Research Article
Development and validation of an ELISA for quantification of soluble IFN-β receptor: assessment in multiple sclerosis
Aim: The soluble isoform of the IFN-β receptor (sIFNAR2) can bind IFN-β and modulate its activity, although its role in autoimmune diseases remains unknown. Methods: A recombinant human sIFNAR2 protein was cloned, expressed and purified after which we developed and validated an ELISA for its quantification in human serum. Serum sIFNAR2 were assessed in multiple sclerosis (MS) patients and healthy controls. Results: The ELISA has a dynamic range of 3.9–250 ng/ml and a detection limit of 2.44 ng/ml. Serum sIFNAR2 were significantly lower in untreated-MS patients than in healthy controls. Conclusion: The ELISA is suitable for quantification of sIFNAR2 in serum and should facilitate the study of sIFNAR2 in neuroimmunological diseases such as MS.
Multiple sclerosis (MS) is a T-cell- mediated autoimmune disorder characterized by inflammation of the central nervous sys- tem, demyelination and axonal damage [1–3]. The cytokine IFN-β is a key molecule in this disease, as it maintains the anti-inflamma- tory status of the immune system [4] and is one of the most widely used treatments for MS patients. The biological activity of IFN-β is medi-
ated through interaction with the IFN α/β cell surface receptor (IFNAR), composed of two subunits, IFNAR1 and IFNAR2 [5]. IFNAR2 bears the IFN-β-binding domain and IFNAR1 is needed to form and stabilize the high-affinity IFN-β-receptor complex [6]. The IFNβ/IFNAR interaction activates a complex intracellular pathway that involves the JAK–STAT family proteins and con- cludes with a variety of biological responses, including antiviral, antiproliferative and immunomodulatory effects [7,8]. Soluble cytokine receptors participate in
the control of cytokine activity by modu- lating their binding to anchored membrane receptors and generating the biological response. These receptors can act as bio- logical agonists, protecting the ligand from proteolysis, improving its stability, modu-
10.4155/bio.15.208 © Begoña Oliver-Martos et al.
lating ligand pharmacokinetics or decreas- ing clearance, or by acting as antagonists in competition with the cell surface recep- tor for ligand binding [9,10]. There are two major mechanisms for the generation of soluble receptors, alternative splicing of the RNA that encodes the cytokine receptor and cleavage of the membrane receptor [11]. The IFNAR2 subunit has three isoforms result- ing from alternative splicing; the short form (IFNAR2.1/IFNAR2b)
is a nonfunctional
transmembrane protein, whereas the long form (IFNAR2.2/IFNAR2c) is a transmem- brane protein that composes the functional receptor together with IFNAR1 [12]. The other transcript encodes the soluble recep- tor form, sIFNAR2 (IFNAR2.3/IFNAR2a). This protein, which lacks the transmembrane and cytoplasmic domains [13], is detected in body fluids [14] and bind type I IFN [15]. A modulatory function has been postulated for sIFNAR2 as an agonist or antagonist of IFN-β bioactivity, depending on sIFNAR2 concentration [16,17]. The contribution of sIF- NAR2 in MS and other autoimmune diseases nonetheless remains unknown, although it might modulate not only endogenous IFN-β but also exogenously administered therapeutic IFN-β [17].
Bioanalysis (2015) 7(22), 2869–2880 ISSN 1757-6180
Teresa Órpez-Zafra1 Pavía2
, Jose
Isaac Hurtado-Guerrero1 Jose L Rodriguez-Bada1 Martín Montañez2 Fernández1
Unidad de Gestión Clínica de
, Maria J Pinto-Medel1 ,
, Óscar , Laura Leyva‡,1
& Begoña Oliver-Martos*,‡,1 1
Neurociencias. Instituto de Biomedicina de Málaga (IBIMA), Hospital Regional
Universitario de Málaga, Spain 2
Departamento de Farmacología y
Pediatría, Facultad de Medicina, Instituto de Biomedicina de Málaga (IBIMA). Universidad de Málaga, Spain *Author for correspondence:
begoliver@gmail.com ‡
Authors contributed equally , , Elisa
part of
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