PROCEDIMIENTO

Electrochemiluminescence (ECL) based assays are known to be highly sensitive, exhibit a broad dynamic range and require small sample volume; the technology has demonstrated the ability to quantify levels of nucleic acids, recombinant proteins and bacterial and viral components in the sub-picogram range with increased precision compared to conventional enzyme-linked immunosorbent assay (ELISA) (143-148).

Highly sensitive methods for detecting soluble biomarkers for neuro-axonal damage are needed in neurodegenerative diseases. I have previously developed an ECL solid- phase sandwich immunoassay on the Meso Scale Discovery (MSD, Gaithersburg, MD, USA) to measure the soluble fraction of neurofilament heavy chain (NfHSMI35) in

cerebrospinal fluid (CSF) employing the same commercially available antibodies used in a conventional ELISA (cELISA) (149-151). Adhering to a previously proposed nomenclature, the soluble fraction of NfH measured is indicated with the capture antibody in the superscript (149).

The NfHSMI35 assay protocol was optimised and validated for reproducibility, precision, accuracy and parallelism. The analytical sensitivity (background plus three standard deviation (SD)) of this assay was 2.4 pg/ml. The mean intra-assay coefficient of variation (CV) was 4.8% and the inter-assay CV 8.4% (151). Patients with ALS (160.1 pg/ml, n=50), mild cognitive impairment (MCI)/AD (65.6 pg/ml, n=20), GBS (91.0 pg/ml, n=20) or subarachnoid haemorrhage (SAH) (345.0 pg/ml, n=20) had higher CSF NfHSMI35 values than the reference cohort (27.1 pg/ml, n=73, p<0.0001 for each comparison). The reference cohort included patients who, based on extensive diagnostic evaluation, had no objective clinical or paraclinical signs of a neurological disease: tension type headache (n=17), lower back pain (n=5), psychiatric disorders (n=30) or miscellaneous diseases for which no neurological explanation could be found (n=21) (151).

In a next step, employing this ECL-based immunoassay, we measured levels of the NfHSMI35 protein in the CSF of healthy controls (HC) and in patients with a clinically isolated syndrome (CIS) or Multiple Sclerosis (MS) (151, 152). In particular, we

examined whether NfHSMI35 levels differ between MS patients and controls and between specific stages (relapsing-remitting versus progressive forms) or states (relapsing

44 versus stable) of disease. The main findings of this study were that CSF levels of NfHSMI35 increase in the course of disease evolution from CIS to definite MS and correlate with the Expanded Disability Status Scale score (EDSS) as clinical measure of disability in CIS and relapsing remitting MS (RRMS), but not in progressive stages (secondary progressive MS (SPMS) and primary progressive MS (PPMS)). In contrast, none of the CSF measures related to the immune response (CSF cell count, intrathecal IgG, IgM or IgA production, CSF total protein or albumin quotient as measures of the blood CSF barrier integrity) correlated with EDSS at any time point of MS evolution. In summary our results supported the utility of NfHSMI35 as a specific biomarker for on- going neuroaxonal damage that can be quantified with high sensitivity and a broad dynamic measuring range, a pre-requisite for use in clinical practice (153).

These findings were important, because we anticipated that further development of the assay in serum/plasma samples could have provided tools to measure longitudinally NfHSMI35 levels during disease progression as well as in clinical trials of potential neuroprotective drugs in diseases like MS and ALS.

To date there are only very few studies on NfH in the blood compartment (154-156). These results await validation and I and others have previously experienced analytical difficulties with reliable and reproducible quantification of NfH levels in blood samples (characterised by the so called “hook effect” or other matrix interferences and thus lack of parallelism between plasma samples and standards in serial dilutions). This effect is most likely based on either the formation of aggregates or endogenous binding of Nf by antibodies and poses an important pre-analytical problem for a quantitative

immunoassay of Nf levels (157, 158). A method for solubilising Nf aggregates by urea preincubation of samples has been recently proposed (159).

Unfortunately, during the following months in 2011 we were not able to adapt the NfHSMI35 ECL immunoassay to measurements in serum or plasma samples due to insufficient recovery and lack of dilutional linearity. Despite several attempts to reach acceptable analytical performance, I finally decided not to follow this development for NfHSMI35 further.

Nevertheless, an assay including the benefits of the ECL technology seemed a promising approach forNf measurements in the blood compartment. Such an assay could potentially provide a sufficiently sensitive tool for blood measurements in several chronic neurodegenerative diseases.

45 1.9. Specific aims

Chapter 2 aims to compare CSF levels of NfL (UmanDiagnostics NF-light® assay) with those of (previously determined) NfHSMI35 in a well characterised group of 148 CIS/MS patients and 72 controls (152). Second, I evaluated the analytical and clinical

performance of the UmanDiagnostics NF-light® assay and stability of its analyte (160).

In chapter 3A, I aimed to determine CSF NfHSMI35 levels using the NfHSMI35 assay I have developed in a subset of MS patients who had previously shown reduced NfL (UmanDiagnostics NF-light®) levels after natalizumab treatment (101).

In chapter 3B, I aimed to assess the ability of CSF NfL (by UmanDiagnostics NF-light®) as a therapeutic biomarker in RRMS, by comparing levels in fingolimod-treated patients versus placebo, and correlating NfL levels with clinical and MRI outcomes.

In chapter 4 I aimed to develop and validate a sensitive ECL-based NfL assay suitable for the quantification of NfL in serum at concentrations relevant to clinical settings.

In chapter 5, I aimed to analyse NfL levels in longitudinally collected serum samples from subjects enrolled in a phase II clinical trial investigating the utility of minocycline to attenuate neurological deficits after spinal cord injury (SCI) (161). I report the

correlation of serum NfL with acute and long-term clinical outcome in these patients. Further, I investigated the potential of serum NfL as drug response marker of the therapeutic effect of minocycline in SCI.

Finally, in chapter 6, my objective was to assess the ability of serum NfL to predict the risk of conversion from adult CIS to CDMS (defined by occurrence of a second clinical attack) using the largest cohort of adult CIS cases ever studied to date (n=1,047). I decided to use a two steps strategy by initially measuring serum NfL in the 100 patients with the shortest time to conversion to CDMS (fast converters (FC)), the 100 patients with the longest follow-up time in the absence of conversion to CDMS (non converters (NC)) and 100 healthy controls. I decided that only if I saw a difference in serum NfL levels between FC and NC serum NfL, I would have measured serum NfL in the rest of the cohort.

46 2. A comparative study of CSF NfL and NfH protein in MS (104)