Development of an Electrochemical CCL5 Chemokine Immunoplatform for Rapid Diagnosis of Multiple Sclerosis

Abstract

Serum level of CCL5 chemokine is considered an emerging biomarker for multiple sclerosis (MS). Due to the lack of specific assays for this disease, the development of a point-of-care test for rapid detection of MS could lead to avoiding diagnostics delays. In this paper, we report the first electrochemical immunoplatform for quantification of the CCL5 biomarker at the clinically required levels, able to discriminate between patients diagnosed with MS and healthy individuals. The immunosensing device involves protein capture from biological samples by complexation with biotinylated specific antibodies immobilized onto neutravidin-functionalized microparticles and sandwich assay with anti-CCL5 antibody and IgG labelled with horseradish peroxidase (HRP) for the enzyme-catalyzed amperometric detection of H2O2 using hydroquinone (HQ) as the redox mediator. The method shows excellent analytical performance for clinical application with a wide linear range of concentrations (0.1–300 ng·mL−1 CCL5, R2 = 0.998) and a low detection limit (40 pg·mL−1 CCL5). The biosensing platform was applied to the determination of the CCL5 endogenous content in 100-fold diluted sera both from healthy individuals and patients diagnosed with MS, with no further sample treatment in just two hours. The results were successfully compared with those obtained by the ELISA methodology.

Identification of candidate biomarkers and pathways associated with multiple sclerosis using bioinformatics and next generation sequencing data analysis

“…GO and REACTOME pathway enrichment analyses were used to explore the molecular mechanisms of the genes involved in the occurrence and development of MS. It is well known that the pathways include immune system [55], TCR signaling [56], cytokine signaling in immune system [57], degradation of the extracellular matrix [58], extracellular matrix organization [58], metabolism of lipids [59] and metabolism [60] plays an important role only in MS. CCL18 [61], FCRL3 [62], EOMES (eomesodermin) [63], CCR2 [64], IL2RB [65], CCL4 [66], FASLG (Fas ligand) [67], CD24 [68], IKZF3 [69], CD2 [70], CD28 [71], IL7R [72], HLA-DRB5 [73], ICOS (inducible T cell costimulator) [74], CCL5 [75], CTLA4 [76], IRF4 [77], C6 [78], NCR1 [79], CHIT1 [80], CD52 [81], CD163 [82], HGF (hepatocyte growth factor) [83], DIO3 [84], SIGLEC1 [85], TTR (transthyretin) [86], IL9 [87], VEGFA (vascular endothelial growth factor A) [88], CR2 [89], ANGPTL4 [90], CHI3L1 [91], MAG (myelin associated glycoprotein) [92], CNP (2’,3’-cyclic nucleotide 3’ phosphodiesterase) [93], CMTM5 [94], SEMA4D [95], NGFR (nerve growth factor receptor) [96], TF (transferrin) [97], MYRF (myelin regulatory factor) [98], MOG (myelin oligodendrocyte glycoprotein) [99], ADAMTS4 [100], BMP2 [101], HTRA1 [102], PNPLA3 [103], DYSF (dysferlin) [104], NINJ2 [105], LRP2 [106], ADAMTS14 [107] and DHCR7 [108] might play an important role in regulating the occurrence and development of MS. The expression of CCL18 [109], SLAMF7 [110], GPR174 [111], CCR4 [112], POU4F2 [113].…”

“…ERBB3 [479], HGF (hepatocyte growth factor) [469] and CCL5 [465] genes are a potential biomarkers for the detection and prognosis of spinal cord injury. A previous study reported that HGF (hepatocyte growth factor) [83], CCL5 [75] and NGFR (nerve growth factor receptor) [96] are altered expressed in MS. HGF (hepatocyte growth factor) [585] and NGFR (nerve growth factor receptor) [593] are a key initiators of Parkinson’s disease. This investigation suggests that LCK (LCK proto-oncogene, Src family tyrosine kinase), PYHIN1, DOK2, EGLN3, SDC1 and FSCN1 might be a novel therapeutic targets for MS, and the related molecular mechanism is worthy of further exploration.…”

Immunosensors for Autoimmune-Disease-Related Biomarkers: A Literature Review

“…An electrochemical immunosensor for the MS-associated protein biomarker chemokine (C-C motif) ligand 5 (CCL5) was developed. CCL5 was captured from biological samples through biotinylated anti-CCL5 antibodies (immobilized onto magnetic microparticles pre-functionalized with neutravidin) and subsequently detected with anti-CCL5 antibodies along with HRP-labeled secondary antibody (through the amperometric signal produced in the presence of H 2 O 2 and using hydroquinone as the redox mediator) in a sandwich-assay setting [78]. One more electrochemical immunosensor has been developed that can serve simultaneous determination of the CXCL7 chemokine and the MMP3 enzyme/metalloproteinase, both of which are present at elevated levels in serum of RA patients.…”