Improved blood diagnostics

Abstract

A method for quantifying the amount of neurofilament light chains (NFLs) in a blood sample, comprising the steps of adding a composition containing polyanionic molecules and reacting it with at least one antibody conjugated to a detection system that specifically binds to one epitope of an NFL, and its use for diagnostic purposes and a corresponding diagnostic kit.

Description

Technical Field

[0001] The present invention relates to the use of neurofilament light chain (NFL) in blood diagnosis and corresponding detection and / or quantification kits.

Background Art

[0002] A variety of markers reflecting neurodegenerative diseases or inflammatory diseases affecting the central nervous system have been established over many years. These are essential for diagnosis and disease monitoring and are key in measuring the target involvement of disease-modifying therapies. Diagnosis of neurodegenerative diseases mainly requires invasive examinations such as functional brain imaging techniques and lumbar puncture for evaluating cerebrospinal fluid (CSF).

[0003] In recent years, easily accessible and cost-effective blood-based biomarkers for detecting these conditions have been developed and have the potential to revolutionize the diagnostic process. These markers are usually identified using approaches based on immunoassay or mass spectrometry and are further precisely quantified.

[0004] In other words, such markers reflecting central nervous system damage are abundant in cerebrospinal fluid. However, since lumbar puncture is considered an invasive examination, diagnostic methods using blood (whole blood, plasma, serum, and other purified fractions) are welcomed. This poses additional challenges. This is because these biomarkers are present at low concentrations in blood. Furthermore, degradation by proteolytic enzymes, non-specific binding to other proteins, and the presence of candidate biomarkers not limited to the central nervous system but also having peripheral-derived sources further complicate accurate measurement. Therefore, precise and sensitive quantification tools and robust methods are required.

[0005] Due to the complexity of the neurodegenerative processes in the central nervous system and the multiplicity of overlapping clinical diagnoses, it is not always easy to identify individual neurodegenerative diseases. Therefore, there is a need for new markers that improve diagnostic accuracy and enable early diagnosis.

[0006] As a novel marker of biological impairment, neurofilament light chains (NFLs) have been proposed as a cerebrospinal fluid (CSF) and blood-based diagnostic tool (Gaetani et al., 2019, J. Neurol. Neurosurg Psychiatry, pp870-881). Neurofilaments are major structural components of the long axon, and high concentrations in the CSF generally reflect rapidly progressive neurodegenerative processes. In contrast to FTD and atypical Parkinson's syndrome, CSF concentrations of NFLs are normal in AD. Due to this characteristic, it can be used in the diagnosis of differentiating Alzheimer's disease (AD) from other diseases associated with dementia (Blennow, K.; Zetterberg, H.; Fagan, AMFluid Biomarkers in Alzheimer's 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 cerebrospinal fluid (CSF) and blood after central and peripheral nerve damage. However, accurate blood measurement of NFL remains extremely difficult, even with the most advanced and sensitive immunoassays, including fluorescence or luminescence-based techniques. In fact, in addition to the need for a sufficiently strong signal, the aforementioned literature reports an analytical variability of 6%, while inter-test variability of up to 11% has been observed in serum samples. In these tests, linearity is only observed at serum concentrations of 20 pg / ml (high concentration) when comparing cerebrospinal fluid with serum. Therefore, in addition to sensitivity, the specificity of the quantitative method must also be sufficiently high to enable comprehensive monitoring.

[0007] U.S. Patent No. 9958460 proposes separating exosomes from blood samples for the quantification of analytes including NFLs.

[0008] Patent application WO2019 / 199871 and the corresponding U.S. patent application disclose the measurement of analytes containing NFL on samples subjected to various physical or chemical treatments.

[0009] In a separate body of literature unrelated to the NFL, Japanese Patent No. 6651794 discloses the addition of dextran sulfate to reduce variability in the quantification of cardiac troponin I, regardless of the type of blood sample. However, the resulting regression line does not always pass through the (0,0) coordinate. The results of this invention suggest that, despite the addition of dextran sulfate, interfering compounds are present in the blood or plasma.

[0010] On the other hand, European Patent Application No. 4130741 describes an immunoassay method for measuring amyloid peptides Aβ40 and Aβ42 in blood samples, which can be improved by adding polyanionic molecules such as dextran sulfate or heparin, preferably at a concentration of 0.5 to 2.5 g / l. From the results of the present invention, it is possible to mask blood components that capture Aβ peptides with dextran sulfate.

[0011] In other words, the same polyanionic molecule can have different effects on the reliability of detection and / or quantification of different analytes in a blood sample. [Overview of the project]

[0012] A first aspect of the present invention is a method for quantifying the amount of neurofilament light chains (NFLs) in a blood sample, the method comprising the steps of adding a composition containing a polyanionic molecule to a blood sample, and reacting the added blood sample with at least one antibody or a 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 having a negative charge of 1 to 20 per 1,000 Da at pH 7, the polyanionic molecule is present in the added blood sample in an amount of 0.01% to 10% by weight, preferably 0.1% to 5% by weight, more preferably 0.5% to 1% by weight (weight of polyanionic molecule: weight of the blood sample after addition), and the antibody or antibody fragment is conjugated to a detection system and specifically binds to one epitope of the NFLs.

[0013] Preferably, the polyanion molecule is a peptide portion covalently bonded to multiple sulfate or phosphate groups, or a linear or branched sugar portion, and is preferably heparin sulfate or a fragment thereof, dextran sulfate, or chondroitin sulfate.

[0014] Preferably, the detection system to which the antibody or fragment of the antibody binds is an antibody or fragment of the antibody bound to a chemiluminescent system.

[0015] Preferably, or additionally, the detection system is selected from fluorescence immunoassay (FIA), enzyme immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLIA), and chemiluminescent immunoassay (CLIA), and is preferably configured for the Lumipulse® or SIMOA® platform.

[0016] Advantageously, this method includes the step of reacting the added sample with a first antibody or fragment thereof immobilized on a support, thereby specifically immobilizing the NFL on the support and separating the NFL-immobilized support from contaminating molecules. The first antibody or fragment thereof does not interfere with the binding of the detection antibody or fragment thereof, and preferably the support is a plurality of magnetic beads.

[0017] Advantageously, the method includes the step of selecting (i) an antibody or fragment thereof conjugated to a chemiluminescent system and / or (ii) a first antibody, such that the detection limit is less than 5 pg NFL per ml and no nonspecific binding occurs to plasma components. Here, the nonspecific binding to plasma components is measured in a composition containing the polyanionic molecule defined above in the amount defined above.

[0018] Preferably, this method is applied to blood samples (plasma or serum) and / or routine examinations from patients after trauma.

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

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

[0021] Preferably, in this application, the polyanionic molecule is a peptide group covalently bonded to multiple sulfate or phosphate groups (preferably heparin sulfate or a fragment thereof, dextran sulfate, or chondroitin sulfate), or a linear or branched sugar group.

[0022] Another related aspect of the present invention is a diagnostic kit, the diagnostic kit is The first antibody, or a fragment thereof, specifically binds to neurofilament light chains (NFLs) and is immobilized on a support. A specific detection antibody against NFL or a fragment thereof, comprising a specific detection antibody bound to a detection system, and a polyanionic molecule having a molecular weight of 5000 to 2000000 Da and having 1 to 20 negative charges per 1000 Da at pH 7, The first antibody or a fragment thereof does not interfere with the binding of the detection antibody or a fragment thereof to NFL.

[0023] Preferably, the support for immobilizing the first antibody is magnetic beads.

Brief Description of the Drawings

[0024] [Figure 1] Figure 1 is a diagram showing the effect of adding a polyanionic molecule to a plasma sample (containing EDTA). [Figure 2] Figure 2 is a diagram comparing the NFL quantification values in serum and plasma (containing EDTA) when no polyanionic molecule is added. [Figure 3] Figure 3 is a diagram comparing the NFL quantification values in serum and plasma (containing EDTA) when a polyanionic molecule is added. [Figure 4] Figure 4 is a diagram showing the effect of adding a polyanionic molecule to a serum sample.

Mode for Carrying Out the Invention

[0025] Quantification of neurofilament light chain (NFL) in plasma, serum, and other blood fractions should yield the same results regardless of the laboratory or hospital where the data was generated or the type of blood sample. However, as a result of comparing a large amount of data, the inventors confirmed significant variations that interfere with comprehensive diagnosis based on NFL detection and / or quantification. This is in contrast to the ideal situation demonstrated in the present invention (diagnostic results that are not affected by potentially present interfering molecules such as molecules that non-specifically bind to any of the antibodies used for detection or molecules that may capture the analyte).

[0026] [[ID=3The inventors discovered that adding polyanionic molecules to plasma samples reduced the detection signal for nerve filament light chains (NFLs). Their first conclusion was that this contradicted European Patent No. 4130741, which advises against the addition of such molecules due to their adverse effect on test sensitivity. However, after testing several conditions, the inventors reached an unexpected conclusion: in antibody-based NFL detection in plasma samples, a significant amount of nonspecific binding was removed by the addition of polyanionic molecules. While this reduces signal intensity, it is beneficial from the perspective of the present invention, particularly in challenging situations with low NFL concentrations or when accurately identifying minute variations in blood.

[0027] On the other hand, when serum samples were analyzed, unexpectedly, the addition of polyanionic molecules did not reduce the NFL signal, and the measured NFL concentrations correlated with those of polyanionic molecule-treated plasma samples.

[0028] Because extremely high sensitivity and specificity are required, and for practical reasons reliable detection and / or quantification must be performed on plasma or serum samples, the inventors concluded that the addition of polyanionic molecules is a simple solution for accurately quantifying NFL in blood samples.

[0029] Therefore, a first aspect of the present invention is a method for reliably detecting and / or quantifying the amount of neurofilament light chains (NFLs) in a blood sample, comprising the steps of adding a composition containing polyanionic molecules to the blood sample (to obtain an added sample) and reacting the blood sample added with the polyanionic molecules with at least one antibody or fragment thereof conjugated to a detection system.

[0030] In the present invention, "blood sample" preferably means a blood sample from a mammalian patient, more preferably from a primate, and even more preferably from a human patient. Patients may be asymptomatic or may suffer from neurological disorders such as neurodegenerative diseases, central nervous system inflammation, or traumatic brain injury. This method is particularly useful in 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 (clinical isolation syndrome, relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), multiple system atrophy, Parkinson's disease, Parkinson's disease dementia, progressive supranuclear palsy, and Down syndrome. In the present invention, "blood sample" is preferably plasma, serum, or any fraction of blood, plasma, or serum containing exosomes (however, the present invention has the advantage of enabling robust quantification without the need to separate exosomes).

[0031] Quantification of NFL can be achieved independently or in combination with the quantification of other markers potentially associated with central nervous system disorders (such as the measurement of various forms of amyloid proteins (Ab40, Ab42), tau protein, and / or various phosphorylated forms of tau protein). Preferably, the quantification of various markers is performed using the same platform, such as the Lumipulse® platform.

[0032] Preferably, the molecular weight of this polyanionic molecule is 5,000 to 2,000,000 Da, and the charge density is 1 to 20 negative charges per 1,000 Da (for example, at pH 7).

[0033] Alternatively, or in addition, the polyanionic molecule may be present in the added blood sample at a concentration of 0.01% to 10% by weight, preferably 0.1% to 5% by weight, and more preferably 0.5% to 1% by weight (weight of polyanionic molecule: weight of added blood sample).

[0034] Preferably, the polyanion molecule includes an anionic polymer having a side chain containing a sulfate group such as dextran sulfate, heparin sulfate, chondroitin A sulfate, chondroitin B sulfate, chondroitin C sulfate, or a sulfo group such as polystyrene sulfonic acid, or an anionic polymer containing a carboxyl group, such as poly(meth)acrylic acid or a salt thereof.

[0035] Among these, anionic polymers containing sulfate or sulfo groups in their side chains are preferred. Dextran sulfate, polystyrene sulfonic acid, and heparin sulfate are more preferred. Dextran sulfate and polystyrene sulfonic acid are particularly preferred. Dextran sulfate is especially preferred. In practice, it is advantageous for these anionic molecules to be in the form of salts. Preferably, they include alkali metal salts such as sodium salts and potassium salts. These anionic polymers can be used alone or in combination of two or more.

[0036] Alternatively, or in addition, preferably, the polyanion molecule is a peptide portion (preferably heparin or a fragment thereof) covalently bonded to multiple sulfate or phosphate groups, or a linear or branched sugar portion, preferably dextran sulfate or chondroitin sulfate.

[0037] Preferably, the antibody or fragment bound to the detection system specifically binds to one epitope of the NFL.

[0038] The preferred antibody (both the capture antibody and / or the detection antibody) has a dissociation constant K D 10 -9 Better (lower), preferably 5 * 10 -10 It is better.

[0039] Preferably, the antibody or fragment bound to the detection system is an antibody or fragment bound to a fluorescence system or a chemiluminescence system (e.g., bound to alkaline phosphatase activity).

[0040] Preferred detection systems are selected from the group consisting of enzyme immunosorbent assay (ELISA), chemiluminescent enzyme immunosorbent assay (CLEIA), and fluorescence immunosorbent assay (FIA). Electrochemiluminescent immunosorbent assay (ECLIA) and chemiluminescent immunosorbent assay (CLIA) are preferably used with the detection platform Lumipulse® or SIMOA®.

[0041] Preferably, the method is a "sandwich method" and includes the step of reacting a first antibody, or a fragment thereof, immobilized on a support with an added (diluted) sample, thereby specifically immobilizing the NFL on the support, and after the immobilization of the NFL, separating the support from the contaminating molecules. Here, the first antibody or fragment thereof does not interfere with the binding of the detection antibody, or a fragment thereof. For example, the two epitopes recognized by the first antibody and the second antibody are sufficiently far apart.

[0042] Preferably, one or two antibodies enable highly specific and sensitive measurement of NFL. Advantageously, the method includes a preliminary step of selecting (i) an antibody or fragment bound to a detection system and / or (ii) a first antibody in order to set the detection limit to at least 3 pg NFL / ml and to avoid or reduce nonspecific binding to plasma components to less than 3 pg NFL / ml. Preferably, this preliminary test is achieved using a composition in the presence of plasma components and / or a composition containing the polyanionic molecules described above.

[0043] In the context of the present invention, the support can be any surface. A preferred support is a magnetic bead.

[0044] Advantageously, this method can be applied to blood samples from the same patient taken regularly, such as after trauma or to monitor the progression of a neurological disease. In fact, progressive diseases can progress in stages. For example, in the case of multiple sclerosis, this method is useful in identifying new attacks and helps physicians decide when to administer specific medications (corticosteroids, interferon, glatiramer acetate, inhibitory antibodies, etc.) to control attacks. In other words, it can identify when these medications are most needed.

[0045] An advantage of this method is that it yields substantially equivalent results for both plasma and serum blood samples. Therefore, it is preferable to perform this method on plasma and / or serum.

[0046] This makes it possible to reliably compare data generated from plasma or serum, depending on local practices.

[0047] Therefore, another relevant aspect of the present invention is the use of polyanionic molecules having a molecular weight of 5,000 to 2,000,000 Da and a negative charge density of 1 to 20 per 1,000 Da at pH 7 for the detection or quantification of NFL in plasma and / or serum samples.

[0048] In this application, the polyanion molecule is described as described above with respect to the method.

[0049] Another related aspect of the present invention is a diagnostic kit, The first antibody, or a fragment thereof, specifically binds to neurofilament light chains (NFLs) and is immobilized on a support. A specific detection antibody against the NFL or a fragment thereof, comprising a specific detection antibody bound to a detection system, It contains polyanionic molecules with a molecular weight of 5000 to 2000000 Da and a negative charge of 1 to 20 per 1000 Da at pH 7, The first antibody or its fragment does not interfere with the binding of the detection antibody or its fragment to the NFL.

[0050] Preferably, these two antibodies are for "sandwich detection" and therefore recognize sufficiently separated NFL epitopes.

[0051] The preferred carrier for the primary antibody is magnetic beads. This allows for easy purification of NFL from the remaining blood (plasma, serum) components.

[0052] Preferably, the kit further includes chemiluminescence detection reagents and / or instructions.

[0053] Other features and advantages of the present invention will become apparent by referring to the following non-limiting description, as well as the drawings and examples.

[0054] Example 1 - Signal reduction in plasma samples by addition of polyanionic molecules The inventors used a sample containing a known amount of NFL.

[0055] The inventors tested specific antibody pairs to obtain a strong signal in plasma even at low concentrations of NFL (Figure 1, Sp2 curve). Specifically, 100 μl of plasma sample was prepared in 35 μl of aqueous medium containing 1 wt% pH buffer (MOPS), NaCl, 0.036 wt% EDTA, 2 wt% BSA, 2 wt% sucrose, and other common reagents. The developed system enables sensitivity of 5 pg / ml (3 pg / ml) or less.

[0056] Next, the inventors reproduced the experimental setup, but added polyanionic molecules (in this case, dextran sulfate; Spa curve) to an aqueous preparation medium (in this case, 3.8 wt%) of dextran 5000 in 35 μl. Surprisingly, the NFL signal was significantly attenuated. This is considered particularly detrimental in NFL measurement in blood samples with extremely low NFL concentrations (typically around 20 pg / ml).

[0057] Example 2 - Comparison of NFL signals in untreated serum and plasma samples The inventors also tested the NFL signal in plasma and serum samples under conditions without the addition of polyanionic molecules (similar preparation to Example 1).

[0058] As shown in Figure 2, the values ​​in serum samples were consistently significantly lower than those in plasma samples.

[0059] Example 3 - Comparison of NFL signals in serum and plasma samples pretreated with polyanionic molecules On the other hand, surprisingly, when the same serum vs. plasma comparison was performed using blood samples with added polyanionic molecules (3.8% by weight of dextran sulfate added to the same prepared medium as in Example 1), the two signals were almost identical (Figure 3).

[0060] Therefore, after comprehensively analyzing these first three examples, the inventors concluded that the addition of polyanionic molecules does not have a detrimental effect (such as a decrease in signal, as in Example 1), but rather removes nonspecific signals from plasma samples.

[0061] This eliminates nonspecific background, enabling more reliable quantification. This also brings a second clear added value: it allows for direct comparison of plasma and serum samples. This gives laboratories the flexibility to handle both sample types according to their internal practices.

[0062] Example 4 - Trend of signal improvement in serum samples due to the addition of polyanionic molecules Next, the inventors compared the quantification of NFL on serum samples with and without pretreatment with polyanionic molecules (using the same pretreatment medium as in Example 1).

[0063] Interestingly, pretreatment with polyanionic molecules slightly increased the signal (Figure 4). Therefore, this pretreatment does not negatively affect quantification, but rather is beneficial.

[0064] It should be understood that the present invention is not limited to the embodiments described, and various modifications are possible without departing from the scope of the claims.

Claims

1. A method for quantifying the amount of neurofilament light chains (NFL) in a blood sample, wherein the method is: The steps include adding a composition containing polyanionic molecules to the blood sample, The step of reacting the added blood sample with at least one antibody or fragment thereof bound to a detection system, 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. The polyanionic molecule is present in the added blood sample in an amount of 0.01% to 10% by weight, preferably 0.1% to 5% by weight, and more preferably 0.5% to 1% by weight (weight of polyanionic molecule: weight of the blood sample after addition). A method comprising: the antibody or antibody fragment being coupled to a detection system and specifically binding to one epitope of the NFL.

2. The method according to claim 1, wherein the polyanionic molecule is a plurality of sulfate groups or phosphate groups, preferably heparin sulfate or a fragment thereof, a peptide group covalently bonded to dextran sulfate or chondroitin sulfate, or a linear or branched sugar group.

3. The method according to claim 1 or 2, wherein the antibody or fragment thereof bound to the detection system is an antibody or fragment thereof bound to a chemiluminescent system.

4. The method according to any one of claims 1 to 3, wherein the detection system is preferably selected from the group consisting of fluorescence immunoassay (FIA), enzyme immunoassay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), electrochemiluminescence immunoassay (ECLIA), and chemiluminescence immunoassay (CLIA) configured for a Lumipulse® or SIMOA® platform.

5. The method according to any one of claims 1 to 4, comprising the steps of reacting the added sample with a first antibody or fragment thereof immobilized on a support to specifically immobilize NFL on the support, and separating the support after NFL immobilization from the contaminating molecules, wherein the first antibody or fragment thereof does not interfere with the binding of the detection antibody or fragment thereof, and preferably the support is a plurality of magnetic beads.

6. The detection limit is set to less than 5 pg / ml and the step of selecting (i) the antibody or a fragment thereof bound to a chemiluminescent system and / or (ii) the first antibody, so as not to cause nonspecific binding to plasma components, The method according to any one of claims 1 to 5, wherein the nonspecific binding with the plasma component is measured in a composition containing the polyanionic molecule according to claim 1 or 2 in the amount described in claim 1.

7. The method according to any one of claims 1 to 6, applicable to blood samples from patients after trauma and / or periodic blood samples.

8. The method according to any one of claims 1 to 7, wherein the blood sample is plasma and / or serum.

9. The use of a polyanionic molecule 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 polyanionic molecule is used for NFL detection in plasma and / or serum samples.

10. The use according to claim 9, wherein the polyanionic molecule is a plurality of sulfate groups or phosphate groups, preferably heparin sulfate or a fragment thereof, a peptide group covalently bonded to dextran sulfate or chondroitin sulfate, or a linear or branched sugar group.

11. It is a diagnostic kit, A first antibody, or a fragment thereof, specifically binds to neurofilament light chains (NFLs) and is immobilized on a support. A specific detection antibody against the NFL or a fragment thereof, comprising a specific detection antibody bound to a detection system, It comprises a polyanionic molecule having a molecular weight of 5,000 to 2,000,000 Da and a negative charge of 1 to 20 per 1,000 Da at pH 7, A diagnostic kit wherein the first antibody or its fragment does not interfere with the binding of the detection antibody or its fragment to the NFL.

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

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