RU2356058C2 - Method of amyloidosis diagnostics in patients with alzheimerts disease and set of means for its realisation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to methods of laboratory diagnostics and concerns method of amyloidosis diagnostics in patients with Alzheimer's disease and set of means for carrying out diagnostics. Method lies in determining quantity of monomer β-amyloid, where in tested aliquot oligomer β-amyloid formations are first dissociated to monomers. If difference of quantity of β-amyloid monomers in tested and control aliquots is determined, conclusion about amyloidosis presence is made. Application of method and set of means ensures high sensibility and specificity of amyloidosis diagnostics in patients with Alzheimer's disease.

EFFECT: ensuring high sensibility and specificity of amyloidosis diagnostics in case of Alzheimer's disease.

11 cl, 1 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of medicine, biotechnology and molecular biology, and is intended for the diagnosis of amyloidosis and related neurodegenerative diseases (NDD).

The term amyloidosis refers to various diseases, a common characteristic of which is the tendency to aggregation of specific proteins (including amyloids) in the form of insoluble fibrils, fibers and plaques. Deposits of proteins in the form of amyloid fibers and plaques are a sign of diseases that are characterized by degenerative processes in the central nervous system, other organs and tissues [Stefani M., Dobson C.M. Mol. Med. 2003, 81, No. 11, 678-699].

All amyloid diseases are recognized, as a rule, in old age and are related to age-related health problems. All NDZ have a number of characteristic common features. Protein deposition is a characteristic property of more than 20 degenerative disorders that affect the central nervous system and a number of peripheral tissues. Well-known amyloid diseases are NDH such as Alzheimer's disease (AD), Parkinson's and Huntington's diseases, Down syndrome, Style-Richardson-Olzhevsky and Reye syndromes, Levi's calf disease, vascular dementia, dementia in the absence of distinctive histopathology, Peak disease, progressive supranuclear palsy, corticobasal degeneration, multiple systemic atrophy, myotropic lateral sclerosis, epilepsy and others. All of them are characterized by amyloidosis - the deposition of amyloid proteins in brain tissue. Since these pathologies include several forms of fatal systemic amyloidosis and at least one disorder associated with medical intervention (hemodialysis), amidoidosis is unusually important in the context of establishing an accurate diagnosis.

The most famous of neurodegenerative diseases is Alzheimer's disease, which, according to WHO in America in 2006, came out on the 4th place in the number of patients and deaths.

The primary cause of AD is generally recognized as the accumulation of soluble β-amyloid proteins (β-amyloid) in the brain as the body ages, which at a certain concentration include a cascade of events leading to neuron death [Maltsev A.V. and others. Almanac "Gerontology and Geriatrics", Moscow, 2003, Issue 2, p. 78-83; Maltsev A.V. and other Almanac "Gerontology and Geriatrics", Moscow, 2004, Issue 3, p.230-232]. In 90-93% of cases, AD occurs as a sporadic disease and about 10% as a hereditary one.

Thus, the task is to diagnose amyloidosis as early as possible.

A known method for the diagnosis of amyloidosis (patent US 6365414) by comparing the number of β-amyloid formations in the whole and a number of dilutions of the cerebrospinal fluid (CSF) of a sick person with the number of β-amyloid formations in the whole and a number of dilutions of the cerebrospinal fluid of a healthy person, which determine by reaction with zinc with the addition of a peptide comprising the 6th to 28th amino acids of the amyloid β-peptide (β-amyloid).

The closest in technical essence to the method we offer is the prototype method for diagnosing amyloidosis (US patent 6114133), which includes determining the amount of β-amyloid (1-42) in a patient’s cerebrospinal fluid sample and comparing it with a predetermined value, when this, the amount of β-amyloid is measured by capturing soluble β-amyloid from the test sample using one antibody, followed by determination of capture using another antibody with an enzymatic label, i.e. enzyme immunosorbent assay, for example ELISA.

The disadvantage of the above methods is the need to use the cerebrospinal fluid of the patient, which is desirable to take only in the presence of serious suspicions of a neurodegenerative disease or for health reasons. At the same time, when trying to use other fluids, such as blood, blood serum, or lymph, the known methods are not sensitive enough.

In addition, [Clark CM. et.al., Arch Neurol, 2003, 60 (12), p. 1696-1702; Burkhard PR. et.al., Clin Chem Lab Med., 2004, 42 (4), p.396-407], that the amount of β-amyloid in the CSF in the control groups of healthy patients varies widely, which reduces the reliability of the above methods, t. to. these methods require comparison with a healthy body.

The technical problem to be solved by the invention is aimed at increasing the sensitivity and diagnostic specificity of the method, which will expand the arsenal of biological fluids that can be used to diagnose amyloidosis.

The problem is solved in that in the known method for the diagnosis of amyloidosis, which includes determining the amount of monomeric β-amyloid in a sample of a liquid biological medium, according to the invention, the previously indicated sample is divided into two aliquots - the experimental and control, in the experimental aliquot, the procedure for dissociation of oligomeric formations of the specified β- amyloid to monomers, and in the presence of a difference in the number of monomers of the indicated β-amyloid in the experimental and control aliquots, it is concluded that amyloidosis is present.

As β-amyloid, β-amyloid Aβ is predominantly determined (1-42).

The determination of the amount of β-amyloid monomers is carried out, for example, by enzyme immunosorbent analysis, for example ELISA, or by high performance liquid chromatography (HPLC), or by electrophoresis.

As a liquid biological medium, the liquid phase of the blood, or lymph, or cerebrospinal fluid is taken and acidified to preserve the oligomers in them.

It is advisable to concentrate a sample of biological fluid before dividing it into aliquots, or the aliquots before performing the procedure of dissociation of oligomeric formations of said β-amyloid to monomers, for example, with methanol or Sephadex, for example G-10, or G-15, or using dialysis, or chromatographic column.

The procedure for dissociation of oligomeric formations of β-amyloid to monomers is carried out by exposure to a chemical agent that disrupts the hydrogen and hydrophobic interactions between the peptide monomers in the oligomers.

As a chemical agent that ensures the destruction of hydrogen bonds between monomers and hydrophobic interactions between monomers, urea is used, for example, which is added to the experimental aliquot to a concentration of, for example, 2.0-5.0 M, followed by incubation at a temperature of, for example, 15 -45 ° C, for 5-45 minutes, while an equal volume of a neutral medium, for example, saline, is added to the control aliquot and incubated under the same conditions as the experimental one.

As the specified chemical means, you can also use reagents that alkalize the medium in the experimental aliquot to a pH of 7.6-9.2, followed by incubation of this aliquot at a temperature, for example, 15-40 ° C, for from 0.5 hour to 25 hours, while in the control aliquot add an equal volume of neutral medium, such as saline, and incubate it under the same conditions as the experimental one.

As a reagent, alkalizing medium, take an alkali or basic buffer solution.

As a liquid biological medium, blood serum is taken, the sample of which is concentrated, divided into two equal aliquots - the experimental and control, in the experimental aliquot, the oligomeric formations of β-amyloid are dissociated to monomers by the addition of urea to a concentration of 2.0-5.0 M, followed by incubation at a temperature of 15-45 ° C for 5-45 minutes, while an equal volume of saline is added to the control aliquot and incubated under the same conditions as the experimental one.

The problem is also solved by the fact that a complex of tools for diagnosing amyloidosis is proposed, including a means for quantitative determination of β-amyloid, which according to the invention further comprises a means for ensuring the dissociation of oligomeric formations of β-amyloid to monomers.

Moreover, the means for ensuring the dissociation of oligomeric formations of β-amyloid to monomers is a reagent that disrupts hydrogen and hydrophobic interactions between the peptide monomers in oligomers, for example urea or alkali, or the main buffer solution, and the means for quantitative determination of β-amyloid is a set for electrophoresis, or a kit for high performance liquid chromatography, or a kit for enzyme-linked immunosorbent assay.

The complex may further comprise an agent for concentrating the body fluid, for example methanol, or Sephadex, for example G-10, or G-15, or a dialysis device.

The invention is based on the following.

It is known [Maltsev A.V. and others. Almanac "Gerontology and Geriatrics", Moscow, 2003, Issue 2, p. 78-83; Maltsev A.V. et al. “Gerontology and Geriatrics” Almanac, Moscow, 2004, Issue 3, pp. 96-101], that in a normally functioning neuron as a result of proteolytic processing of the precursor of amyloid protein (APP) with α, β and γ type secretases a number of non-pathogenic peptides are formed, including β-amyloid. The main share of APP is cleaved by β-secretase, which is a non-specific enzyme for it and can bind to other substrates in the cell. The rest of the APP molecules are exposed to β-secretase with the formation of β-amyloid in low concentrations at which they acquire a water-soluble conformation. Vibrational changes in α- and β-processing preserve within a certain framework the “normal” concentration of APP, and the functioning of the neuron is not disturbed.

In a normal neuron under the action of γ-secretase, β-amyloid nanoparticles are formed, which, as a result of folding, are reversibly transformed into the intermediate structure, then into fully unfolded protein molecules, from which nanoparticles of normally functioning β-amyloids are formed. Probably, such a reversible conformational rearrangement of nanoparticles is necessary for the transition of the formed β-amyloid nanoparticles from the postreaction conformation to water-soluble in order to perform its functions and exit the neuron.

However, with a chronic deficiency of α-secretase, there is an increase in the proportion of β-processing and an increase in the concentration of β-amyloids, which stimulate the synthesis of APP.

Currently, it is generally accepted that the accumulation of β-amyloid products of APP processing is a key pathogenetic factor [Maltsev A.V. and other Neurochemistry, 2005, 22, No. 2, p.97-101]. At certain concentrations, amyloid peptides (β-amyloids) have the ability to spontaneously form oligomers, fibrils and aggregate into insoluble conglomerates with high neurotoxicity.

As a result of the lack of control in the neuron of increasing synthesis of APP and an increase in the concentration of β-amyloid to a critical level from intermediate nanoparticles (intermediate conformation of partially braided post-reaction β-amyloids), molecular β-sheets are first formed, and then oligomeric β-structures are soluble in aqueous solutions [ Maltsev A.V. and other Biophysics, 2005, T.50, No. 3, p. 470-474]. Subsequently, prefibrillar oligomers as a result of conformational rearrangements form fibrils of various complexity. The neurotoxicity of the early oligomeric structures of β-amyloid is due to their ability to interact with intracellular membranes and with divalent metal ions with the formation of active amyloid radicals.

Thus, in neurons with impaired functions, the synthesis of APP is enhanced and its processing is switched from a non-amyloidogenic to an amyloidogenic path — by activating the synthesis of APP and β-secretase with the formation of Aβ (1-40) and Aβ (1-42) nanoparticles. The subsequent stages are characterized by a change in a number of parameters of homeostasis in the neuron, leading to oxidative stress, and favorable conditions are created for conformational molecular rearrangements (modifications) of pathogenic neuropeptides. At the key stage of amyloidosis, oligomeric structures are formed from modified amyloid nanoparticles, and at the final stage, as a result of intermolecular interactions of the oligomers, fibrils of various complexity are created, which accumulate and, ultimately, remain in place of dead neurons.

Experimentally, we have selected the conditions described in this application, allowing to carry out in an experimental aliquot of a sample of biological fluid the dissociation of oligomeric formations (structures) of β-amyloid to monomers. Moreover, the control is the same sample of biological fluid, namely its second, control aliquot.

In this case, when establishing approximately equal amounts of β-amyloid in the experimental and control aliquots, a conclusion is drawn about the absence of amyloidosis. In the case when a greater amount of β-amyloid is determined in the experimental aliquot than in the control, a conclusion is made about the presence of amyloidosis and its severity.

The proposed method allows the use of blood, lymph and blood serum of patients for analysis, while the analysis of the obtained results does not require comparison of results from a healthy group of patients (comparison group).

The proposed method is simple and economical when using the proposed set of tools for its implementation, which, in turn, can be performed using domestic reagents and equipment.

The proposed invention is illustrated by a drawing, which shows the results of a typical experiment for the determination of β-amyloid Aβ (1-42) with varying degrees of dilution of the experimental aliquot obtained from the blood of Wistar rats in which Alzheimer's disease was simulated.

Curve 1 shows the amounts of Aβ (1-42) in diluted experimental aliquots prior to the dissociation of oligomers of β-amyloid.

Curve 2 shows the quantities of Aβ (1-42) in the corresponding diluted samples of the experimental aliquot after the dissociation of oligomers of β-amyloid to monomers (incubation at a temperature of 37 ° C and pH 8.0).

Curve 3 (dashed line) shows the amounts of Aβ (1-42) in diluted samples of the experimental aliquot after incubating it at 0 ° C and pH 8.0.

The invention is illustrated by the following examples:

Example 1

When selecting optimal conditions for the implementation of the proposed method on the example of various β-amyloids [Aβ (1-42), Aβ (1-40), Aβ (25-35)], it was found that the most informative is the definition of β-amyloid Aβ (1- 42).

Using the proposed method, three series of studies were carried out with three replicates in each.

To perform the proposed method used the proposed set of tools for the diagnosis of amyloidosis, containing a means for ensuring the dissociation of oligomeric formations of β-amyloid to monomers in the form of a citrate buffer [Kalinin F.L. et al. Handbook of biochemistry, “Naukova Dumka”, Kiev, 1971] and 0.1% NaOH alkali solution, a tool for the quantitative determination of β-amyloid in the form of an enzyme-linked immunosorbent assay (ELISA) [Crowther J.R. ELISA Theory and Practice. Humana Press, Totowa, 1995, 223 p.] And a means for the concentration of body fluid in the form of Sephadex G-10.

The studies used Wistar rats (males) 3-4 months old. The experimental and control groups consisted of 11 animals each. Experimental rats were injected into the ventricles of the brain with 20 nmol of the commercial preparation β-amyloid Aβ (1-42) to simulate Alzheimer's disease [Maltsev A.V. and other Almanac "Gerontology and Geriatrics", Moscow, 2006, Issue 5, p.70-74].

After 15 days, animals of the experimental and control groups were taken 9 ml of blood in test tubes with 0.5 ml of the known citrate buffer pH 6.0 [Kalinin F.L. et al. Handbook of biochemistry, "Naukova Dumka", Kiev, 1971] for the preservation of amyloid oligomers and in the cold received serum. Blood was taken from experimental animals in a standard way, subject to modern requirements [Balakhovsky SD, Balakhovsky I.S. Methods of a chemical analysis of blood, Moscow, Medgiz, 1953. P.143].

The obtained serum samples were concentrated by Sephadex G-10 10 times and divided into 2 aliquots - experimental and control. In the experimental aliquot, alkalization of the medium was carried out using a pH 6.0 citrate buffer and 0.1% NaOH alkali solution, adjusting the pH to 8.0.

To dissociate amyloid oligomers into individual monomeric molecules of β-amyloid Aβ (1-42), experimental aliquots were incubated at 37 ° C for 6 hours in a thermostat. An appropriate amount of saline was added to the control aliquots and incubated under the same conditions as the experimental ones. After incubation, the experimental and control aliquots were placed for 2 minutes in dry ice, and then stored at 0 ° C.

To determine the amount of monomeric β-amyloid Aβ (1-42) [Maltsev A.V. and others. Almanac "Gerontology and Geriatrics." Moscow. 2004. Issue 3. S.230-232] in the experimental and control aliquots used the known method for conducting enzyme immunoassay ELISA [Crowther JR ELISA. Theory and Practice. Humana Press, Totowa, 1995, 223 p.], Which consists in the fact that the analyzed samples of experimental and control aliquots were diluted in phosphate buffer solution (PBS) containing 138 mm NaCl, 2.7 mm KCl and 10 mm NaH ₂ PO ₄ , pH 7.4, to a final protein concentration of 0.32 mg / ml. 50 μl of a sample of the corresponding aliquot was added to each well of a microtiter plate, diluted several times with PBS and incubated at 37 ° C for 2 hours. After this, the plate was washed in a washing solution - PBS containing 0.05% Tween 20, and then 0.2 ml of blocking solution - PBS containing 1% calf embryo blood serum was coated at 37 ° C. After 60 minutes, the blocking solution was removed and, after washing, 50 μl of anti-β-amyloid Aβ antibody (1-42) (1: 1000) was added to each plate and incubated for 1 hour at 37 ° C. After washing the plate, 50 μl of peroxidase conjugated anti-IgG antibody (1: 250) was added to the wells and incubated for 1 hour at 37 ° C. 100 μl of a color-developing reagent (4 mg of o-phenylenediamine (o = PDA) diluted in 10 ml of pH 5.0 citrate buffer was added to which 38 μl of 1.3 M H ₂ O _{2 was} added). After 10 min, the reaction was stopped by adding 50 μl of 1 M H ₂ SO ₄ and light absorption was measured at 492 nm. At the same time, a positive control was set with a known concentration of amyloid Aβ (1-42) of known concentration, as well as a negative control that did not contain amyloid.

The drawing shows the results of a typical experiment of Aβ dissociation (1-42) with different dilutions of the experimental aliquot before incubation (curve 1), after incubation at 37 ° C and pH 8.0 (curve 2) and after incubation at 0 ° C and a pH of 8.0 (curve 3).

The results of studies conducted on Wistar rats are also presented in table 1. Statistical analysis was performed using a computer program.

Table 1
Investigation of the determination of β-amyloid Aβ monomers (1-42) in animal blood samples Experimental animals (rats) Animal sex and age (months) Number of animals The content of Aβ (1-42) in the blood after dissociation of oligomers The content of Aβ (1-42) in the blood without dissociation Healthy control Males 3-4 eleven 18 ± 7 13 ± 9 Operated (Alzheimer's disease) Males 3-4 8 154 ± 96 Not found

The experimental data shown in Table 1 and in the drawing indicate the dissociation of Aβ (1-42) oligomers to monomers in blood samples. This confirms that the proposed method has high sensitivity and specificity (determines only monomers of β-amyloids Aβ (1-42).

Example 2

We found that the determination of β-amyloid Aβ (1-42) in blood samples can also be carried out using high performance liquid chromatography (HPLC). This method turned out to be simpler in execution and less expensive than the ELISA method, and also showed good results when using the domestic Milichrom A-02 device.

When analyzing cognitive impairment in the initial stage of AD and other forms of damage to brain matter (cerebrovascular disease, NDD), difficulties arise in making a correct diagnosis. Differential diagnosis of cognitive impairment of different genesis and varying severity is based on neuropsychiatric symptoms, consisting of various tests and criteria. Along with this, additional neuroimaging methods are used (magnetic resonance imaging, computed tomography, positron emission tomography) and electrophysiological (electroencephalography) methods.

To perform the proposed method according to example 2, we used the proposed complex of tools for diagnosing amyloidosis, containing a means for ensuring the dissociation of oligomeric formations of β-amyloid to monomers in the form of urea, a means for quantitative determination of β-amyloid in the form of a kit for high performance liquid chromatography (HPLC) and means for concentration of biological fluid in the form of Sephadex G-15.

Patients with an established diagnosis of AD took 30 ml of blood in a standard way, subject to modern requirements [Balakhovsky SD, Balakhovsky I.S. Blood chemistry methods. Moscow, Medgiz, 1953, p.143], from which serum was obtained in the usual way. In order to stabilize amyloid oligomers, the obtained serum samples were adjusted to pH 6.8 with citrate buffer pH 6.0, incubated for 2 hours at 37 ° C, and then concentrated by 10 times with Sephadex G-15.

After concentration, the liquid phase was collected and divided into 2 aliquots - experimental and control. To dissociate amyloid oligomers into individual monomer molecules of β-amyloid Aβ (1-42), urea was added to the experimental aliquot to a total concentration of 2 M and incubated at 37 ° C for 20 minutes in an incubator.

An appropriate amount of saline was added to control aliquots and incubated under the same conditions without urea. After incubation, the experimental and control aliquots were placed for 2 minutes in dry ice, and then stored at 0 ° C. To determine the amount of the monomeric preparation of β-amyloid Aβ (1-42), experimental and control aliquots were analyzed on a Milichrom A-02 high performance liquid chromatograph.

The chromatograph was preliminarily calibrated with samples of β-amyloid Aβ (1-42) of known concentration (positive control), and a negative control (sample without amyloid) was set. The analysis of chromatograms showed that β-amyloid Aβ oligomers (1-42) efficiently dissociate in urea into monomeric β-amyloid Aβ molecules (1-42), which enter the calibration zone of the commercial standard β-amyloid Aβ (1-42). When chromatography of a control aliquot (blood sample not treated with urea) in the standard zone (positive control), peak β-amyloid Aβ (1-42) is absent.

Table 2 presents comparative data of studies conducted on patients at the stage intermediate between the weakening of cognitive functions and dementia of "Alzheimer's type", using the diagnosis of amyloidosis by the proposed method and using other modern methods.

table 2
The number of patients with Alzheimer's disease in different age groups of risk identified by the proposed method for the diagnosis of amyloidosis and other modern methods of instrumental diagnostics Age
Number of patients Research methods Magnetic resonance
snapping
tomography CT scan Electroencephalo
grafia Diagnosis of amyloidosis by the proposed method 45-65 years old (6 people) 6 5 - 6 65-74 years (4 people) four 3 one four After 75 years (2 people) 2 2 one 2

Thus, the experimental results obtained by HPLC to determine the number of β-amyloid Aβ monomers (1-42) in patients' blood samples should be used to diagnose amyloidosis in neurons in patients with age-related cognitive changes and mild dementia, which becomes AD.

The boundaries between normal aging and pathological changes in cognitive functions in NDD are not easy to determine. Early diagnosis of amyloidosis is a primary concern. Its widespread use will help change the tactics of effective prevention and treatment of these diseases.

The present invention can be used in the development of diagnostics of some NDD for practical healthcare, as well as to study the mechanism of occurrence, for example, neurodegenerative diseases. Our COMPLEX, which allows one to determine monomeric and indirectly oligomeric amyloids in the blood, can be effectively used in the pharmaceutical industry for the initial screening of compounds that inhibit the development of amyloidosis of neurons, as well as for the search for substances that prevent the onset and development of amyloidosis in neurons, and the identification of cellular factors that control the process of amyloidogenesis, at the body level, when there is a blood-brain barrier.

Claims (11)

1. A method for diagnosing amyloidosis in Alzheimer's disease, including determining the amount of monomeric β-amyloid in a sample of a liquid biological medium, characterized in that the previously indicated sample is divided into two aliquots - the experimental and control, in the experimental aliquot, the oligomeric formations of the specified β-amyloid are dissociated to monomers and in the presence of a difference in the number of monomers of the specified β-amyloid in the experimental and control aliquots conclude the presence of amyloidosis, while taking as a biological liquid medium t of blood serum, and Aβ (1-42) and / or Aβ (1-40) are determined as β-amyloid. 2. The method according to claim 1, characterized in that the determination of the number of monomers β-amyloid Aβ (1-42) and / or Aβ (1-40) is carried out, for example, by enzyme immunosorbent analysis, or by high performance liquid chromatography, or using electrophoresis. 3. The method according to claim 1, characterized in that said sample is pre-concentrated, for example, with methanol, or Sephadex, for example G-10 or G-15, either by dialysis or on a chromatographic column. 4. The method according to claim 1, characterized in that the dissociation of oligomeric formations of β-amyloid Aβ (1-42) and / or Aβ (1-40) to monomers is carried out using a chemical agent that disrupts the hydrogen and hydrophobic interactions between the monomers of the peptides in oligomers. 5. The method according to claim 4, characterized in that as a chemical agent, for example, urea is used, which is added to the experimental aliquot to a concentration of, for example, 2.0-5.0 M, followed by incubation of this aliquot at a temperature, for example , 15-45 ° С, for 5-45 min, while an equal volume of a neutral medium, for example, saline, is added to the control aliquot, and it is incubated under the same conditions as the experimental one. 6. The method according to claim 4, characterized in that an alkaline solution or basic buffer is used as the chemical agent, which is added to the test aliquot until a pH of 7.6 to 9.2 is reached, followed by incubation at a temperature of, for example, 15-40 ° C, for 0.5-25 hours, while in the control aliquot add an equal volume of neutral medium, for example saline, and incubate it under the same conditions as the experimental one. 7. The method according to claim 1, characterized in that the blood sample is concentrated, oligomeric formations of β-amyloid Aβ (1-42) and / or Aβ (1-40) are dissociated to monomers in the experimental aliquot by adding urea to a concentration of 2.0 -5.0 M, followed by incubation at a temperature of 15-45 ° C for 5-45 min, while an equal volume of saline is added to the control aliquot and incubated under the same conditions as the experimental one. 8. A set of tools for diagnosing amyloidosis in Alzheimer's disease according to claim 1, comprising a means for quantifying β-amyloid, characterized in that it further comprises a means for providing dissociation of oligomeric formations of β-amyloid Aβ (1-42) and / or Aβ (1-40) to monomers. 9. The kit of claim 8, characterized in that the means for ensuring the dissociation of oligomeric formations of β-amyloid Aβ (1-42) and / or Aβ (1-40) to monomers is urea, or an alkaline solution, or a main buffer. 10. The kit of claim 8, characterized in that it further comprises a means for concentrating the biological fluid, for example methanol, or Sephadex, for example G-10 or G-15, or a dialysis device, or a chromatographic column. 11. The kit of claim 8, characterized in that the means for the quantitative determination of β-amyloid Aβ (1-42) and / or Aβ (1-40) is a kit for electrophoresis, or a kit for high performance liquid chromatography, or enzyme immunoassay kit.

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