Reagents and methods for diagnosing thrombotic events

Abstract

The present disclosure relates to reagents, kits, and methods for the detection of anti-PF4 antibodies and the diagnosis of non-heparin-induced thrombotic events including vaccine-induced thrombotic thrombocytopenia. The methods provided by the present disclosure include (i) contacting a whole blood, plasma, or serum sample obtained from a subject with a reagent comprising (a) a binding molecule selected from the group consisting of platelet factor 4 protein (PF4), a fragment of PF4 capable of binding to an anti-PF4 antibody, and an anti-idiotype antibody of an anti-platelet factor 4 antibody (anti-PF4 antibody), and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and the binding molecule (a) does not contain heparin or a heparin surrogate, and (ii) analyzing the sample to detect a complex formed by the reagent and the anti-PF4 antibody, wherein detecting the complex indicates that the sample contains an anti-PF4 antibody, and includes.

Description

Technical Field

[0001] This application claims the benefit of European Patent Application EP22382635.5, filed on July 1, 2022. The present invention relates to the field of clinical diagnosis, in particular to the measurement of anti-platelet factor 4 (anti-PF4) antibodies that enable the diagnosis of non-heparin related thrombotic thrombocytopenia, such as vaccine-induced thrombotic thrombocytopenia (VITT).

Background Art

[0002] Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most important measure to fight the ongoing coronavirus disease 2019 (Covid-19) pandemic. One of the first vaccines approved by the European Medicines Agency is the recombinant chimpanzee adenovirus (ChAdOx1-S) vector encoding the spike glycoprotein of SARS-CoV-2, the COVID-19 vaccine AstraZeneca (also known as ChAdOx1 nCoV-19, AZD1222, Vaxzevria), which is an adenovirus vector-based vaccine.

[0003] Unfortunately, after ChAdOx1 nCoV-19 vaccination, cases of cerebral venous sinus thrombosis (CVST) and visceral vein thrombosis with moderate to severe thrombocytopenia have been reported in multiple countries in healthy individuals. This novel disease, "vaccine-induced thrombotic thrombocytopenia (VITT)", is associated with high titers of immunoglobulin G class antibodies against the cationic platelet chemokine platelet factor 4 (PF4; CXCL4). These antibodies activate platelets via the Fc γ IIa receptor, causing PF4-dependent platelet activation. Another adenovirus-based vaccine, the COVID-19 vaccine Janssen (Johnson & Johnson), may also be associated with the formation of anti-PF4 antibodies.

[0004] PF4 opsonizes the polyanionic surfaces of pathogens and promotes the binding of anti-PF4 antibodies. This is likely an evolutionarily ancient immune defense mechanism, and anti-PF4-producing B cells are found in almost all individuals and are also present in neonatal cord blood. However, when a strong anti-PF4 antibody response is misdirected, it forms the basis for the thrombotic thrombocytopenic disorder immune heparin-induced thrombocytopenia (HIT), and its most severe form, autoimmune heparin-induced thrombocytopenia (aHIT). Autoimmune heparin-induced thrombocytopenia is characterized by the formation of anti-PF4 antibodies that activate platelets, and these antibodies have a very high affinity and react even in the absence of heparin. Severe heparin-induced thrombocytopenia is also characterized by "pancellular" activation (platelets, neutrophils, monocytes, endothelial cells). Vaccine-induced immune thrombotic thrombocytopenia (VITT) is clinically and serologically very similar to autoimmune heparin-induced thrombocytopenia (HIT) (Greinacher et al, "Thrombotic Thrombocytopenia after ChAdOxInCov-19 Vaccination". DOI:10.1056 / NEJMoa2104840). One of the major risk factors in the formation of anti-PF4 antibodies is inflammation and tissue damage, which significantly increase the risk of forming pathogenic anti-PF4 antibodies.

[0005] In addition to VITT, other thromboembolic events unrelated to heparin have also been observed (Favaloro et al, 2022 “Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT / HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT / TTS” DOI: 10.3390 / antibl 101000). The new thromboembolic events include arterial vascular occlusion. The factors causing these pathologies are mostly unknown, but they still share the diagnostic difficulties with VITT. As a result, these pathologies are currently underdiagnosed in current clinical protocols, mainly using rapid HIT assays, commercially available anti-PF4 ELISA assays, and functional assays for heparin-dependent antibodies.

[0006] Currently, several commercially available immunoassays are available for the diagnosis of HIT, including classical IgG-specific and multi-specific ELISA assays (e.g., Asserachrom HPIA IgG, Lifecodes PF4 IgG, Hyphen Biomed ZymutestHIA IgG, AESKULISA HiT II), and rapid assays (e.g., HemosIL® AcuStar HIT-IgG(PF4-H), HemosIL® HIT-Ab(PF4-H), Diamed PaGIA gel, STic Expert). All assays use PF4 protein complexed with a heparin surrogate such as heparin or polyvinyl sulfate to detect anti-PF4 antibodies induced by heparin (anti-PF4 / H antibodies) in patients suspected of having HIT. However, these assays are not suitable for the diagnosis of VITT. Previous studies have shown that rapid assays have very low sensitivity for the diagnosis of VITT and are not suitable for the diagnosis of this particular condition. On the other hand, state-of-the-art anti-PF4 / heparin or anti-PF4 / PVS ELISA assays can detect only some VITT cases, and there is no ELISA method that can detect all cases of VITT (Platton et al, “Evaluation of laboratory assays for anti- platelet factor 4 antibodies after ChAdOxI nCOV- 19 vaccination”. DOI: 10.1111 / jth.15362). Even more problematically, some assays that recognize VITT-like antibodies also recognize heparin-dependent HIT-like antibodies, and the antibody specificities of the two cannot be distinguished. Therefore, when the ELISA result is negative and VITT is strongly suspected clinically, it is recommended to perform a second ELISA or platelet activation assay, but this is a routine that significantly complicates the diagnosis. Even at this step, if the functional test is performed with heparin enhancement instead of PF4, false-negative results will occur. However, ELISA assays widely available in diagnostic laboratories have a long turnaround time of 6 to 8 hours or more, and only a few laboratories can perform platelet activation assays.

[0007] As a further problem, currently available immunoassays are designed to measure the presence of anti-PF4 / H antibodies, i.e., they are not specific for the anti-PF4 antibodies seen in VITT patients. Therefore, these immunoassays cannot distinguish between HIT patients and VITT patients (even if VITT could be detected). Thus, there is no way to identify whether a thrombotic event in a heparin-administered patient was caused by heparin or by vaccination or other unknown factors. Due to this lack of differential diagnosis between VITT and HIT, it is not possible to determine which patients can continue heparin administration and which patients should discontinue heparin administration, and in many cases, it will be replaced by another much more expensive antithrombotic therapy.

[0008] Furthermore, regardless of the presence or absence of heparin or vaccination, the number of patients with thrombotic diseases is increasing. Among these patients, heparin-dependent and heparin-independent anti-PF4 antibodies may be detected at various ratios. Currently, these antibodies can be distinguished by functional assays. There is a need for a new widely applicable assay that can distinguish these different anti-PF4-related diseases, and it is necessary to systematically evaluate the morbidity of different patient groups. This is clinically important, and for patients with anti-PF4 heparin-independent antibodies, (in addition to anticoagulants,) auxiliary measures are required to suppress FcγRIIa-dependent cell activation, which is a factor causing severe hypercoagulable states.

[0009] Therefore, there is an urgent need to provide a highly sensitive and specific assay for detecting anti-PF4 heparin-independent antibodies that is cost-effective, rapid, and easily implementable in the clinical setting for the diagnosis of VITT and other thrombotic events not induced by heparin or vaccination. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] The inventors of the present invention have developed a novel method for detecting heparin - independent anti - PF4 antibodies (hereinafter also referred to as "anti - PF4 antibodies") and have overcome the above - mentioned problems. The method of the present invention specifically detects anti - PF4 antibodies and does not detect heparin - dependent HIT - like antibodies. Therefore, by this novel method, it becomes possible to diagnose thromboembolic events not induced by heparin, such as VITT or other types. This novel method involves a molecule capable of binding to anti - PF4 antibodies from a human patient who has or has had a thromboembolic event not induced by heparin, and a solid support, where the molecule capable of binding to anti - PF4 antibodies is covalently bound to the surface of the solid support, and is based on the solid support.

[0011] Accordingly, in a first aspect of the present disclosure, there is provided a reagent comprising: (a) a molecule for binding to an anti - PF4 antibody, and (b) a solid support, wherein the molecule is covalently bound to the surface of the solid support.

[0012] A schematic diagram of the reagent according to the first aspect of the present disclosure is shown in FIG. 1. The covalent binding of the molecule for binding to an anti - PF4 antibody (hereinafter also referred to as "binding molecule") to the solid support is important. This is because the inventors have found that when the binding molecule is fixed by other means, almost no anti - PF4 antibodies can be detected in a small number of samples from VITT patients (see FIGS. 4 and 5). Also, it has been found that the binding molecule should not form a complex with heparin or a heparin surrogate (e.g., PVS). This is shown in FIG. 3, where when using plasma from VITT patients, only the reagent of the present disclosure containing PF4 alone as the sole binding molecule showed a high signal. Therefore, the binding molecule (a) of the reagent according to the first aspect does not contain heparin or a heparin surrogate, that is, does not form a complex with heparin or a heparin surrogate.

[0013] As a second aspect, the present disclosure provides a method for manufacturing the reagent defined in the first aspect. The method includes (i) contacting a solid support with a solution containing a molecule capable of binding to an anti-PF4 antibody in the presence of a crosslinking agent and optionally a catalyst. Alternatively, a method for producing a reagent defined in the first aspect may include contacting a solid support with a solution containing a crosslinking agent and optionally a catalyst, and (ii) subsequently adding a molecule capable of binding to a human anti-PF4 antibody.

[0014] As described above, for the purpose of this reagent, it is important that a molecule capable of binding to an anti-PF4 antibody, such as PF4 protein, does not form a complex with heparin or a heparin surrogate before covalently binding to the support.

[0015] A third aspect of the present disclosure relates to a reagent obtained by the method defined in the second aspect.

[0016] A fourth aspect of the present disclosure provides a composition comprising the reagent defined in the first or third aspect of the present disclosure.

[0017] A fifth aspect of the present disclosure relates to the use of the reagent defined in the first or third aspect, or the composition defined in the fourth aspect, for the detection of anti-PF4 antibody.

[0018] A sixth aspect of the present disclosure relates to the use of the reagent defined in the first or third aspect, or the composition defined in the fourth aspect, for the differential detection of anti-PF4 antibody (which does not detect anti-PF4 / heparin antibody) and / or the diagnosis of a thromboembolic event not induced by heparin, such as VITT.

[0019] A seventh aspect of the present disclosure relates to an in vitro method for detecting a human anti-PF4 antibody, which includes (i) contacting the reagent defined in the first or third aspect, or the composition defined in the fourth aspect, with a biological sample obtained from a subject, and (ii) analyzing the sample to detect an immune complex formed by the reagent and the anti-PF4 antibody, wherein the formation of the immune complex indicates the presence of the anti-PF4 antibody in the sample.

[0020] Since anti-PF4 antibodies are typically found in thrombotic events not induced by heparin, the detection of anti-PF4 antibodies enables the diagnosis of these conditions. Thus, an eighth aspect relates to an in vitro method for the diagnosis of heparin-uninduced thrombotic events, comprising: (i) contacting a reagent defined in the first or third aspect, or a composition defined in the fourth aspect, with a biological sample obtained from a subject; and (ii) analyzing the sample to detect an immune complex formed by the reagent and an anti-PF4 antibody, wherein the formation of the immune complex indicates a heparin-uninduced thrombotic event. Heparin-uninduced thrombotic events include, in addition to VITT, thrombotic events not induced by heparin or vaccination, such as spontaneous HIT-like syndrome. The presence of anti-PF4 antibodies in these heparin- or vaccination-uninduced thrombotic events has been confirmed, for example, in patients suffering from spontaneous HIT-like syndrome, as shown in FIG. 7. The presence of anti-PF4 antibodies has also been observed in patients with severe or recurrent thrombosis or thrombocytopenia that is independent of heparin and / or vaccination.

[0021] The method described in the present disclosure overcomes many drawbacks of conventional methods for diagnosing heparin-uninduced thrombotic events. First, the method has high sensitivity. As shown in the following examples, in the tests by the method of the present disclosure, all samples of VITT patients were correctly determined to be positive, and all control samples of healthy patients were negative (Figure 2). The relative light unit (RLU) signals observed in VITT samples were very high, indicating the high sensitivity of the method. Importantly, the method of the present disclosure specifically detects heparin-uninduced thrombotic events (Figure 6) and does not show cross-reactivity with HIT (Figure 2). As is also clear from the following examples, all samples of HIT patients tested by this method were negative. As a result, the method of the present disclosure can differentially diagnose VITT and other heparin-uninduced thrombotic events from HIT. This method becomes a very useful tool for clinicians and can assist in the selection of appropriate treatment methods. For example, when a patient's sample is determined to be positive by this method, it suggests a VITT or a severe thrombotic event not induced by heparin. This information is useful for recommending adjuvant therapies to suppress FcγRIIa-dependent cell activation and reduce massive hypercoagulable states. Typically, this purpose is achieved by administration of intravenous immunoglobulin (IVIG). Furthermore, when it is negative in methods for specifically detecting anti-PF4 / heparin antibodies (e.g., the HemosIL® AcuStar HIT-IgG(PF4-H) assay or the HemosIL® HIT-Ab(PF4-H) assay), the thrombotic event is very likely not induced by heparin, and thus clinicians do not need to discontinue heparin treatment. On the other hand, when a patient's sample is negative by this method but positive in a method for specifically detecting anti-PF4 / heparin antibodies, e.g., the HemosIL® AcuStar HIT-IgG(PF4-H) assay or the HemosIL® HIT-Ab(PF4-H) assay, the thrombotic event is very likely induced by heparin, and thus clinicians should consider discontinuing heparin treatment and need to consider switching to another anticoagulant therapy.Furthermore, if a patient's sample is positive by this method and also positive for anti-PF4 / heparin antibodies, in addition to an adjunctive method that suppresses FcγRIIa-dependent cell activation that causes massive hypercoagulable states such as the administration of IVIG, the clinician should consider discontinuing heparin treatment and consider switching to another anticoagulant therapy.

[0022] A ninth aspect of the present invention relates to a method of recommending or initiating a medical regimen in a subject suspected of having a thrombotic event, the method comprising detecting a human anti-PF4 antibody in a biological sample obtained from the subject by the method defined in the seventh aspect, and, if a human anti-PF4 antibody is detected, recommending or initiating an appropriate medical regimen. An appropriate medical regimen includes measures (such as administration of IVIG) to suppress FcγRIIa-dependent cell activation and reduce hypercoagulable states, and may also include heparin depending on the presence or absence of anti-PF4 / heparin antibodies.

[0023] As a tenth aspect of the present invention, there is provided a kit comprising the reagent defined in the first or third aspect. As an eleventh aspect of the present invention, there is provided a cartridge comprising the reagent defined in the first or third aspect. As a twelfth aspect of the present invention, the kit or cartridge of the present disclosure is used for the detection of anti-PF4 antibodies not induced by heparin or the diagnosis of thrombotic events not induced by heparin.

Brief Description of the Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] All terms used in this specification shall be understood to have their ordinary meanings commonly recognized in the relevant technical field, unless otherwise expressly stated. For specific terms, definitions are provided below and shall be uniformly applied throughout this specification and the entire scope of the claims, unless otherwise expressly stated. However, if a specifically stated definition has a broader meaning, that definition shall apply.

[0026] In this specification, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more". Also, unless otherwise expressly stated, the definite article "the" shall include the plural forms of nouns.

[0027] A first aspect of the present disclosure relates to a reagent comprising: (a) a binding molecule for binding to an anti-PF4 antibody, and (b) a solid support, wherein the binding molecule is covalently bonded to the surface of the solid support.

[0028] Platelet factor 4 protein (PF4) is a small cytokine belonging to the CXC chemokine family and is also known as chemokine (C-X-C motif) ligand 4 (CXCL4). This chemokine is released from platelet α-granules during platelet aggregation and promotes blood coagulation by regulating the action of heparin-like molecules. In one embodiment of the present disclosure, the mature human PF4 protein has the following amino acid sequence: EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYK KIIKKLLES (SEQ ID NO: 1).

[0029] In the present disclosure, an "anti-PF4 antibody" is an immunoglobulin G (IgG) antibody that binds to an epitope of platelet factor 4 protein (PF4) without depending on heparin. An "anti-PF4 / heparin antibody or anti-PF4 / H" is an antibody (IgG, IgA, IgM) that recognizes platelet factor 4 (PF4) associated with heparin or a heparin surrogate. In this specification, the terms "anti-PF4 antibody (or simply anti-PF4)" and "heparin-independent anti-PF4 antibody" are used interchangeably. The terms "anti-PF4 / heparin antibody or anti-PF4 / H antibody" and "anti-PF4 heparin-dependent antibody" are used interchangeably. Antibodies associated with HIT and VITT bind to different epitopes on PF4. Anti-PF4 / H is the antibody found in classical HIT and requires the co-presence of PF4 and a pharmacologically relevant polyanion to exert pathogenicity. Antibodies that bind to PF4 alone (heparin-independent antibodies) are associated with VITT and other thrombotic events not caused by heparin or vaccination. Any pathogenic IgG antibody activates platelets via FcγRIIa. The method of the present invention enables the identification of patients who develop thrombocytopenia and thrombosis in relation to the presence of anti-PF4 antibodies. In particular, the method can diagnose thrombotic events that occur even in the absence of any exposure to heparin. As described above, some of these thrombotic events have been confirmed to occur not only in the absence of heparin but also in the absence of vaccination. This suggests that factors other than pharmacological heparin and COVID-19 vaccines may cause thrombotic diseases associated with anti-PF4.

[0030] A "molecule that binds to anti-PF4 antibody" (also referred to as a "binding molecule" herein) generally refers to a molecule that can bind to an anti-PF4 antibody in a manner similar to the PF4 protein. It is usually a polypeptide, but is not limited thereto. The binding molecules in the present disclosure include related epitopes of PF4. These epitopes may be linear epitopes consisting of a continuous amino acid sequence or conformational epitopes composed of non-continuous amino acids that are brought close together by protein folding. Conformational epitopes may include both continuous and non-continuous amino acids.

[0031] The binding molecule may be a full-length PF4 protein, preferably a human PF4 or a protein having high homology thereto. In one embodiment, it is PF4 derived from a mammal, and in another embodiment, it is PF4 derived from a primate. In a specific embodiment, human PF4 is used as the binding molecule. In one embodiment, the binding molecule is mature PF4. The PF4 protein can be of natural or synthetic origin. Also, in one embodiment, it is an isolated naturally occurring PF4, and in another embodiment, it is recombinant PF4. In one embodiment, the binding molecule is a polypeptide having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homology with human PF4 (SEQ ID NO: 1). In particular, the binding molecule is the human PF4 shown in SEQ ID NO: 1.

[0032] As used herein, "identity" refers to the percentage of amino acid residues that match when two sequences are optimally aligned. In an optimal alignment, positions are homologous when the amino acid residue at a position in the first sequence matches the residue at the corresponding position in the second sequence. The percentage of identity is calculated as the ratio of the residues that match to the total length compared (i.e., identity (%) = (number of matching positions / total number of positions compared) × 100). Gaps, i.e., positions that are present in one sequence but not in the other, are counted as non-matching positions.

[0033] There are numerous mathematical algorithms known for rapidly determining the optimal alignment between arrays and calculating homology, and they are implemented in multiple software programs. In the present disclosure, the homology between amino acid sequences is determined using an algorithm based on global alignment (Needleman-Wunsch algorithm) (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453). In particular, it is carried out using the default settings of the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) or the BLAST Global Alignment tool (Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410). When the sequence lengths are approximately the same, local alignment can also be used.

[0034] In one embodiment, the binding molecule is a polypeptide capable of binding to an anti-PF4 antibody, and can, for example, bind to a PF4-reactive antibody in a patient having a thrombotic event not induced by heparin (e.g., VITT). For example, the binding molecule is a fragment of the PF4 protein, and as long as it maintains the ability to bind to the anti-PF4 antibody, such fragment is applicable. It has been reported that a conformational epitope within the PF4 protein binds to an anti-PF4 antibody (Huynh, A. et al). This conformational epitope is defined by the residues R22, H23, E28, K46, N47, K50, K62, and K66 of SEQ ID NO: 1. In one embodiment, the binding molecule is a polypeptide comprising the residues R22, H23, E28, K46, N47, K50, K62, and K66 of SEQ ID NO: 1. This conformational epitope is also denoted as -RH-E-KN-K-K-K-. In another embodiment, the binding molecule is a PF4 fragment capable of binding to an anti-PF4 antibody (hereinafter, "antibody-binding PF4 fragment"). Assays for evaluating the binding of an anti-PF4 antibody are known in the art and can be applied by those skilled in the art to evaluate whether a PF4 fragment or a binding molecule can bind to an anti-PF4 antibody.

[0035] In another embodiment, the binding molecule is an anti-idiotype antibody of an anti-PF4 antibody. An anti-idiotype antibody recognizes the antigen-binding site of an antibody and mimics its structure and / or function. Methods for obtaining anti-idiotype antibodies against a specific antibody are known to those skilled in the art. In yet another embodiment, the binding molecule is an aptamer.

[0036] In certain embodiments, the reagent according to the first aspect of the present disclosure consists essentially of (a) a molecule that binds to an anti-PF4 antibody and (b) a solid support. Here, the binding molecule is covalently bound to the surface of the solid support. In particular, the binding molecule is selected from PF4, an antibody-binding PF4 fragment, and an anti-idiotype antibody of the anti-PF4 antibody. The binding molecule does not contain heparin or a heparin surrogate (i.e., does not form a complex with heparin or its surrogate). In another particular embodiment, the reagent according to the first aspect of the present disclosure consists essentially of (a) PF4 and (b) a solid support. Here, PF4 is covalently bound to the surface of the solid support. Again, the PF4 in (a) does not form a complex with heparin or a heparin surrogate. That is, the molecule recognized and bound by the anti-PF4 antibody does not contain heparin or its surrogate.

[0037] The term "heparin surrogate" is understood by those skilled in the art to be any anionic compound that binds to the heparin-binding site within the PF4 protein. An example of a heparin surrogate is polyvinyl sulfonic acid (PVS).

[0038] In the reagent according to the first aspect of the present disclosure, there are embodiments where the solid support is a particle. Here, a "particle" is generally understood as a small local object having physical or chemical properties such as shape, volume, density, mass, etc. Particles can have a variety of sizes and shapes, ranging from subatomic particles like electrons, microscopic particles like atoms and molecules, to macroscopic particles like powders and other granular materials. Therefore, the solid support in the reagent of the present disclosure can be considered a particulate solid support.

[0039] In one embodiment of the present disclosure, the solid support is nanoparticles or microparticles. The composition of the nanoparticles and microparticles can be diverse, such as metals, organic substances, metal-organic compounds, polymers, quantum dots, carbon structures, etc., but is not limited thereto. The nanoparticles and microparticles have at least two dimensions in the nanoscale, preferably all three dimensions are in the nanoscale. Also, their shapes and sizes can take various forms such as spherical, rod-shaped, disk-shaped, tubular, hemispherical, etc. In one embodiment, the particulate solid support is spherical or bead-shaped, rod-shaped, or tubular.

[0040] In one embodiment of the present disclosure, the particulate solid support contains or is mainly composed of a metal or a metal alloy. In particular, there are embodiments that contain or are mainly composed of a magnetic metal or a magnetic metal alloy. Here, as the magnetic material, any magnetic material widely known to those skilled in the art can be used. Examples include, but are not limited to, Fe2O3, Fe3O4, FePt. Also, metals suitable for the solid support according to the present disclosure include gold, platinum, silver, titanium, zinc, cerium, iron, copper, thallium, and combinations and alloys thereof. In another embodiment, the particulate solid support contains or is mainly composed of silica. Furthermore, in another embodiment, the particulate solid support contains or is mainly composed of a metal-organic structure. There are also embodiments that contain or are mainly composed of quantum dots. Furthermore, embodiments in which the particulate solid support contains (e.g., graphite) or is mainly composed of carbon are also included.

[0041] In another embodiment of the present disclosure, the solid support contains or is mainly composed of a polymer. Polymer-based nanoparticles are widely known to those skilled in the art and can also be used in the reagents of the present disclosure. Polymers suitable in the present disclosure include latex, polystyrene, alginic acid, gelatin, polylactic acid, chitosan, poly(lactic-co-glycolic acid) copolymer, and polycaprolactone. In a specific embodiment, the solid support contains or is mainly composed of latex or polystyrene.

[0042] Binding molecules, such as PF4, may be covalently attached to a solid support and can be attached, for example, by an amide bond, an amine bond, or a thioether bond. In certain embodiments of the first aspect of the present disclosure, PF4, which is a binding molecule, is attached to the solid support via an amide bond. In some embodiments, the reagent of the present disclosure further includes a spacer. Here, a "spacer" is a branched or unbranched carbon chain containing at least one carbon, with one side attached to the solid support and the other side attached to the binding molecule. Preferably, PF4, which is a binding molecule, is attached to the spacer via an amide bond. In certain embodiments, the reagent of the present disclosure has the following structure: SS-(A)-CO-NH-(A)-PF4 SS-(A)-S-(A)-PF4 SS-(A)-CO-S-(A)-PF4 Here, SS is the solid support, A is the spacer, which may or may not be present in the reagent. PF4 is a binding molecule capable of binding to an anti-PF4 antibody.

[0043] The type of spacer in the present disclosure is not limited as long as it does not adversely affect the function of the reagent, particularly the binding to the anti-PF4 antibody. Non-limiting spacers suitable for the reagent of the present disclosure include amino acids, peptides, polyethylene glycol, alkyl groups, and the like. In certain embodiments, the reagent according to the first aspect has a structure of SS-CO-NR-PF4. Here, R is hydrogen or an alkyl group having 1 to 4 carbon atoms. In a further particular embodiment, the reagent has a structure of SS-CO-NH-PF4.

[0044] Methods for attaching polypeptides and proteins to solid supports, particularly various types of particles such as beads, are widely known to those skilled in the art. Di Marco et al. (Int J Nanomedicine. 2010; 5:37-49) and Biju (Chem. Soc. Rev., 2014, 43, 744-764) have shown an overview of such methods, which are hereby incorporated by reference in this disclosure. In one method, bifunctional crosslinker molecules can be used to chemically attach polypeptides to solid supports. These methods are available for producing the reagents of the present disclosure.

[0045] As a second aspect of the present disclosure, a method for producing the aforementioned reagent is provided. The method includes (i) contacting a solid support with a solution containing a crosslinker and adding a catalyst if necessary, and (ii) adding a molecule capable of binding to an anti-PF4 antibody. (i) and (ii) may be carried out in sequence (i.e., (ii) after (i)), or simultaneously (i.e., contacting the solid support with a solution containing both the crosslinker and the binding molecule and adding a catalyst if necessary). All embodiments regarding the aforementioned binding molecule and solid support are also applicable to the method according to the second aspect of the present disclosure.

[0046] The solid support may contain a functional group capable of reacting with the crosslinker. In certain embodiments, the solid support contains a functional group of any of carboxyl group, hydroxyl group, sulfhydryl group, tosyl group, and amino group. In another embodiment, the solid support is modified with a negatively charged functional group. In a specific embodiment, the solid support is carboxylated.

[0047] There are many types of crosslinkers available in the market, and they are selected according to specific requirements such as chemical specificity, spacer arm length, reversibility, etc. Table 1 shows non-limiting examples of crosslinkers that can be used in the present disclosure. Table 1: Crosslinkers for covalently binding polypeptides and proteins to particles based on each function [Table 1] Abbreviations: SIAB is N-succinimidyl-(4-iodoacetyl)aminobenzoate, SMCC is succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, MBS is m-maleimidobenzoyl-N-hydroxysuccinimide ester, SPDP is succinimidyl 3-(2-pyridyldithio)propionate, SPMB is succinimidyl (4-p-maleimidophenyl)butyrate, EDC is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, DCC is dicyclohexylcarbodiimide, NHS is N-hydroxysuccinimide, EGS is ethylene glycol bis(succinimidyl succinate), BS3 is bis-(succinimidyl) suberate, BMME is bis(maleimidomethyl) ether, DSS is disuccinimidyl suberate, DSP is dithiobis(succinimidyl propionate), -NHS is N-hydroxysuccinimide.

[0048] In certain embodiments of the present disclosure, the crosslinking agent is a carbodiimide. In certain embodiments, the method is carried out in the presence of a catalyst (e.g., NHS).

[0049] In other embodiments, the binding molecule may be immobilized on a solid support using molecular engineering or genetic engineering techniques. For example, intein-mediated protein ligation, click chemistry, and other similar techniques may be used.

[0050] The method according to the second aspect of the present disclosure is carried out under conditions for covalently bonding a binding molecule to a solid support. Appropriate conditions can be set by those skilled in the art. In certain embodiments, it includes incubating the solid support with a solution containing a crosslinking agent (including a catalyst if necessary) and the binding molecule for a sufficient time (e.g., 20 minutes to 5 hours). In certain specific embodiments, the incubation time is 1 hour to 4 hours. When the method of the present disclosure is carried out in two independent steps, in certain embodiments, the incubation time of (i) is at least 15 minutes (e.g., 15 minutes to 1 hour), and the incubation time of (ii) is 1 hour to 5 hours. In certain specific embodiments, the incubation time of (i) is 20 minutes to 45 minutes, and the incubation time of (ii) is 1 hour to 3 hours. In certain specific embodiments, the incubation is carried out at room temperature. In another embodiment, the incubation is carried out under continuous stirring. The method may be carried out in the presence of a buffer. In certain specific embodiments, (i) and (ii) are carried out at a slightly acidic pH (e.g., pH 5 to 6.5).

[0051] In some embodiments of the present disclosure, after (ii), it further includes (iii) for blocking the PF4 non-binding sites of the solid support to prevent non-specific binding. Examples of this blocking agent include, but are not limited to, albumin, polyethylene glycol (PEG), and casein. In certain specific embodiments, the blocking agent is albumin.

[0052] The method may further include a step of activating the solid support before the step of (i). The activation is carried out, for example, by washing with a mildly acidic buffer at pH 5 to 6.

[0053] As described above, in most embodiments, the solid support contains a functional group. Solid supports with functional groups are commercially available, and functionalized particles of various sizes and shapes are readily available. If the solid support does not contain a functional group, a step of functionalizing the solid support may be included as the first step of the method of the present disclosure. This step can be carried out by those skilled in the art using standard techniques depending on the desired functional group.

[0054] As a third aspect of the present disclosure, a reagent obtained by the aforementioned method is provided. This reagent can bind to an anti-PF4 antibody.

[0055] Furthermore, the present disclosure provides a composition containing the reagent of the present invention. In certain embodiments, the composition can further contain components such as a buffer, a stabilizing compound, a preservative (e.g., an antibacterial agent).

[0056] As a further aspect of the present disclosure, there is provided the use of a reagent as defined in the first or third aspect. These reagents are used for the detection of anti-PF4 antibodies, or for the diagnosis of thromboembolic events not induced by heparin (e.g., vaccine-induced thrombotic thrombocytopenia (VITT) and other thromboembolic events not induced by heparin or vaccination). "Thromboembolic events not induced by heparin" means thromboembolic events that occur when anti-PF4 antibodies caused by factors other than heparin activate platelets, other blood cells, or vascular endothelium. Non-limiting examples of thromboembolic events not induced by heparin include VITT, autoimmune HIT-like syndrome, and idiopathic thrombosis. "Spontaneous HIT-like syndrome" refers to a disease in which a patient develops thrombocytopenia and thrombosis without heparin exposure and has platelet-activating anti-PF4 antibodies. It has been shown that the sera of these patients can activate platelets even in the absence of heparin, that is, they contain anti-PF4 heparin-independent antibodies (anti-PF4). The present disclosure shows that some patients with autoimmune HIT-like syndrome may have both anti-PF4 heparin-dependent antibodies (anti-PF4 / H) and anti-PF4 heparin-independent antibodies (anti-PF4), while some patients have only anti-PF4 (see FIG. 7). In certain embodiments of the present disclosure, the reagent as defined in the first or third aspect is used for the diagnosis of VITT. In another embodiment, this reagent is used for the diagnosis of VITT caused by COVID-19 vaccination. The reagent is also used for the diagnosis of thromboembolic events not induced by heparin or vaccination (e.g., spontaneous HIT-like syndrome). In certain embodiments, the reagent of the first or third aspect of the present disclosure is also used for the diagnosis of other unclear thrombotic diseases. In all of the above-described embodiments, the use of the reagents of the first and third aspects of the present disclosure is applicable.

[0057] In patients presenting with thrombocytopenia and catastrophic thrombosis in veins and arteries, anti-PF4 heparin-dependent antibodies and heparin-independent antibodies should be considered as potential causes, and this possibility should be borne in mind even if the patient has not been exposed to heparin. This is extremely important because these patients require adjuvant therapies (e.g., IVIG administration). The methods of the present disclosure enable rapid identification of diseases that have heretofore been difficult to diagnose.

[0058] Furthermore, severe atypical cases of HIT ("autoimmune HIT") have been shown to be associated with both HIT-like (heparin-dependent) antibodies and VITT-like (heparin-independent) antibodies (see Figure 8). The important concept here is that in the presence of anti-PF4 heparin-independent antibodies, adjuvant therapies such as high-dose administration of IVIG are required in addition to anticoagulant therapy. Administration of IVIG is essential to suppress the severe hypercoagulable state caused by anti-PF4 IgG. In certain embodiments, the reagents of the present disclosure are used for the diagnosis of atypical HIT.

[0059] As a seventh and eighth aspect of the present disclosure, in vitro methods for detecting anti-PF4 antibodies and in vitro methods for diagnosing thromboembolic events not induced by heparin are provided. These methods involve (i) contacting a biological sample derived from a subject with a reagent or a composition containing the reagent defined in the first or third aspect, and (ii) analyzing the sample to determine the presence or absence of a complex formed between the reagent and the anti-PF4 antibody. The formation of this complex indicates that the sample contains anti-PF4 antibodies not induced by heparin, which serves as an indicator of heparin-independent thromboembolic events. Non-limiting examples of thromboembolic events not induced by heparin include VITT, autoimmune HIT-like syndrome, and idiopathic thrombosis. In certain embodiments, the method is used for the diagnosis of VITT. In another embodiment, the method is used for the diagnosis of VITT caused by COVID-19 vaccination. The method is also used for the diagnosis of thromboembolic events not induced by heparin and vaccination (e.g., autoimmune HIT-like syndrome). Furthermore, the method is applicable to the diagnosis of idiopathic thrombotic complications or atypical HIT.

[0060] In this method, whole blood or its components (e.g., plasma or serum) are used as biological samples. In certain embodiments, the biological sample is citrated plasma or serum. In other embodiments, the biological sample is plasma treated with an anticoagulant such as EDTA, hirudin, PPACK, or plasma containing an anticoagulant aptamer.

[0061] The diagnostic method of the present disclosure is based on the specific binding of the reagent to the anti-PF4 antibody, forming a detectable complex when the anti-PF4 antibody is present in the sample. The reagent contains a binding molecule (a) capable of recognizing and capturing the anti-PF4 antibody. As shown in the following examples, the reagent of the present disclosure does not detect anti-PF4 / H antibodies (heparin-induced anti-PF4 antibodies) (see Figure 2), thereby enabling differential diagnosis between HIT and heparin-independent thromboembolic events.

[0062] In one embodiment, the method is used for the differential diagnosis of heparin-uninduced thrombotic events. For example, the method is for differentiating HIT from heparin-uninduced thrombotic events. In certain embodiments, the method is for differentiating HIT from VITT, particularly for the purpose of distinguishing VITT induced by COVID-19 vaccination from HIT.

[0063] As a ninth aspect of the present disclosure, there is provided a method for recommending or initiating a medical regimen in a patient suspected of having a thrombotic event. The method includes performing the method of the present disclosure on a biological sample obtained from a subject and recommending or initiating an appropriate medical regimen when a human anti-PF4 antibody is detected. As described above, the ability to detect heparin-independent anti-PF4 antibodies is very beneficial to clinicians and is important in selecting an optimal treatment strategy. If a patient's sample is positive by the method of the present disclosure, this suggests the possibility of VITT or other heparin-uninduced thrombotic events, which are often severe. This information helps in recommending adjuvant therapies (e.g., IVIG administration) to suppress FcγRIIa-dependent cell activation and reduce the hypercoagulable state. Furthermore, if the patient sample is positive by the method of the present disclosure but negative by methods specifically detecting anti-PF4 / heparin antibodies (e.g., HemosIL(R) AcuStar HIT-IgG(PF4-H) assay and HemosIL(R) HIT-Ab(PF4-H)), the thrombotic event is very likely not induced by heparin. In this case, the clinician can continue heparin treatment. On the other hand, if the sample is negative by the method of the present disclosure but positive by the HemosIL(R) AcuStar HIT-IgG(PF4-H) assay or the HemosIL(R) HIT-Ab(PF4-H) assay, the thrombotic event is likely to be induced by heparin, and discontinuation of heparin treatment is recommended. It is required to consider IVIG administration or other adjuvant therapies as needed. Furthermore, if positive by both the method of the present disclosure and the HIT-specific test, in addition to discontinuing heparin treatment, it is necessary to combine adjuvant therapies such as IVIG administration. The present disclosure also provides a method for treating a patient having a thrombotic event. The method includes performing the method described in the eighth aspect of the present disclosure, determining whether the patient has a heparin-uninduced thrombotic event, and performing an appropriate medical regimen accordingly. The appropriate medical regimen may include heparin administration.

[0064] As another aspect of the present disclosure, an in vitro method for differential diagnosis between HIT and heparin-independent thrombotic events is provided. The method includes (1) performing the in vitro method described in the seventh aspect of the present disclosure, and (2) performing an in vitro diagnosis of HIT. Here, when the result of (1) is positive and the result of (2) is negative, it is highly likely that the thrombotic event is not related to heparin, and the possibility of HIT is excluded. In this case, the continuation of heparin treatment is possible. On the other hand, when (1) is negative and (2) is positive, HIT is suspected and it is necessary to discontinue the administration of heparin. The diagnosis of HIT can be performed by specific tests such as, for example, the HemosIL(R) AcuStar HIT-IgG(PF4-H) assay or the HemosIL(R) HIT-Ab(PF4-H) assay. The diagnosis of HIT can also be performed by using a functional assay.

[0065] The method of the present disclosure is based on the binding of a reagent and an anti-PF4 antibody to form a detectable immune complex. In certain embodiments, the in vitro method of the present disclosure includes (i) contacting a biological sample with a reagent described in the first or third aspect, or a composition containing the reagent described in the fourth aspect, to form an immune complex, and (ii) analyzing whether an immune complex has been formed. The formation of the immune complex indicates that the sample contains a heparin-independent anti-PF4 antibody.

[0066] The immune complex formed by the reagent and the anti-PF4 antibody may be directly detectable. For example, when a detectable signal changes when the reagent forms an immune complex with the anti-PF4 antibody. This can be achieved by selecting a specific solid support. Solid supports that emit detectable signals are known to those skilled in the art and can be used in the preparation of the reagents of the present disclosure. Representative examples of these solid supports include gold nanoparticles and quantum dots. As another example, the immune complex formed by the reagent and the anti-PF4 antibody may be directly detectable by turbidimetry (turbidimetry assay). This can be achieved by using latex particles as the solid support. It is also possible to achieve this by using polystyrene particles as the solid support.

[0067] In another embodiment of the present disclosure, the aforementioned in vitro method further comprises contacting the mixture obtained in step (i) with a tracer capable of binding to the captured anti-PF4 antibody. In certain embodiments, the tracer comprises a label. In another embodiment, the label is a detectable molecule selected from the following: chemiluminescent molecules, chromogenic molecules, fluorescent molecules, radioactive molecules, quantum dots, colloidal gold, gold nanoparticles. In one embodiment, the label is a molecule that becomes detectable upon contact with a specific trigger. In another embodiment, the tracer comprises an enzyme and is capable of causing a detectable change to a substrate in the sample. In yet another embodiment, the tracer comprises a substrate that exhibits a detectable change in the presence of an appropriate trigger.

[0068] In certain embodiments, the tracer is a labeled anti-human IgG antibody. In particular, there are embodiments where the tracer is an anti-human IgG antibody labeled with a chemiluminescent molecule. Chemiluminescent molecules are widely known to those skilled in the art and include, for example, luminol, isoluminol, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), acridinium, and the like. In certain embodiments, the anti-PF4 antibody or the in vitro method for detecting heparin-uninduced thrombotic events further comprises contacting the mixture obtained in step (i) with a tracer capable of binding to the captured anti-PF4 antibody and a trigger that induces a detectable change in the tracer.

[0069] As a tenth aspect of the present disclosure, there is provided a kit for performing the aforementioned in vitro methods (e.g., detection of anti-PF4 antibodies, diagnosis of heparin-uninduced thrombotic events, or recommendation of an appropriate medical regimen for patients suspected of having a thrombotic event). In certain embodiments, the kit comprises the following components in separate containers: (i) a reagent defined in the first or third aspect, or a composition comprising the reagent defined in the fourth aspect, (ii) a tracer capable of binding to the anti-PF4 antibody. In another embodiment, the kit further comprises a trigger solution for inducing a detectable change in the tracer. In yet another embodiment, the kit comprises a buffer solution, a diluent, a stabilizer, a preservative, and instructions for use regarding the detection of anti-PF4 antibodies or the diagnosis of heparin-uninduced thrombotic events.

[0070] The present disclosure also provides a cartridge for detecting anti-PF4 antibodies, diagnosing thrombotic events, or recommending an appropriate medical regimen. In certain embodiments, the cartridge comprises the following components in separate containers: (i) a reagent defined in the first or third aspect, or a composition comprising the reagent defined in the fourth aspect, (ii) a tracer capable of binding to the anti-PF4 antibody. In another embodiment, the cartridge further comprises a trigger solution for inducing a detectable change in the tracer. In yet another embodiment, the cartridge comprises a buffer solution, a diluent, a stabilizer, a preservative, and instructions for use regarding the aforementioned methods.

[0071] In certain embodiments, the reagents included in the kit or cartridge include particles (e.g., magnetic particles) as solid supports. In another embodiment, the cartridge is for automating the detection of anti-PF4 antibodies in a sample or the diagnosis of thrombotic events and is used to make recommendations for appropriate treatment. The cartridge can include all of the components necessary for the test. For example, the cartridge can include a reagent as a particle suspension, an assay buffer, a tracer, and a diluent. The components within the cartridge are released from individual containers and mixed with the sample to enable an automated test. One of the great advantages of the methods of the present disclosure is the applicability of this automation technology. In certain embodiments, the cartridge is used in combination with an automated analyzer. The automated analyzer is compatible with the cartridge and automatically detects a signal released from the tracer if an anti-PF4 antibody is present in the sample. In certain embodiments, the automated analyzer is an ACL AcuStar (Werfen).

[0072] For the sake of completeness, the present disclosure is further defined by the following numbered embodiments. 1. A reagent comprising: (a) A molecule (binding molecule) for binding to an anti-PF4 antibody, and (b) A solid support,[[]] wherein the binding molecule (a) is covalently attached to the surface of the solid support (b), and the binding molecule (a) does not contain heparin or a heparin surrogate, the reagent. 2. A reagent consisting of:[[]] (a) A molecule (binding molecule) for binding to an anti-PF4 antibody, and (b) A solid support,[[]] wherein the binding molecule (a) is covalently attached to the surface of the solid support (b), and the binding molecule (a) does not contain heparin or a heparin surrogate, the reagent. 3. The reagent according to any one of 1 or 2, wherein the binding molecule is a PF4 protein. 4. The reagent according to 3, wherein the binding molecule is a human PF4 protein. 5. The reagent according to any one of 1 or 2, wherein the binding molecule is a polypeptide having at least 80%, 85%, or 90% sequence identity with human PF4 (SEQ ID NO: 1). 6. The reagent according to 5, wherein the binding molecule is a polypeptide having at least 95%, 97%, 98%, or 99% sequence identity with human PF4 (SEQ ID NO: 1). 7. The reagent according to any one of 1 or 2, wherein the binding molecule is an antibody-binding fragment of PF4 protein. 8. The reagent according to any one of 1 or 2, wherein the binding molecule is an anti-idiotype antibody of an anti-PF4 antibody. 9. The reagent according to any one of 1 or 2, wherein the binding molecule is a polypeptide containing a conformational epitope defined by the residues of R22, H23, E28, K46, N47, K50, K62, and K66 in SEQ ID NO: 1. 10. The reagent according to any one of 1 to 9, wherein the solid support is a particle, particularly a microparticle or a nanoparticle. 11. The reagent according to any one of 1 to 10, wherein the solid support contains a metal or consists of a metal. 12. The reagent according to 11, wherein the metal is a magnetic metal. 13. The reagent according to 12, wherein the metal is selected from Fe2O3, Fe3O4, or FePt. 14. The reagent according to any one of 1 to 10, wherein the solid support contains a polymer or consists of a polymer. 15. The reagent according to 14, wherein the polymer is selected from latex or polystyrene. 16. The reagent according to any one of 1 to 15, wherein the PF4 is bound to the solid support via an amide bond, an amine bond, or a thioether bond. 17. The reagent according to 16, wherein the PF4 is bound to the solid support via an amide bond. 18. The reagent according to any one of 1 to 17, wherein the reagent further comprises a spacer. 19. The reagent according to any one of 1 to 18, wherein the reagent has the following structure: SS-(A)-CO-NH-(A)-PF4, or SS-(A)-S-(A)-PF4, or SS-(A)-CO-S-(A)-PF4 Here, SS is a solid support, A is a spacer (which may or may not be present), and PF4 is a binding molecule capable of binding to an anti-PF4 antibody. 20. The reagent according to 19, wherein the reagent has the following structure: SS-CO-NR-PF4 (wherein R is hydrogen or alkyl having 1 to 4 carbon atoms). 21. A method for producing a reagent, the method comprising the following steps: (i) contacting a solid support with a solution containing a crosslinking agent and optionally a catalyst, (ii) adding a molecule capable of binding to an anti-PF4 antibody (binding molecule), or, (i) contacting a solid support with a solution containing a molecule capable of binding to an anti-PF4 antibody (binding molecule) in the presence of a crosslinking agent and optionally a catalyst. 22. The method according to 21, wherein the solid support comprises a functional group selected from a carboxyl group, a hydroxyl group, a sulfhydryl group, and an amino group. 23. The method according to 21, wherein the solid support comprises a negatively charged functional group. 24. The method according to 23, wherein the solid support is carboxylated. 25. The method according to any one of 21 to 24, wherein the crosslinking agent is carbodiimide. 26. The method according to any one of 21 to 24, wherein the crosslinking agent is an N-hydroxysuccinimide (NHS) ester. 27. The method according to any one of 21 to 24, wherein the crosslinking agent is maleimide. 28. The method according to any one of 21 to 27, wherein the binding molecule is a PF4 protein. 29. The method according to 28, wherein the binding molecule is a human PF4 protein. 30. The method according to any one of 21 to 27, wherein the binding molecule is a polypeptide having at least 80%, 85%, or 90% sequence identity with human PF4 (SEQ ID NO: 1). 31. The method according to 30, wherein the binding molecule is a polypeptide having at least 95%, 97%, 98%, or 99% sequence identity with human PF4 (SEQ ID NO: 1). 32. The method according to any one of 21 to 27, wherein the binding molecule is an antibody-binding fragment of a PF4 protein. 33. The method according to any one of 21 to 27, wherein the binding molecule is an anti-idiotype antibody of an anti-PF4 antibody. 34. The method according to any one of 21 to 27, wherein the binding molecule is a polypeptide comprising a conformational epitope defined by the residues R22, H23, E28, K46, N47, K50, K62, and K66 in SEQ ID NO: 1. 35. The method according to any one of 21 to 34, wherein the solid support is a particle, particularly a microparticle or a nanoparticle. 36. The method according to any one of 21 to 35, wherein the solid support contains or consists of a metal. 37. The method according to 36, wherein the metal is a magnetic metal. 38. The method according to 37, wherein the metal is selected from Fe2O3, Fe3O4, or FePt. 39. The method according to any one of 21 to 35, wherein the solid support comprises a polymer or consists of a polymer. 40. The method according to 39, wherein the polymer is selected from latex or polystyrene. 41. The method according to any one of 21 to 40, wherein the method is carried out in the presence of a catalyst. 42. The method according to 41, wherein the catalyst is N-hydroxysuccinimide (NHS). 43. The method according to any one of 21 to 42, further comprising the step of adding a blocking agent. 44. The method according to any one of 21 to 43, further comprising an initial step of activating the solid support. 45. A reagent obtained by the method according to any one of 21 to 44. 46. A composition comprising the reagent according to any one of 1 to 20 or 45. 47. The composition according to 46, further comprising a buffer, a stabilizing compound, and a preservative (e.g., an antibacterial agent). 48. A method for detecting an anti-PF4 antibody using the reagent according to any one of 1 to 20 or 45, or the composition according to any one of 46 to 47. 49. A method for diagnosing a heparin-independent thrombotic event or atypical HIT using the reagent according to any one of 1 to 20 or 45, or the composition according to any one of 46 to 47. 50. An in vitro method for detecting an anti-PF4 antibody, the method comprising the following steps. (i) Contacting the reagent according to any one of 1 to 20 or 45, or the composition according to any one of 46 to 47, with a biological sample obtained from a subject. (ii) Analyzing the sample to detect a complex formed by the reagent and the anti-PF4 antibody. Detection of the complex indicates that the sample contains the anti-PF4 antibody. 51. The method according to 50 that does not detect anti-PF4 / H antibodies. 52. An in vitro method for diagnosing heparin-uninduced thrombotic events, the method comprising the following steps. (i) Contacting the reagent according to any one of 1 to 20 or 45, or the composition according to any one of 46 to 47, with a biological sample obtained from a subject. (ii) Analyzing the sample and detecting the complex formed by the reagent and the anti-PF4 antibody. The detection of the complex serves as an indicator of heparin-uninduced thrombotic events. 53. The method according to any one of 50 to 52, further comprising the step of contacting the mixture obtained in the step (i) with a tracer capable of binding to the anti-PF4 antibody. 54. The method according to 53, wherein the tracer comprises a detectable label. 55. The method according to 54, wherein the detectable label is selected from a chemiluminescent molecule, a chromogenic molecule, and a fluorescent molecule. 56. The method according to 55, wherein the detectable label is a chemiluminescent molecule. 57. The method according to any one of 53 to 56, wherein the tracer comprises a labeled anti-human IgG antibody. 58. The method according to any one of 50 to 57, wherein the biological sample is selected from whole blood, plasma, or serum. 59. The method according to 58, wherein the biological sample is plasma or serum. 60. The method according to any one of 52 to 59, which is used for differential diagnosis between heparin-uninduced thrombotic events and heparin-induced thrombotic events. 61. The method according to 60, wherein the heparin-induced thrombotic event is HIT. 62. The method according to 60, wherein the heparin-uninduced thrombotic event is selected from VITT or vaccine-uninduced thrombotic events. 63. The method according to 62, wherein the heparin- or vaccine-uninduced thrombotic event is autoimmune HIT or thrombosis of unknown cause. 64. The method according to any one of 60 to 63, further comprising the step of performing an in vitro diagnosis of HIT. 65. The method according to 64, wherein the in vitro diagnosis of HIT is performed using a HemosIL(R) Acustar HIT-IgG(PF4-H) assay or a HemosIL(R) HIT-Ab(PF4-H) assay. 66. The method according to 64, wherein the in vitro diagnosis of HIT is performed using a functional assay. 67. A kit comprising the following. (i) The reagent according to any one of 1 to 20 or 45, or the composition according to any one of 46 to 47. (ii) Optionally, an additional component selected from a tracer capable of binding to an anti-PF4 antibody, an assay buffer, a diluent, and combinations thereof. 68. A cartridge comprising the following. (i) The reagent according to any one of 1 to 20 or 45, or the composition according to any one of 46 to 47. (ii) Optionally, an additional component selected from a tracer capable of binding to an anti-PF4 antibody, an assay buffer, a diluent, and combinations thereof. 69. A method for detecting an anti-PF4 antibody or diagnosing a heparin-uninduced thrombotic event using the kit according to 65 or the cartridge according to 66. 70. A method for recommending or initiating a medical regimen for a subject suspected of having a thrombotic event, the method comprising the following steps. (a) Detecting a human anti-PF4 antibody in a biological sample obtained from the subject using the method according to any one of 50 to 51. (b) Recommending or initiating an appropriate medical regimen when a human anti-PF4 antibody is detected. 71. The method according to any one of 70, which comprises measures for the medical regimen to suppress FcyRIIa-dependent cell activation and reduce the hypercoagulable state. 72. The method according to 71, wherein the measure comprises administration of intravenous immunoglobulin (IVIG). 73. The method according to any one of 70 to 72, further comprising the step of detecting anti-PF4 / H antibody in a sample of a subject, (a) When the sample is positive for anti-PF4 antibody and negative for anti-PF4 / H antibody, administer heparin. (b) When the sample is positive for anti-PF4 / H antibody, discontinue heparin Method.

[0073] In this specification and the claims, the term "comprise" and its derivatives do not exclude other technical features, additives, components, or steps. Further, the term "comprise" shall be taken to include "consisting of". Additional objects, advantages, and features of the present invention will be apparent to those skilled in the art by reviewing this specification, or may be learned by practicing the present invention. The following examples and drawings are provided for the purpose of illustrating the present invention and are not intended to limit the present invention. The reference signs related to the drawings described in parentheses within the scope of the claims are for facilitating the understanding of the claims and are not intended to limit the scope of the claims. Further, the present invention encompasses all combinations of the specific embodiments and preferred embodiments described herein.

Examples

[0074] 1. Preparation of magnetic particles 1.1. Preparation of carboxyl-modified magnetic particles Wash the carboxyl-modified magnetic particles (Dynabeads M-270 Carboxylic Acid) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.8 for 10 minutes with uniform mixing. After removing the supernatant using a magnet, suspend the particle pellet in the working solution of EDAC and activate it at room temperature (20 - 25 °C) for 30 minutes while maintaining constant mixing. Wash the activated particles with coupling buffer (MES or PBS saline buffer) and mix with the PF4 solution. Incubate this mixture at room temperature (20 - 25 °C) for 2 - 3 hours with continuous mixing. Immediately afterwards, place the PF4-bound particles on a magnet, remove the supernatant, and rinse with phosphate buffer containing BSA as a blocking molecule. After the final rinse cycle is completed, separate the particles using a magnetic separator, suspend them in PBS saline buffer containing BSA, and store at 2 - 8 °C. These particles are called "VITT P3" or "carboxyl PF4 particles", and PF4 is covalently bound to the magnetic particles.

[0075] 1.2. Preparation of magnetic particles adsorbed with PF4 Protein adsorption is carried out at pH 7.5 (VITT P1) and pH 5.8 (VITT P2). Wash the carboxyl-modified magnetic particles (Dynabeads M-270 Carboxylic Acid) in 50 mM phosphate buffer (pH 7.5, VITT P1 particles) or 50 mM MES buffer (pH 5.8, VITT P2 particles) for 10 minutes with uniform mixing. After removing the supernatant using a magnet, suspend the particle pellet in the PF4 solution (PF4 diluted in 50 mM phosphate buffer pH 7.5 for VITT P1, PF4 diluted in 50 mM MES pH 5.8 for VITT P2). Incubate this mixture at room temperature (20 - 25 °C) for 2 - 3 hours with continuous mixing. Immediately afterwards, place the PF4-binding particles on a magnet, remove the supernatant, and rinse with phosphate buffer containing BSA as a blocking molecule. After the final rinse cycle is completed, separate the particles using a magnetic separator, suspend them in PBS saline buffer containing BSA, and store at 2 - 8 °C. Table 1. Main characteristics of the magnetic particles used in the examples [Table 2]

[0076] 2. VITT samples Plasma samples from suspected VITT patients were provided through the kindness of Dr. Andreas Greinacher at the University Hospital of Greifswald, the reference laboratory for VITT and HIT. Cases suspected of VITT were identified based on clinical symptoms, radiological findings of thrombosis, in-hospital test results of platelet count and coagulation parameters, and positive test results for PF4-dependent platelet activation antibodies. The case definition is as follows. · Onset within 5 to 28 days after ChAdOx1 nCoV-19 vaccination · Thrombosis and thrombocytopenia (platelet count < 150×10 9 / L), or isolated thrombocytopenia · Evidence of extreme activation of the coagulation system (D-dimer > 4000 pg / L, or > 2000 pg / L with strong clinical suspicion) These cases are classified into three categories: "low probability", "possible", and "high probability" of VITT. All samples analyzed in this study were collected before VITT treatment.

[0077] Plasma samples from patients definitively diagnosed with HIT were provided through the kindness of Dr. Andreas Greinacher. These patients were diagnosed with HIT at the reference laboratory for VITT and HIT at the University Hospital of Greifswald.

[0078] Plasma samples from patients with thrombosis of unknown origin were also provided through the kindness of Dr. Andreas Greinacher. These patients are those in whom HIT and VITT were ruled out after appropriate tests for HIT and VITT. Plasma from healthy donors was used as a control sample.

[0079] 3. Measurement of VITT Using the ACL AcuStar (trademark) system, samples were analyzed by the following assays. · Commercially available anti-PF4 ELISA (LIFECODES PF4 IgG assay) · HemosIL (registered trademark) AcuStar (trademark) HIT-IgG (PF4-H) (Werfen) · VITT assay using the magnetic particles prepared in Section 1 For commercially available reagent kits, tests were conducted according to the manufacturer's instructions. For the VITT assay, the following protocol was applied. 1. 15 μL of sample containing anti-PF4 antibody was diluted 10-fold with sample diluent (135 μL) and then mixed with the assay reagent. 2. 15 μL of the diluted sample was dispensed into a cuvette and mixed with 70 μL of assay buffer (AB) and 20 μL of magnetic particles (MPs). The mixture was incubated for 8.5 minutes. 3. The formed complex (particles containing anti-PF4 antibody) was separated using a magnet, the complex was aspirated and removed, and washed with the rinse solution. This washing step was repeated 3 times. 4. 160 μL of tracer was dispensed into the complex and incubated for 9.5 minutes. The obtained complex (particles containing antibody and tracer) was separated using a magnet and washed 4 times with the rinse solution. 5. 190 μL of trigger solution A and 200 μL of trigger solution B were dispensed to initiate the chemiluminescence reaction. 6. The results of relative luminescence intensity (RLU) were reported.

[0080] The RLU values in the ACL AcuStar (trademark) system showed values proportional to the concentration of anti-PF4 antibody (the analyte) in the sample. In HemosIL (registered trademark) AcuStar HIT-IgG (PF4-H), the signal (RLU) was proportional to the anti-PF4 antibody concentration in the VITT assay sample.

[0081] 4. Results: Detection performance of VITT by the reagent of the present invention As shown in Figure 2, the reagent of the present invention (VITT P3) is obtained by covalently binding only PF4 protein to magnetic particles, accurately detecting anti-PF4 antibodies in VITT-positive plasma, but hardly detecting antibodies in samples from HIT patients. Similarly, no reactivity was observed in plasma samples from healthy donors. It was possible to set a threshold (dashed line) at which the VITT assay was positive only for VITT samples. These results indicate that the VITT assay based on the reagent of the present invention is highly specific for the detection of VITT and can distinguish VITT from HIT. Furthermore, as expected, the HemosIL(R) Acustar HIT-lgG(PF4 / H) assay using PF4 / PVS complex on magnetic particles was unable to detect vaccine-induced anti-PF4 antibodies in VITT samples. This is shown in Figure 3, where plasma samples from VITT patients showed a high RLU (relative light unit) response only when VITT P3 was used.

[0082] Also, it was found that it is essential to covalently bind PF4 to magnetic particles in order to detect anti-PF4 antibodies in all VITT samples. As is also clear from the results shown in Figure 4, when VITT P3 (covalent binding of PF4 protein to carboxylated magnetic particles) was used, plasma from all VITT patients showed a high RLU response. On the other hand, VITT P1 and VITT P2 (adsorption of PF4 protein to carboxylated magnetic particles) recognized only one of the VITT patient samples tested. Furthermore, no VITT plasma was recognized in the HemosIL® Acustar HIT-lgG(PF4 / H) assay. These results were also confirmed by the results of an assay comparing VITT P3 particles and VITT P1 particles using plasma samples from more suspected VITT patients (Figure 5).

[0083] Figure 6 shows the results of analyzing two plasma samples from patients with thrombotic complications of unknown cause, in which HIT and VITT were ruled out, using HemosIL® Acustar HIT-lgG(PF4 / H) (HemosIL® HIT) and the VITT P3 assay / prototype. As shown in Figure 6, the anti-PF4 antibodies in these samples could not be detected by the HemosIL® Acustar HIT IgG assay, but the anti-PF4 antibodies in the two plasma samples could be accurately detected by the VITT assay.

[0084] 5. Detection performance of heparin-independent thrombotic events by the reagent of the present invention Figure 7 shows a VITT-like pattern (thrombocytopenia, thrombosis, strongly positive anti-PF4 / heparin IgG EIA, and PIPA), and shows the test results of six patients without a history of heparin or vaccination in the HemosIL® AcuStar HIT-lgG(PF4-H) assay and the new VITT P3 assay / prototype. Three patients had both anti-PF4 heparin-dependent antibodies and anti-PF4 heparin-independent antibodies. On the other hand, the other three patients had only anti-PF4 heparin-independent antibodies. These results are very important and suggest that these patients may benefit from additional treatment with IVIG.

[0085] 6. Serum analysis of patients with autoimmune HIT (aHIT) Serum from patients clinically suspected of aHIT was analyzed using the Acustar HIT-lgG(PF4 / H) assay and the VITT P3 assay / prototype. As a result, it was revealed that 63% of aHIT patients had anti-PF4 antibodies (Figure 8).

[0086] 7. Conclusion VITT-like anti-PF4 antibodies are associated with severe immune-mediated thrombotic disorders independent of heparin. Many of these patients have been undiagnosed because they test negative in rapid HIT assays and heparin-dependent functional assays. The method of the present invention enables the identification of these patients (also in future clinical studies), the discrimination between HIT-like antibodies and VITT-like antibodies, and rapid clinical decision-making.

[0087] Cited References Greinacher et al, "Thrombotic Thrombocytopenia after ChAdOxI nCov-19 Vaccination". DOI: 10.1056 / NEJMoa2104840. Platton et al, “Evaluation of laboratory assays for anti- platelet factor 4 antibodies after ChAdOxI nCOV- 19 vaccination”. DOI: 10.1111 / jth.15362. Favaloro et al, 2022 “Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT / HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT / TTS” DOI: 10.3390 / antibl 101000 Di Marco M, Shamsuddin S, Razak KA, et al. Overview of the main methods used to combine proteins with nanosystems: absorption, bioconjugation, and encapsulation. Int J Nanomedicine. 2010; 5:37-49. Biju V. Chem. Soc. Rev., 2014,43, 744-764 (D0l:10.1039 / C3CS60273G) Huynh, A. et al. Nature volume 596, pages 565-569 (2021) (DOI: 10.1038 / s41586-021-03744-4)

Claims

1. An in vitro method for detecting an anti-PF4 antibody, comprising: (i) contacting a whole blood, plasma or serum sample obtained from a subject with a reagent comprising: (a) a binding molecule selected from the group consisting of platelet factor 4 protein (PF4), a fragment of PF4 capable of binding to an anti-PF4 antibody, and an anti-idiotype antibody of an anti-platelet factor 4 antibody (anti-PF4 antibody); and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and the binding molecule (a) does not contain heparin or a heparin surrogate; and (ii) analyzing the sample to detect a complex formed by the reagent and the anti-PF4 antibody, wherein detection of the complex indicates that the sample contains an anti-PF4 antibody. A method comprising the above steps.

2. An in vitro method for diagnosing a non-heparin-induced thrombotic event, comprising: (i) contacting a whole blood, plasma or serum sample obtained from a subject with a reagent comprising: (a) a binding molecule selected from the group consisting of platelet factor 4 protein (PF4), a fragment of PF4 capable of binding to an anti-PF4 antibody, and an anti-idiotype antibody of an anti-platelet factor 4 antibody (anti-PF4 antibody); and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and the binding molecule (a) does not contain heparin or a heparin surrogate; and (ii) analyzing the sample to detect a complex formed by the reagent and the anti-PF4 antibody, wherein detection of the complex indicates a non-heparin-induced thrombotic event. A method comprising the above steps.

3. The in vitro method according to claim 1 or 2, wherein the non-heparin-induced thrombotic event is selected from vaccine-induced immune thrombotic thrombocytopenia (VITT) and non-vaccine-induced immune thrombotic thrombocytopenia.

4. The in vitro method according to any one of claims 1 to 3 for differential diagnosis between a non-heparin-induced thrombotic event and heparin-induced thrombocytopenia (HIT).

5. The in vitro method according to any one of claims 1 to 4, further comprising determining that there is no anti-PF4 heparin-dependent antibody (anti-PF4 / H) in the sample.

6. ​ ​ The in vitro method according to any one of claims 1 to 5, further comprising contacting the mixture obtained in (i) with a tracer capable of binding to the anti-PF4 antibody.

7. The in vitro method according to any one of claims 1 to 6, wherein the tracer comprises a labeled anti-human IgG antibody.

8. The reagent consists essentially of (a) a binding molecule selected from the group consisting of platelet factor 4 protein (PF4), a fragment of PF4 capable of binding to an anti-PF4 antibody, and an anti-idiotype antibody of an anti-platelet factor 4 antibody (anti-PF4 antibody), and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and the binding molecule (a) does not contain heparin or a heparin surrogate. The in vitro method according to any one of claims 1 to 7.

9. The in vitro method according to any one of claims 1 to 8, wherein the binding molecule is PF4, particularly human PF4.

10. The in vitro method according to any one of claims 1 to 9, wherein the PF4 is bound to the solid support via an amide bond.

11. The reagent is SS-(A)-CO-NH-(A)-PF4, or SS-(A)-S-(A)-PF4, or SS-(A)-CO-S-(A)-PF4 (wherein SS is a solid support, A is a spacer that may or may not be present in the reagent, and PF4 is a binding molecule capable of binding to an anti-PF4 antibody). The in vitro method according to any one of claims 1 to 10, having the structure of.

12. The in vitro method according to any one of claims 1 to 11, wherein the solid support is a particle, particularly a microparticle or a nanoparticle.

13. The in vitro method according to any one of claims 1 to 12, wherein the solid support contains a magnetic metal or consists essentially of a magnetic metal.

14. A method for recommending or initiating a medical regimen for a subject suspected of suffering from a thrombotic event, the method comprising: (a) detecting a human anti-PF4 antibody in a biological sample obtained from the subject by the method according to any one of claims 1 and 6 to 13, and (b) recommending or initiating an appropriate medical regimen when a human anti-PF4 antibody is detected. The method comprising.

15. The medical regimen according to claim 14, comprising means for reducing FcγRIIa-dependent cell activation that causes a hypercoagulable state, in particular, the means comprising administering intravenous immunoglobulin (IVIG).

16. further comprising detecting anti-PF4 / H antibody in a sample of the subject, wherein (a) heparin may be administered when the sample is positive for anti-PF4 antibody and negative for anti-PF4 / H antibody, and (b) heparin is discontinued when the sample is positive for anti-PF4 / H antibody The method according to claim 14 or 15.

17. (i) a reagent according to any one of claims 1 to 6 to 13, (ii) a further component selected from a tracer, an assay buffer, a diluent, and combinations thereof that can bind to an anti-PF4 antibody, and (iii) instructions for use for detecting an anti-PF4 antibody, diagnosing a thromboembolic event not induced by heparin, or recommending or initiating a medical regimen in a subject suspected of having a thromboembolic event A kit or cartridge comprising.

18. The kit or cartridge according to claim 17, wherein the solid support of the reagent is a particle.

19. Use of a reagent according to any one of claims 1 to 6 to 13, or a kit or cartridge according to claim 17 or 18, for detecting an anti-PF4 antibody, diagnosing a thromboembolic event not induced by heparin, or recommending or initiating a medical regimen for a subject suspected of having a thromboembolic event.

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