2 Reagents & buffers The reagents and buffers are summarized in Supplementary Table 1.
Serum samples Human serum samples were obtained from 137 untreated patients with relapsing-remitting MS defined according to the revised McDonald criteria [18], recruited from the Málaga Regional University Hospi- tal (Malaga, Spain). The samples were always obtained when the patients were in clinical remission. As con- trols (HC), 88 unrelated age-matched healthy indi- viduals were selected. Samples and HC were processed following standard procedures and frozen immedi- ately after reception by the Málaga Hospital-IBIMA Biobank, as part of Andalusian Public Health System Biobank. All patients who participated in the study gave informed consent and protocols were approved by institutional ethical committees (Comité de Ética de la Investigación Málaga Nordeste). Recombinant sIFNAR2 was quantified by den-
sitometry analysis using a bovine serum albu- min standard and analyzed with ImageJ software (Supplementary Figure 2). The sIFNAR2 standard curve was generated, and prepared freshly for each assay. The curve covered a range from 3.9 to 250 ng/ml, using six serial dilutions of recombinant sIFNAR2. The reference standard throughout the validation process was a single lot of protein with >95% purity.
Definition of quality control/nominal value As quality controls (QC1
samples derived from the study population, as recom- mended by Valentin et al. [21]. These samples were ana- lyzed in three duplicate assays and the mean of the three experiments was used as nominal value for intra- and inter-assay accuracy and imprecision assessments.
–QC5 future science group ) we used five neat serum
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154