ENHANCED BLOOD TEST

Abstract

A method to quantify the abundance of Neurofilament light chain (NFL) in a blood sample, comprising the steps of adding a composition comprising a polyanionic molecule and of reacting it with at least one antibody coupled to a detection system, specifically binding to one epitope of the NFL, its use for a diagnostic application and the corresponding diagnostic kit.

Description

1 / 15 ENHANCED BLOOD TEST Technical Field

[001] The present invention relates to a blood test application for Neurofilament Light Chain (NFL) and to the corresponding detection and / or quantification kit. Previous Technique

[002] A variety of markers reflecting neurodegenerative diseases, or inflammatory diseases affecting the central nervous system, have been established over the years. They are critical for diagnosis, disease monitoring, and will be essential for measuring the target involvement of disease-modifying therapies. Diagnosis of neurodegenerative diseases primarily requires functional brain imaging techniques or invasive examinations such as lumbar puncture to assess cerebrospinal fluid (CSF).

[003] In recent years, readily available and low-cost blood biomarkers have been developed that detect these pathologies, which could revolutionize diagnosis. These markers are generally identified and even accurately quantified using immunoassay and mass spectrometry approaches.

[004] In other words, such markers, which often reflect damage to the central nervous system, are abundant in CSF. However, lumbar puncture is considered an invasive diagnostic method, and blood diagnostics (whole blood, plasma, serum, other purified fractions) are welcome. This presents an additional challenge since these biomarkers are less abundant in blood. Furthermore, degradation by proteolytic enzymes, nonspecific binding to other proteins, and the existence of a peripheral, and not exclusively central nervous system, source of candidate biomarkers further complicates accurate measurement. There is, therefore, a need for robust tools and methods for quantification. Petition 870250073510, dated 08 / 20 / 2025, page 9 / 34 2 / 15 accurate and sensitive.

[005] Due to the complexity of neurodegenerative processes in the central nervous system and the large number of overlapping clinical diagnoses, the identification of individual neurodegenerative diseases is not always straightforward, so new markers are welcome to increase accuracy, as well as to enable early diagnoses.

[006] Among the new markers for biological disorders, the Neurofilament Light Chain (NFL) has been proposed (Gaetani et al., 2019, J. Neurol. Neurosurg Psychiatry, pp 870-881), for both CSF-based and blood-based diagnosis. Neurofilaments are the main structural components of myelinated axons. High concentrations of them in the CSF generally reflect rapidly progressing neurodegenerative processes. Unlike FTD and atypical parkinsonian syndromes, their concentrations in the CSF are normal in AD. Therefore, they can be used in differential diagnoses to distinguish AD from other diseases accompanied by dementia (Blennow, K.; Zetterberg, H.; Fagan, AM Fluid Biomarkers in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 2012, 2, a00622; Sjogren, M.; Rosengren, L.; Minthon, L.; Davidsson, P.; Blennow, K.; Wallin, A. Cytoskeleton proteins in CSF distinguish frontotemporal dementia from AD. Neurology 2000, 54, 1960-1964).NfL is released into CSF ​​and blood after damage to both central and peripheral neurons. However, accurate measurement of NfL in blood remains a significant challenge, even with the use of the most advanced and sensitive immunoassays, such as fluorescent or luminescent technologies. In fact, besides the need for a very strong signal, an analytical variation of 6% is mentioned in the document cited above, while interassay variations of up to 11% have been observed in serum samples. In these tests, the comparison with CSF... Petition 870250073510, dated 08 / 20 / 2025, page 10 / 34 3 / 15 versus serum shows linearity starting only from 20 pg / ml in serum, which is a high level. Therefore, in addition to sensitivity, the specificity of the quantification must be high enough to allow comprehensive monitoring.

[007] US patent 9958460 suggests the isolation of exosomes from a blood sample for the quantification of analytes, including NFL.

[008] Patent application WO2019 / 199871 and corresponding US patent applications disclose the measurement of analytes, including NFL, in a sample subjected to different physical or chemical treatments.

[009] In another group of publications, unrelated to the NFL, patent JP6651794 discloses the addition of dextran sulfate to reduce differences, regardless of blood sample types, in the quantification of cardiac troponin I. However, the regressions obtained consistently lack the (0,0) coordinate. In view of the results of the present invention, this suggests the presence in blood or plasma of interfering compounds, despite the addition of dextran sulfate.

[0010] Conversely, patent application EP 4130741 describes an immunoassay for amyloid peptides Ab40 and Ab42 in blood samples, which is enhanced by the addition of polyanionic molecules, such as dextran sulfate or heparin, at a preferred concentration between 0.5 and 2.5 g / l. In view of the results of the present invention, dextran sulfate allows masking of a blood component that sequesters the Ap peptide.

[0011] In other words, the same polyanionic molecule affects the reliability of detection and / or quantification of different analytes in blood samples differently. Brief Summary of the Invention Petition 870250073510, dated 08 / 20 / 2025, page 11 / 34 4 / 15

[0012] A first aspect of the present invention is a method for quantifying the abundance of Neurofilament Light Chain (NFL) in a blood sample, comprising the steps of supplementing this blood sample with a composition comprising a polyanionic molecule and reacting this supplemented blood sample with at least one antibody, or a fragment thereof, coupled to a detection system, wherein this polyanionic molecule has a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, wherein this polyanionic molecule is present in the supplemented blood sample in an amount between 0.01% by weight and 10% by weight, preferably between 0.1% by weight and 5% by weight, more preferably between 0.5% by weight and 1% by weight (weight of the polyanionic molecule:weight of the blood sample after supplementation), and in which this antibody, or antibody fragment,When coupled with a detection system, it specifically links to an epitope of this NFL.

[0013] Preferably the polyanionic molecule is a peptide moiety, or a linear or branched sugar moiety, covalently coupled to a plurality of sulfate or phosphate groups, preferably heparin sulfate or fragments thereof, dextran sulfate or chondroitin sulfate.

[0014] Preferably the antibody, or a fragment thereof, is coupled to a detection system; it is an antibody or a fragment thereof coupled to a chemiluminescent system.

[0015] Preferably, or additionally, the detection system is selected from the group consisting of fluorescent immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLIA), chemiluminescent immunoassay (CLIA), Petition 870250073510, dated 08 / 20 / 2025, page 12 / 34 5 / 15, preferably configured for Lumipulse® or SIMOA® platforms.

[0016] Advantageously, this method comprises the step of reacting the sample supplemented with a first antibody, or a fragment thereof, fixed on a support so that it specifically fixes the NFL to said support, and of separating the support after fixation of the NFL from contaminating molecules, wherein this first antibody or fragment thereof does not interfere with the binding of the detection antibody, or fragment thereof, and preferably wherein this support is a plurality of magnetic beads.

[0017] Advantageously, this method comprises the step of selecting (i) the antibody or a fragment thereof coupled to a chemiluminescent system and / or (ii) the first antibody in order to achieve a detection limit of less than 5 picograms of NFL per milliliter and no specific binding to plasma components, wherein this specific binding to said plasma components is measured in a composition comprising the polyanionic molecule defined above, in the quantity defined above.

[0018] Preferably, this method is applied to a blood sample (plasma or serum) from a patient after trauma and / or on a regular basis.

[0019] Preferably, in this method, the sample is plasma and / or serum.

[0020] Another related aspect of the present invention is the use of a polyanionic molecule, having a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, for the detection of NFL in plasma and / or serum samples.

[0021] Preferably, in this usage, the polyanionic molecule is a peptide moiety, or a linear or branched sugar moiety, covalent Petition 870250073510, dated 08 / 20 / 2025, page 13 / 34 6 / 15 strongly coupled to a plurality of sulfate or phosphate groups, preferably heparin sulfate or fragments thereof, dextran sulfate or chondroitin sulfate.

[0022] Another related aspect of the present invention is a diagnostic kit comprising: - a first antibody, or a fragment thereof, that specifically binds to the neurofilament light chain (NFL) and is fixed to a support, - an antibody specifically designed to detect NFL, or a fragment thereof, coupled to a detection system, - a polyanionic molecule having a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, wherein this first antibody or fragments thereof do not interfere with the binding of this detection antibody, or of the fragment thereof, to the NFL.

[0023] Preferably, the support that holds the first antibody is magnetic beads. Brief description of the drawings

[0024] Figure 1 shows the effect of adding a polyanionic molecule to plasma samples (with EDTA).

[0025] Figure 2 compares the quantification of NFL in serum and plasma (with EDTA) without the addition of the polyanionic molecule.

[0026] Figure 3 compares the quantification of NFL in serum and plasma (with EDTA) when the polyanionic molecule was added.

[0027] Figure 4 shows the effect of adding a polyanionic molecule to serum samples. Detailed description of an embodiment of the invention.

[0028] Quantification of neurofilament light chain (NFL) in plasma, serum, or other blood fractions should yield results Petition 870250073510, dated 08 / 20 / 2025, page 14 / 34 7 / 15 of the identical data, regardless of the laboratory and hospitals where the data were generated or the type of blood sample. However, the inventors compared large amounts of data and observed significant variations, compromising a comprehensive diagnosis based on the detection and / or quantification of NFL. This contrasts with an ideal situation in which the diagnostic outcome is not influenced by any potentially present interfering molecule, as demonstrated in the present invention: molecules that bind specifically to one of the antibodies used for detection, or molecules that potentially sequester the analyte.

[0029] The inventors found that adding a polyanionic molecule to plasma samples reduced the signal obtained for Neurofilament Light Chain (NFL). As a first conclusion, this is contrary to what is shown in document EP 4130741, which teaches to avoid incorporating such molecules, since the sensitivity of the test is detrimentally affected. However, after examining a plurality of conditions, the inventors reached the unexpected conclusion that much of the specific binding for antibody-based detection of NFL in plasma samples was removed thanks to the addition of the polyanionic molecule: the signal intensity is reduced, but, in light of the present invention, this is beneficial, even in the difficult context of a low abundance of NFL and / or the need to accurately identify small variations in the blood.

[0030] Conversely, when serum samples were analyzed, unexpectedly, the NFL signal was not reduced with the addition of the polyanionic molecule and the measured NFL concentrations are now correlated with plasma samples treated with the polyanionic molecule.

[0031] Given that very high sensitivity and very high specificity are required, and given that, for practical reasons, the Petition 870250073510, dated 08 / 20 / 2025, page 15 / 34 8 / 15 reliable detection and / or quantification is performed in plasma or serum samples, the inventors concluded that the addition of the polyanionic molecule is a simple solution to enable accurate quantification of NFL in blood samples.

[0032] Therefore, a first aspect of the present invention is a method for the reliable detection and / or quantification of Neurofilament Light Chain (NFL) abundance in a blood sample, comprising the steps of adding to said blood sample a composition comprising a polyanionic molecule (thus obtaining a supplemented sample) and reacting this blood sample supplemented with this polyanionic molecule with at least one antibody, or a fragment thereof, coupled to a detection system.

[0033] In the context of the present invention, a blood sample is preferably understood to be a blood sample from a mammalian patient, preferably a primate, more preferably a human patient. The patient may be asymptomatic, or affected by a neurological problem, such as a neurodegenerative disease, inflammation of the central nervous system, or brain trauma.The present method is especially useful in the context of HIV-associated dementia, Alzheimer's disease (both prodromal AD and AD-associated dementia), amyotrophic lateral sclerosis, corticobasal degeneration, Creutzfeldt-Jakob disease, Lewy body dementia, frontotemporal dementia, HIV-associated dementia, mild traumatic brain injury, multiple sclerosis (clinically isolated syndrome, relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), multiple system atrophy, Parkinson's disease, Parkinson's disease-associated dementia, progressive supranuclear palsy, and Down syndrome. A blood sample, in the context of the present invention, is preferred. Petition 870250073510, dated 08 / 20 / 2025, page 16 / 34 9 / 15 blood plasma, serum or any fraction of blood, plasma or serum, including exosomes (although the present invention advantageously enables robust quantification even without isolation of exosomes).

[0034] NFL quantification can be obtained specifically, or it can be obtained together with the quantification of other markers potentially associated with diseases affecting the central nervous system, such as the measurement of different forms of amyloid protein (Ab 40, Ab 42), Tau and / or the different phosphorylated forms of Tau protein. Advantageously, the same platform, such as the Lumipulse® platform, is used for the quantification of the different markers.

[0035] Preferably, the polyanionic molecule has a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da (for example, at a pH of 7).

[0036] Alternatively or additionally, this polyanionic molecule is present in the supplemented blood sample in an amount between 0.01% by weight and 10% by weight, preferably between 0.1% by weight and 5% by weight, more preferably between 0.5% by weight and 1% by weight (weight of the polyanionic molecule:weight of the supplemented blood sample).

[0037] Preferably, the polyanionic molecule comprises: - a sulfate group, such as dextran sulfate, heparin sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, or - an anionic polymer comprising a side chain containing a sulfo group, such as polystyrene sulfonic acid, or - an anionic polymer comprising a carboxyl group, such as poly(meth)acrylic acid or a salt thereof.

[0038] Among these, an anionic polymer comprising a Petition 870250073510, dated 08 / 20 / 2025, page 17 / 34 A 10 / 15 side chain containing a sulfate group or a sulfo group is preferred. Dextran sulfate, polystyrene sulfonic acid, and heparin sulfate are most preferred. Dextran sulfate and polystyrene sulfonic acid are especially preferred. In particular, dextran sulfate is preferred. In practice, these anionic molecules are advantageously in the form of salts; preferably, the salt includes alkali metal salts such as sodium salt and potassium salt. These anionic polymers can be used individually or in combination with two or more of them.

[0039] Alternatively, or additionally, preferably, the polyanionic molecule is a peptide moiety (preferably heparin or a fragment thereof), or a linear or branched sugar moiety, covalently coupled to a plurality of sulfate or phosphate groups, preferably dextran sulfate or chondroitin sulfate.

[0040] Preferably, the antibody, or a fragment thereof, coupled to a detection system, binds specifically to an epitope of said NFL.

[0041] Preferred antibodies (capture antibodies and / or detection antibodies) have a dissociation constant Kd better (smaller) than 10-9, preferably better than 5*10-10.

[0042] Preferably the antibody, or a fragment thereof, coupled to a detection system is an antibody or a fragment thereof coupled to a fluorescent system or a chemiluminescent system (e.g., coupled to an alkaline phosphatase activity).

[0043] The preferred detection system is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), fluorescent immunoassay (FIA), electrochemiluminescent immunoassay (ECLIA). Petition 870250073510, dated 08 / 20 / 2025, p. 18 / 34 11 / 15 chemiluminescent immunoassay (CLIA), such detection systems being preferably used on the Lumipulse® or SIMOA® detection platform.

[0044] Preferably, the method is a sandwich method comprising the step of reacting the supplemented (diluted) sample with a first antibody, or a fragment thereof, fixed to a support so as to specifically fix the NFL to said support, and separating the support after fixation of the NFL from contaminating molecules, wherein said first antibody or fragment thereof does not interfere with the binding of the detection antibody, or fragment thereof. For example, the two epitopes recognized by the first and second antibodies are sufficiently far apart.

[0045] Preferably, the antibody or two antibodies enable a very specific and sensitive measurement of NFL. Advantageously, the method thus comprises a preliminary step of selecting (i) the antibody or a fragment thereof coupled to a detection system and / or (ii) the first antibody in order to achieve a detection limit of at least 3 picograms of NFL per milliliter and no specific binding to plasma components, or specific binding of less than 3 picograms of NFL / milliliter. Preferably, this preliminary examination is obtained using compositions in which plasma components are present and / or using compositions comprising the polyanionic molecule described above.

[0046] In the context of the present invention, the support can be any surface. A preferred support is magnetic beads.

[0047] Advantageously, this method is applied to a patient's blood sample after trauma or to blood samples from the same patient on a regular basis, for example, to monitor the progression of a neurological disease. In fact, progressive diseases often evolve in stages. For example, in the case of sclerosis. Petition 870250073510, dated 08 / 20 / 2025, page 19 / 34 12 / 15 multiple, the present method is useful for identifying new flushes, which will help the physician administer specific drugs (corticosteroids, interferons, Glatiramer acetate, blocking antibodies,...) to control the flushes; therefore, when such drugs are most needed.

[0048] Advantageously, the present method produces substantially the same results for blood samples whether plasma or serum. Therefore, the present method is preferably performed on plasma and / or serum.

[0049] This allows reliable comparisons of data, generated in plasma or serum, depending on local practice.

[0050] Another related aspect of the present invention is, therefore, the use of a polyanionic molecule having a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7 for the detection, or quantification, of NFL in plasma and / or serum samples.

[0051] In this use, the polyanionic molecule is as described above for the method.

[0052] Another related aspect of the present invention is a diagnostic kit comprising: - a first antibody, or a fragment thereof, that specifically binds to an epitope of the Neurofilament Light Chain (NFL) and is fixed to a support, - a detection antibody for NFL, or a fragment thereof, coupled to a detection system, - a polyanionic molecule having a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, this first antibody, or fragments thereof, does not Petition 870250073510, dated 08 / 20 / 2025, p. 20 / 34 13 / 15 interferes with the binding of this antibody to the NFL, or a fragment thereof.

[0052] Advantageously, these two antibodies are for sandwich detection and therefore recognize quite distant NFL epitopes.

[0053] A preferred support for the first antibody is magnetic beads: this allows for easy purification of the NFL from the other blood components (plasma, serum).

[0054] Preferably, this kit also includes reagents for chemiluminescent detection and / or explanatory notes.

[0055] Other features and advantages of the present invention will be perceived from the non-limiting description that follows, and with reference to the drawings and examples. Example 1 - Reduced signal in plasma samples due to the addition of the polyanionic molecule.

[0056] The inventors used samples with known amounts of NFL.

[0057] The inventors tested specific antibody pairs to enable a strong signal in plasma, even at low NFL concentrations (Figure 1, Sp2 curve). In practice, 100 μl of the plasma sample was conditioned with 35 μl of an aqueous medium comprising 1 wt% pH buffer (MOPS), NaCl, 0.036 wt% EDTA, 2 wt% BSA, 2 wt% sucrose and other common reagents. The developed system allows sensitivity even below 5 picograms per milliliter (3 pg / ml).

[0058] Next, the inventors reproduced the experimental settings, but with the addition of a polyanionic molecule (here, dextran sulfate; Spa curve) to the aqueous conditioning medium (here, 3.8 wt%; dextran 5000 in these 35 μl). To their surprise, the NFL signal was markedly reduced, which is conceivable. Petition 870250073510, dated 08 / 20 / 2025, page 21 / 34 14 / 15 considered harmful, particularly in the context of measuring NFL in blood samples, where the concentration of NFL is very low (typically around 20 picograms per milliliter). Example 2 - Comparison of the NFL signal in untreated serum and plasma samples.

[0059] The inventors also tested the NFL signal in plasma samples and in serum samples, in the absence of the polyanionic molecule (same conditioning medium as in Example 1).

[0060] As shown in Figure 2, the values ​​in the serum samples were always lower than in the plasma samples. Example 3 - Comparison of the NFL signal in serum and plasma samples pre-treated with the polyanionic molecule.

[0061] On the other hand, surprisingly, when the same serum versus plasma comparison was achieved in blood samples supplemented with the polyanionic molecule (same conditioning medium as in Example 1, supplemented with 3.8 wt% dextran sulfate), the two signals were virtually identical (Figure 3).

[0062] Therefore, by analyzing these first three examples together, the inventors concluded that the addition of the polyanionic molecule, instead of being detrimental (i.e., reduced signal as in Example 1), removes the specific signal from plasma samples.

[0063] This allows for more reliable quantification, since the specific background is now removed. This also allows for a direct comparison between plasma and serum samples, which is a clear second added value, as it offers laboratories more flexibility to work with both types of samples, depending on their own internal practice. Example 4 - an improved signal trend in serum samples due to the addition of the polyanionic molecule. Petition 870250073510, dated 08 / 20 / 2025, page 22 / 34 15 / 15

[0064] Next, the inventors compared the quantification of NFL in serum samples, pre-treated or not with the polyanionic molecule (same conditioning medium as in Example 1).

[0065] Interestingly, the signal was slightly increased (Figure 4) thanks to pretreatment with the polyanionic molecule, which, in this way, is not detrimental to quantification and is even an advantage.

[0066] It should be understood that the present invention is not limited to the embodiments described and that variations may be made without departing from the scope of the claims. Petition 870250073510, dated 08 / 20 / 2025, page 23 / 34

Claims

1 / 3 CLAIMS 1. A method for quantifying the abundance of Neurofilament Light Chain (NFL) in a blood sample, characterized in that it comprises the steps of supplementing said blood sample with a composition comprising a polyanionic molecule and reacting said supplemented blood sample with at least one antibody, or a fragment thereof, coupled to a detection system, wherein said polyanionic molecule has a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, wherein said polyanionic molecule is present in the supplemented blood sample in an amount between 0.01% by weight and 10% by weight, preferably between 0.1% by weight and 5% by weight, more preferably between 0.5% by weight and 1% by weight (weight of the polyanionic molecule:weight of the NFL). (blood sample after supplementation), and in which the aforementioned antibody,or the aforementioned antibody fragment, coupled to a detection system, binds specifically to an epitope of the aforementioned NFL.

2. Method according to claim 1, characterized in that the polyanionic molecule is a peptide moiety, or a linear or branched sugar moiety, covalently coupled to a plurality of sulfate or phosphate groups, preferably heparin sulfate or fragments thereof, dextran sulfate or chondroitin sulfate.

3. Method according to claim 1 or 2, characterized in that the antibody, or a fragment thereof, coupled to a detection system is an antibody or a fragment thereof coupled to a chemiluminescence system.

4. Method according to any of the preceding claims, characterized in that the detection system is selected from the group consisting of fluorescent immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLIA), chemiluminescent immunoassay (CLIA), preferably configured for the Lumipulse® or SIMOA® platforms.

5. A method according to any of the preceding claims, characterized in that it comprises the step of reacting the sample supplemented with a first antibody, or a fragment thereof, fixed on a support so as to specifically fix the NFL to said support, and of separating the support after fixation of the NFL from contaminating molecules, wherein said first antibody or fragment thereof does not interfere with the binding of the detection antibody, or fragment thereof, and preferably wherein said support is a plurality of magnetic beads.

6. A method according to any of the preceding claims, characterized in that it comprises the step of selecting (i) the antibody or a fragment thereof coupled to a chemiluminescent system and / or (ii) the first antibody to achieve a detection limit below 5 picograms of NFL per milliliter and no specific binding to plasma components, wherein said specific binding to said plasma components is measured in a composition comprising the polyanionic molecule defined in claim 1 or 2 in the amount defined in claim 1.

7. A method according to any of the preceding claims, characterized in that it is applied to a blood sample from a patient after trauma and / or on a regular basis.

8. Method according to any of the preceding claims in Petition 870250073510, dated 08 / 20 / 2025, page 25 / 34 3 / 3, characterized by the fact that the blood sample is plasma and / or serum.

9. Use of a polyanionic molecule having a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, characterized by the fact that said polyanionic molecule is used for the detection of NFL in plasma and / or serum sample(s).

10. Use according to claim 9, characterized in that the polyanionic molecule is a peptide moiety, or a linear or branched sugar moiety, covalently coupled to a plurality of sulfate or phosphate groups, preferably heparin sulfate or fragments thereof, dextran sulfate or chondroitin sulfate.

11. Diagnostic kit, characterized in that it comprises: - a first antibody, or a fragment thereof, that specifically binds to the Neurofilament Light Chain (NFL) and is fixed to a support, - a specific detection antibody for NFL, or a fragment thereof, coupled to a detection system, - a polyanionic molecule having a molecular weight between 5000 and 2000000 Da, and a charge density between 1 and 20 negative charges per 1000 Da at a pH of 7, wherein said first antibody or fragments thereof do not interfere with the binding of said detection antibody, or the fragment thereof, to NFL.

12. Diagnostic kit according to claim 11, characterized in that the support is magnetic beads. Petition 870250073510, dated 20 / 08 / 2025, pp. 26 / 34

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