Apolipoprotein detection method

Abstract

The present invention relates to methods for detecting and quantifying apolipoproteins and isoforms thereof in a sample, and methods for predicting the probability of development of neurodegenerative or cardiovascular diseases based on the apolipoprotein levels determined according to the detection methods described in the invention.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680077646.7, filed on November 2, 2016, and entitled “Apolipoprotein Detection Method”. Technical Field

[0002] The present invention relates to methods for detecting and quantifying apolipoprotein and its isoforms in a sample, and a predictive method for estimating the probability of developing a neurodegenerative or cardiovascular disease based on the apolipoprotein level determined by the detection method of the present invention. Background Art

[0003] Apolipoprotein E (apoE) is a 34kDa glycoprotein involved in lipid metabolism. The human APOE gene encoding this protein is polymorphic and is located on chromosome 19. Three common codominant alleles (ε2, ε3 and ε4) encode three apoE protein isoforms: E2, E3 and E4. These isoforms differ in amino acid residues 112 and 158. Isoform E2 has cysteine ​​residues at both sites, E4 has arginine residues at both sites, and E3 (the most common form) has cysteine ​​at position 112 and arginine at position 158. These differences have a profound impact on the biological function of apoE. The lipid binding activity of these isoforms is different: E2 and E3 preferentially bind to high-density lipoprotein (HDL), while E4 prefers very low-density lipoprotein (VLDL). These biochemical differences may be the cause of the association of isoforms with different pathological processes. Isoform E4 is associated with higher levels of cholesterol and an increased risk of coronary heart disease and Alzheimer's disease (Alzheimer and coronary heart disease and Alzheimer). In contrast, isoform E2 shows a protective effect against Alzheimer's disease, but it is associated with familial type III hyperlipoproteinemia. Therefore, interest in APOE genotypes or apoE isoforms is of high interest for epidemiological studies, patient stratification, and identification of those at increased risk for clinical trials and prevention.

[0004] Several methods are commonly used to genotype the three major APOE haplotypes. The more commonly used methods are: PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism), capillary electrophoresis, PCR plus sequencing or mass spectrometry, ARMS-PCR (amplification refractory mutation system-PCR) and SSP-PCR (simple sequence-specific primers-PCR), real-time PCR detection of fluorescent melting curves, FRET (fluorescence resonance energy transfer), allele-specific RT-PCR and However, all of these APOE gene-based methods require informed consent for DNA extraction and genetic information analysis and cannot be easily implemented in clinical analysis routines.

[0005] Several optional biochemical (non-genetic) methods mainly for research purposes are used for sensitive characterization of apoE isotypes. The most commonly used are isoelectric focusing (IEF) and sandwich ELISA, which rely on the characterization of apoE from biological fluids (such as plasma or CSF). For the analytical IEF of apoE, the isotypes separated by immunodetection of anti-ApoE antibodies after isoelectric focusing in an immobilized pH gradient. The specific apoE band pattern allows the detection of different apoE isotypes present in the sample. This technology has the advantage of being able to detect rare apoE variants except apoE E2, E3 and E4 isotypes; however, this is technically demanding and time-consuming.

[0006] The ELISA (enzyme-linked immunosorbent assay) technique uses antibodies to capture the target protein. Sandwich ELISA can be used to examine the presence of the apoE E4 isoform and relies on the use of two antibodies (capture and reporter antibodies), one of which must be an E4 isoform-specific antibody. There are several commercially available ELISA kits for detecting apoE E4 (e.g., ApoE4 / Pan-ApoE ELISA Kit, MBL#7635); however, this approach has not been implemented in a routine clinical setting, likely due to inherent technical limitations.

[0007] Methods for predicting disease progression based on apolipoprotein levels have been described in the art. US5945289Approx relates to assessing a subject's propensity to develop prostate cancer based on genotyping of ApoE alleles by PCR methods.

[0008] However, there is still a need in the art to develop reliable and accurate methods for apolipoprotein detection and apolipoprotein isoform determination, which allow rapid and reliable determination of apolipoprotein in a sample. Such methods are of interest for predicting the development of certain diseases associated with the presence or absence of apolipoprotein or its specific isoforms. Summary of the invention

[0009] The authors of the present invention have developed a method for detecting, quantifying and analyzing apolipoproteins and their isoforms. This method is based on the specific binding properties of apolipoproteins to surfaces, especially polystyrene surfaces, which avoids the need for detection or quantification using capture antibodies or prior separation procedures. The authors have demonstrated that apolipoproteins, especially apoE, are specifically captured by polystyrene surfaces (including ELISA plates, luminex beads or turbidity beads). Once a specific apolipoprotein isoform, such as apoEE4, is bound to the surface, it can be detected by anti-apoE4 antibodies, thereby achieving detection and quantification of apoE4 isoforms in the sample. This quantification can be associated with the presence of the APOEε4 genotype, whether it is heterozygous (2 / ε4, ε3 / ε4) or homozygous (ε4 / ε4). Considering that this binding property is common to other apolipoproteins, this method can also be applied to other apolipoproteins, including apoAI, apoAIV, apoCI, apoCII, apoCIII and apoJ (clustered protein).

[0010] Thus, in a first aspect, the present invention relates to an in vitro method for detecting and / or quantifying an apolipoprotein selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) or an isoform thereof in a sample, the method comprising:

[0011] (i) contacting a sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein or an isoform thereof to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein or an isoform thereof.

[0012] (ii) contacting the surface bound to the apolipoprotein or its isoform formed in step (i) with an antibody specific for the apolipoprotein or its isoform under conditions suitable for the formation of a complex between the antibody and the apolipoprotein or its isoform, and

[0013] (iii) detecting the complex formed in step (ii),

[0014] The polystyrene surface or the polycarbonate surface is not treated before contacting with the sample or is blocked with albumin before contacting with the sample.

[0015] In yet another aspect, the present invention relates to an in vitro method for determining the relative amount of an isoform of a given apolipoprotein selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) relative to the total content of said apolipoprotein in a sample, the method comprising:

[0016] (i) contacting the sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein,

[0017] (ii) contacting the apolipoprotein-bound surface formed in step (i) with a first antibody specific for an isoform of the apolipoprotein and with a second antibody capable of binding to all isoforms of the apolipoprotein present in the sample, wherein the contacting is performed under conditions suitable for the formation of a first complex comprising the first antibody and the isoform of the apolipoprotein and a second complex comprising the second antibody and all isoforms of the apolipoprotein,

[0018] (iii) detecting the first and second complexes formed in step (ii) and

[0019] (iv) determining the relative amount of the isoform relative to the total apolipoprotein content based on the levels of the first and second complexes obtained in step (iii),

[0020] The polystyrene surface or the polycarbonate surface is not treated before contacting with the sample or is blocked with albumin before contacting with the sample.

[0021] In another aspect, the present invention relates to a method for determining the allele dosage of a haplotype associated with the expression of an apolipoprotein isoform of an apolipoprotein selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) in a subject, the method comprising,

[0022] (i) contacting a protein-containing sample derived from a tissue of the subject expressing the apolipoprotein isoform with a polystyrene surface or a polycarbonate surface under conditions suitable for binding the apolipoprotein isoform to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein isoform.

[0023] (ii) contacting the apolipoprotein-bound surface formed in step (i) with an antibody specific for at least one apolipoprotein isoform under conditions suitable for formation of a complex between the antibody and the apolipoprotein isoform,

[0024] (iii) determining the allelic dosage of the apolipoprotein isoform by correlating the amount of the complex formed in step (ii) with the number of alleles encoding the gene variant

[0025] The polystyrene surface or the polycarbonate surface is not treated before contacting with the sample or is blocked with albumin before contacting with the sample.

[0026] In another aspect, the present invention relates to a method for determining the probability of a subject developing a neurodegenerative disease, the method comprising determining the level of isoform apoE E4 in a sample from the subject by one of the methods described herein, wherein if the apoE E4 level is above a reference value, it indicates that the subject has a high probability of developing a neurodegenerative disease.

[0027] In another aspect, the present invention relates to a method for determining the probability of a subject developing a neurodegenerative disease, the method comprising determining the allele dosage of a haplotype encoding an apoE4 isoform in the subject by a method according to any of the methods of the present invention, wherein the presence of one or two apoE4 alleles in the genome of the subject indicates that the subject has a high probability of developing the neurodegenerative disease, and wherein the absence of an apoE4 allele in the genome of the subject indicates that the subject has a low probability of developing the neurodegenerative disease.

[0028] In another aspect, the present invention relates to a method for determining the probability of a subject developing a cardiovascular disease, the method comprising determining the level of an apolipoprotein isoform in a sample from the subject by one of the methods described herein, wherein if the apolipoprotein level is above a reference value, it indicates that the subject has a high probability of developing a cardiovascular disease.

[0029] In another aspect, the present invention relates to a method for determining the probability of a subject developing a cardiovascular disease, the method comprising determining the allele dosage of a haplotype encoding an apolipoprotein isotype in the subject by a method according to any of the methods of the present invention, wherein the presence of one or two alleles of the apolipoprotein isotype in the genome of the subject indicates a higher probability of the subject developing a cardiovascular disease, and wherein the absence of the allele of the apolipoprotein isotype in the genome of the subject indicates a lower probability of the subject developing a cardiovascular disease.

[0030] In another aspect, the present invention relates to a kit comprising a polystyrene surface or a polycarbonate surface and an antibody specific for apolipoprotein or an isoform thereof, wherein the kit does not comprise a second antibody specific for the apolipoprotein or an isoform thereof.

[0031] In another aspect, the present invention relates to the use of a kit as described above for detecting and / or quantifying apolipoproteins or their isoforms selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin), wherein the polystyrene surface or the polycarbonate surface is blocked with albumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Shown is the analysis of the presence of apoE E4 isoform in 157 plasma samples that did not contain apoE4 isoform and 73 samples that contained apoE4.

[0033] Figure 2 The distribution of apoE E4 (from Figure 1 ) were measured by absorbance ELISA readings of 73 samples.

[0034] Figure 3 Shown are the average absorbance readings for the 230 samples analyzed, distributed according to APOE genotype. Error bars show the standard error of the mean.

[0035] Figure 4 Shown are the ratios of apoE E4 / total apoE absorbance (4E4 / pan-apoE) at different plasma dilutions in APOE e4 non-carriers (E3 / E3) and heterozygous (E3 / E4) and homozygous (E4 / E4) APOE e4 carriers.

[0036] Figure 5 Binding of apolipoprotein from plasma samples (1:200) to a polystyrene surface is shown on an ELISA plate format.

[0037] Figure 6 The effect of different buffers containing salt (NaCl 0-2.4 M) or detergents (0-0.5% of polysorbate 20-Tween20- or Triton X-100) on the binding of ApoE to ELISA plates is shown. Dark grey and light grey bars represent samples analyzed in wells blocked with BSA or Superblock blocking solution, respectively.

[0038] Figure 7 The effect of pH (2-10) on the binding of ApoE to ELISA plates is shown. Light grey and dark grey bars represent samples analyzed in wells blocked with BSA or Superblock blocking solution, respectively.

[0039] Figure 8 The effect of stringent conditions on ApoE binding to ELISA plates is shown.

[0040] Fig. 9 Preliminary analysis of apoE e3 / e3 and apoE e4 / e4 samples by LEIT at different times is shown. DETAILED DESCRIPTION

[0041] The author of the present invention finds that apolipoprotein from biological fluid, particularly apolipoprotein apoE, more particularly isoform 4 (apoE E4) of apoE, has stable high affinity binding capacity to polystyrene surface.Based on this property, the inventor has developed a method for detecting and quantifying apolipoprotein without isoelectric focusing or capture antibody.The present invention describes a simple, reliable and cost-effective method, which can be easily implemented in research laboratories and clinical analysis settings.Because apolipoprotein is related to cardiovascular and neurovascular risks and neurodegeneration, the method provided herein is that patient stratification and identification provide valuable information for the object of development Alzheimer's disease (AD) and cardiovascular disease risk increase.This method can also be used to characterize other apolipoproteins as protein biomarkers, i.e. apoCI, apoCII, apoCIII, apoAI and clustered proteins, and the method is related to the analysis of biological fluids such as blood or urine that are easily available.

[0042] Definition of terms

[0043] As used herein, the term “ Allele ” Pertaining to one of two or more forms of a gene, genetic locus, or genetic polymorphism. Sometimes, different alleles will result in different traits; however, in other cases, different alleles will have the same result in the expression of the gene. Most multicellular organisms have two sets of chromosomes, that is, they are diploid. These chromosomes are called homologous chromosomes. Diploid organisms have one copy of each gene (and one allele) per chromosome. If the two alleles are identical, they are homozygous. If the alleles are different, they are heterozygous.

[0044] As used herein, the term “ Allele dosage ” refers to the number of copies of a particular allelic variant, which in diploid organisms ranges from 0 to 2.

[0045] As used herein, the term “ Alzheimer's disease ” or “AD” refers to mental deterioration associated with a specific degenerative brain disease characterized by senile plaques, neuritic tangles, and progressive neuronal loss, which manifests clinically as progressive memory loss, confusion, behavioral problems, inability to care for oneself, gradual physical deterioration, and ultimately death. As used herein, this term is intended to include all stages of the disease.

[0046] the term" Antibody", "immunoglobulin" and other terms refer to polypeptides that specifically bind and recognize analytes (antigens) that are essentially encoded by one or more immunoglobulin genes or fragments thereof. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as countless immunoglobulin variable region genes. Light chains are divided into κ or λ. Heavy chains are divided into γ, μ, α, δ or ε, which define the immunoglobulin IgG, IgM, IgA, IgD and IgE categories, respectively. An exemplary immunoglobulin (antibody) structural unit is composed of 2 pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The N-terminal portion of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light chains and heavy chains, respectively. The The C-terminals are linked together by disulfide bonds and form the constant region of the antibody. According to the amino acid sequence of the constant domain of its heavy chain, antibodies can be divided into different categories. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The full-length immunoglobulin "light chain" (about 25kDa or about 214 amino acids) contains a variable region of about 1-10 amino acids at the NH2 end and a kappa or lambda constant region at the COOH end. The full-length immunoglobulin "heavy chain" (about 50kDa or about 446 amino acids) similarly contains a variable region (about 116 amino acids) and one of the above-mentioned heavy chain constant regions or categories, such as gamma (about 330 amino acids). The subunit structure and three-dimensional conformation of different classes of immunoglobulins are well known.

[0047] As used herein, the term “ Apolipoprotein "Related to proteins that bind lipids to form lipoproteins. Such proteins can function as enzyme cofactors, receptor ligands, and lipid transport carriers that regulate protein metabolism and protein uptake into tissues. This term includes many classes of apolipoproteins, including apoA (including isoforms apoA-I, apoA-II, apoA-IV, and apoA-V), apoB (including isoforms apoB48 and apoB100), apoC (including isoforms apoC-I, apoC-II, apoC-III, and apoC-IV), apoD, apoE (including isoforms apoE2, apoE3, and apoE4), apoH and apoJ (clusterin), as well as apoF, apoG, apoL, apoM, apoN, and apoO. In a specific embodiment, the apolipoprotein is apoE.

[0048] As used herein, the term “ Apolipoprotein AI” or “Apo AI” or “ApoAI” refers to a protein that is the major protein component of high-density lipoprotein (HDL) in plasma. ApoAI is a cofactor of phosphatidylcholine cholesterol acyltransferase (LCAT), which is responsible for the formation of most plasma cholesterol esters. Human apoAI is encoded by the APOAI gene. ApoA-I can come from any source, such as human, bovine, mouse, horse, dog, etc. In a specific embodiment, apo AI is a human protein with UniProt accession number P026547 (published on May 16, 2014).

[0049] As used herein, the term “ Apolipoprotein A-IV " or "Apo A-IV" or "ApoAIV" refers to a protein obtained by proteolysis of the primary translation product of the 306-residue proprotein. ApoA-IV can be from any source, such as human, bovine, mouse, horse, dog, etc. In a specific embodiment, Apo A-IV is a human protein with UniProt accession number P06727 (published on May 16, 2014). Human apoA-IV isoforms include apoA-IV-1a (T347S) and apoA-IV-2 (Q360H).

[0050] As used herein, the term “ Apolipoprotein CI " or "Apo CI" or "ApoCI" refers to a protein that is normally present in plasma and is responsible for the activation of esterified phosphatidylcholine cholesterol, which plays an important role in the exchange of esterified cholesterol between apolipoproteins and the removal of cholesterol from tissues. Human apoC-I is encoded by the APOC1 gene. ApoC-I can be from any source, such as human, bovine, mouse, horse, dog, etc. In a specific embodiment, apoC-I is a human protein with UniProt accession number P02654 (published on May 16, 2014). Based on their calculated isoelectric points, ApoC-I isoforms include acidic (apoC-IA) and basic (apoC-IB).

[0051] As used herein, the term “ Apolipoprotein C-II " or "Apo C-II" or "Apo CII" refers to a protein normally present in plasma, where it is a component of very low density lipoprotein (VLDL) and chylomicrons. Human apoC-II is encoded by the APOC2 gene. ApoC-II can be from any source, such as human, bovine, mouse, horse, canine, etc. In a specific embodiment, apoC-II is the human protein with UniProt accession number P02655 (published on May 16, 2014).

[0052] As used herein, the term “ Apolipoprotein C-III" or "Apo C-III" or "Apo CIII" refers to a protein found as a component of very low density lipoprotein (VLDL). ApoCIII is a relatively small protein containing 79 amino acids that can be glycosylated at threonine-74. It inhibits lipoprotein lipase and hepatic lipase. Human apoC-III is encoded by the APOC3 gene. ApoC-I can be from any source, such as human, bovine, murine, equine, canine, etc. In a specific embodiment, apo C-III is the human protein with UniProt accession number P02656 (published on May 16, 2014). ApoC-III exists in three isoforms, referred to as apoC-III0, apoC-III1, and apoC-III2, depending on the number of sialic acid molecules (0 to 2) terminating the oligosaccharide portion of the protein. Each isoform has been found to account for approximately 10, 55, and 35%, respectively, of the total apoC-III levels in circulation.

[0053] As used herein, the term “ Apolipoprotein-E " or "Apo-E" or "ApoE" refers to a protein found in chylomicrons and intermediate density lipoproteins (IDL) that is important for the normal catabolism of triglyceride-rich lipoprotein components. Apolipoprotein E in humans is encoded by the gene ApoE, which is a polymorphic gene with three major alleles, ε2, ε3 and ε4, encoding the isoforms ApoE2 (cys112, cys158), ApoE3 (cys112, arg158) and ApoE4 (argll2, arg158). Apolipoprotein E can be from any source, such as human, bovine, murine, equine, canine, etc. In a specific embodiment, Apo E is the human protein of UniProt accession number P02649 (published on May 16, 2014).

[0054] As used herein, the term “ Apolipoprotein E type 2 isoform ” or “apo E2” refers to isoform E2 of the protein apolipoprotein E, which is defined by the presence of cysteines at residues 112 and 158 of the amino acid sequence.

[0055] As used herein, the term “ Apolipoprotein E type 3 isoform ” or “apo E3” refers to isoform E3 of the protein apolipoprotein E, which is defined by the presence of cysteine ​​at residue position 112 and arginine at residue position 158 of the amino acid sequence.

[0056] As used herein, the term “ Apolipoprotein E4 isoform ” or “apo E cod variable” refers to isoform E4 of the protein apolipoprotein E, which is defined by the presence of arginine residues at positions 112 and 158 of the amino acid sequence.

[0057] As used herein, the term “ Apolipoprotein-J " or "Apo-J" or "ApoJ" or "clusterin", or "complement lysis inhibitor or "CLI", or "sulfated glycoprotein 2" or "SGP2" or "SP-40,40" or "testosterone-inhibiting prostate messenger 2 ketone" or "TRPM2", refers to a secreted heterodimeric glycoprotein that affects immune regulation, cell adhesion, transformation, lipid trafficking, tissue remodeling, membrane recycling and cell-cell interactions. Clusterin is synthesized as a 449 amino acid polypeptide that is post-translationally cleaved at the internal bond between Arg227 and Ser228. Apolipoprotein J can be from any source, such as human, bovine, murine, equine, canine, etc. In a specific embodiment, ApoJ is the human protein with UniProt accession number P10909 (published on May 16, 2014).

[0058] As used herein, the term “ Cardiovascular disease or “cardiovascular disorders” refers to diseases affecting the heart or blood vessels or both or related to the cardiopulmonary and circulatory systems, including but not limited to ischemia, angina, edema, atherosclerosis, coronary heart disease, LDL oxidation, monocyte adhesion to endothelial cells, foam cell formation, fatty streak development, platelet adhesion and aggregation, smooth muscle cell proliferation, reperfusion injury, hypertension, thrombotic disease, arrhythmias (atrial or ventricular or both); heart rhythm disturbances; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysms; vasculitis, stroke; peripheral occlusive arterial disease of a limb, organ or tissue; reperfusion injury to the brain, heart or other organs or tissues following ischemia, endotoxin, surgical or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; shock; vasoconstriction (including vasoconstriction associated with migraine); vascular abnormalities, inflammation and dysfunction limited to a single organ or tissue.

[0059] As used herein, the term “ Cleaners ", also known as "surfactants", refer to amphiphilic surfactants, which, when added to a liquid, reduce the surface tension of the liquid compared to the same liquid without the detergent. Detergents can also prevent aggregation of proteins and prevent non-specific interaction or binding of contaminants to target proteins. Detergents according to the present invention include, but are not limited to, nonionic (neutral), anionic, cationic or zwitterionic detergents.

[0060] Examples of nonionic or neutral detergents include, but are not limited to, detergents from the Tween series (polysorbates), such as 20 (polysorbate 20), 21 (polysorbate 21), 40 (polysorbate 40), 60(Polysorbate 60), 61 (polysorbate 61), 65 (polysorbate 65), 80 (polysorbate 80), 81 (polysorbate 81), 85 (polysorbate 85); Series of cleaning agents, such as 20; cleaning agents of the Tergitol series, such as 15-S-12 type Tergitol; Series of cleaning agents, such as 35. 56. 72. 76. 92V, 97. 58P; Tween series of cleaning agents, such as 20. twenty one, 40. 60. 61. 65. 80. 81. 85; Series of cleaning agents, such as X-100, X-114, CF-21, CF-32, DF-12, DF-16, GR-5M, X-102, X-15, X-151, X-207, X-165, X-305, X-405, X-45, X-705-70, or at least one nonionic conservative variant of said detergent.

[0061] Examples of anionic detergents include, but are not limited to, cholic acid and its derivatives, taurocholic acid, Triton X-200, Triton W-30, Triton-30, Triton-770, dioctyl sulfosuccinate, N5N-dimethyldodecylamine N-oxide, sodium 1-alkyl sulfonate, N-lauroylsarcosine or fatty acid salts.

[0062] Examples of cationic detergents include, but are not limited to, monomethyl and dimethyl fatty amines, alkyl trimethyl ammonium salts, dialkyl dimethyl ammonium salts, alkyl amine acetates, trialkyl ammonium acetates, alkyl dimethyl benzyl ammonium salts, dialkyl methyl benzyl ammonium salts, alkyl pyridinium halides and alkyl (alkyl substituted) pyridinium salts, alkyl thiomethyl pyridinium salts, alkyl amidomethyl pyridinium salts, alkyl quinoline salts, alkyl isoquinoline salts, N,N-alkyl methyl pyrrole salts, 1,1-dialkyl piperidine salts, 4,4-dialkyl thiomorpholine salts, 4,4-dialkyl thiomorpholine salts. Morpholine-1-oxide salts, methylbis(alkylethyl)-2-alkylimidazoline methylsulfate (and other salts), methylbis(alkylaminoethyl)-2-hydroxyethylammonium methylsulfate (and other salts), alkylaminopropyl-dimethylbenzylammonium salts, carboxyalkyl-alkyldimethylammonium salts, alkylamine oxides, alkyldimethylamine oxides, poly(vinylmethylpyridine) salts, poly(vinylpyridine) salts, polyethyleneimine, trialkylphosphonium bicarbonates (and other salts), trialkylmethylphosphonium salts, alkylethylmethylsulfonium salts and alkyldimethylsulfonium oxide salts.

[0063] Examples of zwitterionic detergents include, but are not limited to: 3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonate (CHAPS); 3-[(3-cholesterol aminopropyl) dimethylamino]-2-hydroxy-1-propanesulfonic acid (CHAPSO); N-(alkyl C10-C16)-N,N-dimethylglycine betaine (EMPIGEN BB); octanoyl sulfobetaine (SB3-10); 3-[N,N-dimethyl(3-myristylaminopropyl)amino]propane sulfonate (amidosulfobetaine-14; ASB-14); N-tetradecyl-N,N-dimethyl-3-amino-1-propane sulfonate (3-14 detergent; amphoteric detergent); N-dodecyl-N,N'-dimethyl-3-amino-1-propane sulfonate; N-octadecyl-N,N-dimethyl-3-amino-1-propane sulfonate; N-decyl-N,N-dimethyl-3-ammonium-1-propane sulfonate; Mirataine CB; Mirataine BB; Mirataine CBR; Mirataine ACS; Miracare 2MHT and Miracare 2MCA.

[0064] As used herein, the expression “ Probability determination" or "probability prediction" or similar terms, expressing synonyms of "assessing probability" or "assessment of probability", and meaning that the present invention allows the prediction, estimation or assessment of the probability of a patient developing a disease, particularly a neurodegenerative disease or a cardiovascular disease. The prediction of probability generally indicates an increase or decrease in probability. As will be appreciated by those skilled in the art, while the prediction is preferably 100% correct for the subject being assessed, this is not a requirement. However, the term requires that a statistically significant portion of the patients can be identified as having an increased probability of developing a disease, particularly a neurodegenerative disease or a cardiovascular disease. Those skilled in the art can readily determine whether a subject is statistically significant by using various well-known statistical assessment tools, such as determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc. The statistical significance of the subject can be determined in Dowdy and Wearden's Statistics for Research, John Wiley & Sons, New York, NY. Details can be found in 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is preferably 0.05, 0.025, 0.001, 0.0001 or less.

[0065] As used herein, the term “ Haplotype ” refers to a specific set of genes that offspring inherit from one parent. In particular, a haplotype encompasses a specific set of alleles on a chromosome that are likely to be inherited together. A haplotype can contain two or more alleles.

[0066] As used herein, the term “ Immunoturbidimetry " relates to a technique for detecting an analyte in a sample based on a reaction of the analyte with an antibody, which results in the formation of an antibody-antigen immune complex between the analyte and the antibody that precipitates, increasing the turbidity of the sample. As a result, when light passes through the reaction solution, part of the light is scattered by the sample, part of the light is absorbed by the sample, and the rest of the light passes through the sample. The amount of light absorbed is proportional to the concentration of the analyte, or in other words, the transmitted light signal is proportional to the concentration of the analyte. According to the present invention, the analyte to be detected by immunoturbidimetry is an apolipoprotein or an isoform thereof, in particular an apolipoprotein selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) and isoforms thereof, more particularly apoE and / or apoE isoforms apoE2, apoE3 and apoE4, more preferably apoE4. In a specific embodiment of the present invention, the immunoturbidimetric assay is LEIT (latex enhanced immunoturbidimetric technique).

[0067] As used herein, the term “ Neurodegenerative diseases"Relates to diseases caused by degeneration or degeneration of neural tissue, particularly neurons, which over time leads to functional impairment or disability; the term "degeneration" includes loss of cell viability, loss of cell function and / or loss of cell (neuronal or other) number. Exemplary, non-limiting examples of neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, etc. In a specific embodiment, the neurodegenerative disease is AD.

[0068] As used herein, the term “ Oxidants "Involves a chemical substance that removes electrons from another substance in a redox (oxidation-reduction) reaction.

[0069] As used herein, the term “ Polycarbonate " refers to a group of thermoplastic polymers containing carbonate groups (-O-(C=O)-O-) in their chemical structure.

[0070] As used herein, the term “ Polystyrene "Pertaining to synthetic aromatic thermoplastic polymers made from the monomer styrene. Polystyrene may be solid or foamed.

[0071] As used herein, the term “ Protease ", also known as "peptidase", refers to an enzyme that catalyzes the hydrolysis of peptide bonds in proteins. Illustrative, non-limiting examples of proteases include: serine peptidases, threonine peptidases, cysteine ​​peptidases, aspartyl peptidases, metallopeptidases, and glutamyl peptidases. In a specific embodiment, the protease is pepsin, trypsin, or chymotrypsin. As used in this specification, the term "pepsin" refers to a protease that catalyzes the hydrolysis of peptide bonds between hydrophobic amino acids, preferably aromatic amino acids (e.g., phenylalanine, tryptophan, and tyrosine).

[0072] As used herein, the term “ reducing agent "Involves a compound that loses electrons to another chemical species (the oxidant) in a redox (oxidation-reduction) chemical reaction. The loss of electrons by the reducing agent results in its oxidation.

[0073] As used herein, the term “ Reference value"Relates to a predetermined standard used as a reference for evaluating values ​​or data obtained from a sample collected from a subject. A reference value or reference level can be an absolute value; a relative value; a value with an upper or lower limit; a range of values; an average value; a median value; a mean; or a value relative to a specific control or baseline value. A reference value can be based on an individual sample value at an earlier time point, such as a value obtained from a sample from a test subject. A reference value can be based on a large number of samples, such as from a matched group of chronological age, or on a pool of samples that include or exclude the sample to be tested.

[0074] As used herein, the term "salt" relates to a compound in any form in ionic form, or charged and coupled to a counterion (cation or anion) or in solution. The definition specifically includes pharmaceutically acceptable salts.

[0075] As used herein, the term “ sample " refers to biological material separated from a subject, and thus includes biological samples. The sample may contain any biological material suitable for detecting a desired marker, and may include cells and / or non-cellular material from a subject. In general, the sample can be isolated from any suitable biological tissue or fluid. Particularly preferred samples according to the present invention include, but are not limited to, blood, plasma, serum, saliva, urine, and cerebrospinal fluid (CSF). In one embodiment of the present invention, the sample is a plasma sample.

[0076] the term" Object " or "individual" or "animal" or "patient" includes any subject in need of treatment, particularly a mammalian subject. Mammalian subjects include humans, livestock, farm animals, and zoo animals or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc. In a specific embodiment, the subject is a human.

[0077] Apolipoprotein detection and quantification method of the present invention

[0078] In a first aspect, the present invention relates to an in vitro method for detecting and / or quantifying apolipoprotein or an isoform thereof in a sample (first method of the present invention), the method comprising:

[0079] (i) contacting a sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein or an isoform thereof to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein or an isoform thereof.

[0080] (ii) contacting the surface bound to the apolipoprotein or its isoform formed in step (i) with an antibody specific for the apolipoprotein or its isoform under conditions suitable for the formation of a complex between the antibody and the apolipoprotein or its isoform, and

[0081] (iii) detecting the complex formed in step (ii).

[0082] In a specific embodiment, the present invention relates to an in vitro method for detecting and / or quantifying an apolipoprotein or an isoform thereof selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) in a sample, the method comprising:

[0083] The sample is contacted with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein or an isoform thereof to the surface, wherein the surface is free of specific antibodies that bind to the apolipoprotein or an isoform thereof.

[0084] (i) contacting the surface bound to the apolipoprotein or its isoform formed in step (i) with an antibody specific for the apolipoprotein or its isoform under conditions suitable for the formation of a complex between the antibody and the apolipoprotein or its isoform, and

[0085] (ii) detecting the complex formed in step (ii),

[0086] The polystyrene surface or the polycarbonate surface is not treated before contacting with the sample or is blocked with albumin before contacting with the sample.

[0087] Apolipoprotein to be detected and / or quantified according to the first method of the present invention includes apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clustered protein). Apolipoprotein isotype to be detected and / or quantified includes apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clustered protein) isotype. In a specific embodiment, according to the first method of the present invention, apolipoprotein to be detected and / or quantified is apoE. In a specific embodiment, according to the first method of the present invention, apolipoprotein isotype to be detected and / or quantified is apoE isotype selected from apoE2, apoE3 and apoE4. In a more specific embodiment, the apolipoprotein is apoE, and more particularly, the apolipoprotein isotype is apoE4.

[0088] Therefore, in The first step of the first method of the present invention The sample is contacted with the polystyrene or polycarbonate surface under conditions suitable for binding apolipoprotein or its isoform in the sample to the polycarbonate surface or polystyrene surface, wherein the surface does not contain a specific antibody that specifically binds to the apolipoprotein or its isoform.

[0089] The contact between the sample and the polystyrene surface or the polycarbonate surface can be carried out by direct contact under suitable conditions to bind the apolipoprotein or its isoform in the sample to the polystyrene surface or the polycarbonate surface. As will be appreciated by those skilled in the art, the conditions will be suitable for the interaction between the apolipoprotein or its isoform and the polystyrene surface or the polycarbonate surface, preferably suitable for the electrostatic and / or hydrophobic interaction between the apolipoprotein or its isoform and the polystyrene surface or the polycarbonate surface. In a particularly preferred embodiment, the surface is a polystyrene surface. Suitable conditions for binding can be determined by those skilled in the art and include suitable temperature, incubation time, pH, sample concentration, etc. In a specific embodiment, the temperature range is 4 to 40°C, particularly 10 to 35°C, more particularly 15 to 30°C, preferably 20 to 25°C (room temperature). In a specific embodiment, the pH range during the contact step is pH2 to pH 10, preferably pH 4 to pH 10. In a specific embodiment, the sample is contacted with a surface, preferably a polystyrene surface, and incubated for at least 1 minute, preferably at least 5 minutes, more preferably at least 30 minutes, and even more preferably at least 60 minutes. The sample containing apolipoprotein according to the present invention can be contacted with a surface, preferably a polystyrene surface, without prior dilution or with dilution, wherein the dilution is at least 1:2, preferably at least 1:5, more preferably at least 1:10, even more preferably at least 1:100, and even more preferably at least 1:200. As will be appreciated by those skilled in the art, different buffers are suitable for diluting the sample. In a specific sample, the dilution of the sample is carried out in phosphate buffered saline (PBS) or carbonate / bicarbonate buffer, more preferably in PBS.

[0090] In a specific embodiment, the binding of apolipoprotein or an isoform thereof to a polystyrene surface or a polycarbonate surface is not mediated by any peptide, any polypeptide, any lipid or any cross-linking agent. In particular, the peptide that does not mediate the binding is a peptide that is capable of binding to apolipoprotein or an isoform thereof, and includes but is not limited to amyloid peptides. Amyloid peptides include, but are not limited to, amyloid precursor protein (APP), β-amyloid peptide [a key peptide involved in Alzheimer's disease, containing 36-43 amino acids, and is the main component of amyloid plaques present in the brains of patients with Alzheimer's disease, including Αβ(1-5), Αβ(1-6), Αβ(1-12), Αβ(1-40), Αβ(1-42), Αβ(12-28), Αβ(13-28), Αβ(25-35), Αβ(35-42), Αβ(33-42) and Αβ(33-40) (wherein, the expression "Αβ(X-Y)" refers to a peptide derived from β-amyloid peptide, wherein the β-amyloid peptide consists of the amino acid at position X to the amino acid at position Y); Αβ40 and Αβ42. Lipids that do not mediate binding include, but are not limited to, phospholipids and fatty acids.

[0091] In a specific embodiment, the sample containing the apolipoprotein or its isoform to be determined and / or quantified is a biological sample. Particularly preferred biological samples according to the present invention include, but are not limited to, biological fluids such as blood, plasma, serum, saliva, urine and cerebrospinal fluid (CSF). Methods for separating samples are well known to those skilled in the art. In a specific embodiment, the sample is a plasma sample. According to the first method described in the present invention, the suitable dilution range of the plasma sample is 1:10 to 1:100000, preferably 1:100 to 1:1000, and more preferably the dilution of the plasma sample is 1:200.

[0092] The polystyrene surface or polycarbonate surface (preferably a polystyrene surface) according to the present invention does not contain any specific antibodies for apolipoprotein or its isoform that is bound to it and is detected and / or quantified. The polystyrene surface according to the present invention includes any polystyrene surface that is easily contacted with a sample, especially a biological sample, and includes but is not limited to plates, tubes, beads and arrays, all of which are commercially available, and generally includes any polystyrene surface suitable for microfluidic determination. In a specific embodiment, the polystyrene surface is composed of ELISA plates, turbidimetric beads, luminex beads or generally consists of any polystyrene surface suitable for microfluidic determination. The polycarbonate surface according to the present invention includes any polycarbonate surface that is easily contacted with a sample, especially a biological sample, and includes but is not limited to plates, tubes, beads and arrays, all of which are commercially available. In any case, the polystyrene surface or polycarbonate surface does not contain specific antibodies for apolipoprotein or its isoform that is bound to the surface.

[0093] By way of example, polystyrene plates according to the present invention include ELISA polystyrene plates commercially available from Nunc, Fisher Scientific, VWR, Greiner, and Corning.

[0094] The polystyrene beads according to the present invention include but are not limited to beads commercially available from Sigma Aldrich. In a specific embodiment, the average particle size of the polystyrene beads ranges from 50 nm to 1 μm, preferably from 100 nm to 500 nm, more preferably from 150 nm to 400 nm, and even more preferably from 200 to 300 nm. In a specific embodiment, the polystyrene beads are amine-modified polystyrene beads. In a specific optional embodiment, the polystyrene beads are carboxylate-modified polystyrene beads. The polystyrene beads according to the present invention include but are not limited to Luminex beads are microspheres commercially available from Luminex.

[0095] In a specific embodiment, the polystyrene surface or polycarbonate surface is not treated before contacting with the sample. In a preferred optional embodiment, the polystyrene surface or polycarbonate surface is blocked before contacting with the sample, that is, the polystyrene surface or polycarbonate surface is blocked before the binding step (i) of the first method of the present invention. In a specific embodiment, the polystyrene surface or polycarbonate surface is blocked by a protein other than apolipoprotein and / or a detergent before the binding step. Proteins suitable for blocking the surface, preferably the polystyrene surface, before the binding step include but are not limited to albumins such as bovine serum albumin (BSA), casein and gelatin and commercially available blocking solutions such as Superblock (Pierce). Particularly preferred proteins for blocking are BSA, casein and gelatin, more preferably BSA, even more preferably in the range of 1% to 3% BSA, more preferably 1% BSA. In a specific embodiment, the polystyrene surface or polycarbonate surface is blocked by albumin before contacting with the sample, and more preferably the albumin is BSA. In a specific embodiment, the surface is blocked by 1% BSA. Additionally or alternatively, in addition to non-apolipoprotein proteins, the surface, preferably a polystyrene surface, is blocked with a detergent prior to the binding step. Detergents suitable for blocking the polystyrene surface prior to the binding step include, but are not limited to, polyvinylpyrrolidone-40 (PVP-40), polysorbate 20 (Tween 20), Nonidet-P40, and Triton X-100. In a specific embodiment, the detergent is polysorbate 20. In a specific embodiment, the surface is blocked with polysorbate 20.

[0096] In one embodiment of the present invention, the polystyrene or polycarbonate, preferably polystyrene surface is treated with a washing solution after contact with the sample, that is, the surface is treated with a washing solution after the binding step (i) of the first method of the present invention. Preferably, the surface is treated with a washing solution after the binding step (i) and before step (ii) of the first method of the present invention. In a specific embodiment, the washing solution contains one or more salts. Preferably, the salt contained in the washing solution is NaCl, composed of Tris buffer (TBS) or phosphate buffered saline (PBS). Additionally or alternatively, the washing solution contains at least one detergent. Preferably, the detergent is selected from polysorbate 20 (Tween 20) and Triton X-100. Additionally or alternatively, the washing solution contains an acid. Preferably, the acid is selected from HCl and formic acid. More preferably, the acid is selected from 2M HCl and 70% formic acid. Additionally or alternatively, the washing solution contains a base. Preferably, the base is NaOH, more preferably 2M NaOH. Additionally or alternatively, the washing solution contains a reducing agent. The reducing agent is preferably 2-mercaptoethanol. Additionally or alternatively, the washing solution does not contain an enzymatic cleaning agent (eg, protease), an oxidizing agent (eg, sodium hypochlorite), or a combination thereof.

[0097] exist The second step of the first method of the present invention In the method, as a result of the first step, a polystyrene or polycarbonate surface (preferably a polystyrene surface) bound to apolipoprotein or an isoform thereof is contacted with an antibody, which is a specific antibody against the apolipoprotein or an isoform thereof bound to the polystyrene surface or the polycarbonate surface. This step is carried out under suitable conditions to form a complex between the antibody bound to the polystyrene surface or the polycarbonate surface and the apolipoprotein or an isoform thereof.

[0098] Apolipoprotein-specific antibodies are known to those skilled in the art and are commercially available. Exemplary, non-limiting antibodies that can be used in the context of the present invention include the following:

[0099] -Used for apoAIDetection and / or quantification: Rabbit polyclonal antibody for apoAI from Santa Cruz Biotechnology (FL-267, catalog #sc-30089), mouse monoclonal anti-apoAI antibody from Santa Cruz Biotechnology (069-01, catalog #sc-58320), mouse monoclonal anti-apoAI antibody from Santa Cruz Biotechnology (B10, catalog #sc-376818), rabbit polyclonal anti-apoAI antibody from Abcam (catalog #ab64308), and goat polyclonal anti-ApoAI antibody from Abcam (catalog #ab7613).

[0100] - For apoAII detection and / or quantification: Mouse monoclonal anti-apoAII antibody from LifeSpan Biosciences (Catalog #LS-B4281) and rabbit polyclonal anti-apoAII antibody from OriGene (Catalog #TA328111).

[0101] -Used for apoAIV Detection and / or quantification: Goat polyclonal anti-human apoAIV from Abcam (Catalog # ab59036) and rabbit polyclonal anti-human apoAIV from Sigma-Aldrich (Catalog # HPA001352).

[0102] -Used for apoCI Detection and / or quantification: Rabbit polyclonal anti-ApoCI antibody from Abcam (Catalog # ab20793) and goat polyclonal anti-ApoCI antibody from Abcam (Catalog # ab104446).

[0103] -Used for apoCII Detection and / or quantification: Rabbit polyclonal anti-apoCII antibody from Abcam (Catalog # ab76452), Rabbit polyclonal anti-apoCII antibody from Sigma-Aldrich (Catalog # SAB1300917), and Mouse monoclonal anti-apoCII antibody from Novus-Biologicals (3E4, Catalog # H00000344-M01).

[0104] -Used for apoCIII Detection and / or quantification: Goat polyclonal anti-apoCIII antibody from Abcam (Catalog # ab7619) and rabbit polyclonal anti-apoCIII antibody from Sigma-Aldrich (Catalog # A0734).

[0105] -Used for apoEDetection and / or quantification: Rabbit polyclonal antibody for total apoE (Santa Cruz Biotechnology, Inc., #sc-98573) and rabbit IgG anti-human apoE from TaKaRa Clontech (Catalog #18171A and #18171B) and rabbit polyclonal anti-human apoE antibody from Abcam (Catalog #ab72398).

[0106] -Used for apoE2 Isotype detection and / or quantification: Mouse monoclonal anti-human apoE2 from Biolegend (clone 3C2, catalog #815001; clone 8G3, catalog #812701).

[0107] -Used for apoE3 Isotype detection and / or quantification: Rabbit polyclonal anti-human apoE3 from Preprotech (Catalog #350-02).

[0108] -Used for apoE4 Isotype detection and / or quantification: Mouse monoclonal anti-human apoE4 (clone 4E4, catalog #MABN43) from Merck Millipore, mouse monoclonal anti-human apoE4 (clone 5B5, catalog #811601) from BioLegend, a specific mouse monoclonal antibody to apoE E4, clone 4E4 (Novus Biologicals, #NBP1-49529), a specific mouse monoclonal antibody to apoE E4, clone 5B5 (IBL #10025), a specific mouse monoclonal antibody to apoE E4, clone 1F9 (MBL, #M067-3) and a specific mouse monoclonal antibody to apoE4, clone 4E4 (catalog #MA5-16146) from Thermo Scientific.

[0109] -Used for apoJ Detection and / or quantification: Mouse monoclonal anti-apoJ antibody from Abeam (CLI-9, catalog # ab16077) and goat polyclonal anti-ApoJ antibody from Sigma-Aldrich (catalog # SAB2500251).

[0110] The contact between the surface bound to apolipoprotein or its isoform obtained in step (i) and the specific antibody for the apolipoprotein or its isoform is carried out under suitable conditions to form a complex between the antibody and the apolipoprotein or its isoform. Conditions suitable for forming the complex can be determined by a person skilled in the art and include suitable temperature, incubation time and pH. In a specific embodiment, the temperature range is 4 to 40°C, particularly 10 to 35°C, more particularly 15 to 30°C, preferably 20 to 25°C (room temperature). In a preferred embodiment, the temperature in steps (i) and (ii) of the first method of the present invention is substantially the same. In a specific embodiment, the pH range is pH 2 to pH 10, preferably pH 4 to pH 10. In a preferred embodiment, the pH in steps (i) and (ii) of the first method of the present invention is substantially the same. In a specific embodiment, the antibody is cultured with the apolipoprotein or its isoform bound to the surface for at least 1 minute, preferably at least 5 minutes, more preferably at least 30 minutes, more preferably at least 60 minutes.

[0111] exist The third step of the first method of the present invention In the method, the complex obtained in the second step is detected, wherein the complex is a complex between the apolipoprotein or an isoform thereof bound to the polystyrene surface or the polycarbonate surface and the antibody specific for the apolipoprotein or an isoform thereof.

[0112] Since apolipoprotein or its isoform itself is not a detectable molecule, in a specific embodiment, the detection step is performed by a detectable molecule or complex. Apolipoprotein-antibody complex or apolipoprotein isoform-antibody complex can be detected by different methods known to those skilled in the art. In a specific embodiment of the present invention, the detection of apolipoprotein-antibody complex or apolipoprotein isoform-antibody complex is performed by a detectable agent that specifically binds to the apolipoprotein-antibody complex or apolipoprotein isoform-antibody complex, wherein the detectable agent promotes the detection of the complex by generating a signal that can be measured. Methods for labeling biomolecules such as polypeptides and antibodies are well known in the art.

[0113] Any of a variety of detectable agents may be used in the practice of the present invention. Suitable detectable agents include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., enzymes for ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and haptens and proteins for which biotin, digoxigenin, or other antisera or monoclonal antibodies are available.

[0114] Detectable agents for optical imaging include, for example, fluorescein, fluorescein derivatives, indocyanine green, Oregon green, Oregon green derivatives, rhodamine green, rhodamine green derivatives, eosin, erythrosine, Texas red, Texas red derivatives, malachite green, nanogold sulfosuccinimidyl esters, Cascade Blue, coumarin derivatives, naphthalene, pyridoxazole derivatives, Cascade Yellow dyes, dapoxyl dyes, and various other fluorescent compounds, such as Cy3, Cy2, Cy5, Alexa Fluor ... A family of fluorescent markers (Molecular Probes, Inc.), carboxyfluorescein (FAM) and fluorescein isothiocyanate (FITC).

[0115] In a specific embodiment, the detectable agent is a protein. In the context of the present invention, the term "protein" refers to a macromolecule composed of one or more amino acid chains. Based on its ability to specifically bind to other molecules, proteins are responsible for performing a variety of cellular functions. Proteins can bind to other proteins as well as small substrate molecules. Non-limiting examples of proteins suitable as detectable agents include, but are not limited to, enzymes, fluorescent proteins, luminescent proteins, and antigens.

[0116] In a preferred embodiment, the protein is an enzyme. In the context of the present invention, the term "enzyme" refers to a protein that acts as a highly selective catalyst that accelerates the speed and specificity of a specific metabolic reaction. Non-limiting examples of enzymes suitable for the present invention include, but are not limited to, horseradish peroxidase (HRP) and alkaline phosphatase. As will be appreciated by those skilled in the art, enzymes suitable for the present invention are indirectly detectable due to their ability to catalyze modifications of substrates in compounds that can be detected by colorimetry, chemiluminescence or fluorometry. Examples of suitable substrates include, but are not limited to, p-nitrophenyl phosphate (PNPP), 2,2'-azobis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB).

[0117] Bioluminescent proteins or photoproteins are special cases of oxidases that are able to carry out chemical reactions of their specific prosthetic groups, thereby emitting light without prior excitation. Non-limiting examples of bioluminescent proteins include firefly luciferase, Renilla luciferase, and aequorin.

[0118] In another embodiment, the protein is a fluorescent protein. In the context of the present invention, the term "fluorescent protein" refers to a protein that has the ability to emit light when excited at a wavelength suitable for excitation. Non-limiting examples of fluorescent proteins that can be used in the complexes of the present invention include, but are not limited to, GFP, GFPuv, BFP, CFP, YFP, EBFP2, mCerulean, mCerulean3, mVenus, mTurquoise, T-Sapphire, citrine, amFP486, zFP506, zFP538, drFP, DsRed, mCherry, dTomate, mTFP1, TagRFP-T, mKO2, mRuby, mKate, mAmetrine, REACh, R-phycoerythrin (R-PE) and allophycocyanin (APC).

[0119] In another embodiment, the protein is a photoprotein.In the context of the present invention, the term "photoprotein" refers to a protein that is able to emit light when it is excited at a wavelength suitable for excitation.

[0120] In another embodiment, the protein is an antigen. In the context of the present invention, the term "antigen" refers to a molecule that induces an immune response in vivo. Thus, an antigen can be used to generate antibodies that recognize it and specifically bind to it. Non-limiting examples of antigens include, in particular, tumor antigens such as carcinoembryonic antigen (CEA), HER2, prostate-specific antigen (PSA), and tissue plasminogen activator and recombinant variants thereof such as and bacterial antigens, allergens, etc. As will be appreciated by those skilled in the art, antigens suitable for use in the present invention are indirectly detectable due to their ability to be specifically recognized by antibodies.

[0121] In another embodiment, the detectable agent is a hapten. In the context of the present invention, the term "hapten" refers to a class of compounds with a small molecular weight (<10,000 Da) that are antigenic but cannot induce a specific immune response by themselves. The hapten is chemically coupled to a large immunogenic protein called a carrier to produce a hapten-immunogenic carrier conjugate capable of inducing a specific immune response. Non-limiting examples of vitamins include biotin (vitamin B7), digoxin, dinitrophenol (DNP) and nitroiodophenol (NIP). In a specific embodiment, the vitamin is biotin. In the context of the present invention, the term "biotin" refers to a water-soluble and alcohol-soluble heat-stable vitamin, also known as vitamin H and vitamin B7, characterized by specific binding to avidin with the highest affinity to date, Kd=10-15M. As understood by those skilled in the art, biotin can be detected indirectly due to its ability to be specifically recognized by avidin or avidin variants (such as streptavidin and neutravidin).

[0122] Detection of the detectable agent can be performed fluorometrically or colorimetrically using equipment appropriate to the type of agent and sample known to those skilled in the art.

[0123] For example, apolipoprotein-antibody complex (wherein, apolipoprotein in the complex is attached to a surface, preferably a polystyrene surface) is co-cultured with a second antibody (reporter antibody), which is a specific antibody for an anti-apolipoprotein antibody pre-compounded with apolipoprotein. Therefore, in a specific embodiment of the first method of the present invention, the apolipoprotein-antibody complex formed in step (ii) of the method is detected by a specific reporter antibody of an anti-apolipoprotein antibody. In a specific embodiment, the anti-apolipoprotein antibody and the reporter antibody are the same antibody. In a preferred embodiment, under conditions similar to the culture surface and apolipoprotein and / or the specific antibody of the culture apolipoprotein and the apolipoprotein bound to the surface, the second antibody is conjugated to an enzyme. By adding a substrate that can be converted into a detectable product by an enzyme, the complex antibody-apolipoprotein bound to the surface is detected, for example, by a fluorescence determination method in a fluorescence microscope or by a colorimetric method in a spectrophotometer. In an optional embodiment, detection can be performed in a similar manner by apolipoprotein-specific probe appropriately labeled with a detectable agent.

[0124] Therefore, according to the present invention, as mentioned above, apolipoprotein-antibody complex or apolipoprotein isotype-antibody complex can be detected by detectable molecules or complexes. In an optional embodiment of the present invention, apolipoprotein-antibody or apolipoprotein isotype-antibody complex is detected by turbidimetry, particularly by immunoturbidimetry. When the apolipoprotein or apolipoprotein isotype is present in the sample to be analyzed, the formation of apolipoprotein-antibody complex or apolipoprotein isotype-antibody complex makes turbidity rise relative to the sample turbidity lacking apolipoprotein or apolipoprotein isotype, wherein the turbidity change can be measured (for example, by spectrophotometer). In a preferred embodiment of the present invention, the immunoturbidimetry assay is LEIT (latex enhanced immunoturbidimetry technique).

[0125] In a specific embodiment, the detection of the complex is performed by ELISA. In an alternative embodiment, the detection of the complex is performed by immunoturbidimetric assay.

[0126] Method for determining the relative amounts of apolipoprotein isoforms

[0127] The inventors of the present invention have developed a method for determining the presence or absence of a specific apolipoprotein isoform in a subject based on a dual ELISA assay of a plasma sample from the subject, wherein one assay determines the level of the specific apolipoprotein isoform and the other assay determines the level of total apolipoprotein. The ratio of apolipoprotein isoform / total apolipoprotein is then calculated. See Examples 1 and Figure 4 : "Identification of heterozygous / homozygous APOE ε4 carriers in plasma samples". The apolipoprotein isoform content of apoE has also been determined by immunoturbidimetric assay. See Example 2: "Detection of ApoE4 by turbidimetric assay.

[0128] Therefore, in another embodiment, the present invention relates to an in vitro method for determining the relative amount of an isoform of a given apolipoprotein relative to the total amount of said apolipoprotein in a sample (second method of the present invention), said method comprising:

[0129] (i) contacting the sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein,

[0130] (ii) contacting the apolipoprotein-bound surface formed in step (i) with a first antibody specific for an isotype of the apolipoprotein and with a second antibody capable of binding to all isotypes of the apolipoprotein present in the sample, wherein the contacting is performed under conditions suitable for the formation of a first complex comprising the first antibody and the isotype of the apolipoprotein and a second complex comprising the second antibody and all isotypes of the apolipoprotein,

[0131] (iii) detecting the first and second complexes formed in step (ii), and

[0132] (iv) determining the relative amount of the isoform relative to the total apolipoprotein content based on the levels of the first and second complexes obtained in step (iii).

[0133] In a specific embodiment, the present invention relates to an in vitro method for determining the relative amount of an isoform of a given apolipoprotein selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) in a sample relative to the total content of said apolipoprotein in the sample.

[0134] (i) contacting a sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein to the surface, wherein the surface does not contain antibodies that specifically bind to the apolipoprotein,

[0135] (ii) contacting the apolipoprotein-bound surface formed in step (i) with a first antibody specific for an isoform of the apolipoprotein and with a second antibody capable of binding to all isoforms of the apolipoprotein present in the sample, wherein the contacting is performed under conditions suitable for the formation of a first complex comprising the first antibody and the isoform of the apolipoprotein and a second complex comprising the second antibody and all isoforms of the apolipoprotein,

[0136] (iii) detecting the first and second complexes formed in step (ii), and

[0137] (iv) determining the relative amount of the isoform relative to the total apolipoprotein content based on the levels of the first and second complexes obtained in step (iii),

[0138] The polystyrene surface or the polycarbonate surface is not treated before contacting with the sample or is blocked with albumin before contacting with the sample.

[0139] According to the second method of the present invention, the apolipoprotein isotype to be determined includes apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (cluster protein) isotypes. In a specific embodiment, the apolipoprotein isotype to be determined is an apoE isotype selected from apoE2, apoE3 and apoE4, preferably apoE4. In a more specific embodiment, the apolipoprotein is apoE and the apolipoprotein isotype is apoE4.

[0140] exist The first step of the second method of the present invention is In the method, the sample is contacted with the surface under conditions suitable for binding apolipoprotein or an isoform thereof in the sample to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein or an isoform thereof.

[0141] Suitable conditions for binding apolipoprotein or an isoform thereof in a sample to a polystyrene or polycarbonate surface, preferably a polystyrene surface, have been previously described in the context of the first method of the invention and are introduced here.

[0142] In a specific embodiment, the sample containing the apolipoprotein to be determined or its isoform is a biological sample. Suitable biological samples have been mentioned in the context of the first method of the present invention and are introduced herein. Preferred biological samples include biological fluids such as blood, plasma, serum, saliva, urine and cerebrospinal fluid (CSF). In a specific embodiment, the sample is a plasma sample. The suitable dilution range of the plasma sample is 1:10 to 1:100000, preferably 1:100 to 1:1000, and more preferably the dilution of the plasma sample is 1:200.

[0143] The polystyrene surface or polycarbonate surface according to the present invention does not contain any specific antibodies for apolipoprotein or its isoforms that bind to and are detected therefrom. The polystyrene surface or polycarbonate surface according to the present invention has been mentioned above and is introduced herein. In a specific embodiment, the polystyrene surface comprises an ELISA plate, luminex beads or turbidimetric beads. In any case, the polystyrene surface or polycarbonate surface does not contain specific antibodies for apolipoprotein or its isoforms that are bound to the surface.

[0144] In a specific embodiment, the polystyrene surface or polycarbonate surface according to the second method of the present invention is formed by a single entity, so that the single entity provides the surface necessary for binding apolipoprotein, such as a single plate such as an ELISA plate, or a single hole of an ELISA plate. It should be understood that this embodiment can be used in the case of detecting the first and second antibodies using different detectable labels, thereby allowing the signal generated by each antibody to be accurately determined without interference from the signal provided by the second antibody. In an optional embodiment, the polystyrene surface or polycarbonate surface according to the second method of the present invention is formed by multiple entities, that is, by providing an entity of at least two units of the surface that can be used to bind apolipoprotein, such as a plurality of beads, such as Luminex beads or turbidity beads. In a specific embodiment of the second method of the present invention, the surface contacted with the sample under conditions suitable for binding apolipoprotein in the sample to the polystyrene surface or polycarbonate surface is formed by multiple entities. Preferably, the surface is formed by Luminex beads or turbidity beads. When the complex formed in step (i) is not further treated with a detectable reagent, this embodiment is useful, and the two antibodies can be distinguished by the signal provided by each detectable reagent. For example, in the case of turbidimetric detection, the formation of a complex between apolipoprotein and an antibody is detected by an increase in the turbidity of the sample. In this case, it is not possible to determine whether the increase in turbidity is due to the formation of a complex between an apolipoprotein isotype and a first antibody or a complex between an apolipoprotein and a second antibody. In this case, a surface formed by a plurality of entities (e.g. in the form of microparticles) must be used for contact, whereby the entities contacted with each type of antibody can be detected separately by placing them in different containers.

[0145] In a specific embodiment, the polystyrene surface or polycarbonate surface is not treated before contact with the sample. In an alternative preferred embodiment, the polystyrene surface or polycarbonate surface is blocked before contact with the sample, that is, the surface is blocked before the binding step (i) of the second method of the present invention. In a specific embodiment, the polystyrene surface or polycarbonate surface is blocked with proteins and / or detergents other than apolipoprotein before the binding step. Suitable proteins and detergents for blocking surfaces according to the present invention have been described above and are incorporated herein by reference. Particularly preferred proteins for blocking are BSA, casein and gelatin, more preferably BSA, even more preferably BSA in the range of 1% to 3%, more preferably 1% BSA. Suitable detergents for blocking surfaces before the binding step include, but are not limited to, polyvinylpyrrolidone-40 (PVP-40), polysorbate 20 (Tween 20), Nonidet-P40 and Triton X-100.

[0146] In a particular embodiment, the polystyrene surface or polycarbonate surface is treated with a washing solution after contact with the sample, that is, the polystyrene surface or polycarbonate surface is treated with a washing solution after the binding step (i) of the second method of the present invention. Preferably, the polystyrene surface or polycarbonate surface is treated with a washing solution after the binding step (i) and before step (ii) of the second method of the present invention. Suitable washing solutions have been described above and are introduced here. In a particular embodiment, the washing solution contains one or more salts. Preferably, the salt contained in the washing solution is NaCl. Additionally or alternatively, the washing solution contains at least one detergent. Preferably, the detergent is selected from polysorbate 20 (Tween 20) and Triton X-100. Additionally or alternatively, the washing solution contains an acid. Preferably, the acid is selected from HCl and formic acid. More preferably, the acid is selected from 2M HCl and 70% formic acid. Additionally or alternatively, the washing solution contains a base. Preferably, the base is NaOH, more preferably 2M NaOH. Additionally or alternatively, the washing solution contains a reducing agent. The reducing agent is preferably 2-mercaptoethanol. Additionally or alternatively, the washing solution does not contain an enzymatic cleaning agent (such as a protease), an oxidizing agent (such as sodium hypochlorite) or a combination thereof.

[0147] exist The second step of the second method of the present inventionThe apolipoprotein-bound surface formed in step (i) is contacted with a first antibody specific for an isotype of the apolipoprotein and with a second antibody capable of binding to all isotypes of the apolipoprotein present in the sample, wherein the contacting is performed under conditions suitable for the formation of a first complex comprising the first antibody and an isotype of the apolipoprotein and a second complex comprising the second antibody and all isotypes of the apolipoprotein,

[0148] Apolipoprotein-specific antibodies and antibodies specific for a particular apolipoprotein isoform have already been cited above in the context of the first method of the invention and are incorporated herein.

[0149] According to the second method of the present invention, the surface bound to apolipoprotein formed in step (i) is contacted with the first antibody and the second antibody, wherein the first antibody is a specific antibody for the apolipoprotein isotype to be determined, and wherein the second antibody is capable of binding to all isotypes of the apolipoprotein. In a specific embodiment, the surface bound to apolipoprotein and contacted with the first antibody is the same as the surface bound to apolipoprotein and contacted with the second antibody, that is, the contact is carried out in a single container, for example, a single hole in a plate (such as an ELISA plate). Therefore, the contact between the surface bound to apolipoprotein and the first antibody is carried out in the same container as the contact with the second antibody. In an optional implementation, the first surface bound to apolipoprotein is contacted with the first antibody, and the second surface bound to apolipoprotein is contacted with the second antibody, wherein the first and second surfaces are different surfaces, for example, different holes in a plate (such as an ELISA plate), different beads (such as turbidimetric beads or luminex beads) or different particles. Therefore, the contact of the surface bound to apolipoprotein and the first antibody, the contact of the surface bound to apolipoprotein and the second antibody are carried out in different containers, that is, different containers are used. In a more specific embodiment, when different containers are used, each of said containers comprises a plurality of entities, such as two different sets of beads (such as Luminex beads or nephelometric beads) or two different sets of particles.

[0150] In a preferred embodiment of the second method of the present invention, the surface used in step (i) is formed by multiple entities, and the contact with the first antibody and the contact with the second antibody in step (ii) are carried out in different containers, each container containing a part of the multiple entities forming the surface.

[0151] The surface bound to the apolipoprotein formed in step (i) is contacted with a first antibody specific for an isotype of the apolipoprotein, and with a second antibody capable of binding to all isotypes of the apolipoprotein present in the sample, under conditions suitable for forming a first complex comprising the first antibody and an isotype of the apolipoprotein, and a second complex comprising the second antibody and all isotypes of the apolipoprotein. Conditions suitable for forming the complex can be determined by a person skilled in the art and include suitable temperature, incubation time and pH. In a particular embodiment, the temperature range is 4 to 40°C, particularly 10 to 35°C, more particularly 15 to 30°C, preferably 20 to 25°C (room temperature). In a preferred embodiment, the temperature in steps (i) and (ii) of the second method of the present invention is substantially the same. In a particular embodiment, the pH range is pH 2 to pH 10, preferably pH 4 to pH 10. In a preferred embodiment, the pH in steps (i) and (ii) of the second method of the present invention is substantially the same. In a specific embodiment, the first antibody is cultured with the apolipoprotein isoform to be determined that is bound to the surface for at least 1 minute, preferably at least 5 minutes, more preferably at least 30 minutes, even more preferably at least 60 minutes. In a specific embodiment, the second antibody is cultured with the apolipoprotein isoform to be determined that is bound to the surface for at least 1 minute, preferably at least 5 minutes, more preferably at least 30 minutes, even more preferably at least 60 minutes.

[0152] exist The third step of the second method of the present invention In the method, the first and second complexes formed in the second step are detected.

[0153] As previously described, the surface bound to the apolipoprotein and contacted with the first antibody may be the same as or different from the surface bound to the apolipoprotein and contacted with the second antibody.

[0154] In a specific embodiment, when the surfaces are different surfaces and the contacting is carried out in different containers, the detection in step iii) of the second method of the present invention is carried out in each container separately.

[0155] In a preferred embodiment of the second method of the present invention, the surface used in step (i) is formed by a plurality of entities, the contact with the first antibody and the contact with the second antibody in step (ii) are carried out in different containers, each container comprising a portion of the plurality of entities forming the surface, and the detection in step (iii) is carried out in each container separately. The above embodiment is particularly preferred when the polystyrene surface or the polycarbonate surface consists of beads, preferably Luminex beads or turbidimetric beads and the detection is carried out by immunoturbidimetry. Therefore, according to this embodiment described in the present invention, the first and second complexes formed in the second step of the method are detected by turbidimetry, in particular by immunoturbidimetric assay. When the apolipoprotein or apolipoprotein isoform is present in the sample to be analyzed, the formation of an apolipoprotein (all its isoforms)-antibody complex or an apolipoprotein isoform-antibody complex causes an increase in turbidity relative to a sample lacking an apolipoprotein or an apolipoprotein isoform, wherein the turbidity change can be measured (e.g., by a spectrophotometer). In a preferred embodiment of the present invention, the immunoturbidimetric assay is LEIT.

[0156] In a specific optional embodiment, the first and second complexes formed in the second step of the method are detected by a detectable reagent. Suitable detectable reagents in the context of the present invention have been described above in the context of the first method of the present invention, and are introduced herein. In a preferred embodiment, the first and second complexes formed in the second step of the method can be detected by a specific reporter antibody of an anti-apolipoprotein antibody and / or an anti-apolipoprotein isotype antibody. In a preferred embodiment, the second antibody (reporter antibody) is coupled to an enzyme under culture conditions similar to those for culturing the surface with an apolipoprotein isotype, and / or under culture conditions similar to those for culturing a specific antibody of the apolipoprotein isotype with an apolipoprotein isotype bound to the surface. The above embodiment is particularly preferred when a polystyrene surface or a polycarbonate surface comprises a plate, preferably an ELISA plate, and is detected by an ELISA assay.

[0157] exist The fourth step of the second method of the present invention is In step (iii), the relative amounts of the isoforms relative to the total apolipoprotein content are determined based on the levels of the first and second complexes obtained in step (iii).

[0158] In a specific embodiment, the ratio between the detection signals from the first and second complexes detected in step (iii) of the second method of the present invention is calculated. Thus, the relative amount of the isoform of a specific apolipoprotein relative to the total content of the apolipoprotein is determined. As known to those skilled in the art, the ratio range can be between 0 (when a specific apolipoprotein isoform is not present in the sample to be analyzed) and 1 (when a specific apolipoprotein isoform is the only isoform of the apolipoprotein present in the sample to be analyzed).

[0159] Allele dosage determination method of the present invention

[0160] The inventors of the present invention have developed a method for determining whether a subject is a homozygous or heterozygous carrier of a specific apolipoprotein isoform based on a dual assay of a subject's sample, wherein one assay determines the level of a specific apolipoprotein isoform and the other assay determines the level of total apolipoprotein. The ratio of apolipoprotein isoform / total apolipoprotein is then calculated and the allele dosage is inferred from this ratio. See Example 1. The apolipoprotein isoform content is also determined by immunoturbidimetric assay. See Example 2.

[0161] Therefore, in another aspect, the present invention relates to a method for determining the allele dosage of a haplotype associated with the expression of an apolipoprotein isoform in a subject (third method of the invention), the method comprising:

[0162] (i) contacting a protein-containing sample derived from a tissue of the subject expressing the apolipoprotein isoform with a polystyrene surface or a polycarbonate surface under conditions suitable for binding the apolipoprotein isoform to the polystyrene surface or the polycarbonate surface, wherein the surface does not contain an anti-specific antibody that binds to the apolipoprotein isoform.

[0163] (ii) contacting the apolipoprotein-bound surface formed in step (i) with an antibody specific for at least one apolipoprotein isoform under conditions suitable for formation of a complex between the antibody and the apolipoprotein isoform,

[0164] (iii) determining the allelic dosage of the apolipoprotein isoform by correlating the amount of the complex formed in step (ii) with the number of alleles encoding the gene variant.

[0165] In a specific embodiment, the present invention relates to a method for determining the allele dosage of a haplotype associated with the expression of an apolipoprotein isoform of an apolipoprotein selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin) in a subject, the method comprising,

[0166] (i) contacting a protein-containing sample derived from a tissue of the subject expressing the apolipoprotein isoform with a polystyrene surface or a polycarbonate surface under conditions suitable for binding the apolipoprotein isoform to the polystyrene surface or the polycarbonate surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein isoform,

[0167] (ii) contacting the apolipoprotein-bound surface formed in step (i) with an antibody specific for at least one apolipoprotein isoform under conditions suitable for formation of a complex between the antibody and the apolipoprotein isoform,

[0168] (iii) determining the allelic dosage of the apolipoprotein isoform by correlating the amount of the complex formed in step (ii) with the number of alleles encoding the gene variant,

[0169] Wherein, the polystyrene surface or the polycarbonate surface is not treated before contacting with the sample or is blocked with albumin before contacting with the sample.

[0170] According to the third method described in the present invention, the apolipoprotein isotype to be determined includes apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (cluster protein) isotypes. In a specific embodiment, according to the third method described in the present invention, the apolipoprotein isotype to be determined is selected from the isotypes apoE2, apoE3 and apoE4 of apoE apolipoprotein. In a more specific embodiment, the apolipoprotein isotype to be determined according to the third method described in the present invention is apoE4.

[0171] exist The first step of the third method of the present invention is In the method, a protein-containing sample is contacted with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein or an isoform thereof in the sample to the surface, wherein the sample is derived from a tissue of the subject that expresses an apolipoprotein isoform, wherein the surface does not contain specific antibodies that bind to the apolipoprotein or an isoform thereof.

[0172] Suitable conditions for binding apolipoprotein or an isoform thereof in a sample to a polystyrene or polycarbonate surface, preferably a polystyrene surface, have been described above in the context of the first method of the invention and are introduced here.

[0173] The sample is a sample derived from a tissue expressing an apolipoprotein isoform containing the protein. As previously mentioned, suitable biological samples include biological fluids such as blood, plasma, serum, saliva, urine, and cerebrospinal fluid (CSF). In a specific embodiment, the sample is a plasma sample. The suitable dilution range of the plasma sample is 1:10 to 1:100000, preferably 1:100 to 1:1000, and more preferably the dilution of the plasma sample is 1:200.

[0174] The polystyrene surface or polycarbonate surface according to the present invention does not contain any specific antibodies for the apolipoprotein or its isoform to be bound and detected thereto. The polystyrene surface or polycarbonate surface according to the present invention is as described above and is introduced herein. In a specific embodiment, the polystyrene surface comprises an ELISA plate, luminex beads or turbidimetric beads. In any case, the polystyrene surface or polycarbonate surface does not contain specific antibodies for the apolipoprotein or its isoform to be bound to the surface.

[0175] In a specific embodiment, the polystyrene surface or polycarbonate surface according to the third method described in the present invention is formed by a single entity, so that the single entity provides the surface necessary for binding apolipoprotein, for example, a single flat plate such as a single ELISA plate. In an optional embodiment, the polystyrene surface or polycarbonate surface according to the third method described in the present invention is formed by multiple entities, that is, at least two entity units provide a surface that can be used for apolipoprotein to bind to the surface, such as multiple beads, such as Luminex beads or turbidimetric beads. In a specific embodiment of the third method of the present invention, under conditions suitable for binding apolipoprotein in the sample to a polystyrene surface or a polycarbonate surface, the surface in contact with the sample is formed by multiple entities. Preferably, the surface is formed by Luminex beads or turbidimetric beads.

[0176] In a specific embodiment, the polystyrene surface or polycarbonate surface is not treated before contact with the sample. In another optional preferred embodiment, the polystyrene surface or polycarbonate surface is blocked before contact with the sample, that is, the surface is blocked before the binding step (i) of the third method of the present invention. In a specific embodiment, the polystyrene surface or polycarbonate surface is blocked with proteins and / or detergents other than apolipoprotein before the binding step. Suitable proteins and detergents for blocking surfaces according to the present invention have been described above and are introduced herein. Particularly preferred proteins for blocking are BSA, casein and gelatin, more preferably BSA, more preferably BSA in the range of 1% to 3%, more preferably 1% BSA. Suitable detergents for blocking surfaces before the binding step include, but are not limited to, polyvinylpyrrolidone-40 (PVP-40), polysorbate 20 (Tween 20), Nonidet-P40 and Triton X-100.

[0177] In a particular embodiment, the polystyrene or polycarbonate surface is treated with a washing solution after contact with the sample, that is, the polystyrene surface or polycarbonate surface is treated with a washing solution after the binding step (i) of the third method of the present invention. Preferably, the polystyrene surface or polycarbonate surface is treated with a washing solution after the binding step (i) and before step (ii) of the third method of the present invention. Suitable washing solutions have been described above and are introduced here. In a particular embodiment, the washing solution contains one or more salts. Preferably, the salt contained in the washing solution is NaCl. Additionally or alternatively, the washing solution contains at least one detergent. Preferably, the detergent is selected from polysorbate 20 (Tween 20) and Triton X-100. Additionally or alternatively, the washing solution contains an acid. Preferably, the acid is selected from HCl and formic acid. More preferably, the acid is selected from 2M HCl and 70% formic acid. Additionally or alternatively, the washing solution contains a base. Preferably, the base is NaOH, more preferably 2M NaOH. Additionally or alternatively, the washing solution contains a reducing agent. The reducing agent is preferably 2-mercaptoethanol. Additionally or alternatively, the washing solution does not contain an enzymatic cleaning agent (such as a protease), an oxidizing agent (such as sodium hypochlorite) or a combination thereof.

[0178] exist The second step of the third method of the present invention In the step (i), the apolipoprotein-bound surface formed in step (i) is contacted with an antibody specific for at least one apolipoprotein isoform under conditions suitable for formation of a complex between the antibody and the apolipoprotein isoform.

[0179] In a specific preferred embodiment, the second step of the third method of the present invention also includes contacting the surface with a second antibody capable of binding to all isoforms of the apolipoprotein in the sample, wherein the contact is carried out under conditions suitable for forming a first complex and a second complex, the first complex comprising the first antibody and the apolipoprotein isoform, and the second complex comprising the second antibody and all isoforms of the apolipoprotein.

[0180] Antibodies specific for apolipoprotein and antibodies specific for a particular apolipoprotein isoform have already been cited above in the context of the first method of the invention and are incorporated herein.

[0181] Conditions suitable for forming a first complex comprising a first antibody and an apolipoprotein isotype and suitable for forming a second complex comprising a second antibody and all isotypes of the apolipoprotein can be determined by a person skilled in the art and as described above, include suitable temperature, incubation time and pH. In a specific embodiment, the temperature range is 4 to 40°C, particularly 10 to 35°C, more particularly 15 to 30°C, preferably 20 to 25°C (room temperature). In a preferred embodiment, the temperature in steps (i) and (ii) of the third method of the present invention is substantially the same. In a specific embodiment, the pH range is pH2 to pH 10, preferably pH 4 to pH 10. In a preferred embodiment, the pH in steps (i) and (ii) of the third method of the present invention is substantially the same. In a specific embodiment, the first antibody is cultured with the apolipoprotein isotype to be determined that is bound to the surface for at least 1 minute, preferably at least 5 minutes, more preferably at least 30 minutes, and even more preferably at least 60 minutes. In a specific embodiment, the secondary antibody is incubated with the surface-bound apolipoprotein isoform to be determined for at least 1 minute, preferably at least 5 minutes, more preferably at least 30 minutes, even more preferably at least 60 minutes.

[0182] In a specific embodiment, the surface that is combined with apolipoprotein and contacts with the first antibody is the same as the surface that is combined with apolipoprotein and contacts with the second antibody, that is, contact is carried out in a single container, for example, a single hole in a plate (such as an ELISA plate). Therefore, the contact between the surface combined with apolipoprotein and the first antibody is carried out in the same container that contacts the second antibody. In an optional preferred embodiment, the first surface combined with apolipoprotein contacts the first antibody, and the second surface combined with apolipoprotein contacts the second antibody, wherein the first and second surfaces are different surfaces, for example, different holes in a plate (such as an ELISA plate), different beads (such as turbidimetric beads or luminex beads) or different particles. Therefore, the contact of the surface combined with apolipoprotein and the first antibody and the contact of the surface combined with apolipoprotein and the second antibody are carried out in different containers, that is, different containers are used. In a more specific embodiment, when using different containers, each of the containers includes multiple entities, such as two groups of different beads (such as Luminex beads or turbidimetric beads) or two groups of different particles.

[0183] In a preferred embodiment of the second method of the present invention, the surface used in step (i) is formed by multiple entities, and the contact with the first antibody and the contact with the second antibody in step (ii) are carried out in different containers, each container containing a part of the multiple entities forming the surface.

[0184] exist The third step of the third method of the present invention The allelic dosage of the apolipoprotein isoform is determined by relating the amount of complex formed in step (ii) to the number of alleles encoding the gene variant.

[0185] In a specific embodiment, the relative amount of the isoform relative to the total apolipoprotein content is determined based on the levels of the first and second complexes obtained in step (ii), wherein the allele dosage of the haplotype associated with the expression of the apolipoprotein isoform is determined from the relative amounts determined in (iii).

[0186] In a specific embodiment, the ratio between the detection signals from the first and second complexes detected in step (iii) is calculated. Thus, the relative amount of the isotype of a specific apolipoprotein relative to the total content of the apolipoprotein is determined. As known to those skilled in the art, the ratio range can be between 0 (when there is no specific apolipoprotein isotype in the sample to be analyzed) and 1 (when a specific apolipoprotein isotype is the only isotype of the apolipoprotein present in the sample to be analyzed). Therefore, a ratio equal to zero indicates that there is no apolipoprotein isotype in the analyzed sample. A ratio equal to 1 indicates a homozygous object of an apolipoprotein isotype, wherein 1 is an arbitrary value of arbitrary units. A ratio equal to 0.5 indicates a heterozygous object of an apolipoprotein isotype, wherein 0.5 is an arbitrary value of arbitrary units. The method for detecting a complex is as described above and incorporated herein.

[0187] In a preferred embodiment of the second method of the present invention, the surface used in step (i) is formed by a plurality of entities, the contact with the first antibody and the second antibody in step (ii) is carried out in different containers, each container comprising a portion of the plurality of entities forming the surface, and the detection in step (iii) is carried out in each container separately. The above embodiment is particularly preferred when the polystyrene surface or the polycarbonate surface comprises beads, preferably Luminex beads or turbidimetric beads and the detection is carried out by immunoturbidimetry. Therefore, the first and second complexes formed in the second step of the method are detected by turbidimetry, in particular by immunoturbidimetric assay. When the apolipoprotein or apolipoprotein isoform is present in the sample to be analyzed, the formation of an apolipoprotein (all its isoforms)-antibody complex or an apolipoprotein isoform-antibody complex causes an increase in turbidity relative to a sample lacking an apolipoprotein or an apolipoprotein isoform, wherein the turbidity change can be measured (e.g., by a spectrophotometer). In a preferred embodiment of the present invention, the immunoturbidimetric assay is LEIT.

[0188] In a specific optional embodiment, the first and second complexes formed in the second step of the method are detected by a detectable reagent. Suitable detectable reagents in the context of the present invention have been described above and are introduced herein. In a preferred embodiment, the first and second complexes formed in the second step of the method can be detected by a specific reporter antibody of an anti-apolipoprotein antibody and / or an anti-apolipoprotein isotype antibody. In a preferred embodiment, the second antibody (reporter antibody) is coupled to an enzyme under culture conditions similar to culture surfaces with apolipoprotein isotypes, and / or under culture conditions similar to culture specific antibodies of apolipoprotein isotypes with apolipoprotein isotypes bound to the surface. When a polystyrene surface or a polycarbonate surface comprises a plate, preferably an ELISA plate, and when detection is performed by an ELISA assay, the above embodiment is particularly preferred.

[0189] Thus, a ratio of zero indicates the absence of the apolipoprotein isoform in the analyzed sample, that is, the dosage of the specific allele encoding the gene variant is zero. A ratio of 1 indicates homozygosity for the apolipoprotein isoform, that is, the dosage of the specific allele encoding the gene variant is 2. A ratio of 0.5 relates to a subject heterozygous for the apolipoprotein isoform, that is, the dosage of the specific allele encoding the gene variant is 1.

[0190] In a particularly preferred embodiment of the third method of the present invention, the surface used in step (i) is formed by a plurality of entities, and step (ii) further comprises contacting the surface with a second antibody capable of binding to all isoforms of the apolipoprotein present in the sample, wherein the contact is carried out under conditions suitable for forming a first complex comprising the first antibody and the apolipoprotein isoform and suitable for forming a second complex comprising the second antibody and all isoforms of the apolipoprotein, wherein the contacting of the first antibody and the second antibody is carried out in different containers, each container comprising a portion of the plurality of entities forming the surface, wherein based on the levels of the first and second complexes obtained in step (ii), the relative amount of the isoform relative to the total apolipoprotein content is determined, and based on the relative amount determined in step (iii), the allele dose of the genotype associated with the expression of the apolipoprotein isoform is determined.

[0191] Methods to predict the development of neurodegenerative diseases

[0192] It is known that the presence of the ApoEε4 gene on chromosome 19 of a subject increases the risk of the subject developing Alzheimer's disease (AD). Thus, it has been determined that a subject with one apoE4 allele has a four-fold increased probability of developing AD compared to a subject that does not carry the apoE4 allele, and a subject with two apoE4 alleles has a 15-20-fold increased probability of developing AD compared to a subject that does not carry the apoE4 allele. Therefore, the method of the present invention for determining apolipoprotein isoforms (including apoE4) can be applied to predict the development of AD in a subject.

[0193] Therefore, in another aspect, the present invention relates to a method for determining a filter for a subject to develop a neurodegenerative disease, the method comprising determining the level of apoE4 isoform in a sample from the subject by any of the methods of the present invention described above (the first, second and third methods), wherein if the apoE4 level is higher than a reference value, it indicates that the subject has a high probability of suffering from a neurodegenerative disease.

[0194] The reference value for the method for determining the probability of developing a neurodegenerative disease according to the present invention is the level of apoE4 determined in a sample from a subject not carrying the apoE4 allele.

[0195] The reference value or reference level of the method according to the present invention can be an absolute value; a relative value; a value with an upper or lower limit; a range of values; an average value; a median value; a mean; or a value compared to a specific control or baseline value. The reference value can be based on the value of a single sample, such as a value obtained from a sample from the subject being tested, but at an earlier time point. The reference value can be based on a large number of samples, such as a population of subjects from an age-matched group, or based on a sample pool that includes or excludes the sample to be tested. Various factors need to be considered when determining the reference value of a marker. Factors to consider include age, weight, sex, the patient's overall physical condition, etc. For example, a group of at least 2, at least 10, at least 100, preferably at least 1000 objects of equal size, preferably classified according to various age categories, is used as a reference group according to the aforementioned considerations.

[0196] When the subject shows a 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% probability of developing or contracting the disease over time, there is a high probability of developing a neurodegenerative disease. In a specific embodiment, the high probability is at least 100%. In other embodiments, the high probability is at least 200%, at least 300%, at least 400%, at least 500%, at least 700%, at least 800%, at least 900% and at least 1000%. However, other critical points or ranges that are considered suitable for characterizing the present invention by those skilled in the art may also be included, and these critical points or ranges are also within the scope of the present invention. According to the prediction method of the present invention, the high probability of a subject developing a neurodegenerative disease is associated with the presence of one or two apoE4 alleles in the genome of the subject, wherein the apoE4 allele determination is performed by any of the first, second or third methods of the present invention as described above.

[0197] A low probability of developing a neurodegenerative disease exists when the subject exhibits a 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% probability of developing or contracting the disease over time. In a specific embodiment, the low probability is at least 100%. In other embodiments, the low probability is at least 200%, at least 300%, at least 400%, at least 500%, at least 700%, at least 800%, at least 900% and at least 1000%. However, other critical points or ranges deemed suitable for characterizing the present invention by those skilled in the art may also be encompassed, and these critical points or ranges are also within the scope of the present invention. According to the prediction method of the present invention, the low probability of a subject developing a neurodegenerative disease is associated with the absence of an apoE4 allele in the genome of the subject, wherein the apoE4 allele determination is performed by any of the first, second or third methods of the present invention as described above.

[0198] In a specific embodiment, the neurodegenerative disease is selected from Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Down syndrome-related dementia, vascular dementia, Parkinson's disease, dementia with Lewy bodies, and Creutzfeldt-Jakob disease. In a more specific embodiment, the neurodegenerative disease is Alzheimer's disease (AD).

[0199] In yet another aspect, the present invention relates to a method for determining the probability of a subject developing a neurodegenerative disease, the method comprising determining the allele dosage of a haplotype encoding an ApoE4 isoform in the subject by the third method according to the present invention, wherein the presence of one or two ApoE4 alleles in the genome of the subject indicates that the subject has a high probability of developing a neurodegenerative disease, and wherein the absence of an ApoE4 allele in the genome of the subject indicates that the subject has a low probability of suffering from a neurodegenerative disease.

[0200] In a specific embodiment, the neurodegenerative disease is selected from Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Down syndrome-related dementia, vascular dementia, Parkinson's disease, dementia with Lewy bodies and Creutzfeldt-Jakob disease. In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease (AD).

[0201] Methods for predicting the development of cardiovascular disease

[0202] Apolipoprotein defects are associated with the development of cardiovascular diseases. Therefore, the methods of the present invention for determining apolipoproteins and apolipoprotein isoforms can also be used to predict the risk of developing cardiovascular diseases.

[0203] Therefore, in another aspect, the present invention relates to a method for determining the probability of a subject developing cardiovascular and cerebrovascular diseases, the method comprising determining the level of apoE4 isoform in a sample from the subject by any of the methods of the present invention described above (the first, second and third methods), wherein if the apoE4 level is higher than a reference value, it indicates that the subject has a high probability of developing cardiovascular diseases.

[0204] In another aspect, the present invention relates to a method for determining the probability of a subject developing cardiovascular disease, the method comprising determining the allele dosage of a haplotype encoding an apolipoprotein isotype in the subject by the third method according to the present invention, wherein the presence of one or two of the apolipoprotein isotype alleles in the genome of the subject indicates that the subject has a higher probability of developing cardiovascular disease, and wherein the absence of the ApoE4 allele in the genome of the subject indicates a lower probability of the subject developing cardiovascular disease.

[0205] In one embodiment of the cardiovascular disease prediction method of the present invention, the apolipoprotein isoform to be determined is an apoE isoform, particularly selected from apoE isoforms apoE E2, apoE E3 and apoE E4, and the cardiovascular disease is selected from type III hyperlipoproteinemia (also known as aspergillusemia or generalized beta cell disease, a rare genetic disease characterized by poor lipid metabolism, especially cholesterol and triglycerides, leading to abnormal accumulation of lipids in the body or hyperlipidemia), aspergillusemia caused by apolipoprotein ed defect, familial hyperbetalipoproteinemia (increased accumulation of low-density lipoprotein or beta lipoprotein in the blood) and probetalipoproteinemia (excess very low-density lipoprotein or probetalipoprotein in the blood), familial hypercholesterolemia with hyperlipidemia, hyperlipidemia with familial hypercholesterolemia xanthomatosis (deposition of cholesterol-rich accumulations in the body), generalized beta cholelipoproteinemia (a condition characterized by the development of excess fat areas in different parts of the body, which causes fat to be stored in abnormal locations throughout the body, including the palms, fingers, knees, elbows and under the arms), floating beta lipoproteinemia and coronary artery disease (hardening and narrowing of the arteries supplying blood to the heart muscle, leading to atherosclerosis and even angina or heart attack).

[0206] In another embodiment of the cardiovascular disease prediction method of the present invention, the apolipoprotein to be determined is apoAI, and the cardiovascular disease is hypoalphalipoproteinemia (HDL deficiency, inherited in an autosomal dominant manner).

[0207] In another embodiment of the cardiovascular disease prediction method of the present invention, the apolipoprotein to be measured is apoCII, and the cardiovascular disease is type Ib hyperlipoproteinemia (a rare genetic disease characterized by high levels of chylomicrons caused by low apoCII levels).

[0208] In another embodiment of the cardiovascular disease prediction method of the present invention, the apolipoprotein to be determined is apoCIII and the cardiovascular disease is apoCIII deficiency associated with hypertriglyceridemia (elevated blood levels of triglycerides).

[0209] Kit of the present invention and its use

[0210] In yet another aspect, the present invention relates to a kit comprising a polystyrene surface or a polycarbonate surface and a specific antibody against apolipoprotein or an isoform thereof, wherein the kit does not comprise a second specific antibody against the apolipoprotein or an isoform thereof.

[0211] Therefore, the first element of the kit of the present invention is a polystyrene surface or a polycarbonate surface. Polystyrene and polycarbonate surfaces according to the present invention have been described above in the context of the first method of the present invention, and are introduced herein. Preferably, the surface is a polystyrene surface. In a specific embodiment, the polystyrene surface or the polycarbonate surface is composed of ELISA plates, luminex beads, turbidity beads or protein arrays. Particularly preferred surfaces are polystyrene surfaces, including but not limited to flat plates such as ELISA plates, beads such as Luminex beads and turbidity beads and particles.

[0212] The second element of the kit of the present invention is a specific antibody for apolipoprotein or its isotype. According to the kit of the present invention, the apolipoprotein includes apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clustering protein). The apolipoprotein isotype according to the kit of the present invention includes apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clustering protein) isotype. The specific antibody of apolipoprotein or its isotype has been described above, and is introduced herein. In a specific embodiment, the apolipoprotein is apoE, and the antibody is an anti-apoE antibody. In a specific embodiment, the apolipoprotein isotype is an apoE isotype selected from apoE2, apoE3 and apoE4, and the antibody is selected from anti-apoE2, anti-apoE3 and anti-apoE4 antibodies.

[0213] In a particular embodiment, the specific antibody against apolipoprotein or its isoform of the kit of the present invention is bound to a polystyrene surface or a polycarbonate surface, preferably a polystyrene surface.

[0214] The kit of the present invention does not contain a second antibody specific for a specific apolipoprotein or a specific isoform thereof to which the antibody contained in the kit specifically binds. However, in a specific embodiment, when the antibody contained in the kit is a specific antibody for a specific apolipoprotein isoform, the kit may further contain a second antibody capable of binding to all isoforms of the specific apolipoprotein.

[0215] In a specific embodiment, the kit of the present invention further comprises a reporter antibody, wherein the reporter antibody is specific for the apolipoprotein or its isoform specific antibody contained in the kit. In a specific optional embodiment, the apolipoprotein or its isoform specific antibody of the kit of the present invention comprises a detectable agent. Suitable detectable agents in the context of the present invention have been described above in the context of the first method of the present invention and are introduced herein.

[0216] In yet another embodiment, the present invention relates to the use of a kit as described above for detecting and / or quantifying apolipoprotein or its isoform in a sample, for determining the relative amount of an isoform of a given apolipoprotein relative to the total content of the apolipoprotein in a sample, for determining the allele dosage of a haplotype associated with the expression of an apolipoprotein isoform in a subject, or for determining the probability of a subject developing a neurodegenerative disease or for determining the probability of a subject developing a cardiovascular disease. In a specific embodiment, the present invention relates to the use of a kit as described above for detecting and / or quantifying apolipoprotein or its isoform selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clustering proteins). In a more specific embodiment, the present invention relates to the use of a kit as described above for detecting and / or quantifying apolipoprotein or its isoform selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clustering proteins), wherein the polystyrene surface or the polycarbonate surface is blocked by albumin, more preferably wherein the albumin is BSA. The specific details of the assay have been described sequentially in the present specification and are incorporated herein.

[0217] Therefore, the present invention relates to the following aspects:

[0218] 1. An in vitro method for detecting and / or quantifying apolipoprotein or an isoform thereof in a sample, the method comprising:

[0219] (i) contacting a sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein or an isoform thereof to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein or an isoform thereof,

[0220] (ii) contacting the surface bound to the apolipoprotein or its isoform formed in step (i) with an antibody specific for the apolipoprotein or its isoform under conditions suitable for formation of a complex between the antibody and the apolipoprotein or its isoform, and

[0221] (iii) detecting the complex formed in step (ii).

[0222] 2. The method according to aspect 1, wherein the apolipoprotein is selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin).

[0223] 3. An in vitro method for determining the relative amount of a given apolipoprotein isoform in a sample relative to the total amount of said apolipoprotein, the method comprising:

[0224] (i) contacting the sample with a polystyrene surface or a polycarbonate surface under conditions suitable for binding apolipoprotein to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein,

[0225] (ii) contacting the apolipoprotein-bound surface formed in step (i) with a first antibody specific for an isoform of the apolipoprotein and with a second antibody capable of binding to all isoforms of the apolipoprotein present in the sample, wherein the contacting is performed under conditions suitable for the formation of a first complex comprising the first antibody and the isoform of the apolipoprotein and a second complex comprising the second antibody and all isoforms of the apolipoprotein,

[0226] (iii) detecting the first and second complexes formed in step (ii) and

[0227] (iv) determining the relative amount of the isoform relative to the total apolipoprotein content based on the levels of the first and second complexes obtained in step (iii).

[0228] 4. In a preferred embodiment of the second method of the present invention, the surface used in step (i) is formed by multiple entities, wherein the contact with the first antibody and the second antibody in step (ii) is carried out in different containers, each container containing a portion of the multiple entities forming the surface, and the detection in step (iii) is carried out in each container separately.

[0229] 5. The method according to aspect 3 or 4, wherein the apolipoprotein isoform is an apolipoprotein isoform selected from the group consisting of apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin).

[0230] 6. The method according to aspect 5, wherein the apolipoprotein is apoE and the apoE isoform is selected from apoE2, apoE3 and apoE4.

[0231] 7. A method for determining the allele dosage of a haplotype associated with expression of an apolipoprotein isoform in a subject, the method comprising:

[0232] (i) contacting a protein-containing sample derived from a tissue of the subject expressing the apolipoprotein isoform with a polystyrene surface or a polycarbonate surface under conditions suitable for binding the apolipoprotein isoform to the surface, wherein the surface does not contain specific antibodies that bind to the apolipoprotein isoform,

[0233] (ii) contacting the apolipoprotein-bound surface formed in step (i) with at least one antibody specific for an apolipoprotein isoform under conditions suitable for formation of a complex between the antibody and the apolipoprotein isoform,

[0234] (iii) determining the allelic dosage of the apolipoprotein isoform by correlating the amount of complex formed in step (ii) with the number of alleles encoding the gene variant.

[0235] 8. A method according to aspect 7, wherein the surface used in step (i) is formed by multiple entities, and step (ii) further comprises: contacting the surface with a second antibody capable of binding to all isoforms of the apolipoprotein present in the sample, wherein the contact is carried out under conditions suitable for forming a first complex and suitable for forming a second complex, the first complex comprising the first antibody and the apolipoprotein isoform, and the second complex comprising the second antibody and all isoforms of the apolipoprotein, wherein the contact with the first antibody and the contact with the second antibody are carried out in different containers, each container comprising a portion of the multiple entities forming the surface, wherein the relative amount of the isoform relative to the total apolipoprotein content is determined based on the levels of the first and second complexes obtained in step (ii), and the allele dose of the genotype associated with the expression of the apolipoprotein isoform is determined based on the relative amount determined in step (iii).

[0236] 9. The method according to aspect 7 or 8, wherein the gene variant encoding an apolipoprotein isoform is a gene encoding an apolipoprotein isoform selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin).

[0237] 10. The method according to aspect 9, wherein the gene encoding the apolipoprotein isoform is the gene encoding apoE4.

[0238] 11. The method according to any one of the preceding aspects, wherein the sample is selected from the group consisting of blood, plasma, serum, saliva, urine and cerebrospinal fluid (CSF).

[0239] 12. The method according to the preceding aspect, wherein the sample is plasma, preferably in the dilution range of 1:10 to 1:100000, preferably 1:100 to 1:1000, more preferably 1:20.

[0240] 13. The method according to any one of the preceding aspects, wherein the polystyrene surface or the polycarbonate surface is blocked prior to the bonding step (i).

[0241] 14. The method according to the preceding aspect, wherein the surface is blocked with a protein other than apolipoprotein and / or a detergent.

[0242] 15. The method according to the preceding aspects, wherein the surface is blocked with a protein selected from the group consisting of BSA, casein and gelatin, and wherein the detergent is selected from the group consisting of polyvinylpyrrolidone-40 (PVP-40), polysorbate 20, Nonidet-P40 and Triton X-100.

[0243] 16. The method according to the preceding aspect, wherein the surface is blocked with BSA, preferably 1% BSA.

[0244] 17. The method according to any one of the preceding aspects, wherein the polystyrene surface is treated with a washing solution after the bonding step (i).

[0245] 18. The method according to the preceding aspect, wherein the washing solution contains one or more salts.

[0246] 19. A method according to any one of the preceding claims, wherein the salt is NaCl.

[0247] 20. The method according to any one of aspects 17 to 19, wherein the washing solution contains at least one cleaning agent.

[0248] 21. The method according to the preceding aspect, wherein the at least one detergent is selected from polysorbate 20 and Triton X-100.

[0249] 22. The method according to any one of aspects 17 to 21, wherein the washing solution contains at least one acid.

[0250] 23. The method according to the preceding aspect, wherein the acid is 2M HCl or 70% formic acid.

[0251] 24. The method according to any one of aspects 17 to 21, wherein the washing solution contains at least one base.

[0252] 25. A method according to any preceding claim, wherein the base is 2M NaOH.

[0253] 26. The method according to any one of aspects 17 to 25, wherein the washing solution contains at least one reducing agent.

[0254] 27. A method according to any preceding claim, wherein the reducing agent is 2-mercaptoethanol.

[0255] 28. The method according to any one of aspects 17-27, wherein the wash solution does not contain a protease, an oxidizing agent, or a combination thereof.

[0256] 29. The method according to any one of the preceding aspects, wherein the polystyrene surface or polycarbonate surface consists of an ELISA plate, luminex beads or nephelometry beads.

[0257] 30. The method according to any one of the preceding aspects, wherein the detection of the complex in step (iii) is performed by ELISA or by immunoturbidimetry.

[0258] 31. The method according to the preceding aspect, wherein the immunoturbidimetric assay is LEIT (latex enhanced immunoturbidimetric technique).

[0259] 32. The method according to any one of aspects 1 to 30, wherein the detection of the complex formed in step (iii) is performed by a reporter antibody specific for the anti-apolipoprotein antibody.

[0260] 33. A method for determining the probability of a subject developing a neurodegenerative disease, the method comprising determining the level of an apoE4 isoform in a sample from the subject by a method according to any one of aspects 1 to 32, wherein if the apoE4 level is higher than a reference value, it indicates that the subject has a high probability of suffering from the neurodegenerative disease.

[0261] 34. A method for determining the probability of a subject developing a neurodegenerative disease, the method comprising determining the allele dosage of a haplotype encoding an apoE E4 isoform in the subject by a method according to any one of aspects 7 to 32, wherein the presence of one or two apoE E4 alleles in the genome of the subject indicates that the subject has a high probability of developing the neurodegenerative disease, and wherein the absence of an apoE4 allele in the genome of the subject indicates that the subject has a low probability of developing the neurodegenerative disease.

[0262] 35. The method of any one of aspects 33 or 34, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Down syndrome-related dementia, vascular dementia, Parkinson's disease, Lewy body dementia and Creutzfeldt-Jakob disease.

[0263] 36. The method according to the preceding aspects, wherein the degenerative disease is Alzheimer's disease (AD).

[0264] 37. A method for determining the probability of a subject developing cardiovascular disease, the method comprising determining the level of an apolipoprotein isoform in a sample from the subject by the method described in any one of aspects 1 to 30, wherein if the apolipoprotein level is higher than a reference value, it indicates that the subject has a high probability of developing cardiovascular disease.

[0265] 38. A method for determining the probability of a subject developing cardiovascular disease, the method comprising determining the allele dosage of a haplotype encoding an apolipoprotein isotype in the subject by a method according to any one of aspects 7 to 30, wherein the presence of one or two of the apolipoprotein isotype alleles in the genome of the subject indicates that the subject has a higher probability of developing cardiovascular disease, and wherein the absence of the ApoE4 allele in the genome of the subject indicates that the subject has a lower probability of developing cardiovascular disease.

[0266] 39. The method of aspect 37 or 38, wherein the apolipoprotein isoform is an apoE isoform and the cardiovascular disease is selected from type III hyperlipoproteinemia, ascorbylemia caused by apolipoprotein ed deficiency, familial high beta and pro-beta lipoproteinemia, familial hypercholesterolemia with hyperlipidemia, hyperlipidemia with familial hypercholesterolemia xanthomatosis, generalized beta lipoproteinemia, floating beta lipoproteinemia and coronary artery disease.

[0267] 40. The method according to aspect 37 or 38, wherein the apolipoprotein isoform is apoAI and the cardiovascular disease is hypoalphalipoproteinemia.

[0268] 41. The method of aspect 37 or 38, wherein the apolipoprotein isoform is apoCII and the cardiovascular disease is type Ib hyperlipoproteinemia.

[0269] 42. The method of aspect 37 or 38, wherein the apolipoprotein isoform is apoCIII and the cardiovascular disease is apoCIII deficiency associated with hypertriglyceridemia.

[0270] 43. A kit comprising a polystyrene surface or a polycarbonate surface and an antibody specific for apolipoprotein or an isoform thereof, wherein the kit does not comprise a second antibody specific for the apolipoprotein or an isoform thereof.

[0271] 44. A kit according to aspect 43, wherein, if the antibodies present in the kit are specific for an apolipoprotein isoform, the kit further comprises a second antibody capable of binding to all isoforms of said apolipoprotein.

[0272] 45. A kit according to aspect 43 or 44, wherein the apolipoprotein is selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin), and wherein the apolipoprotein isoform is an apolipoprotein isoform selected from apoAI, apoAIV, apoCI, apoCII, apoCIII, apoE and apoJ (clusterin).

[0273] 46. ​​The method of aspect 45, wherein the apolipoprotein is apoE and the apoE isoform is selected from the group consisting of apoE2, apoE3 and apoE4.

[0274] 47. The method of any one of aspects 43 to 46, wherein the polystyrene surface or polycarbonate surface consists of an ELISA plate, luminex beads, or nephelometry beads.

[0275] 48. The kit according to any one of aspects 43 to 47, further comprising a reporter antibody, wherein the reporter antibody is specific for the antibody specific for apolipoprotein or an isoform thereof comprised by the kit.

[0276] 49. The kit according to any one of aspects 43 to 47, wherein the antibody specific for apolipoprotein or an isoform thereof comprises a detectable agent.

[0277] 50. Use of the kit according to any one of aspects 43 to 49 for detecting and / or quantifying apolipoprotein or its isoform in a sample, for determining the relative amount of a given apolipoprotein isoform relative to the total amount of said apolipoprotein in a sample, for determining the allele dosage of a haplotype associated with the expression of an apolipoprotein isoform in a subject, for determining the probability of a subject developing a neurodegenerative disease or for determining the probability of a subject developing a cardiovascular disease.

[0278] The present invention is described in detail below by the following examples, which should be construed as merely illustrative purposes and not limiting the scope of the present invention.

[0279] Example

[0280] Example 1. Detection of ApoE4 by ELISA

[0281] Materials and Equipment

[0282] High Binding ELISA Plates ( Flat bottom 96 well plates). Alternative ELISA plate types and brands can be used with similar results.

[0283] Superblock in TBS (Thermo-Fisher, #PI-37535). Alternative blocking agents including gelatin, BSA, casein, Tween-20, etc. have been used with similar results.

[0284] • apoE E4, clone 4E4 (Novus Biologicals, #NBP1-49529) specific mouse monoclonal antibody.

[0285] Specific surrogate antibodies for apoE E4 were used (specific mouse monoclonal antibody for apoE E4, clone 5B5 (IBL #10025); specific mouse monoclonal antibody for apoE E4, clone 1F9 (MBL, #M067-3)).

[0286] • Rabbit polyclonal antibody for total apoE (Santa Cruz Biotechnology, Inc., #sc-98573).

[0287] • Polysorbate-20 (Tween-20) from Sigma-Aldrich.

[0288] Polyclonal antibody anti-mouse IgG peroxidase conjugate.

[0289] TMB Peroxidase EIA Substrate (Biorad)

[0290] ELISA cleaning station (TECAN)

[0291] ELISA spectrophotometer (TECAN)

[0292] Detection Methods

[0293] - Block the ELISA plate by adding 200 μl Superblock (SB) in Tris-buffered saline (TBS) for 16 hours at room temperature (RT).

[0294] Wash twice with 300 μl TBS 0.1% Tween20 in the ELISA washing station.

[0295] - Incubate 100 μl of diluted plasma 1:200 in TBS + 20% SB for 1 hour at RT.

[0296] - Wash 4 times with 300 μl TBS 0.1% Tween20 in the ELISA washing station.

[0297] - Incubate with 50 μl of reporter antibody: 4E4 antibody diluted 1:2000 in TBS + 20% SB for 45 min at RT.

[0298] - Wash 4 times with 300 μl TBS 0.1% Tween20 in the ELISA washing station.

[0299] Incubate with 50 μl of TBS + 20% SB containing anti-mouse IgG-peroxidase (1:10,000) for 30 minutes.

[0300] - Wash 4 times with 300 μl TBS 0.1% Tween20 in the ELISA washing station.

[0301] • Color was developed by adding 100 μl of de TMB for 10 minutes in dark conditions.

[0302] Stop by adding 100 μl H2SO4 2.15N.

[0303] Read the plate with a spectrophotometer at 450 nm, using 750 nm as reference.

[0304] Plasma samples were analyzed at different dilutions, ranging from 1:20 to >1:80000. The best discrimination results were obtained in the range between 1:100 and 1:1,000. For the assays presented herein, the plasma dilution used was 1:200 unless otherwise stated.

[0305] Alternatively, other specific monoclonal antibodies for apoE E4 were used, such as 5B5 and 1F9, but the best results were obtained with the 4E4 clone.

[0306] The incubation time can be shortened without significant changes in the results.

[0307] Different biological fluids can be analyzed by the protocol described here, but the optimal dilution of the sample must be determined based on the starting apoE concentration.

[0308] result

[0309] Distinguishing the presence of apoE E4 in plasma samples

[0310] Using the above protocol, the presence / absence of ApoE4 was detected by using polystyrene plates and E4 isoform-specific monoclonal antibodies. 230 plasma samples from individuals previously genotyped by real-time PCR (e2 / e3, n=16; e2 / e4, n=4; e3 / e3, n=141; e3 / e4, n=59; e4 / e4, n=10) were analyzed. Validation of the assay (73 ApoE4 carriers, 157 non-ApoE4 carriers) showed 100% concordance with ApoE genotyping by real-time PCR (see Figure 1 and Table 1).

[0311] Table 1. Genotype distribution and ELISA readouts of 230 plasma samples used for apoE4 isotype analysis

[0312] ApoE genotype n average value Standard Deviation Minimum Maximum 2 / 3 16 0.0010 0.0025 -0.001 0,008 3 / 3 141 0.0020 0.0045 -0.004 0.036 2 / 4 4 3.087 0.2189 2.713 3.097 3 / 4 59 3.093 0.3787 1.322 3.471 4 / 4 10 3.226 0.2124 2.788 3.557

[0313] According to Table 1 and Figure 2As shown, plasma samples from homozygous APOE e4 / 4 tended to have higher absorbance readings than APOE e3 / e4 ​​and e2 / e4 heterozygous, indicating that although there is some overlap between the groups, it is possible to distinguish between e4 homozygotes and heterozygotes (see Figure 3 ).

[0314] Differentiation of heterozygous / homozygous APOEε4 carriers in plasma samples

[0315] Alternatively, further differentiation between heterozygous and homozygous samples can be accomplished by performing a dual ELISA assay on each sample, one specific for apoE E4 and the other determining total apoE levels (reporter antibody: pan-apoE antibody, e.g. polyclonal pan-apo, SantaCruz Biotech., H-223, #sc-98573). Figure 4 As shown, the ratio of apoEE4 / total apoE is a good indicator of APOEε4 allele dosage. This discrimination power is sufficient for the three dilutions studied ( Figure 4 ).

[0316] Binding of different apolipoproteins to polystyrene surfaces

[0317] To explore the binding of other apolipoproteins to the polystyrene surface, an ELISA protocol as described above was performed, wherein different reporter antibodies specific for certain apolipoproteins were used. Analysis of the results showed that apoCI, apoAI, apoCIII, total apoE and clusterin were able to bind to the plate in a similar manner to apoE E4 (see Figure 5 ).

[0318] ApoE stably binds to the polystyrene surface

[0319] To further characterize the nature of apoE-polystyrene binding, the ability of different buffers containing salt (NaCl 0-2.4 M) or detergents (polysorbate 20 or 0-0.5% Triton X-100) to remove apoE bound to polystyrene plates was analyzed. For this purpose, after binding to apoE present in plasma (1:200 dilution), the wells were incubated with the different target buffers for 1 hour at room temperature, washed and the assay continued as described above. Figure 6 As shown, increasing concentrations of NaCl or detergent enhanced the detection of apoE compared to controls without salt or detergent. These results suggest that binding is mediated by a combination of electrostatic and hydrophobic forces ( Figure 6 ).

[0320] The stability of bound apoE-polystyrene was also analyzed as a function of pH (range pH 2 to 10) in BSA-based blocking solutions or Superblock-blocked wells ( Figure 7 The binding of apoE to BSA-blocked polystyrene pores was largely unaffected by pH within the range studied. Interestingly, at pH below 5, the binding of apoE to Superblock-blocked polystyrene pores was unstable ( Figure 7 ), indicating that the presence of BSA may help maintain a more stable microenvironment due to its own buffering capacity. Therefore, further stability analysis was performed on the pores blocked with BSA solution.

[0321] More stringent tests have also been performed (see Figure 8 ), wherein after treatment at 56°C for 1 hour as follows:

[0322] - strong acid (2M HCl or 70% formic acid),

[0323] - strong base (2M NaOH),

[0324] - a combination of anionic detergent and reducing agent (2% SDS and 0.7% 2-mercaptoethanol, "Membrane Regeneration Solution"),

[0325] - sodium hypochlorite (20,000 ppm), and

[0326] - Enzyme cleaners (Coulter Cleaning agent, Beckman Coulter)

[0327] Interestingly, the only reagents that were able to completely remove plate-bound apoE were those that destroyed the protein either by digestion (enzymatic detergents) or by oxidation (sodium hypochlorite). Figure 8 ).

[0328] These results indicate that the apoE-polystyrene binding interaction is highly stable and resistant to strong detergents, acidic and alkaline washing. These properties can allow immunoassays, protein-microarray analysis, turbidimetric assays, etc. to be performed under conditions of high stringency and high specificity, without the need for additional purification, fractionation or concentration procedures.

[0329] Similar results have been observed for other apolipoproteins binding to polystyrene; therefore, similar conclusions translate to experiments with other different apolipoproteins.

[0330] Example 2. Detection of ApoE4 by turbidimetric assay

[0331] Materials and Equipment

[0332] - Serum samples: 11 APOE e3 / e3 and 6 APOE e4 / e4 samples.

[0333] -LEIT white microspheres coated with Superblock-NH2, Prot.20141222143CM, #143CM1

[0334] -Spectrophotometer, Model UV-3100PC (VWR), No.: PDX-1004

[0335] -Pipette Finnpipette F1 2-20μL, Model 4641060 (Thermoscientific, VWR), No.: PDX-1009

[0336] - Pipette Finnpipette F1 20-200 μL, model 4641080 (Thermoscientific, VWR), number: PDX-1010

[0337] -MILI-Q ReferenceA+, model Z00QSVC01

[0338] -Combi fridge Liebherr, model CN4056, code: PDM-1000

[0339] - Vortex mixer, model VX200 (Labnet, Era biotech), number: PDX-1013

[0340] - Optimal Conditions Heating Circulator, Model TC120-ST12 (VWR), Code No.: PDX-1028

[0341] Turbidimetric assays based on the latex enhanced immunoturbidimetric technique (LEIT) rely on the use of polystyrene beads. Therefore, the inventors used this technique to analyze apoE E4 and other apolipoproteins, taking advantage of their highly stable high affinity interaction with polystyrene as shown in ELISA plates.

[0342] LEIT is not as sensitive as ELISA, but interestingly, offers a great advantage for relatively high concentrations of analytes such as apolipoprotein. LEIT allows the test to be run in 5-20 minutes and can be run using a fully automated random access universal clinical chemistry analyzer. The higher cost of the raw materials used by LEIT compared to ELISA is more than compensated by the time saving, ease of use, and practicality of the technique in the clinical laboratory. LEIT is then considered a useful technique for apoE determination.

[0343] Initial analysis of APOE e3 / e3 and 6APOE e4 / e4 plasma samples using LEIT technology and monoclonal 4E4 demonstrated that this method was able to distinguish APOE e4 carriers from non-carriers ( Fig. 9 ).

[0344] Detection method (test 1)

[0345] Prepare working solution:

[0346] - 0.5 mL of a 0.176 mg / mL solution of monoclonal antibody anti-ApoE 4 in 10 mM pH 7.3 PBS.

[0347] - Dissolve 88 μL of monoclonal antibody in 412 antibody.

[0348] Experimental protocol:

[0349] 1. Transfer 14.6 μL of 7.3 white microspheres-NH2 to an Eppendorf tube and add 131.4 μL of PBS containing 0.01% Tween 20. Homogenize with a micropipette and incubate at 37°C for 2 minutes.

[0350] 2. Add 3.85 μL of apoE sample solution to the same Eppendorf tube, homogenize with a micropipette and incubate again at 37°C for 2 minutes.

[0351] 3. Add 50 μL of monoclonal antibody solution, homogenize using a micropipette and transfer the entire volume of the solution to a quartz cuvette.

[0352] 4. The minimum time for reading absorbance is 5 minutes and the maximum time is 10 minutes.

[0353] Detection method (test 2)

[0354] Prepare working solution:

[0355] - 0.5 mL of a 0.044 mg / mL solution of monoclonal antibody anti-ApoE 4 in 10 mM pH 7.3 PBS.

[0356] - Dissolve 22 μL of monoclonal antibody in 478 μL.

[0357] Experimental protocol:

[0358] 1. Transfer 14.6 μL of white microsphere-NH2 to an Eppendorf tube and add 131.4 μL of PBS (#144CM1) containing 0.01% Tween 20. Homogenize with a micropipette and incubate at 37°C for 2 minutes.

[0359] 2. Add 3.85 μL of ApoE sample solution to the same Eppendorf tube, homogenize with a micropipette and incubate again at 37°C for 2 minutes.

[0360] 3. Add 50 μL of monoclonal antibody solution, homogenize using a micropipette and transfer the entire volume of the solution to a quartz cuvette.

[0361] 4. The minimum time for reading absorbance is 5 minutes and the maximum time is 10 minutes.

[0362] result

[0363] Test 1

[0364] The results of tests 1 and 2 for LEIT analysis of apoE at different time points are shown in Tables 2 and 3, respectively.

[0365] Table 2. Analysis of 3 e4 / e4, 3 e3 / e4 ​​and 3 e3 / e3 samples by LEIT at different times in Test 1

[0366]

[0367] Table 3. Analysis of 6 e4 / e4 and 11 e3 / e3 samples by LEIT at different times in Test 2

[0368]

[0369]

[0370] Based on the data from Test 1, the authors of the present invention believe that the LEIT assay is able to distinguish between different apoE plasma samples, except for one 3 / 4 plasma sample (see Table 2). Therefore, optimal experimental conditions have been developed for the ApoE4 latex turbidimetric assay (see Table 3):

[0371] o 2,75μg anti-ApoE4 / test

[0372] o 3,85μl serum sample

[0373] o Total assay volume is 200 μl.

Claims

1. An in vitro method for determining the relative amount of apoE4 isoform in a sample relative to the total amount of said apolipoprotein in the sample, the method comprising: (i) contacting the sample with the polystyrene surface under conditions suitable for direct electrostatic and / or hydrophobic interaction of the apolipoprotein with the polystyrene surface, (ii) contacting the apolipoprotein-bound surface formed in step (i) with a first antibody specific for the apoE4 isoform and with a second antibody capable of binding to all isoforms of the apolipoprotein present in the sample, wherein the contacting is performed under conditions suitable for the formation of a first complex comprising the first antibody and the apoE4 isoform and a second complex comprising the second antibody and all isoforms of the apolipoprotein, (iii) detecting the first and second complexes formed in step (ii), and (iv) determining the relative amount of said apoE4 isoform relative to the total apolipoprotein content based on the levels of the first and second complexes obtained in step (iii), wherein the polystyrene surface is treated with a washing solution after the combining step (i) and before the step (ii), wherein the washing solution comprises one or more salts and at least one detergent, the salt is NaCl, the detergent is Tween 20, the washing solution further comprises HCl and formic acid, or NaOH, or 2-mercaptoethanol, and the washing solution does not contain protease, sodium hypochlorite or a combination thereof, wherein the pH of step (i) and step (ii) is pH 5 to pH 10, wherein the polystyrene surface is blocked with bovine serum albumin before contacting the sample; and The sample is selected from human plasma, and the dilution range of the plasma sample is 1:

200.

2. The method according to claim 1, characterized in that The surface used in step (i) is formed by a plurality of entities, the contacting with the first antibody and the second antibody in step (ii) is carried out in different containers, each container containing a portion of the plurality of entities forming the surface, and the detection in step (iii) is carried out in each container separately.

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