RU2779197C9 - Method for determination of apolipoprotein e4 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to a method for detection of apolipoprotein E isotype 4 (hereinafter – ApoE4) or its fragments in a patient’s blood sample and its use. The method for in vitro detection of apolipoprotein E isotype 4 (ApoE4) or its fragments in a subject’s blood sample includes stages of: contact of the specified sample with a solid phase, where the solid phase is not covered with any binding agent or capturing molecule; contact of the specified sample simultaneously or sequentially with at least one binding agent, which specifically binds to ApoE4 to form, thereby, an ApoE4-binding agent complex, where the specified binding agent is an antibody, or a functional fragment of the antibody, or frame protein other than IgG, where ApoE4 or its fragments are immobilized on the specified solid phase, and the binding agent binds to ApoE4, and where binding of ApoE4 or the ApoE4-binding agent complex with the specified solid phase occurs in the presence of a detergent, and detection of the ApoE4-binding agent complex, where ApoE4 fragments contain at least 6 amino acids in length, provided that amino acid 112, which is arginine, is included in the specified fragment, where a sequence including amino acid 112 relates to SEQ ID NO:1. A kit for detection of ApoE4 in vitro in a subject’s blood sample is described. The use of the kit is described. The use of a method is also described for stratification of the risk or stratification of the use of therapeutic measures for a treatment or a pathological condition associated with increased levels of ApoE4 and for detection of the risk of development of Alzheimer’s disease, where the increased level of ApoE4 above the determined threshold value is prognostic for an increased risk of development of Alzheimer’s disease.

EFFECT: above-mentioned group of solutions allows for simple and reliable specific detection of apolipoprotein E isotype 4 or its fragment in a subject’s blood sample.

22 cl, 4 tbl, 8 ex, 7 dwg

Description

The present invention provides a simple and cost-effective method for determining apolipoprotein E (ApoE) status in subjects. Such subjects include, but are not limited to, patients with cognitive impairment, dementia, and/or Alzheimer's disease (AD).

Among various susceptibility genes associated with the risk of developing late-onset Alzheimer's disease (LOAD) and other neurological disorders, ApoE has been identified as a strong genetic determinant (Leduc et al., 2011). The human APOE gene is found as three polymorphic alleles ε2, ε3, and ε4, with a worldwide frequency of 8.4%, 77.9%, and 13.7%, respectively. Apolipoprotein E (ApoE) consists of 299 amino acids and is associated with lipoproteins in plasma and cerebrospinal fluid (Leduc et al., 2011). Differences between the three isoforms of ApoE are limited to amino acids 112 and 158 (SEQ ID NO: 1) where cysteine or arginine is present: ApoE2 (cys112, cys158), ApoE3 (cys112, arg158) and ApoE4 (arg112, arg158). In addition, the risk of developing AD is highly correlated with the ApoE4 allele. Subjects with one ApoE4 allele have a five to six times higher risk of developing AD, and subjects with two ApoE4 alleles have a more than 20-fold risk of developing AD. In addition, the presence of one (heterozygous) or two copies (homozygous) ApoE4 allele correlates with an earlier age of onset of about 10-20 years compared to non-carriers in patients with late onset disease (Verghese et al., 2011, Weisgraber & Mahley 1996, Leduc et al., 2011). Research results indicate that subjects who meet the clinical criteria for mild cognitive impairment (MCI) and who are also ApoE4 positive are more likely to progress to AD over several years than subjects who are ApoE4 negative (Aggarwal et al. ., 2005). Therefore, ApoE genotyping can be used to identify MCI patients at higher risk of progression from MCI to AD. ApoE4 carriers also respond differently to several AD treatments, such as the acetylcholinesterase inhibitor, tacrine, reinforcing the importance of patients knowing their genotype (Cacabelos et al. 2012).

In addition, ApoE status can be used to predict other diseases (subjects carrying the ApoE4 isoform have a higher risk of developing cardiovascular disease (CVD) and also have a higher risk of developing prostate cancer (US patent No. 5945289)). On the other hand, the ApoE4 genotype has been shown to protect against severe liver damage during hepatitis C, increase viral load and accelerate disease progression in HIV-infected subjects, and increase the risk of developing AD as a result of herpes simplex virus infection (WO 03 /060158 A2). Treatment of age-related macular degeneration is also more effective in the ApoE4 isoform (Bakbak et al., 2015).

The ApoE genotype as a risk marker for developing Alzheimer's disease was first described in Mayeux et al., 1998 and US Pat. No. 5,508,167. There are many possibilities to determine the genetic status of ApoE in subjects using various genotyping methods.

ApoE4 has been shown to be much more sensitive to proteolysis than ApoE3 and ApoE2, and carboxyl-truncated forms of ApoE4 have been found in the brains of AD patients and ApoE4 transgenic mice. ApE4 fragmentation has been suggested to be an early event in the pathogenesis of AD (Brecht WJ et al., 2004. Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice. PNAS 24 , 2527-2534).

Several specific, possibly neurotoxic, carboxyl-truncated fragments of ApoE4 have been found in the brains of AD patients:

a 19 kDa fragment of ApoE4 1-165 (Huang Y. et al., 2001. Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons. 98, 8838-8843);

ApoE4 fragment 1-185 (Dafnis I. et al., 2010. An apolipoprotein E4 fragment can promote intracellular accumulation of amyloid peptide beta 42. 115, 873-884);

fragments of ApoE4 1-191 and ApoE4 1-260 (Tanaka M., 2006. Effect of carboxyl-terminal truncation on structure and lipid interaction of human apolipoprotein E4. Biochemistry, 45(13):4240-7);

ApoE4 fragment 1-272 (Chang S. et al., 2005. Proc. Natl. Acad. Sci. USA. Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity. 102(51) :18694-9);

fragments of ApoE4 1-259, ApoE4 1-229 and ApoE4 1-202 (Dafnis I. et al., 2015. Influence of Isoforms and Carboxyl-Terminal Truncations on the Capacity of Apolipoprotein E To Associate with and Activate Phospholipid Transfer Protein. Biochemistry, 54(38):5856-66 Vezeridis et al., 2011 Domains of apoE4 required for the biogenesis of apoE-containing HDL Ann Med., 43(4):302-11);

ApoE4 fragment 1-231 (Chou C.Y. et al., 2006. Structural and functional variations in human apolipoprotein E3 and E4. J. Biol. Chem., 281(19):13333-44);

fragments of ApoE4 1-251 and ApoE4 1-266 (Dong L.M. 1994. Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms. J. Biol. Chem., 269(35):22358-65 ).

In addition, N-terminally truncated ApoE4 fragments were examined:

fragment ApoE4 72-299 (Chou C.Y. et al. 2006. Structural and functional variations in human apolipoprotein E3 and E4. J Biol Chem. 281(19):13333-44; Chou C.Y. et al. 2005. Structural variation in human apolipoprotein E3 and E4: secondary structure, tertiary structure, and size distribution Biophys J. 88(1):455-66).

Standard genotyping methods use DNA isolated from peripheral venous blood or oral epithelial cells to analyze the APOE gene polymorphism. The more commonly used method has traditionally been PCR-RFLP (Restriction Fragment Length PCR Polymorphism) analysis. However, this is a time-consuming and error-prone method due to the possible incomplete digestion by restriction enzymes. PCR plus sequencing or mass spectrometry are effective methods, but they require expensive specialized equipment. The ARMS-PCR (Refractor Mutation System-PCR Amplification) and SSP-PCR (Specific Primer Sequence-PCR) methodologies require analysis with agarose gels, thus limiting the number of samples that can be tested simultaneously. Real-time PCR detection from fluorescent melting curves is a simple and fast method, but primer-dimer formation can complicate the interpretation of melting curves. Real-time PCR detection from fluorescence melting curves, the use of FRET (fluorescence resonance energy transfer) or ^TaqMan® are very effective, albeit expensive, methods. More and more rapid DNA-based ApoE assays are emerging (e.g., Calero et al., 2009; Zhong et al., 2016), but they are all based on the above methods.

Phenotype-based assays are also available, such as isoelectric focusing or 2D gel electrophoresis, which are quite prone to errors due to post-translational changes in ApoE isoforms. Another method is immunochemical analysis, which is described in US patent No. 6027896 and is based on the presence of recoverable cysteine residues in the E2 and E3 isoforms, but not in E4. ApoE levels in biological fluids can also be measured using commercially available immunoassays, usually sandwich-type ELISA. These assays measure either all ApoE in body fluids, or only ApoE4, or both.

ApoE from cerebrospinal fluid (CSF) has been shown to bind to polypropylene, polystyrene, polyethylene and glass tubes (Hesse et al., 2000, Holmquist, 1982). Based on such results, Hesse et al. explained the differences in the results of the analysis of levels of ApoE in CSF in different experiments and identified the binding of ApoE to these materials as an interfering factor for immunoassays. Holmquist, 1982, describes the binding of isolated and purified serum ApoC and ApoE to plastic tubes and glass pipettes and highlights the potential for errors in ApoE immunoassays that occur during sample preparation. The fact that delipidated human serum apolipoproteins are highly adsorbed on glass and plastic surfaces is the cause of errors in double antibody immunoassays (eg Sandwich ELISA; Holmquist 1982, Høgåsen et al. 1993).

Høgåsen et al., 1993 showed that clusterin binds strongly to polystyrene microplates. This ability to non-specifically bind clusterin is enhanced by the addition of tween-20. They developed a single antibody immunoassay in which they pre-incubated plasma or serum samples in PBS-T (PBS with 0.2% Tween-20) on a polystyrene microplate. They then detected and quantified the immobilized clusterin using a specific antibody to clusterin.

The present invention exploits this ability of ApoE, in particular ApoE4, to bind strongly to polymeric and glass surfaces and thus provides a simple and cost effective single antibody immunoassay method using blood, plasma or serum samples.

As described above, Hesse et al., 2000 showed that ApoE from directly used CSF binds to polymer or glass tubes and can be detected after elution (eg, by SDS elution followed by Western blotting). However, in CSF, the average concentration of total protein is 0.15-0.45 mg/ml. In contrast, in plasma and serum, the protein concentration is 100-500 times higher than in CSF, and is 60-80 mg/ml. It is common for immunoassays to saturate binding surfaces with blocking proteins (eg, bovine serum albumin) at concentrations of 0.5 to 2 mg/mL to inhibit non-specific binding of sample proteins to surfaces. Serum ApoE concentrations range from 30 to 400 μg/ml (Bury et al., 1986). Therefore, the estimated amount of ApoE4 in plasma/serum is only 0.02-0.5% of total plasma/serum proteins, and it should not be expected that a signal specific for ApoE4 can be measured. Surprisingly, applicants can show that ApoE4 from plasma, serum, and even whole blood binds specifically and quantitatively to solid surfaces, even in the presence of more than 200-5000 times the total protein concentration in the sample.

Also unexpectedly white found, ApoE4 specifically and quantitatively binds to solid surfaces in the presence of blocking proteins (eg, 0.5-2 mg/ml bovine serum albumin (BSA)) in reaction buffer. The presence of blocking proteins such as BSA may even enhance the binding of ApoE4 to the solid phase.

A factor that enhances the specific binding of ApoE4 to polymeric and glass surfaces is the use of a detergent. Detergents are commonly used to prevent non-specific binding of sample proteins to solid phases in immunoassays. Hesse et al. showed that ApoE binds to polystyrene, polypropylene, polyethylene and glass tubes in the presence of 0.2% tween-20 (Hesse et al., 2000).

The subject of the present invention is a method for (a) identifying ApoE4 status and/or (b) quantifying ApoE4 levels in blood samples. Determining whether a subject is ApoE4 positive or ApoE4 negative (a) is based on the fact that the subject's blood sample has an ApoE4 level above a certain threshold, which is limited by an internal cutoff control. Quantification of ApoE4 levels in blood samples (b) is done by analyzing internal standards along with the samples and calculating the ApoE4 concentration using a standard curve.

Detailed description of embodiments of the present invention

The subject of the present invention is a method (in vitro) for detecting apolipoprotein E isotype 4 (ApoE4) or fragments thereof in a blood sample of a subject, comprising the steps:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, wherein said ApoE4-binding agent complex is immobilized on said solid phase by ApoE4 or ApoE4 is immobilized on said solid phase, and the binding agent to ApoE4.

The subject of the present invention is a method (in vitro) for detecting apolipoprotein E isotype 4 (ApoE4) or fragments thereof in a blood sample of a subject, comprising the steps:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, where said ApoE4-binding agent complex is immobilized on said solid phase, where the solid phase is not coated with any binding agent or capture molecule, and where the binding of ApoE4 or the ApoE4-binding agent complex to said solid phase occurs in the presence of detergent.

In the context of the present invention, the ApoE4-binding agent complex is immobilized through ApoE4 on the solid phase, in particular ApoE4 is immobilized on the solid phase and the binding agent is bound to ApoE4.

The subject of the present invention is also an assay based on the binding of an analyte (ApoE4 or fragments thereof) to a solid phase in the presence of a detergent and the detection of this immobilized analyte using an ApoE4-specific binding agent with a detectable label.

The ApoE4 specific binding agent can be labeled directly or indirectly.

The subject of the present invention is also a method for detecting ApoE4 or fragments thereof, wherein the subject's blood sample is contacted with a solid phase, thereby immobilizing ApoE4 on said solid phase.

The blood sample is selected from a group of whole blood, serum and plasma. Plasma is herein defined as EDTA plasma, heparinized plasma and/or citrated plasma.

In the context of the present invention, the term "ApoE4 fragment" includes shorter spliced ApoE4 variants and truncated ApoE4 proteins or peptides or ApoE4 regions (Rohn et al., 2013; Love et al., 2015).

These fragments are at least 6 amino acids in length, provided that amino acid 158 (arginine, R) is included in said fragment, where the sequence including amino acid 158 refers to SEQ ID NO:1.

In a specific embodiment of the present invention, the fragments are selected from the group consisting of SEQ ID sequences. NO: 1-15.

As used herein, the term "subject" refers to a living human or non-human organism, preferably a human, where the subject is healthy, presumably healthy, suffering from cognitive impairment, suffering from dementia, in particular Alzheimer's disease (AD).

In one embodiment of the method of the present invention, the binding of ApoE4 or fragments thereof to the solid phase occurs without the interaction of a connecting molecule that is capable of binding/linking ApoE4 or its fragments to said solid phase.

In yet another embodiment of the method of the present invention, for the duration of contact of the solid phase with the sample, said contact occurs without interaction of said connecting molecule.

In yet another embodiment of the method of the present invention, the binding of ApoE4 or fragments thereof to the solid phase occurs in the presence of a detergent.

As used herein, the detergent is selected from the group of anionic detergents (eg, sodium dodecyl sulfate (SDS)), cationic detergents (eg, cetyltrimethylammonium bromide (CTAB)), zwitterionic detergents (eg, CHAPS), and non-ionic detergents (eg, Tween-20 , triton X-100).

Examples of non-ionic detergents include epoxy fatty acid esters, e.g. , Brij 35 and Brij 58 (polyoxyethylene lauryl ether), Nonidet P-40 (polyoxyethylene octylphenol ether), Lubrol PX (polyethylene oxide-alkyl ether adduct), Berol EMU 043 (C16, C18 fatty alcohol with 10 oxyethylene chains); deoxy-BIGCHAP, digitonin, HECAMEG, ND-gluco-N-methylalkanamide (MEGA-8, 9, 10), n-octyl-D-glucopyranoside, rac.-1- ^{oleoylglycerin} , Pluronic® F-68, sucrose monolaurate, saponin from quillaya; etc.

Zwitterionic detergents include, but are not limited to, CHAPS, CHAPSO, n-octyl-β-D-glucopyranoside, sulfobetaine SB 12, and sulfobetaine SB 14.

Examples of cationic detergents include benzethonium chloride, cetylpyridinium chloride monohydrate, cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and the like.

Examples of anionic detergents include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LiDS), sodium cholate, sodium deoxycholate, N-lauroylsarcosine sodium salt, 1-decanesulfonic acid sodium salt, 1-dodecanesulfonic acid sodium salt, 1-heptanesulfonic acid sodium salt. anhydrous salt, 1-hexane sulfonic acid sodium salt anhydrous, 1-nonane sulfonic acid sodium salt, 1-octane sulfonic acid sodium salt, 1-pentane sulfonic acid sodium salt anhydrous, and so on.

In one embodiment of the method of the present invention, the concentration of detergent in the reagent is preferably from 0.0001 to 10% (w/v) and particularly preferably from 0.01 to 1% (w/v). The person skilled in the art knows that it is possible to provide for the use of a certain range of concentrations for each particular detergent.

In a preferred embodiment of the method of the present invention, Tween-20 is used at a concentration of 0.001-10%, preferably 0.01-1%, most preferably 0.2-0.5%. In yet another embodiment of the method of the present invention, Triton X-100 is used at a concentration of 0.001-0.5%, preferably 0.01-0.2%, most preferably 0.05-0.1%.

In the present invention, detergents are required to bind ApoE4 or fragments thereof to the solid phase, but they do not function as bonding or binding molecules. Detergents must be present in the reaction buffer since ApoE4 binding to the solid phase does not occur in sample buffer without detergent (see example 4).

In a preferred embodiment of the method of the present invention, the binding agent is a specific binding agent for ApoE4 or fragments thereof.

In a further embodiment of the method of the present invention, the binding agent is an antibody or a functional fragment of an antibody or a non-IgG scaffold protein with a binding affinity constant of at least 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 , the preferred affinity constant being higher 10 ⁹ M ^-1 , most preferably above 10 ¹⁰ M ^-1 .

In the context of the present invention, "binding agent molecules" are molecules that can be used to bind target molecules or molecules of interest, i.e. analytes (in the context of the present invention ApoE4 and its fragments) from the sample. Thus, the binding agent molecules must have an adequate shape, both spatially and in terms of surface properties, such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and/or acceptors, in order to specifically bind target or presenting molecules. molecule interest. Thus, binding may be mediated, for example, by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic, or hydrogen-bonded interactions, or a combination of two or more of the above interactions between capturing molecules and target molecules or molecules of interest. . In the context of the present invention, the binding agent molecules may, for example, be selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the binding agent molecules are antibodies, including fragments thereof with sufficient affinity for the target molecule or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibodies or fragments derived from a variant chain length of at least 12 amino acids.

The anti-ApoE4 antibody may be in formats known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment of the present invention, the antibodies of the present invention are recombinantly produced antibodies, such as IgG, conventional full-length immunoglobulin, or antibody fragments containing at least the F-(fragment) of the heavy and/or light chain variable domain, for example, chemical cross-linked antibodies (antigen-binding fragment), including, but not limited to, Fab fragments, including Fab minibody, single chain Fab antibody, epitope tagged monovalent Fab antibody, eg Fab-V5S×2; a bivalent Fab (minibody) dimerized with a CH3 domain; a bivalent Fab or a multivalent Fab, eg obtained by multimerization with a heterologous domain, eg by dimerization of dHLX domains, eg Fab-dHLX-FS×2; F(ab') ₂ fragments, scFv fragments, multimerized, multivalent and/or multispecific scFv fragments, bivalent and/or bispecific dimeric antibodies (diabodies), ^BITE® (bispecific T cell activator), trifunctional antibodies, polyvalent antibodies , for example, from a class other than G; single-domain antibodies, such as nanobodies derived from camelid or fish immunoglobulins, and many other formats.

In addition to anti-ApoE4 antibodies, other biopolymer scaffolds that complex with a target molecule are well known in the art and are used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins.

Scaffolds other than Ig can represent protein scaffolds and can be used as antibody mimetics because they are able to bind to ligands or antigens. Non-Ig scaffolds can be selected from the group consisting of tetranectin-based non-Ig scaffolds (e.g., described in US Patent Application No. 2010/0028995), fibronectin-based scaffolds (e.g., described in EP 1266025), scaffolds lipocalin-based (for example, described in WO 2011/154420), ubiquitin-based scaffolds (for example, described in WO 2011/073214), transferrin-based scaffolds (for example, described in US patent application No. 2004/0023334), scaffolds based on Protein A backbone (e.g. described in EP 2231860), ankyrin repeat scaffolds (e.g. described in WO 2010/060748), scaffolds based on microproteins (preferably "cystine knot" forming microproteins) (e.g. described in EP 2314308) , Fyn kinase SH3 domain scaffolds (e.g. described in WO 2011/023685), EGFR-A domain scaffolds (e.g. described in WO 2005/040229) and Kunitz domain scaffolds (e.g. described in EP 1941867) . Frameworks other than Ig may be peptide or oligonucleotide aptamers. Aptamers are usually generated by selection from a large pool of random sequences, and they are short oligonucleotide chains (DNA, RNA or XNA; Xu et al., 2010, Deng et al., 2014) or short peptide variable domains attached to a protein backbone (Li et al., 2011).

Binding agents that can be used to determine the level of ApoE4 or fragments thereof have an affinity constant for ApoE4 of at least 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 , the preferred affinity constant is above 10 ⁹ M ^-1 , most preferably above 10 ¹⁰ M ^-1 . The person skilled in the art knows that it may be possible to compensate for lower affinity by using a higher dose of compounds, and this measure will not lead to a departure from the scope of the invention.

In a more preferred embodiment of the method of the present invention, the binding agent is labeled with a detectable label for detection after binding to ApoE4.

In a preferred embodiment of the present invention, the binding agent label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label. The amount of labeled antibody bound to the analyte is then measured by an appropriate method.

In yet another embodiment of the method of the present invention, the binding agent label is selected from the group consisting of rare earth metal cryptates or rare earth metal chelates in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine type dye.

Preferred methods for detecting the level of ApoE4 or fragments thereof include immunoassays in various formats, such as radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA), luminex-based beads, protein microarrays, and rapid test formats, for example such as immunochromatographic methods with test strips.

In the context of the present invention, fluorescence-based assays include the use of dyes which, for example, may be selected from the group consisting of FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700/800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4 ',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrodamine (TAMPA), 6-carboxy-X-rhodamine (ROX), 5- carboxyrodamine-6G (R6G5), 6-carboxyrodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, oregon green, coumarins such as umbelliferone, benzimides such as Hoechst 33258 ; phenanthridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

In the context of the present invention, chemiluminescence-based assays involve the use of dyes that operate on the physical principles described for chemiluminescent materials in the Kirk-Othmer Encyclopedia of Chemical Technology. Preferred chemiluminescent dyes are acridinium esters.

The chemiluminescent label may be an acridinium ester label, steroid labels including isoluminol labels, and the like.

Enzyme labels may represent lactate dehydrogenase (LDH), creatinine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, and the like.

In yet another embodiment of the process of the present invention, the solid phase is selected from the group consisting of polymeric surface (eg polystyrene), glass surface, cellulose and cellulose derivatives (eg nitrocellulose, polyvinylidene fluoride, nylon). In yet another specific embodiment of the method of the present invention, the solid phase is selected from the group consisting of glass or polymeric surfaces, where the polymeric surface includes, but is not limited to, polystyrene, poly(divinylbenzene), styrene-acrylic copolymer, styrene-butadiene copolymer, styrene- divinylbenzene copolymer, poly(styrene-oxyethylene), polymethylmethacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, N,N'-methylene-bis-acrylamide, polyolefins, polyethylene, polytetrafluoroethylene, polyethylene terephthalate, polypropylene, polyvinyl chloride, polyvinyl acetate, polyacrylonitrile, polysulfone, poly (sulfonic ether), polydimethylsiloxane, pyrolyzed materials, block copolymers and copolymers of the above compounds, silicones or silicon dioxide.

Any material is suitable for such a surface/solid phase, provided that it serves to achieve the objectives of the present invention. Preferred examples of surfaces include polymeric surfaces and glass surfaces. The group of polymer surfaces includes, but is not limited to: polystyrene, poly(divinylbenzene), styrene-acrylic copolymer, styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene-oxyethylene), polymethylmethacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, N, N'-methylene-bis-acrylamide, polyolefins, polyethylene, polytetrafluoroethylene, polyethylene terephthalate, polypropylene, polyvinyl chloride, polyvinyl acetate, polyacrylonitrile, polysulfone, poly(sulfonic ether), polydimethylsiloxane, pyrolyzed materials, block copolymers and copolymers of the above compounds, silicones or silicon dioxide , cellulose and cellulose derivatives (eg nitrocellulose, polyvinylidene fluoride, nylon). The preferred surface material is polystyrene.

Polystyrene is a hydrophobic polymer that is easily derivatized by the manufacturer. EIA microtiter plates are typically made from: 1) unmodified polystyrene (eg pureGrade BRANDplates ^® ), 2) modified polystyrene that has an ionized and hydrophobic surface (eg lipoGrade BRANDplates ^® , Thermo Scientific Microlite 1+ plates), 3) polystyrene optimized for IgG binding (hydrophilic and hydrophobic; eg Greiner High Binding Plates, immuneBRANDplates ^® ) or 3) polystyrene with a hydrophilic or low binding surface (eg Thermo Scientific MultiSorp, hydroGrade BRANDplates ^® ). Polymer surfaces, including polystyrene microplates, are modified by physical bombardment with energetic particles (ions, electrons, fast neutrals and/or radicals) and/or ultraviolet radiation photons, or by chemical reactions at or near the surface (e.g., acid treatment or treatment with organosilanes) (Hegemann et al., 2003, WO 9203732, DE 2018523 A1). The preferred solid phase for the present invention is a highly binding polystyrene microplate.

In yet another embodiment of the present invention, the solid phase is unmodified (pure) or modified to be hydrophilic, hydrophobic or optimized for IgG binding (high binding: hydrophilic and hydrophobic) by physical bombardment with energetic species (ions, electrons, fast neutrals and/or radicals) and ultraviolet radiation photons and/or chemical reactions at or near the surface (e.g. acid treatment or treatment with organosilanes).

In yet another specific embodiment of the method of the present invention, the surface of the solid phase is used in suitable forms such as plates, multiwell plates, tubes or beads, plates, films, particles, magnetic or non-magnetic particles. In a preferred embodiment of the present invention, the shape of the solid phase surface is a microtiter plate.

In a more specific embodiment of the method of the present invention, the subject's blood sample is contacted with an unsaturated solid phase. This means that the solid phase, at the time it is in contact with the sample, is not saturated/coated with blocking agents and/or binding molecules and/or stabilizing proteins and/or connecting molecules.

However, blocking agents or stabilizing proteins may maintain the existing binding of ApoE4 or fragments thereof to the solid phase and the binding agent when the sample has already been in contact with the solid phase, which means that stabilizing/blocking molecules/proteins may not necessarily be present at the time of detection of the immobilized ApoE4-binding complex. agent (at the stated stage b).

In the context of the present invention, the term "blocking agents" includes, but is not limited to, protein solutions (eg, bovine serum albumin, human serum albumin, milk proteins, casein, fish gelatin) or whole whey (eg, calf whey).

In one embodiment of the method of the present invention, the solid surface is not coated with any binding agent or capture molecule. As used here, the definition of the term "coupling agent" is given above.

In the context of the present invention, "capture molecules" are molecules that can be used to bind target molecules or molecules of interest, ie. analytes (i.e., in the context of the present invention, peptide(s)) from the sample. Thus, capture molecules must be of adequate shape, both spatially and in terms of surface properties, such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and/or acceptors, in order to specifically bind target molecules or molecules of interest. molecules. Thus, binding may be mediated, for example, by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic, or hydrogen-bonded interactions, or a combination of two or more of the above interactions between capturing molecules and target molecules or molecules of interest. . In the context of the present invention, capture molecules may, for example, be selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide, or a glycoprotein. Preferably, capture molecules are antibodies, including fragments thereof with sufficient affinity for the target or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibodies or fragments derived from a variant chain of length, at least 12 amino acids.

In yet another embodiment of the method of the present invention, the stabilizing protein maintains the existing binding of ApoE4 or fragments thereof from the solid phase blood sample, where said protein may optionally be added to the reaction buffer.

Reaction buffers for immunoassays typically contain stabilizing/blocking proteins (eg, bovine serum albumin, human serum albumin, milk proteins, casein, gelatin, immunoglobulins). These additional proteins prevent non-specific binding to the solid phase and stabilize binding partners (binding agent and antigen) as well as binding agent-antigen interaction. Surprisingly, the addition of such stabilizing/blocking proteins to the reaction enhances the binding of ApoE4 or its fragments to the solid phase (see example 6).

In yet another embodiment of the method of the present invention, the stabilizing protein is selected from the group consisting of bovine serum albumin (BSA), human serum albumin, casein, milk proteins, immunoglobulins, stabilizing amino acids (e.g., alanine, arginine, glycine, isoleucine, lysine, proline , serine) and the like. The preferred stabilizing protein is bovine serum albumin (BSA).

In yet another embodiment of the method of the present invention, the concentration of BSA is in the range of 0-10%, preferably 0.2-5%, most preferably 0.5-2.5%.

In yet another specific embodiment of the method of the present invention, the presence of proteins other than ApoE4 in the blood sample does not affect the detection of ApoE4 or fragments thereof.

In yet another embodiment of the method of the present invention, contacting a blood sample of a subject with a solid phase and contacting said sample with at least one binding agent that specifically binds to ApoE4 or a fragment thereof occurs in a one-step or two-step procedure (see example 5 ).

In the context of the present invention, the term "one-step procedure" means that the immobilization of ApoE4 or its fragments on the solid phase and the binding of ApoE4 or its fragments to a labeled binding agent (eg, antibody) are performed simultaneously in the same vessel. During this kind of procedure, the ApoE4 alone and then the specific binding agent and/or the ApoE4-binding agent complexes are immobilized on the solid phase.

As used here, the term "two-step procedure" means that the immobilization of ApoE4 or its fragments on the solid phase and the binding of ApoE4 or its fragments with a specific binding agent occurs at time points in steps, optionally with a washing step between the incubation steps. For example, ApoE4 may be first immobilized on solid phase and then an ApoE4-specific labeled binding agent will bind to the immobilized ApoE4. Another possibility is that the ApoE4 and the ApoE4-specific labeled binding agent are incubated in the first step to allow formation of the ApoE4-binding agent complexes, and in the second stage the ApoE4-binding agent complexes are immobilized on the solid phase.

In yet another specific embodiment of the method of the present invention, the ApoE4-binding agent complex is detected qualitatively or quantitatively, wherein the qualitative detection of the ApoE4-binding agent complex indicates the presence of ApoE4 or fragments thereof in said sample, and where the quantitative detection of the ApoE4-binding agent complex indicates the concentration of ApoE4 or its fragments in the specified sample.

In yet another embodiment of the method of the present invention, the quantitative determination of the concentration of ApoE4 or its fragments shows how the subjects are homozygous or heterozygous for ApoE4 alleles.

In yet another specific embodiment of the method of the present invention, the presence of the ApoE4-binding agent complex correlates with the risk of developing/morbidity of AD.

In one specific embodiment of the method of the present invention, ApoE4 detection is used to determine the risk of developing Alzheimer's disease (AD).

The subject of the present invention is also a method for predicting the risk of Alzheimer's disease, as well as the medical consequences of all the above diseases and infections.

In one embodiment of the method of the present invention, the detection of immobilized ApoE4 in the ApoE4-binding agent complex is used to classify a subject as ApoE4-positive or ApoE4-negative when the level of detected ApoE4 in the ApoE4-binding agent complex is above a certain threshold value, where the specified threshold value determined by the specific value of the internal positive control.

In yet another embodiment of the method of the present invention, the level of the ApoE4-binding agent complex above a certain threshold is predictive of an increased risk of AD morbidity, where said threshold is determined by an internal cut-off control.

According to the method of the present invention, ApoE4-positive blood samples of subjects have a value higher than a specific level of internal control cut-off (= threshold value). In a more preferred embodiment, the threshold value is about 1.5-100 times lower than the level in ApoE4 positive samples, preferably 1.5-20 times lower, most preferably 1.5-10 times lower. ApoE4-negative samples have a lower value than the internal cut-off control. In a more preferred embodiment, the threshold value is about 10-10000 times higher than the level in ApoE4 negative samples, preferably 50-5000 times higher, most preferably 100-1000 times higher.

In the context of the present invention, an ApoE4 level above this threshold indicates the presence of ApoE4 in the patient and thus a high risk of developing/morbidity with AD. An ApoE4 level below this threshold indicates the absence of ApoE4 in the patient and thus a low risk of developing/incidence of AD.

In a specific embodiment of the present invention, for qualitative detection of the ApoE4-binding agent complex, the level of ApoE4 or fragments thereof is measured by an immunoassay using antibodies or antibody fragments. ApoE4-positive and ApoE4-negative samples are separated by a threshold, which is determined by a specific internal cut-off control, where all ApoE4-negative samples show only background signal, and all ApoE4-positive samples have a signal at least 1.5 times higher than clipping control (see examples 5, 6 and 7).

Another embodiment of the present invention is the evaluation of measured units (i.e., units of fluorescence, luminescence, enzymatic reaction, etc.) that correspond to the level of detected levels of ApoE4, which can be analyzed quantitatively using ApoE4 calibrators with standard concentrations (see. example 7). Through quantitative analysis, the ApoE4-positive group of the present invention can be further divided into two groups, which correspond to subjects that are homozygous or heterozygous for ApoE4 alleles using a certain threshold value. Such a specific threshold can be determined by analysis of previously genotyped homozygous or heterozygous samples.

One embodiment of the present invention is to correlate the presence of ApoE4/ApoE4-binding agent complex with the risk of developing/morbidity of AD. Evaluation of the results indicates a low risk of development/incidence of AD in ApoE4-negative subjects and indicates a high risk of development/incidence of AD in ApoE4-positive subjects.

In one embodiment of the invention, the method is used to stratify risk or stratify the use of therapeutic interventions for a disease or condition associated with elevated levels of ApoE4.

In yet another embodiment of the invention, the disease or pathological condition is selected from the group of cognitive impairment and neurodegenerative diseases, including dementia and/or Alzheimer's disease.

The subject of the invention is also an assay that provides a method for detecting ApoE4 or fragments thereof.

In the context of the present invention, the assay includes all test components that are necessary to carry out the method of the present invention. However, as stated here, "assay" is not only a box or block containing these test components, but rather a functional combination of all interacting components, providing the implementation of the claimed method of the present invention.

Said assay of the present invention enables the implementation of the method of the present invention also as a fully automated assay system, for example on a platform available on the market (Roche cobas ^® , Abbott Architect ^® , Siemens Centauer ^® , Brahms Kryptor ^® , Biomerieux Vidas ^® , Alere Triage ^® ).

In a specific embodiment of the present invention, the assay provides for a method for detecting ApoE4 or fragments thereof in a blood sample of a subject, comprising the following steps:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, wherein said ApoE4-binding agent complex is immobilized on said solid phase via ApoE4 or ApoE4 immobilized on said solid phase, and the binding agent to ApoE4.

In a specific embodiment of the present invention, the assay provides for a method for detecting ApoE4 or fragments thereof in a blood sample of a subject, comprising the following steps:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, wherein said ApoE4-binding agent complex is immobilized on said solid phase via ApoE4 or ApoE4 immobilized on said solid phase, where the solid phase is not coated with any binding agent or capture molecule and where binding of ApoE4 or ApoE4 complex is the binding agent with said solid phase occurs in the presence of a detergent.

In the context of the present invention, said assay provides for the implementation of the method of the present invention wherein the ApoE4-binding agent complex is immobilized on the solid phase via ApoE4 and the binding agent is bound to ApoE4.

In one embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the binding of ApoE4 to a solid phase occurs without the interaction of a linking molecule (which is capable of binding/linking ApoE4 or fragments thereof) to said solid phase).

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention wherein, for the duration of contact of the solid phase with the sample, said contact occurs without interaction of said connecting molecule.

In yet another embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the binding of ApoE4 to the solid phase occurs in the presence of a detergent.

In one embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the concentration of detergent in the reagent is preferably from 0.0001 to 10% (w/v) and particularly preferably from 0.01 to 1% (w/v). about.). The person skilled in the art knows that it is possible to provide for the use of a certain range of concentrations for each particular detergent.

In a preferred embodiment of the present invention, said assay provides for the implementation of the method of the present invention wherein Tween-20 is used at a concentration of 0.001-10%, preferably 0.01-1%, most preferably 0.2-0.5%. In yet another embodiment of the method of the present invention, Triton X-100 is used at a concentration of 0.001-0.5%, preferably 0.01-0.2%, most preferably 0.05-0.1%.

In a preferred embodiment of the present invention, said assay provides for the implementation of the method of the present invention wherein the binding agent specifically binds to ApoE4 or fragments thereof.

In a further embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the binding agent is an antibody or functional antibody fragment or scaffold protein other than IgG with a binding affinity constant of at least 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 preferred affinity constant above 10 ⁹ M ^-1 most preferably above 10 ¹⁰ M ^-1 .

In a more preferred embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the binding agent is labeled with a detectable label for detection after binding to ApoE4.

In a preferred embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the binding agent label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label. The amount of labeled antibody bound to the analyte is then measured by an appropriate method.

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the binding agent label is selected from the group consisting of rare earth metal cryptates or rare earth metal chelates in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine type dye.

In yet another embodiment of the present invention, said analysis provides for the implementation of the method of the present invention, wherein the solid phase is selected from the group consisting of a polymeric surface (e.g., polystyrene), a glass surface, cellulose, and cellulose derivatives (e.g., nitrocellulose, polyvinylidene fluoride, nylon).

In yet another embodiment of the present invention, said analysis provides for the implementation of the method of the present invention, where the solid phase is selected from the group consisting of glass or polymeric surfaces, where the polymeric surface includes, but is not limited to, polystyrene, poly(divinylbenzene), styrene-acrylic copolymer , styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene-oxyethylene), polymethyl methacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, N,N'-methylene-bis-acrylamide, polyolefins, polyethylene, polytetrafluoroethylene, polyethylene terephthalate, polypropylene, polyvinyl chloride , polyvinyl acetate, polyacrylonitrile, polysulfone, poly(sulfonic ether), polydimethylsiloxane, pyrolyzed materials, block copolymers and copolymers of the above compounds, silicones or silicon dioxide.

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the solid phase is unmodified (pure) or modified to be hydrophilic, hydrophobic, or optimized for IgG binding (high binding: hydrophilic and hydrophobic) by physical bombardment with energetic particles. (ions, electrons, fast neutrals and/or radicals) and photons of ultraviolet radiation and/or chemical reactions on or near the surface (for example, acid treatment or treatment with organosilanes).

In yet another specific embodiment of the present invention, said assay provides for the implementation of the method of the present invention wherein the solid phase surface is used in suitable forms such as plates, multiwell plates, tubes or beads, plates, films, particles, magnetic or non-magnetic particles. In a preferred embodiment of the present invention, the shape of the solid phase surface is a microtiter plate.

In a more specific embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the blood sample of the subject is contacted with an unsaturated solid phase. This means that the solid phase, at the time it is in contact with the sample, is not saturated/coated with blocking agents and/or binding molecules and/or stabilizing proteins and/or connecting molecules.

However, blocking agents or stabilizing proteins may maintain the existing binding of ApoE4 or fragments thereof to the solid phase and the binding agent when the sample is already bound to the solid phase, which means that the stabilizing/blocking molecules/proteins may not necessarily be present at the time of detection of the immobilized ApoE4-binding complex. agent (at the stated stage b).

In one embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the solid phase is not coated with any binding agent or capture molecule. As used here, the definition of the term "coupling agent" is given above.

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the stabilizing protein maintains the present binding of ApoE4 from the solid phase blood sample, wherein said protein may optionally be added to the reaction buffer.

In one further embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the stabilizing protein is selected from the group consisting of bovine serum albumin (BSA), human serum albumin, casein, milk proteins, immunoglobulins, stabilizing amino acids (e.g., alanine, arginine , glycine, isoleucine, lysine, proline, serine) and the like. The preferred stabilizing protein is bovine serum albumin (BSA).

In yet another embodiment of the present invention, the specified analysis provides for the implementation of the method according to the present invention, where the concentration of BSA is in the range of 0-10%, preferably 0.2-5%, most preferably 0.5-2.5%.

In yet another specific embodiment of the present invention, said assay provides for the implementation of the method of the present invention, where the presence of other proteins other than ApoE4 in the blood sample does not affect the detection of ApoE4 or fragments thereof.

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein contacting a blood sample of a subject with a solid phase and contacting said sample with at least one binding agent that specifically binds to ApoE4 or a fragment thereof occurs in a single step. or a two-stage procedure (see example 5).

In yet another specific embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the method for detecting ApoE4 or fragments thereof, the ApoE4-binding agent complex, detects qualitatively or quantitatively, wherein the qualitative detection of the ApoE4-binding agent complex indicates the presence of ApoE4 or its fragments in the specified sample, and where the quantitative determination of the complex ApoE4-binding agent indicates the concentration of ApoE4 or its fragments in the specified sample.

In yet another embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the quantitative determination of the concentration of ApoE4 shows how subjects are homozygous or heterozygous for ApoE4 alleles.

In yet another specific embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the presence of an ApoE4-binding agent complex correlates with the risk of developing/incidence of AD.

In one specific embodiment of the present invention, the specified analysis provides for the implementation of the method of the present invention, where the detection of ApoE4 or its fragments is used to determine the risk of developing Alzheimer's disease (AD).

In yet another specific embodiment of the present invention, said assay provides for the implementation of the method of the present invention, where the detection of ApoE4 or fragments thereof is used to predict the risk of Alzheimer's disease, as well as the medical consequences of all of the above diseases and infections.

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention, wherein the detection of immobilized ApoE4 in the ApoE4-binding agent complex is used to classify a subject as ApoE4-positive or ApoE4-negative when the level of detected ApoE4 in the ApoE4-binding agent complex above a certain threshold value, where the specified threshold value is determined by a specific value of the internal positive control.

In yet another embodiment of the present invention, said assay provides for the implementation of the method of the present invention, where the level of the ApoE4-binding agent complex above a certain threshold is predictive of an increased risk of AD morbidity, where said threshold is determined by an internal cut-off control.

According to the method of the present invention, ApoE4-positive blood samples of subjects with a value higher than the cut-off level, where the cut-off is determined by measuring the ApoE4-negative sample and the ApoE4-positive sample and calculating the ratio.

In one embodiment, the ratio is calculated as follows:

[Signal] _neg CO+X factor multiplied by [Signal] _pos CO where the factor is >0 and <1.

According to the method of the present invention, in one embodiment, ApoE4-positive blood samples of subjects have a value greater than a cut-off level (cut-off value), where the cut-off value is determined by measuring the ApoE4 negative control signal and calculating from 1.5- to 100,000-fold signal, greater than preferably 10 to 50,000 times the signal, even more preferably 20 to 20,000 times the signal, most preferably 50 to 10,000 times the signal, most preferably 100 to 1000 times the signal. According to the method of the present invention, ApoE4-positive blood samples from subjects have a value greater than a cut-off level (cut-off value), where the cut-off value is determined by measuring the ApoE4 positive control signal and multiplying the signal value by a factor of >0 and <1, more preferably >0.01 and <0.8, even more preferably >0.05 and <0.5, most preferably >0.1 and <0.25.

In a specific embodiment of the present invention, the assay is used to determine the level of ApoE4 and its fragments containing at least 6 amino acids, where the sensitivity of the determination in the specified analysis allows the detection/quantification of ApoE4 and its fragments in a blood sample of subjects, and it is <1 ng/ml, preferably <5 ng/ml, more preferably <10 ng/ml and even more preferably <25 ng/ml.

The subject of the invention is also an assay that provides for the implementation of the method of the present invention, where the level of ApoE4 or fragments thereof is measured using an immunoassay comprising one or more capture probes directed against one or more ApoE4 epitopes or fragments thereof.

In one embodiment of the present invention, said immunoassay is selected from the group consisting of radioimmunoassay (RIA), homogeneous enzyme immunoassay (EMIT), enzyme-linked immunosorbent assay (ELISA), apoenzyme reactivation immunoassay (ARIS), test strip immunoassay, and immunochromatographic assays.

In yet another embodiment of the present invention, such an assay may be a so-called POC test (point-of-care assay), which is a testing technology that allows the assay to be performed in less than 1 hour at the patient's side without the need for a fully automated assay system. One example of this technology is the immunochromatographic assay technology.

In yet another embodiment of the present invention, such an assay is a single antibody immunoassay using any type of detection technology, including, but not limited to, an enzyme label, a chemiluminescent label, an electrochemiluminescent label, preferably a fully automated assay. Examples of automated or fully automated assays include assays that can be used for one of the following systems: Roche ^Elecsys® , Abbott Architect®, Siemens Centauer®, Brahms ^Kryptor® , ^{Biomerieux Vidas®} , ^{Alere Triage®} .

Another subject of the present invention is an assay that provides for the implementation of the method of the present invention, including test components:

a. one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

b. solid phase (e.g. plate, test tube, beads, plate),

with. reaction buffer containing detergent.

Yet another embodiment of the present invention provides an assay that enables the implementation of the method of the present invention, comprising:

a. one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

b. a polymer or glass solid, preferably a highly binding polystyrene microplate,

with. a reagent that contains a detergent, preferably 0.2% tween-20.

The assay may additionally include controls:

at least one of the following controls is required: a negative control, a cut-off control, and a positive control,

controls can represent samples with a known concentration of ApoE4 (native, purified or recombinant).

Yet another embodiment of the present invention provides an assay that provides for the implementation of the method of the present invention to quantify ApoE4 or fragments thereof in a sample of a subject, comprising:

a. one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

b. a polymer or glass solid, preferably a highly binding polystyrene microplate,

with. a reagent that contains a detergent, preferably 0.2% tween-20.

The assay may optionally include one or more calibrators:

the calibrator can be samples with a known concentration of ApoE4 (native, purified or recombinant).

Yet another embodiment of the present invention provides an assay that provides a method for detecting ApoE4 or fragments thereof, consisting of:

one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments containing at least 6 amino acids, where the binding agent is labeled with a detectable label,

a polymer or glass solid, preferably a highly binding polystyrene microplate,

a reagent that contains a detergent, preferably 0.2% tween-20,

controls and/or calibrators,

wash buffer,

reagents for measurement.

Another subject of the present invention is a kit for detecting ApoE4 or fragments thereof in a blood sample of a subject.

As used herein, a "kit" is a box containing a combination of materials that are necessary to carry out the method of the present invention, in particular the detection of ApoE4 and its fragments in a subject's blood sample.

Another subject of the present invention is a kit for detecting ApoE4 and its fragments in a blood sample of a subject, comprising:

a. one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

b. solid phase (e.g. plate, test tube, beads, plate),

with. reaction buffer containing detergent.

Another embodiment of the present invention provides a kit for detecting ApoE4 and its fragments in a blood sample of a subject, comprising:

one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

a polymer or glass solid, preferably a highly binding polystyrene microplate,

a reagent that contains a detergent, preferably 0.2% tween-20.

The set may additionally contain controls:

at least one of the following controls is required: a negative control, a cut-off control, and a positive control,

controls can be samples with a known concentration of ApoE4 (native, purified or recombinant).

Another embodiment of the present invention provides a kit for the quantification of ApoE4 or fragments thereof in a subject sample, comprising:

one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

a polymer or glass solid, preferably a highly binding polystyrene microplate,

a reagent that contains a detergent, preferably 0.2% tween-20.

The kit may additionally contain one or more calibrators:

the calibrator can be samples with a known concentration of ApoE4 (native, purified or recombinant).

Another embodiment of the present invention is a kit for detecting ApoE4 or fragments thereof in a blood sample of a subject, consisting of:

one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments containing at least 6 amino acids, where the binding agent is labeled with a detectable label,

a polymer or glass solid, preferably a highly binding polystyrene microplate,

a reagent that contains a detergent, preferably 0.2% tween-20,

controls and/or calibrators,

wash buffer,

reagents for measurement.

Another embodiment of the present invention is the use of a binding agent for the detection of ApoE4 or fragments thereof in a blood sample of a subject, comprising the steps of:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, wherein said ApoE4-binding agent complex is immobilized on said solid phase via ApoE4 or ApoE4 is immobilized on said solid phase and binding agent to ApoE4.

Another embodiment of the present invention is the use of a binding agent for the detection of ApoE4 or fragments thereof in a blood sample of a subject, comprising the steps of:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, where said ApoE4-binding agent complex is immobilized on said solid phase via ApoE4 or ApoE4 immobilized on said solid phase, where the solid phase is not coated with said binding agent or capture molecule, and where said ApoE4 or ApoE4-binding agent complex is bound with the specified solid phase occurs in the presence of a detergent.

In the context of the present invention, the application of a binding agent in the method of the present invention is synonymous with the claimed method of the present invention, including all of the above embodiments.

The following embodiments are also the subject of the present invention:

1. A method (in vitro) for detecting apolipoprotein E isotype 4 (ApoE4) or fragments thereof in a blood sample of a subject, comprising the steps of:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, wherein said ApoE4-binding agent complex is immobilized on said solid phase via ApoE4.

2. A method (in vitro) for detecting apolipoprotein E isotype 4 (ApoE4) or fragments thereof in a blood sample of a subject, comprising the steps of:

a. contacting said sample with a solid phase,

contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, and

b. detection of an ApoE4-binding agent complex, wherein said ApoE4-binding agent complex is immobilized on said solid phase, wherein said solid phase is not coated with said binding agent or capture molecule, and wherein binding of ApoE4 or ApoE4-binding agent complex to said solid phase occurs in the presence of a detergent.

3. The method according to embodiments 1 or 2, wherein ApoE4 or fragments thereof are immobilized on said solid phase and the binding agent is bound to ApoE4.

4. The method according to embodiments 1-3, wherein the ApoE4 fragments are at least 6 amino acids in length, provided that amino acid 158 (arginine) is included in said fragment, wherein the sequence including amino acid 158 refers to SEQ ID NO: one.

5. The method according to embodiments 1-4, wherein the binding of ApoE4 or an ApoE4-binding agent complex to said solid phase occurs without interaction of a linking molecule that is capable of binding ApoE4 or fragments thereof to said solid phase.

6. The method according to embodiments 1-5, wherein for the duration of contact of the solid phase with the sample, said contact occurs without interaction of said connecting molecule.

7. The method according to embodiments 1-6, wherein the binding of ApoE4 or the ApoE4-binding agent complex to said solid phase occurs in the presence of a detergent.

8. The method according to embodiments 1-7, wherein the ApoE4-binding agent complex is detected qualitatively or quantitatively.

9. The method according to embodiments 1-8, wherein the quantification of the ApoE4-binding agent complex indicates the concentration of ApoE4 in said sample, wherein the concentration indicates how homozygous or heterozygous the subjects are for ApoE4 alleles.

10. The method according to embodiments 1-9, wherein the binding agent is an antibody or a functional antibody fragment or scaffold protein other than IgG with a binding affinity constant of at least 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 , preferably an affinity constant above 10 ⁹ M ^-1 , most preferably above 10 ¹⁰ M ^-1 .

11. The method according to embodiments 1-10, wherein the binding agent is labeled with a detection label, wherein said label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, or a radioactive iodine label.

12. The method according to embodiments 1-11, wherein ApoE4 detection is used to classify a subject as ApoE4 positive or ApoE4 negative when the detected ApoE4 level is above a certain threshold, where said threshold is determined by a particular internal positive control value.

13. The method according to embodiments 1-12, wherein the solid phase is selected from the group consisting of a polymeric surface (eg, polystyrene), a glass surface, cellulose, and cellulose derivatives (eg, nitrocellulose, polyvinylidene fluoride, nylon).

14. The method according to embodiments 1-13, wherein the solid phase is selected from the group of glass or polymeric surfaces, wherein the polymeric surface includes, but is not limited to, polystyrene, poly(divinylbenzene), styrene-acrylic copolymer, styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene-oxyethylene), polymethylmethacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, N,N'-methylene-bis-acrylamide, polyolefins, polyethylene, polytetrafluoroethylene, polyethylene terephthalate, polypropylene, polyvinyl chloride, polyvinyl acetate, polyacrylonitrile, polysulfone, poly( sulfonic ether), polydimethylsiloxane, pyrolyzed materials, block copolymers and copolymers of the above compounds, silicones or silicon dioxide.

15. The method according to embodiments 1-14, wherein said blood sample is contacted with an unsaturated solid phase.

16. The method according to embodiments 1-15 wherein the solid phase is not coated with any binding agent or capture molecule.

17. The method according to embodiments 1-16, wherein the optional stabilizing protein in the reaction buffer stabilizes the binding of ApoE4 and its fragments in the blood sample to the solid phase by blocking non-specific binding sites and interacting with the ApoE4 conformation, wherein the stabilizing protein is selected from the group consisting of bovine serum albumin (BSA), human serum albumin, casein, milk proteins, immunoglobulins, stabilizing amino acids (eg alanine, arginine, glycine, isoleucine, lysine, proline, serine), and preferably the stabilizing protein is bovine serum albumin (BSA).

18. The method according to embodiments 1-17, wherein the concentration of BSA as a stabilizing protein is in the range of 0-10%, preferably 0.2-5%, most preferably 0.5-2.5%.

19. The method according to embodiments 1-18, wherein the detergent is selected from the group of anionic detergents (eg, sodium dodecyl sulfate (SDS)), cationic detergents (eg, cetyltrimethylammonium bromide (CTAB)), zwitterionic detergents (eg, CHAPS), and/ or non-ionic detergents (eg Tween-20, Triton X-100).

20. The method according to embodiments 1-19, wherein the concentration of tween-20 is selected in the range of 0.001-10%, preferably 0.01-1%, most preferably 0.2-0.5%.

21. The method according to embodiments 1-20, wherein the concentration of Triton X-100 is selected in the range of 0.001-0.5%, preferably 0.01-0.2%, most preferably 0.05-0.1%.

22. The method according to any of the preceding embodiments, wherein the blood sample is selected from the group of whole blood, serum and citrated plasma, heparinized plasma, EDTA plasma.

23. The method according to any of the preceding embodiments, wherein the subject is selected from the group consisting of a living human or human body, preferably a human subject, wherein the subject is healthy, presumably healthy, suffering from cognitive impairment, dementia, in particular AD.

24. A set containing:

a. one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label,

b. solid phase (e.g. plate, test tube, beads, plate),

with. reaction buffer containing detergent.

25. Use of a kit according to any of the preceding embodiments for the in vitro detection and/or quantification of ApoE4 or fragments thereof in a blood sample of a subject.

26. Use of a method according to any of the preceding embodiments for risk stratification or stratification of the use of therapeutic interventions for a disease or condition associated with elevated levels of ApoE4.

27. Use of a method according to any of the preceding embodiments, wherein the disease or condition is selected from the group of cognitive impairment and neurodegenerative diseases, including dementia and/or Alzheimer's disease (AD).

28. A method according to any of the preceding embodiments for detecting the risk of developing AD, wherein an elevated level of ApoE4 above a certain threshold is predictive of an increased risk of developing AD.

29. The method according to any of the preceding embodiments, wherein said presence of ApoE4 or an ApoE4-binding agent complex correlates with the risk of developing/morbidity of AD.

Examples

Example 1

Obtaining antibodies and determining their affinity constants

- Peptides/conjugates for immunization:

A specific ApoE4 peptide for immunization and a specific control peptide ApoE2/3 were synthesized, see Table 1 (JPT Technologies, Berlin, Germany). The ApoE4 peptide was synthesized with an additional N-terminal cysteine residue to allow conjugation of the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using Sulfolink binding gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual.

Table 1 Peptides ApoE region Description Affinity constant
[M ^-one ] Cys-EDVRGRLVQYR 109-119 ApoE4 Immunogen 2.1× ¹⁰⁹ EDVCGRLVQYR 109-119 ApoE2/3 Control peptide -

- Mice Immunization, Immune Cell Fusion and Screening:

Balb/c mice were immunized with 100 μg of peptide-BSA conjugate on days 0 and 14 (emulsified in 100 μl of complete Freund's adjuvant) and 50 μg on days 21 and 28 (in 100 μl of incomplete Freund's adjuvant). Three days before the fusion experiment, the animals received 50 μg of the conjugate dissolved in 100 μl of saline, as one intraperitoneal and one intravenous injection.

Splenocytes from immunized mice and SP2/0 myeloma cell line were fused with 1 ml of 50% polyethylene glycol for 30 seconds at 37°C. After washing, the cells were plated in 96-well cell culture plates. Hybrid clones were selected by cultivation in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT supplement]. Two weeks later, HAT medium was replaced with HT medium for three passages, followed by a return to normal cell culture medium.

Cell culture supernatants were subjected to primary screening for antigen-specific IgG antibodies three weeks after fusion. Positive tested microcultures were transferred to 24-well multiplication plates. After retesting, selected cultures were cloned and recloned using the limiting dilution method and isotypes determined (Lane et al., 1985, Ziegler et., al. 1996).

- Obtaining monoclonal antibodies

Antibodies were prepared using standard antibody preparation methods (Marx et al., 1997) and purified with protein A. Antibody purity was >95% based on SDS gel electrophoresis analysis.

- Affinity constants

To determine the affinity of the antibody to ApoE4, the binding kinetics of the recombinant ApoE4 to the immobilized antibody was studied by unlabeled surface plasmon resonance using the Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible antibody immobilization was performed using an anti-mouse Fc antibody covalently bound to the surface of a CM5 sensor according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare).

- Labeling procedure (indicator)

100 μg (100 μl) of antibody (1 mg/ml in PBS, pH 7.4) was mixed with 10 μl of acridinium NHS ester (1 mg/ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 20 min at room temperature. The labeled antibodies were purified by size exclusion HPLC on a Bio-Sil® SEC ^400-5 (Bio-Rad Laboratories, Inc., USA). Purified labeled antibodies were diluted (300 mmol/l potassium phosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative light units (RLU) of the labeled compound (approximately 20 ng of labeled antibody) per 200 μl. Acridinium ester chemiluminescence was measured using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

Example 2

Analysis of plasma samples with a known genotype. Using appropriate plasma and whole blood, one can genetically analyze ApoE status by genotyping and measuring ApoE4 levels in appropriate plasma using the ApoE4 assay of the present invention.

2.1 Methods

- Genotyping

Genetic analysis of 180 whole blood samples was performed by Medigenomix, Ebersberg, Deutschland. DNA from the samples was isolated and the ApoE gene was sequenced using Sanger sequencing (Sanger & Coulson 1975).

- ApoE4 assay procedure

ApoE4 single antibody assay was performed in a one-step procedure. Plasma samples were directly pipetted into the wells of a 96-well microtiter plate (Greiner, Lumitrac600, high binding polystyrene; 20 μl of sample per well) in duplicate. A low ApoE4 cutoff control (recombinant ApoE4 [4 μg/ml] in ApoE4 negative human plasma, 20 μl per well, in duplicate) was added to each microplate. 200 μl of reaction buffer (PBS with 0.2% tween-20, 1% BSA, 0.1% bovine IgG, 0.1% ovine IgG and 0.02% mouse IgG, pH 7.4) with labeled acridinium ether anti-ApoE4 antibody (indicator). The microplates were incubated for 1 h at room temperature. The unbound indicator was removed by washing 4 times with a wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Luminescence (in relative light units (RLU)) was measured using an MTP luminometer (Centro LB960, Berthold Technologies GmbH, Bad Wildbad).

2.2 Results

In FIG. 1a shows the results of the ApoE4 analysis differentiated by genotype. With clipping control, ApoE4-positive and ApoE4-negative samples can be easily separated by RLU values. All ApoE4 negative samples have RLU values below 3000 RLU and all ApoE4 positive samples have RLU values above 3000 RLU. Negative samples generally show only a background signal in the immune assay, and no ApoE4 signal. In FIG. 1b shows the corresponding ROC curve. Using an area under the curve (AUC) of 1, the assay accurately differentiates between ApoE4 positive and ApoE4 negative samples.

Example 3

Analysis of ApoE4 binding to various polymeric and glass surfaces with/without detergent.

3.1 Methods

- Pre-incubation of plasma samples on polymer or glass surfaces:

Each ApoE4-positive plasma sample was divided into 120 µl aliquots, which were incubated in tubes of different materials (polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET) and glass), two tubes for each material. 0.1% Triton X-100 was added to one half of the aliquots prior to incubation and the other half was treated with an equal volume of PBS. All tubes were incubated at room temperature overnight.

- Analysis by the method of one antibody to ApoE4

20 μl of each preparation, as well as control without pre-incubation, was pipetted into a high-binding Greiner microtiter plate per well. An ApoE4 assay was then performed as described in Example 2. The measured ApoE4 concentration was calculated relative to the concentration in the corresponding control sample (defined as 100%).

3.2 Results

Detergent-treated samples showed a significant decrease in ApoE4 levels after pre-incubation in polymer or glass tubes, the loss was approximately 50% (see Fig. 2). There were no differences between the type of surface on which the samples were preincubated. After pre-incubation of samples not treated with detergent, a slight loss of the ApoE4 signal was observed, but not as significant as when using detergent.

The detergent enhanced the binding of ApoE4 to polymer or glass surfaces.

Example 4

An analysis of how detergents function as bridge molecules between ApoE4 and the solid phase.

4.1 Methods

- Coating of microtiter plates (MTP)

Three high-binding Greiner microtiter plates were loaded with 300 µl per well of either ddH ₂ O, 0.05% Triton X-100 in ddH ₂ O, or 0.4% Triton X-100 in ddH ₂ O (one plate per variant). ). The plates were incubated overnight at room temperature. Thereafter, the MTP was washed three times with 1×PBS and directly used for one-step analysis by a single anti-ApoE4 antibody method.

- Analysis by the method of one antibody to ApoE4

20 μl plasma of ApoE4 positive and ApoE4 negative samples, as well as a cut-off control (4 μg/ml recombinant ApoE4 in human plasma) were pipetted into pretreated Greiner high binding microplates per well. Half of the wells were filled with 200 µl of indicator buffer (PBS with 1% BSA, 0.1% bovine IgG, 0.1% ovine IgG and 0.02% mouse IgG, pH 7.4 + acridinium ester labeled anti-ApoE4 antibody) containing 0.05% Triton X-100 and the other half filled with the same indicator buffer but without detergent (0% Triton X-100). The microplates were incubated for 1 h at room temperature. The unbound indicator was removed by washing 4 times with a wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Luminescence (in relative light units (RLU)) was measured using an MTP luminometer (Centro LB960, Berthold Technologies GmbH, Bad Wildbad).

4.2 Results

If detergents are to function as a bridge molecule between ApoE4 and the solid phase, then there should be a strong signal when ApoE4 assay is run on detergent-treated plates without any detergent in the assay buffer, possibly even depending on the concentration. But that was not the option. All samples, as well as the cutoff control, showed no signal on any pretreated plate when the assay was run in assay buffer without detergent (see FIG. 3). The signals of both ApoE4-positive and ApoE4-negative samples were at the baseline at the same level and, therefore, they could not be differentiated from each other. In contrast, when the assay was performed with the presence of detergent in the assay buffer, a significant signal appeared in the ApoE4 positive samples and the cutoff control. This signal was also independent of the amount of X-100 triton associated with MTP.

Detergent is a necessary buffer component in the ApoE4 assay. Coating with MTP detergent and adding sample and detergent-free buffer does not result in a reliable signal. This shows that the detergent is more needed in its mobile form than in its immobilized form, possibly as a bonding molecule.

Example 5

- One step analysis procedure

Anti-ApoE4 single antibody assay was performed as described in Example 2. In a one-step procedure, samples and indicator antibody were incubated together for 1 hour at room temperature. Luminescence (in relative light units (RLU)) was measured using an MTP luminometer (Centro LB960, Berthold Technologies GmbH, Bad Wildbad). When added to each microplate, there was a low ApoE4 cut-off control (recombinant ApoE4 [4 μg/ml] in human ApoE4 negative plasma; 20 μl per well, in duplicate).

- Two-stage analysis procedure

In a two-step procedure, plasma samples were directly pipetted into the wells of a 96-well microtiter plate (Greiner, Lumitrac600, high binding polystyrene; 20 μl of sample per well) in duplicate. When added to each microplate, there was a low ApoE4 cut-off control (recombinant ApoE4 [4 μg/ml] in human ApoE4 negative plasma; 20 μl per well, in duplicate). The wells were filled with 200 μl of reaction buffer (PBS with 0.2% tween-20, 1% BSA, 0.1% bovine IgG, 0.1% ovine IgG and 0.02% mouse IgG, pH 7.4). The microplates were incubated overnight at room temperature (= stage 1). Thereafter, the microplates were washed 4 times with a wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100) and 200 μl of acridinium ester labeled anti-ApoE4 antibody reaction buffer was added to the wells. The microplates were incubated for 2 hours at room temperature (= stage 2) and washed again 4 times with the wash solution. Luminescence (in relative light units (RLU)) was measured using an MTP luminometer (Centro LB960, Berthold Technologies GmbH, Bad Wildbad).

For a more direct comparison of the two variants of the analysis procedure, the RLU values were converted to relative units "fold change compared to cut-off control".

5.2 Results

In this case, a short (1 h) one-step procedure was compared with a long (overnight/2 h) two-step procedure. The relative light units (RLU) of the one-step assay were about ten times lower than the RLUs of the two-step assay (see Table 2). But this ratio was only true for each ApoE4-positive sample and cut-off control. Values for ApoE4-negative samples remained equally low in either assay format. The sample values relative to the internal control cut-off were approximately the same in each assay (see FIG. 4) and ApoE4-positive from ApoE4-negative samples could be differentiated with each assay.

table 2 ApoE4 status Samples One step analysis Two-stage analysis RLU fold change compared to clipping control RLU fold change compared to clipping control positive P1 16.126 2.75 162.422 3.19 P2 19.917 3.40 217.180 4.26 P3 17.034 2.91 126.767 2.49 P4 9.665 1.65 98.857 1.94 negative P5 79 0.0135 88 0.0017 P6 71 0.0121 62 0.0012 fold change compared to clipping control 5,861 1.00 50.986 1.00

Example 6

Effect of a blocking/stabilizing agent (BSA) on ApoE4 binding to the solid phase.

6.1 Methods

The ApoE4 assay was performed in a two step procedure where the first step (binding of ApoE4 to the solid phase) was performed in the presence or absence of BSA. The second step (binding of anti-ApoE4 antibody to immobilized ApoE4) was performed in assay buffer containing 1% BSA.

Plasma samples and a low ApoE4 cutoff control (recombinant ApoE4 [4 μg/mL] in ApoE4 negative human plasma) were directly pipetted into the wells of a 96-well microtiter plate (Greiner, Lumitrac600 high binding polystyrene; 20 μl sample per well). 200 µl of reaction buffer (PBS with 0.2% tween-20, 0.1% bovine IgG, 0.1% ovine IgG and 0.02% mouse IgG, pH 7.4) containing 1% BSA was added to the wells. or did not contain BSA (0% BSA). The microplates were incubated for 1 hour at room temperature (= stage 1). After that, the microplates were washed 4 times with a wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100) and 200 μl of 1% BSA-containing reaction buffer with labeled acridinium ether anti-ApoE4 antibody was added to the wells . The microplates were incubated for 1 hour at room temperature (= stage 2) and washed again 4 times with the wash solution. Luminescence (in relative light units (RLU)) was measured using an MTP luminometer (Centro LB960, Berthold Technologies GmbH, Bad Wildbad).

6.2 Results

The signals of samples incubated in buffer without BSA were significantly lower than with the presence of BSA in the reaction mixture (see table 3, figure 5). With both assays, it was possible to differentiate between ApoE4 positive and ApoE4 negative samples using cutoff control. Therefore, BSA is not necessary for ApoE4 binding, but surprisingly it significantly enhanced ApoE4 binding to the solid phase and thus it can optionally be added to the reaction buffer to enhance signals. This result is unexpected since BSA is commonly used to block non-specific signals and is often accompanied by lower signals after BSA addition.

Table 3

ApoE4 status Samples One step analysis Two-stage analysis RLU fold change compared to clipping control positive A1 69.708 9.76 253.321 10.15 A2 40.772 5.71 278.431 11.16 A3 20.839 2.92 187.286 7.51 A4 75.446 10.57 271.817 10.89 A5 44.542 6.239 297.033 11.906 A6 84.069 11.776 239.914 9.616 negative A7 472 0.066 2.776 0.111 A8 401 0.056 244 0.010 A9 119 0.017 97 0.004 fold change compared to clipping control 7.139 one 24.949 one

Example 7

Quantification of ApoE4 in plasma samples.

7.1 Methods

ApoE4 assay was performed as described in Example 2. Calibrators were used to quantify plasma ApoE4 levels. Recombinant ApoE4 was serially diluted in ApoE4-negative human plasma to obtain a calibration curve over a concentration range of 0.01-21.87 µg/mL. The concentration of ApoE4 in the samples was calculated using a calibration curve based on linear regression. Samples that showed RLU values above the highest standard value were diluted with ApoE4-negative human plasma and measured again. A cut-off control at a specific concentration of 4 μg/mL was also measured as a control for the standard curve.

7.2 Results

In the concentration range from 0.01 to 21.87 μg/ml ApoE4 linearly diluted (see Fig. 6). Table 4 shows the measured RLU values and the calculated ApoE4 concentrations. ApoE4-positive samples had concentrations in the range of 9.7-30.4 µg/ml. The concentration of all ApoE4-negative samples in all cases was <0.1 μg/ml (baseline value in the analysis). The cutoff control concentration was 4 µg/mL, exactly as measured with the calibrators diluted prior to measurement.

Table 4 ApoE4 status Samples ApoE4 concentration [µg/ml] positive A 22.3 B 25.2 C 9.7 D 21.9 E 30.4 negative F <0.1 G <0.1 H <0.1 fold change compared to clipping control 4.0

Example 8

Effect of protein precoating on ApoE4 binding to the solid phase.

8.1 Methods

A 96-well microtiter plate (Greiner, Lumitrac600 high binding polystyrene) was pre-coated with 300 µl per well of human serum (ApoE4 negative) or a saturated solution containing 6.5 mM KH ₂ PO ₄ , 3.5 mM Na ₂ HPO ₄ ×2 H ₂ O, 3% Karion (sugar) and 0.5% BSA for 18 hours at room temperature. After incubation, the microplates were washed 4 times with a wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

ApoE4 analysis was then performed in a one-step procedure (as described in Example 2).

Plasma samples (ApoE4-positive and ApoE4-negative) and recombinant ApoE4 samples (recombinant ApoE4 in human ApoE4-negative plasma) were pipetted directly into the wells of a 96-well microtiter plate (20 μl of sample per well). The wells were filled with 200 µl of reaction buffer (PBS with 0.2% tween-20, 0.1% bovine IgG, 0.1% ovine IgG and 0.02% mouse IgG, pH 7.4) containing acridinium ester labeled anti- ApoE4 antibody. Thereafter, the microplates were washed 4 times with a wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Luminescence (in relative light units (RLU)) was measured using an MTP luminometer (Centro LB960, Berthold Technologies GmbH, Bad Wildbad).

8.2 Results

As shown in FIG. 7(A) ApoE4-positive EDTA plasma samples gave a significant signal on the untreated solid phase. In contrast, wells pre-treated with a protein-containing solution (either BSA or human serum) produced no signal.

Similarly, as shown in FIG. 7(B), ApoE4-negative EDTA plasma samples containing various concentrations of recombinant ApoE4 gave a significant signal in untreated wells, but no signal when incubated in wells pre-treated with protein.

Links

Aggarwal N.T., Wilson R.S., Beck T.L. et al. The apolipoprotein E epsilon4 allele and incident Alzheimer's disease in persons with mild cognitive impairment. Neurocase. 2005; 11(1):3-7.

Bakbak B. et al., 2015. Association of Apolipoprotein E Polymorphism with Intravitreal Ranibizumab Treatment Outcomes in Age-Related Macular Degeneration. Current eye research, 3683(October), pp. 1-5.

Bury J. et al., 1986. Apolipoprotein E quantified by enzyme-linked immunosorbent assay. Clin Chem., 32(2), pp. 265-70.

Cacabelos R. et al., 2012. Genomics of dementia: APOE - And CYP2D6-related pharmacogenetics. International Journal of Alzheimer's Disease, 2012(518901).

Calero O. et al., 2009. Apolipoprotein E genotyping method by Real Time PCR, a fast and cost-effective alternative to the TaqMan and ^FRET® assays. J. Neurosci. Methods, 183(2), pp. 238-40.

Deng B. et al., 2014. Aptamer binding assays for proteins: The thrombin example-A review. Analytica Chimica Acta, 837, pp. 1-15.

Hegemann D., Brunner H. & Oehr C., 2003. Plasma treatment of polymers for surface and adhesion improvement. Nuclear Instruments and Methods in Physics, 208, pp. 281-286.

Hesse C. et al., 2000. Measurement of Apolipoprotein E (apoE) in Cerebrospinal Fluid. Neurochemical Research, 25(4), pp. 511-517.

Høgåsen K. et al., 1993. Quantitation of vitronectin and clusterin Pitfalls and solutions in enzyme immunoassays for adhesive proteins. Journal of Immunological Methods, 160, pp. 107-115.

Holmquist L., 1982. Loss of human serum apolipoproteins C and E during manipulation of diluted solutions. Journal of Lipid Research, 23, pp. 357-359.

Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, J. I. Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562, incorporated here by reference, including the quotations on pages 551-562.

Lane R.D. (1985). A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secreting hybridomas. J. Immunol. Meth. 81: 223-228

Leduc V. et al., 2011. Function and comorbidities of apolipoprotein e in Alzheimer's disease. International Journal of Alzheimer's disease, 2011(974361).

Li J. et al., 2011. Peptide aptamers with biological and therapeutic applications. Current medicinal chemistry, 18(27), pp. 4215-22.

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Marx et al., Monoclonal Antibody Production, ATLA 25, 121, 1997

Mayeux R. et al., 1998. Utility of the Apolipopreoin E Genotype in the Diagnosis of Alzheimer's Disease. N. Engl. J. Med., 338(8), pp. 506-511.

Rohn T.T., 2013. Proteolytic cleavage of apolipoprotein E4 as the keystone for the heightened risk associated with Alzheimer's disease, Int. J. Mol. Sci., 14(7):14908-22.

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Verghese P.B., Castellano J.M. & Holtzman D.M., 2011. Roles of Apolipoprotein E in Alzheimer's disease and other neurological disorders. Lancet neurology, 10(3), pp. 241-252.

Weisgraber K.H. & Mahley R.W., 1996. Human apolipoprotein E: the Alzheimer's disease connection. Federation of American Societies for Experimental Biology, 10, pp. 1485-94.

Xu Y., Yang X. & Wang E., 2010. Review: Aptamers in microfluidic chips. Analytica chimica acta, 683(1), pp. 12-20.

Ziegler B. et al. (1996) Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28:11-15)

Zhong L. et al., 2016. A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism. Molecular Neurodegeneration, 11(2), pp. 1-8.

Sequences

SEQ ID NO: 1

SEQ ID NO: 2 (human preApoE4 1-317, including signal sequence )

SEQ ID NO: 3 (ApoE4 fragment 1-165)

SEQ ID NO: 4 (ApoE4 fragment 1-185)

SEQ ID NO: 5 (ApoE4 fragment 1-191)

SEQ ID NO: 6 (ApoE4 fragment 1-202)

SEQ ID NO: 7 (ApoE4 fragment 1-229)

SEQ ID NO: 8 (ApoE4 fragment 1-231)

SEQ ID NO: 9 (ApoE4 fragment 1-251)

SEQ ID NO: 10 (ApoE4 fragment 1-259)

SEQ ID NO: 11 (ApoE4 fragment 1-260)

SEQ ID NO: 12 (ApoE4 fragment 1-266)

SEQ ID NO: 13 (ApoE4 fragment 1-271)

SEQ ID NO: 14 (ApoE4 fragment 1-272)

SEQ ID NO: 15 (ApoE4 72-299)

Description of figures

Fig. one:

(A) Analysis of plasma samples with a known ApoE genotype. Plasma ApoE4 concentrations correlate with genetic status (low values for ApoE4-negative samples, high values for ApoE4-positive samples; statistical analysis: Mann-Whitney test).

(B) ROC curve of ApoE4 analysis with genotype (AUC - area under the curve).

Fig. 2:

Results of measuring ApoE4 levels after pre-incubation in test tubes of various materials with and without detergent added (PS - polystyrene, PP - polypropylene, PET - polyethylene terephthalate).

Fig. 3:

Results of measuring ApoE4 levels in MTP after MTP pretreatment with/without detergent (Triton X-100). Assay buffer contained Triton X-100 (0.05%) or no detergent (0%), samples were analyzed in a one-step procedure. Clipping control shown (A), two ApoE4-positive samples D1 (B) and D2 (C) and ApoE4-negative sample D3 (D).

Fig. 4:

Comparison of one-step and two-step ApoE4 assay. The dotted line shows the clipping control value. Each sample with values below this cut-off value is ApoE4-negative, and each sample with values above the cut-off value is ApoE4-positive.

Fig. five:

Analysis of ApoE4 binding to solid phase in the presence (1%)/absence (0%) of BSA. The dotted line shows the cutoff control value (C) in the corresponding assay.

Fig. 6:

Linear regression of ApoE4 standards over the concentration range from 0.01 to 21.87 µg/mL.

Fig. 7:

Results Measurement of ApoE4 levels after pretreatment of the solid phase with a protein solution (bovine serum albumin or human serum).

(A) Measurement results of ApoE4-positive and ApoE4-negative samples in native EDTA plasma.

(B) Measurement results of recombinant ApoE4 solutions.

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Sequence list

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<120> METHOD FOR DETERMINING APOLYPOPROTEIN E4

<130> S75097WO

<150> EP 16 193 587.9

<151> 2016-10-12

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<170>PatentIn version 3.5

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Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala

245 250 255

Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu Asp Met

260 265 270

Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala Val Gly

275 280 285

Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His

290 295

<210> 2

<211> 317

<212> Protein

<213> Homo sapiens

<400> 2

Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys

1 5 10 15

Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu

20 25 30

Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu

35 40 45

Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln

50 55 60

Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala

65 70 75 80

Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu

85 90 95

Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser

100 105 110

Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp

115 120 125

Val Arg Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu

130 135 140

Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg

145 150 155 160

Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg

165 170 175

Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu

180 185 190

Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val

195 200 205

Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg

210 215 220

Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly

225 230 235 240

Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu

245 250 255

Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala

260 265 270

Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu

275 280 285

Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala

290 295 300

Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His

305 310 315

<210> 3

<211> 165

<212> Protein

<213> Homo sapiens

<400> 3

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly

165

<210> 4

<211> 185

<212> Protein

<213> Homo sapiens

<400> 4

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val

180 185

<210> 5

<211> 191

<212> Protein

<213> Homo sapiens

<400> 5

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg

180 185 190

<210> 6

<211> 202

<212> Protein

<213> Homo sapiens

<400> 6

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro

195 200

<210> 7

<211> 229

<212> Protein

<213> Homo sapiens

<400> 7

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu

225

<210> 8

<211> 231

<212> Protein

<213> Homo sapiens

<400> 8

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu

225 230

<210> 9

<211> 251

<212> Protein

<213> Homo sapiens

<400> 9

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg

245 250

<210> 10

<211> 259

<212> Protein

<213> Homo sapiens

<400> 10

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala

245 250 255

Phe Gln Ala

<210> 11

<211> 260

<212> Protein

<213> Homo sapiens

<400> 11

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala

245 250 255

Phe Gln Ala Arg

260

<210> 12

<211> 266

<212> Protein

<213> Homo sapiens

<400> 12

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala

245 250 255

Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu

260 265

<210> 13

<211> 271

<212> Protein

<213> Homo sapiens

<400> 13

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala

245 250 255

Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu Asp

260 265 270

<210> 14

<211> 272

<212> Protein

<213> Homo sapiens

<400> 14

Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln

1 5 10 15

Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg

20 25 30

Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln

35 40 45

Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met

50 55 60

Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu

65 70 75 80

Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu

85 90 95

Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg

100 105 110

Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln

115 120 125

Ser Thr Glu Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu

130 135 140

Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala

145 150 155 160

Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala

165 170 175

Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln

195 200 205

Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg

210 215 220

Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg

225 230 235 240

Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala

245 250 255

Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu Asp Met

260 265 270

<210> 15

<211> 228

<212> Protein

<213> Homo sapiens

<400> 15

Lys Ala Tyr Lys Ser Glu Leu Glu Glu Gln Leu Thr Pro Val Ala Glu

1 5 10 15

Glu Thr Arg Ala Arg Leu Ser Lys Glu Leu Gln Ala Ala Gln Ala Arg

20 25 30

Leu Gly Ala Asp Met Glu Asp Val Arg Gly Arg Leu Val Gln Tyr Arg

35 40 45

Gly Glu Val Gln Ala Met Leu Gly Gln Ser Thr Glu Glu Glu Leu Arg Val

50 55 60

Arg Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu Leu Arg Asp

65 70 75 80

Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr Gln Ala Gly Ala Arg

85 90 95

Glu Gly Ala Glu Arg Gly Leu Ser Ala Ile Arg Glu Arg Leu Gly Pro

100 105 110

Leu Val Glu Gln Gly Arg Val Arg Ala Ala Thr Val Gly Ser Leu Ala

115 120 125

Gly Gln Pro Leu Gln Glu Arg Ala Gln Ala Trp Gly Glu Arg Leu Arg

130 135 140

Ala Arg Met Glu Glu Met Gly Ser Arg Thr Arg Asp Arg Leu Asp Glu

145 150 155 160

Val Lys Glu Gln Val Ala Glu Val Arg Ala Lys Leu Glu Glu Gln Ala

165 170 175

Gln Gln Ile Arg Leu Gln Ala Glu Ala Phe Gln Ala Arg Leu Lys Ser

180 185 190

Trp Phe Glu Pro Leu Val Glu Asp Met Gln Arg Gln Trp Ala Gly Leu

195 200 205

Val Glu Lys Val Gln Ala Ala Val Gly Thr Ser Ala Ala Pro Val Pro

210 215 220

Ser Asp Asn His

225

<---

Claims (30)

1. An in vitro method for detecting apolipoprotein E isotype (ApoE4) 4 or fragments thereof in a blood sample of a subject, comprising the steps of: a. contacting said sample with a solid phase, where the solid phase is not covered by any binding agent or capture molecule, contacting said sample simultaneously or sequentially with at least one binding agent that specifically binds to ApoE4, thereby forming an ApoE4-binding agent complex, where said binding agent is an antibody or a functional fragment of an antibody or a scaffold protein other than IgG, where ApoE4 or fragments thereof are immobilized on the specified solid phase, and the binding agent is associated with ApoE4, and wherein the binding of ApoE4 or the ApoE4-binding agent complex to said solid phase occurs in the presence of a detergent, and b. detection of an ApoE4-binding agent complex, where the ApoE4 fragments are at least 6 amino acids in length, provided that amino acid 112, which is arginine, is included in said fragment, where the sequence including amino acid 112 refers to SEQ ID NO:1. 2. The method of claim 1, wherein the ApoE4-binding agent complex is detected qualitatively and quantitatively. 3. The method of claim 1 or 2, wherein the quantitation of the ApoE4-binding agent complex indicates the concentration of ApoE4 in said sample, where the concentration indicates how homozygous or heterozygous the subjects are for the ApoE4 alleles. 4. The method according to claims 1 to 3, wherein the binding agent is an antibody or functional antibody fragment or scaffold protein other than IgG with a binding affinity constant of at least 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 , the preferred affinity constant above 10 ⁹ M ^-1 , most preferably above 10 ¹⁰ M ^-1 . 5. The method according to claims 1 to 4, wherein the binding agent is labeled with a detection label, wherein said label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, or a radioactive iodine label. 6. The method of claims 1-5, wherein ApoE4 detection is used to classify a subject as ApoE4 positive or ApoE4 negative when the level of detected ApoE4 is above a certain threshold, where said threshold is determined by a particular internal positive control value. 7. The method according to claims 1 to 6, wherein the solid phase is selected from the group consisting of polymeric surface, eg polystyrene, glass surface, cellulose and cellulose derivatives, eg nitrocellulose, polyvinylidene fluoride, nylon. 8. The method according to claims 1-7, where the solid phase is selected from the group of glass or polymer surfaces, where the polymer surface includes, but is not limited to, polystyrene, poly(divinylbenzene), styrene-acrylic copolymer, styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene-oxyethylene), polymethylmethacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, N,N'-methylene-bis-acrylamide, polyolefins, polyethylene, polytetrafluoroethylene, polyethylene terephthalate, polypropylene, polyvinyl chloride, polyvinyl acetate, polyacrylonitrile, polysulfone, poly( sulfonic ether), polydimethylsiloxane, pyrolyzed materials, block copolymers and copolymers of the above compounds, silicones or silicon dioxide. 9. The method according to claims 1 to 8, wherein said blood sample is contacted with an unsaturated solid phase. 10. The method according to claims 1-9, wherein the optional stabilizing protein in the reaction buffer stabilizes the binding of ApoE4 and its fragments in the blood sample to the solid phase by blocking non-specific binding sites and interacting with the conformation of ApoE4, where the stabilizing protein is selected from the group including bovine serum albumin (BSA), human serum albumin, casein, milk proteins, immunoglobulins, stabilizing amino acids eg alanine, arginine, glycine, isoleucine, lysine, proline, serine, and preferably the stabilizing protein is bovine serum albumin (BSA). 11. The method according to claim 10, wherein the concentration of BSA as a stabilizing protein is in the range of 0-10%, preferably 0.2-5%, most preferably 0.5-2.5%. 12. The method according to claims 1 to 11, wherein the detergent is selected from the group of anionic detergents, e.g. sodium dodecyl sulfate (SDS), cationic detergents, e.g. cetyltrimethylammonium bromide (CTAB), zwitterionic detergents, e.g. CHAPS, and/or non-ionic detergents, for example, Tween-20, Triton X-100. 13. The method according to claim 12, wherein the concentration of Tween-20 is selected in the range of 0.001-10%, preferably 0.01-1%, most preferably 0.2-0.5%. 14. The method according to claim 12, wherein the concentration of Triton X-100 is selected in the range of 0.001-0.5%, preferably 0.01-0.2%, most preferably 0.05-0.1%. 15. The method according to any one of the preceding claims, wherein the blood sample is selected from the group of whole blood, serum and citrated plasma, heparinized plasma, EDTA plasma. 16. The method according to any of the preceding claims, wherein the subject is selected from the group consisting of a living human or human body, preferably a human subject, wherein the subject is healthy, presumably healthy, suffering from cognitive impairment, dementia, in particular Alzheimer's disease. 17. A kit for detecting ApoE4 or its fragments in a blood sample of a subject, for the method according to any one of claims 1-16, containing: a. one binding agent directed against an epitope included in the sequence of ApoE4 or its fragments, where the binding agent is labeled with a detectable label, b. the solid phase with. reaction buffer containing detergent. 18. Use of the kit according to claim 17 for in vitro detection and/or quantification of ApoE4 in a blood sample of a subject. 19. Use of the method of any one of claims 1 to 16 for risk stratification or stratification of the use of therapeutic interventions for a disease or condition associated with elevated levels of ApoE4. 20. Use of the method of claim 19, wherein the disease or condition is selected from the group of cognitive impairment and neurodegenerative diseases, including dementia and/or Alzheimer's disease. 21. Use of the method of any one of claims 1 to 16 to detect risk of developing Alzheimer's disease, wherein an elevated level of ApoE4 above a certain threshold is predictive of an increased risk of developing Alzheimer's disease. 22. Use according to claim 20 or 21, wherein said presence of ApoE4 or an ApoE4-binding agent complex correlates with the risk of developing/morbidity of Alzheimer's disease.

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