Improved blood diagnosis

Abstract

A method of quantifying the abundance of neurofilament light chains (NFLs) in a blood sample, comprising the steps of adding a composition comprising polyanionic molecules and reacting it with at least one antibody coupled to a detection system, said antibody specifically binding to one epitope of NFL, for diagnostic applications and corresponding diagnostic kits.

Description

Technical Field

[0001] This invention relates to the blood diagnostic applications of neurofilament light chains (NFL) and corresponding detection and / or quantitative kits. Background Technology

[0002] Over the years, numerous biomarkers have been established to reflect neurodegenerative diseases or inflammatory diseases affecting the central nervous system. These biomarkers are crucial for diagnosis and disease monitoring, and will also be key to measuring the engagement of disease-modifying therapies. Diagnosis of neurodegenerative diseases primarily requires functional brain imaging techniques or invasive tests, such as lumbar puncture, to assess cerebrospinal fluid (CSF).

[0003] In recent years, readily available and cost-effective blood-based biomarkers have been developed for the detection of these diseases, potentially revolutionizing diagnostic work. These biomarkers are typically identified, and even precisely quantified, using methods based on immunoassays and mass spectrometry.

[0004] In other words, these biomarkers typically reflect damage to the central nervous system and are abundant in cerebrospinal fluid. However, lumbar puncture is considered an invasive diagnostic procedure, and blood-based diagnostics (whole blood, plasma, serum, and other purified fractions) are preferred. This presents an additional challenge because these biomarkers are present in lower concentrations in the blood. Furthermore, degradation by proteolytic enzymes, nonspecific binding to other proteins, and the presence of peripheral sources (rather than solely from the central nervous system) of candidate biomarkers further complicate accurate measurement. Therefore, precise and sensitive quantitative tools and robust methods are needed.

[0005] Due to the complexity of neurodegenerative processes within the central nervous system and the large amount of overlapping clinical diagnoses, identifying individual neurodegenerative diseases is not always straightforward. Therefore, new biomarkers are welcome to improve accuracy and allow for earlier diagnosis.

[0006] Neurofilamentary light chains (NFLs) have been proposed as novel biomarkers for biological diseases (Gaetani et al., 2019, J. Neurol. Neurosurg Psychiatry, pp. 870-881) for CSF and blood-based diagnostics. Neurofilaments are the main structural components of myelinated axons. Their high concentrations in CSF typically reflect rapidly progressing neurodegenerative processes. Their CSF concentrations are normal in Alzheimer's disease (AD) compared to FTD and atypical Parkinson's syndrome. Therefore, they can be used for differential diagnosis to distinguish AD from other diseases associated with dementia (Blennow, K.; Zetterberg, H.; Fagan, A.M. 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 following damage to central and peripheral neurons. However, even with the most advanced and sensitive immunoassays, such as fluorescence-based or luminescent techniques, accurate blood measurement of NFL remains very challenging. In fact, besides requiring a sufficiently strong signal, the aforementioned literature also mentions a 6% analytical variability, while the inter-batch variability in serum samples is as high as 11%. In these tests, comparisons between CSF and serum showed linearity only starting from 20 pg / ml in serum, which is a very high level. Therefore, in addition to sensitivity, the specificity of quantification must be sufficiently high for comprehensive monitoring.

[0007] Patent US9958460 proposes the isolation of exosomes from blood samples for the quantitative analysis of analytes, including NFL.

[0008] Patent application WO2019 / 199871 and the corresponding U.S. patent application disclose the measurement of analytes (including NFL) on samples that have undergone various physical or chemical treatments.

[0009] In another set of publications unrelated to the NFL, patent JP6651794 discloses the addition of dextran sulfate to reduce variability in cardiac troponin I quantification, regardless of the type of blood sample. However, the obtained regressions consistently miss the (0,0) coordinates. Given the results of this invention, this suggests that interfering compounds still exist in the blood or plasma despite the addition of dextran sulfate.

[0010] Conversely, patent application EP 4130741 describes an immunoassay for amyloid peptides Aβ40 and Aβ42 in blood samples, which is improved upon the addition of a polyanionic molecule (such as dextran sulfate or heparin, preferably at a concentration between 0.5 and 2.5 g / L). As a result of this invention, dextran sulfate can mask blood components that isolate Aβ peptides.

[0011] In other words, the same polyanionic molecule can have different effects on the reliability of detection and / or quantification of different analytes in blood samples. Summary of the Invention

[0012] A first aspect of the present invention is a method for quantifying the abundance of neurofilament light chains (NFL) in a blood sample, comprising the steps of: supplementing the blood sample with a composition comprising a polyanionic molecule, and reacting the supplemented blood sample with at least one antibody or fragment thereof conjugated to a detection system, wherein the polyanionic molecule has a molecular weight of 5,000 to 2,000,000 Da and a charge density of 1 to 20 negative charges per 1,000 Da at pH 7, wherein the amount of the polyanionic molecule in the supplemented blood sample is 0.01 wt% to 10 wt%, preferably 0.1 wt% to 5 wt%, more preferably 0.5 wt% to 1 wt% (weight polyanionic molecules: weight of the supplemented blood sample), wherein the antibody or antibody fragment conjugated to the detection system specifically binds to an epitope of the NFL.

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

[0014] Preferably, the antibody or fragment thereof conjugated to the detection system is an antibody or fragment thereof conjugated to the chemiluminescence system.

[0015] Preferably, or otherwise, the detection system is selected from fluorescence immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLIA), and chemiluminescent immunoassay (CLIA), and is preferably configured for use with or platform.

[0016] Advantageously, the method includes the step of reacting a supplementary sample with a first antibody or fragment thereof immobilized on a support to specifically immobilize NFL on the support, and separating the support from contaminant molecules after NFL immobilization, wherein the first antibody or fragment thereof does not interfere with the linkage of the detection antibody or fragment thereof, preferably wherein the support is a plurality of magnetic beads.

[0017] Advantageously, the method includes the steps of selecting (i) an antibody or fragment thereof conjugated to a chemiluminescence system and / or (ii) a first antibody to achieve a detection limit of less than 5 picograms of NFL per milliliter and to avoid nonspecific binding to plasma components, wherein the nonspecific binding to said plasma components is measured in a composition comprising the polyanionic molecules described above, in the amount described above.

[0018] Preferably, the method is applied to blood samples (plasma or serum) from trauma patients and / or used periodically.

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

[0020] Another related aspect of the invention is the use of polyanionic molecules in the detection of NFL in plasma and / or serum samples, the polyanionic molecules having a molecular weight between 5,000 and 2,000,000 Da and a charge density of 1 to 20 negative charges per 1,000 Da at pH 7.

[0021] Preferably, in this application, the polyanionic molecule is a peptide moiety covalently coupled to multiple sulfate or phosphate groups, or a straight-chain or branched sugar moiety, preferably heparin sulfate or fragments thereof, dextran sulfate, or chondroitin sulfate.

[0022] Another related aspect of the present invention is a diagnostic kit comprising:

[0023] -A first antibody or fragment thereof that specifically binds to neurofilament light chains (NFL) and is immobilized on a support.

[0024] - NFL-specific detection antibodies or fragments thereof coupled to the detection system

[0025] - Polyanionic molecules with molecular weights ranging from 5,000 to 2,000,000 Da and charge densities of 1 to 20 negative charges per 1,000 Da at pH 7.

[0026] The first antibody or its fragment does not interfere with the binding of the detection antibody or its fragment to NFL.

[0027] Preferably, the support for immobilizing the first antibody is a magnetic bead. Attached Figure Description

[0028] Figure 1 The effect of adding polyanionic molecules on plasma samples (containing EDTA) was shown.

[0029] Figure 2 NFL quantification was compared between serum and plasma (containing EDTA) without the addition of polyanionic molecules.

[0030] Figure 3 The quantification of NFL in serum and plasma (containing EDTA) was compared after the addition of polyanionic molecules.

[0031] Figure 4 The effect of adding polyanionic molecules on serum samples is shown. Detailed Implementation

[0032] Quantification of neurofilament light chains (NFL) in plasma, serum, or other blood components should yield consistent results, regardless of the laboratory and hospital from which the data originates or the type of blood sample. However, the inventors compared a large amount of data and noted significant variations that hinder comprehensive diagnosis based on NFL detection and / or quantification. This contrasts sharply with the ideal situation where diagnostic results are unaffected by any potentially interfering molecules, such as those that nonspecifically bind to the antibody used for detection, or molecules that may isolate the analyte, as illustrated in this invention.

[0033] The inventors discovered that adding polyanionic molecules to plasma samples reduces the signal acquired by neurofilament light chains (NFLs). As a first conclusion, this is contrary to what is shown in EP 4130741, which teaches to avoid introducing such molecules as the sensitivity of the test is adversely affected. However, after testing under various conditions, the inventors reached an unexpected conclusion that many non-specific bindings in antibody-based NFL detection in plasma samples were eliminated due to the addition of polyanionic molecules: the signal intensity was reduced, but according to the invention, this is beneficial even in challenging contexts such as low NFL abundance and / or the need for precise identification of minute changes in the blood.

[0034] Conversely, when analyzing serum samples, surprisingly, the addition of polyanionic molecules did not decrease the NFL signal, and the measured NFL concentrations are now correlated with plasma samples treated with polyanionic molecules.

[0035] Because of the need for very high sensitivity and very high specificity, and for practical reasons, for reliable detection and / or quantification in plasma or serum samples, the inventors concluded that adding polyanionic molecules is a simple solution for accurately quantifying NFL in blood samples.

[0036] Therefore, a first aspect of the present invention is a method for reliably detecting and / or quantifying the abundance of neurofilament light chains (NFL) in a blood sample, comprising the steps of adding a composition containing a polyanion molecule to the blood sample (thereby obtaining a supplemented sample), and reacting the blood sample supplemented with the polyanion molecule with at least one antibody or fragment thereof, said antibody or fragment being coupled to a detection system.

[0037] In the context of this invention, "blood sample" preferably refers to a blood sample from a mammalian patient, preferably a primate, and more preferably a human patient. The patient may be asymptomatic or affected by neurological problems such as neurodegenerative diseases, central nervous system inflammation, or traumatic brain injury. This method is particularly useful in the context of HIV-related dementia, Alzheimer's disease (including prodromal AD and dementia caused by AD), amyotrophic lateral sclerosis (ALS), cortical-basal degeneration, Creutzfeldt-Jakob disease (CJD), Lewy body dementia, frontotemporal dementia, HIV-related dementia, mild traumatic brain injury, multiple sclerosis (clinically isolated syndrome, relapsing-remitting multiple dementia, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), multiple system atrophy, Parkinson's disease, Parkinson's dementia, progressive supranuclear palsy, and Down syndrome. In the context of this invention, "blood sample" is preferably plasma, serum, or any fraction of blood, plasma, or serum, including exosomes (even though this invention advantageously allows for robust quantification even without separating exosomes).

[0038] Quantification of NFL can be achieved specifically or in conjunction with the quantification of other biomarkers that may be associated with diseases affecting the central nervous system, such as measuring different forms of amyloid-beta (Aβ).

[0039] 40, Aβ42), and different phosphorylation forms of Tau and / or Tau proteins. Advantageously, the same platform, such as... The platform is used to quantify different biomarkers.

[0040] Preferably, the polyanionic molecule has a molecular weight of 5,000 to 2,000,000 Da and a charge density of 1 to 20 negative charges per 1,000 Da (e.g., at pH 7).

[0041] Alternatively or additionally, the content of the polyanionic molecule in the supplemented blood sample is between 0.01 wt% and 10 wt%, preferably between 0.1 wt% and 5 wt%, more preferably between 0.5 wt% and 1 wt% (weight of polyanionic molecule: weight of supplemented blood sample).

[0042] Preferably, the polyanionic molecule comprises:

[0043] -Sulfate groups, such as dextran sulfate, heparin sulfate, chondroitin A sulfate, chondroitin B sulfate, chondroitin C sulfate, or

[0044] - Anionic polymers containing sulfonate side chains, such as polystyrene sulfonic acid, or

[0045] - Anionic polymers containing carboxyl groups, such as poly(meth)acrylic acid or its salts.

[0046] Preferably, the anionic polymers contain side chains with sulfate or sulfonyl groups. More preferably, dextran sulfate, polystyrene sulfonic acid, and heparin sulfate are preferred. Dextran sulfate and polystyrene sulfonic acid are particularly preferred. In particular, dextran sulfate is preferred. In practice, these anionic molecules are advantageously in the form of salts; preferably, the salts include alkali metal salts, such as sodium and potassium salts. These anionic polymers can be used alone or in combination of two or more.

[0047] Alternatively, or additionally, preferably, the polyanionic molecule is a peptide moiety (preferably heparin or a fragment thereof), or a straight-chain or branched-chain sugar moiety covalently coupled to a plurality of sulfate or phosphate groups, preferably dextran sulfate or chondroitin sulfate.

[0048] Preferably, the antibody or a fragment thereof conjugated to the detection system specifically binds to an epitope of the NFL.

[0049] The dissociation constant K of the preferred antibody (capture and / or detection antibody) D Better than (less than) 10 -9 Preferred is better than 5*10 -10 .

[0050] Preferably, the antibody or fragment thereof conjugated to the detection system is an antibody or fragment conjugated to a fluorescent or chemiluminescent system (e.g., conjugated to alkaline phosphatase activity).

[0051] Preferred detection systems are selected from enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), and fluorescence immunoassay (FIA). Electrochemiluminescent immunoassay (ECLIA) and chemiluminescent immunoassay (CLIA) systems are preferred for use in detection platforms. or

[0052] Preferably, the method is a "sandwich method," comprising reacting a supplemental (diluted) sample with a first antibody or fragment thereof immobilized on a support to specifically immobilize NFL on the support, and separating the support from contaminant molecules after NFL immobilization, wherein the first antibody or fragment thereof does not interfere with the linkage of the detection antibody or fragment thereof. For example, the two epitopes recognized by the first and second antibodies are sufficiently far apart.

[0053] Preferably, the antibody or two antibodies allow for highly specific and sensitive measurement of NFL. Advantageously, the method therefore includes the preliminary step of selecting (i) an antibody or fragment thereof conjugated to the detection system and / or (ii) a first antibody to achieve a detection limit of at least 3 picograms of NFL / mL and to avoid nonspecific binding to plasma components, or to nonspecific binding of less than 3 picograms of NFL / mL. Preferably, this preliminary test is performed using a composition containing plasma components and / or using a composition comprising polyanionic molecules as described above.

[0054] In the context of this invention, the support can be any surface. A preferred support is a magnetic bead.

[0055] Advantageously, this method can be applied to blood samples from post-traumatic patients or periodically to blood samples from the same patient, for example, to monitor the progression of neurological diseases. Indeed, progressive diseases sometimes develop gradually. For example, in the case of multiple sclerosis, this method can be used to identify new seizures, thus helping doctors to use specific medications (corticosteroids, interferon, glatiramer acetate, blocking antibodies, etc.) to control seizures when they are most needed.

[0056] Advantageously, this method produces substantially the same results for blood samples of plasma or serum. Therefore, this method is preferably performed on plasma and / or serum.

[0057] This allows for reliable comparisons of data generated on plasma or serum, depending on local practices.

[0058] Therefore, another relevant aspect of the present invention is to use polyanionic molecules with a molecular weight between 5,000 and 2,000,000 Da and a charge density of 1 to 20 negatively charged molecules per 1,000 Da at pH 7 to detect or quantify NFL in plasma and / or serum samples.

[0059] In this application, the polyanionic molecule is as described above for this method.

[0060] Another related aspect of the present invention is a diagnostic kit comprising:

[0061] -A first antibody or fragment thereof that specifically binds to neurofilament light chain (NFL) epitopes and is immobilized on a support.

[0062] - NFL detection antibodies or fragments thereof coupled to the detection system

[0063] - Polyanionic molecules with molecular weights ranging from 5,000 to 2,000,000 Da and charge densities of 1 to 20 negative charges per 1,000 Da at pH 7.

[0064] The first antibody or its fragment will not interfere with the binding of the detection antibody to NFL or its fragment.

[0065] Advantageously, these two antibodies are used for "sandwich detection," thus enabling the identification of NFL epitopes that are far enough away.

[0066] The preferred support for the primary antibody is magnetic beads: these allow for easy purification of NFL from remaining blood (plasma, serum) components.

[0067] Preferably, the kit also includes reagents and / or instructions for chemiluminescence detection.

[0068] Other features and advantages of the invention will become apparent from the following non-limiting description and by reference to the accompanying drawings and embodiments.

[0069] Example 1—The signal on the plasma sample was reduced due to the addition of polyanionic molecules.

[0070] The inventors used a known amount of NFL samples.

[0071] The inventors tested multiple pairs of specific antibodies that produced strong signals in plasma even at low concentrations of NFL. Figure 1 (Sp2 curve). In practice, 100 μl of plasma sample is conditioned with 35 μl of an aqueous medium containing 1 wt% pH buffer (MOPS), NaCl, 0.036 wt% EDTA, 2 wt% BSA, 2 wt% sucrose, and other commonly used reagents. The developed system allows for sensitivity even below 5 picograms per milliliter (3 pg / mL).

[0072] The inventors then repeated the experimental setup, but added a polyanionic molecule (here, dextran sulfate; SPAM curve) to the aqueous conditioning medium (here, dextran 5000 containing 3.8 wt% in this 35 μl). To their surprise, the NFL signal was significantly reduced, which is considered detrimental, especially when measuring NFL in blood samples, as the concentration of NFL is very low (typically around 20 picograms per milliliter).

[0073] Example 2—Comparison of NFL signals in untreated serum and plasma samples

[0074] The inventors also tested the NFL signal on plasma and serum samples in the absence of polyanionic molecules (using the same modulating medium as in Example 1).

[0075] like Figure 2 As shown, the values ​​in serum samples are always much lower than those in plasma samples.

[0076] Example 3—Comparison of NFL signals in serum and plasma samples pretreated with polyanionic molecules

[0077] On the other hand, surprisingly, when the same serum and plasma comparisons were performed on blood samples supplemented with polyanionic molecules (using the same conditioning medium as in Example 1, supplemented with 3.8 wt% dextran sulfate), the two signals were almost equal. Figure 3 ).

[0078] Therefore, when analyzing the three initial embodiments together, the inventors concluded that the addition of polyanionic molecules removed nonspecific signals from the plasma sample, rather than harmful ones (i.e., signal reduction as in Example 1).

[0079] This allows for more reliable quantification because nonspecific background is now removed. It also allows for direct comparison of plasma and serum samples, a second significant added value, as it provides laboratories with greater flexibility, allowing them to work with both sample types according to their own internal practices.

[0080] Example 4 – Due to the addition of polyanionic molecules, the signal on the serum sample tended to improve.

[0081] The inventors then compared the quantification of NFL on serum samples, regardless of whether they were pretreated with polyanionic molecules (using the same conditioning medium as in Example 1).

[0082] Interestingly, the signal was slightly increased due to pretreatment with polyanionic molecules. Figure 4 Therefore, this is not detrimental to quantification, and may even be an advantage.

[0083] It should be understood that the present invention is not limited to the described embodiments, and variations may be applied without departing from the scope of the claims.

Claims

1. A method for quantifying the abundance of neurofilament light chains (NFL) in blood samples, comprising the following steps: The blood sample is supplemented with a composition comprising polyanionic molecules, and the supplemented blood sample is reacted with at least one antibody or fragment thereof conjugated to a detection system, wherein the polyanionic molecules have a molecular weight of 5,000 to 2,000,000 Da and a charge density of 1 to 20 negative charges per 1,000 Da at pH 7, wherein the content of the polycationic molecules in the supplemented blood sample is 0.01 wt% to 10 wt%, preferably 0.1 wt% to 5 wt%, more preferably 0.5 wt% to 1 wt% (weight of polyanionic molecules: weight of the supplemented blood sample), wherein the antibody or the antibody fragment is conjugated to the detection system and specifically binds to an epitope of the NFL.

2. The method according to claim 1, wherein the polyanionic molecule is a peptide moiety covalently coupled to a plurality of sulfate groups or phosphate groups, or a straight-chain or branched-chain sugar moiety, preferably heparin sulfate or a fragment thereof, dextran sulfate or chondroitin sulfate.

3. The method according to claim 1 or 2, wherein, The antibody or fragment thereof conjugated to the detection system is the antigen or fragment thereof conjugated to the chemiluminescence system.

4. The method according to any one of the preceding claims, wherein the detection system is selected from fluorescence immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLIA), and chemiluminescent immunoassay (CLIA), preferably configured for use with or platform.

5. The method according to any one of the preceding claims, comprising reacting the supplemented sample with a first antibody or fragment thereof immobilized on a support to specifically immobilize NFL on the support, and separating the support from contaminant molecules after NFL immobilization, wherein the first antibody or fragment thereof does not interfere with the linkage of the detection antibody or fragment thereof, and preferably wherein the support is a plurality of magnetic beads.

6. The method according to any one of the preceding claims, comprising the steps of selecting (i) an antibody or a fragment thereof conjugated to a chemiluminescence system and / or (ii) a first antibody, To achieve a detection limit below 5 picograms of NFL per milliliter and to avoid nonspecific binding to plasma components, wherein nonspecific binding to said plasma components is measured in a composition comprising the polyanionic molecule as defined in claim 1 or 2, said polyanionic molecule being present in the amount defined in claim 1.

7. The method according to any one of the preceding claims, applied to blood samples from post-traumatic patients and / or used periodically.

8. The method according to any one of the preceding claims, wherein the blood sample is plasma and / or serum.

9. Use of polyanionic molecules having a molecular weight of 5,000 to 2,000,000 Da and a charge density of 1 to 20 negative charges per 1,000 Da at pH 7, wherein the polycationic molecules are used for the detection of NFL in plasma and / or serum samples.

10. The use according to claim 9, wherein the polyanionic molecule is a peptide moiety covalently coupled to a plurality of sulfate groups or phosphate groups, or a straight-chain or branched-chain sugar moiety, preferably heparin sulfate or a fragment thereof, dextran sulfate or chondroitin sulfate.

11. A diagnostic kit comprising: -A first antibody or fragment thereof that specifically binds to neurofilament light chains (NFL) and is immobilized on a support. - NFL-specific detection antibodies or fragments thereof coupled to the detection system - Polyanionic molecules with molecular weights ranging from 5,000 to 2,000,000 Da and charge densities of 1 to 20 negative charges per 1,000 Da at pH 7. The first antibody or its fragment does not interfere with the binding of the detection antibody or its fragment to NFL.

12. The diagnostic kit of claim 11, wherein the support is a magnetic bead.

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