RU2680848C1 - Method for assessing character of autoimmune reaction of human body to multiple modified low-density lipoproteins in lytic test - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, in particular to clinical immunology, and can be used to assess the lytic activity of multiply modified low-density lipoproteins. Method involves preparing a precipitate from human serum by incubating the supernatant obtained after preliminary precipitation of multiple modified low-density lipoproteins (mmLDL) with buffer containing 20 % polyvinylpyrrolidone with a molecular weight of 35,000 (PVP-35000) for 30 minutes at 4 °C. Then the formed aggregates of immune complexes containing mmLDL (IC-mmLDL) are precipitated by centrifugation at 4 °C for 10 min at 31,000 g, decanted, the resulting precipitate is dissolved in buffer without PVP-35000 and the cholesterol content in immune complexes is determined. Standardized erythrocytes of ram are added to samples of mmLDL and IC-mmLDL, incubated for 24 hours at 37 °C, the coefficient of specific lytic activity (C) of mmLDL and IC-mmLDL is calculated as the ratio of the degree of lysis to cholesterol in these complexes, the values of Cof mmLDL and IC-mmLDL are compared in the examined person. In the case of a decrease in this coefficient, IC-mmLDL compared with CmmLDL is estimated as neutralization of lytic activity. With the opposite effect, with a tendency to increase of this coefficient of IC-mmLDL, is estimated as an increase in lytic activity. Equal values of C(mmLDL) and C(IC-mmLDL) indicate that autoantibodies, binding to mmLDL in immune complexes, do not affect lytic activity.EFFECT: use of this invention allows to evaluate the nature of the autoimmune reaction of the body to mmLDL for early prediction of the course and monitoring the effectiveness of pathogenetic therapy of atherosclerosis.1 cl, 1 ex, 7 tbl, 1 dwg

Description

The invention relates to clinical immunology and can be used to assess the lytic activity of immune complexes containing multi-modified low-density lipoproteins (IR-mmLDL), for predicting the course of diseases associated with elevated mmLDL (atherosclerosis, gestosis, diabetes mellitus, etc. .d.), as well as for the development of drugs that reduce the pathogenicity of mmLNP and immune complexes containing mmLNP.

Atherosclerosis is a complex, periodically exacerbating chronic inflammatory process of vascular damage that develops against the background of cholesterol metabolism disorders, which leads to structural modification of the vascular endothelium and proliferation of connective tissue, causing the formation of atherosclerotic plaque. Atherosclerosis and its complications - coronary heart disease, stroke, damage to the vessels of the lower extremities continue to be the most common cause of disability and mortality in the economically developed countries of Europe, the USA and especially Russia [European Guidelines on Cardiovascular Disease // Eur. Heart J. - 2003. - V. 24 No. 17. - P. 1601-1610; NCEP. Adult Treatment Panel III Guidelines // Circulation. - 2004. - V. 110. - P. 227-239; GFCF Russian recommendations. Diagnosis and correction of lipid metabolism disorders in order to prevent and treat atherosclerosis. M. - 2004. - 36 p.].

The disease begins with the accumulation of lipoproteins, primarily low-density lipoproteins (LDL), in the extracellular matrix of blood vessels. These LDL particles aggregate and undergo oxidation modification. Oxidation-modified LDL (mLNP) are toxic and damage the blood vessels, causing inflammation and fibrosis [Steinberg D. The LDL modification hypothesis of atherogenesis: an update // J. Lipid Res. - 2009. - V. 50. - P. S376-S381]. In parallel with the oxidation of LDL, they acquire immunogenic properties and autoantibodies to mLNP are synthesized in the body and immune complexes containing mLNP are formed. Autoantibodies formed against neo-epitopes of mLNP are detected in the blood and the titer of these antibodies correlates with the severity of atherosclerosis and is considered as a marker of the disease [Itabe N., Mori M., Higashi Y., and Takano T. Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines // J Biochem. - 2003. - V. 134. - P. 459-465; Tsimikas S. Oxidative biomarkers in the diagnosis and prognosis of cardiovascular disease // Am J Cardiol. - 2006. - V. 98. - P. 9P-17P].

As a prototype, a method for determining the lytic activity of multiple modified low density lipoproteins (LDL) was used [RF Patent No. 2577719 of 03/20/2016]. The essence of the method lies in the fact that mmLDP is first isolated from human blood serum by treatment with a solution of 20% polyvinylpyrrolidone with a molecular weight of 35,000 (PVP-35000), incubated, the resulting mmLNP aggregates are precipitated by centrifugation, decanted, the mmLNP precipitate is dissolved in buffer without PVP-35000, washed standardized sheep erythrocytes are added, incubated for 48 hours at room temperature, the optical density of the samples is measured photometrically at a wavelength of 620 nm, the degree of l is determined from the calibration graph sis and lysis of more than 10% ascertain mmLNP increased lytic activity.

The technical result of the proposed method is to expand the arsenal of laboratory tests for assessing the lytic activity of IR-mmLNP by comparing the values of the coefficients of specific lytic activity of mmLNP and immune complexes containing mmLNP (IR-mmLNP) for early prognosis and monitoring the effectiveness of pathogenetic therapy of atherosclerosis in clinical diagnostic conditions laboratories.

This result is achieved by the fact that the specific precipitation of IR-mmLDL from human blood serum is carried out by incubating the supernatant obtained after preliminary precipitation of the multi-modified low-density lipoproteins (mmLNP) with a buffer containing 20% polyvinylpyrrolidone (PVP) with a molecular weight of 35,000 (PVP-35000 ), for 30 minutes at 4 ° С, then the aggregates formed (IR-mmLNP) are precipitated by centrifugation at 4 ° С for 10 min at 3100g, carefully decanted, the resulting precipitate is dissolved in buffer without PVP-3500 0 and determine the cholesterol content using commercial enzymatic kits. For samples mmLNP and IR mmLNP added standardized sheep red blood cells were incubated for 24 hours at 37 ° C, calculated coefficient specific lytic activity (K _ULA) mmLNP and IR mmLNP as the ratio of the degree of lysis to cholesterol in these complexes is corresponded value K _ULA mmLNP and IR-mmLNP in the examined person, in the case of a decrease in this coefficient of IR-mmLNP compared to K _ULA mmLNP - is assessed as a neutralization of lytic activity, with the opposite effect, with a tendency to increase this coefficient IR-mmLNP - sc enivaetsya as an increase in the lytic activity of equal magnitude K _ULA (mmLNP) and K _ULA (IR mmLNP) suggest that the autoantibody binding to mmLNP in immune complexes do not influence the lytic activity.

The method is as follows: carry out blood sampling on an empty stomach from the human ulnar vein, prepare blood serum. The preparation of mmLNP and IR-mmLNP is described in [Patent No. 2632118 of 02.10.2017]. Briefly: 84 μl of a buffer containing 20% PVP-35000 (final concentration of PVP-35000 in the sample is 9.1%) is added to 100 μl of blood serum, thoroughly mixed and incubated at room temperature for 10 minutes. Aggregated mmLDPs are precipitated by centrifugation, the supernatant is carefully selected and incubated for 30 min at 4 ° C. The resulting aggregates of immune complexes containing IR-mNLP were precipitated by centrifugation at 4 ° C at 3100g for 10 min, thoroughly decanted, and the precipitate was dissolved in buffer without PVP-35000. 50 μl of standardized sheep erythrocytes (1.5 × 10 ⁸ cells / ml) are added to 10 μl of mm LDL and IR-mm LDL preparations prepared from the blood serum of individuals, the total volume is adjusted to 100 μl with VBS ²⁺ buffer, incubated for 24 hours at 37 ° C, the optical density is measured on a photometer at a wavelength of 620 nm and the degree of lysis is determined from the calibration graph. Calculate the coefficient of specific lytic activity (K _ULA) mmLNP and IR mmLNP as the ratio of the degree of lysis to cholesterol in these complexes is corresponded value K _ULA mmLNP and IR mmLNP in human subject, in case of decrease of this ratio IR mmLNP compared with K _ULA mmLNP - assessed as neutralization lytic activity, with the opposite effect, with a tendency to an increase of this ratio IR mmLNP - measured as an increase in the lytic activity of equal magnitude K _ULA (mmLNP) and K _ULA (IR mmLNP) testimony so that autoantibody binding to mmLNP in immune complexes do not influence the lytic activity.

To isolate immune complexes containing mmLNP use:

- Buffer-1 for the precipitation of multi-modified low-density lipoproteins, which contains 20% PVP-35000 in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl;

- Buffer-2 for dissolving the precipitate of immune complexes, which is 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl.

The invention is illustrated by figure 1 which shows a calibration graph for determining the degree of lysis of sheep erythrocytes by optical density at a wavelength of 620 nm.

Determination of the optimal concentration of sheep erythrocytes for the test for determining the lytic activity of IR-mmLDL. Sheep (E) erythrocytes are washed 3 times in Veronal Salt Buffer (VBS ²⁺ ) by centrifugation for 10 minutes at 2500 rpm. A graph is built of the dependence of the optical density of the erythrocyte suspension (at a wavelength of 620 nm) on the concentration of red blood cells. For this, a 1% suspension of red blood cells is progressively diluted in a flat-bottomed 96-well immunological plate in a volume of 100 μl of 0.15 M NaCl. Thoroughly mixed and measured on a photometer for enzyme immunoassay at a wavelength of 620 nm. The results are presented in table 1.

As can be seen from the data presented in table 1, there is a linear dependence of the optical density of the erythrocyte suspension on the concentration in solution to A ₆₂₀ of 0.56 U. Over 0.56 units at A _620, this dependence is non-linear. Therefore, for the test for determining the lytic activity of IR-mmLDL, we chose the concentration of suspension E, which in a volume of 100 μl in a well of a 96-well plate gives an optical density of 0.40-0.56 U (1.5 × 10 ⁸ cells / ml) . To determine the degree of erythrocyte lysis, we proposed a calibration graph in which the degree of erythrocyte lysis is assessed by the decrease in optical density at a wavelength of 620 nm. The optical density of the control of red blood cells for spontaneous lysis (50 μl E + 50 μl of VBS ²⁺ ) is taken as 0% lysis, and the optical density of the control for complete lysis (50 μl E + 50 μl of H ₂ O) is taken as 100% lysis. The degree of erythrocyte lysis is evaluated after 24-hour incubation at 37 ° C according to the calibration graph shown in FIG. one.

As can be seen from FIG. 1, the calibration graph allows you to determine the degree of red blood cell lysis in percent by the optical density of the sample without the centrifugation and hemoglobin measurements in the supernatant and the subsequent calculation of the degree of red blood cell lysis, which greatly simplifies the recording of the results of analysis of the lytic activity of IR-mmLNP in routine studies.

Isolation of immune complexes containing multi-modified low density lipoproteins. IR-mmLNP was isolated as described in [Patent No. 2632118 of 02.10.2017, bull. No. 28]. At the first stage, mNLP is isolated from individual blood serum by adding 84 μl of buffer containing 20% PVP to 100 μl of serum (final concentration of PVP-35000 is 9.1%) and incubation for 10 min at room temperature. Aggregated under these conditions, mNLP is precipitated by centrifugation at 3100g for 10 min at 23 ° C, the supernatant is carefully selected, the mmLNP precipitate is dissolved in 100 μl of Buffer-2. Next, the supernatant prepared as described above is incubated at 4 ° C for 30 minutes. Aggregated immune complexes are precipitated by centrifugation at 3100g for 10 min at 4 ° C. the recipient IR-MLNL is carefully decanted and the resulting precipitate resuspended in the original volume, in 100 μl of buffer-2. After dissolution of the precipitates in the obtained fractions of mNLP and IR-mmLNL, the cholesterol content is determined using commercial enzyme kits. The results are presented in table 2.

As can be seen from the data presented in table 2, the cholesterol content in LDL averaged 53.4 mg / dl (fluctuations from 13.5 mg / dl to 116.4 mg / dl). The cholesterol content in IR-MLNL averaged 22.6 mg / dl (fluctuations from 1.8 mg / dl to 68.8 mg / dl). Moreover, in 15 samples of IR-mmLDL, the cholesterol level was less than 10 mg / dl.

Thus, in all samples, the cholesterol content in mmLDL was increased, while the cholesterol in IR-mmLDL in 29% of the tested samples was less than 10 mg / dl.

Example 1. Determination of the lytic activity of mm LDL and IR-mm LDL prepared from the blood serum of pregnant women.

The lytic activity of IR-mmLDL was determined similarly to the determination of the lytic activity of mmLDL as described in RF patent No. 2577719 dated 03/20/2016. The lytic test was performed in 96-well flat-bottomed immunological plates. First, 10 μl of mm LDL or IR-mm LDL is added to the wells, then 40 μl of VBS ²⁺ buffer and 50 μl of a suspension of standardized (1.5 × 10 ⁸ cells / ml) sheep erythrocytes are added. In parallel, the following controls are set: for complete lysis (50 μl of a sheep erythrocyte suspension + 50 μl of H ₂ O); for spontaneous lysis (50 μl of sheep erythrocytes + 50 μl of VBS ²⁺ buffer). The tablet is sealed with a film and left for 24 hours at 37 ° C. After incubation, the plate is thoroughly mixed and the absorbance of the samples is measured on a photometer for enzyme immunoassay at a wavelength of 620 nm, and the degree of lysis is determined from the calibration graph. The results are presented in table 3.

As can be seen from the data presented in table 3, according to the political activity, the studied samples of mm LDL are divided into three groups:

Group 1 with low lytic activity (% lysis less than 11%), only 5 samples;

Group 2 - with an average lytic activity (% lysis from 12% to 50%), a total of 20 samples;

Group 3 - with high lytic activity (% lysis more than 51%), a total of 26 samples.

In the same way, according to the amount of lytic activity, the IR-MLNP samples are also divided into three groups:

Group 1 with low lytic activity (less than 11%). A total of 15 samples;

Group 2 - with an average lytic activity (%> lysis from 12% to 50%). A total of 30 samples;

Group 3 - with high lytic activity (lysis of more than 51%). Only 6 samples.

Thus, this conditional distribution into groups would be correct if the amount of cholesterol in mmLDL and IR-mmLDL was equal. On the other hand, the test for determining the lytic activity of these physiological macromolecular complexes is functional and, therefore, the standardization of lytic activity is required taking into account the cholesterol content in these complexes through a calculated coefficient, specific lytic activity coefficient (K _ALA ).

Calculation of the coefficient of specific lytic activity (K _ULA) mmLNP and IR mmLNP, for the integrated assessment of lytic activity mmLNP and IR mmLNP proposed coefficient specific lytic activity, which is the ratio of the degree of lysis of erythrocytes to cholesterol in the individual samples mmLNP and IR mmLNP prepared from the blood serum of the same person. The obtained values of K _{ULA are} presented in table 4.

As can be seen from the data presented in table 4, the magnitude of the coefficients of specific lytic activity (K _ULA ) mmLNP samples are conventionally divided into three groups:

Group 1 - 24 samples with low K _ULA (less than 1.0 PIECES);

Group 2 - 23 samples with an average value of K _ULA (from 1.0 UNITS to 2.0 UNITS);

Group 3 - 4 samples with high K _ULA (more than 2 units).

Similarly, IR-mmLNP samples in terms of K _{ULA are} also divided into three groups:

Group 1 - 19 samples with low K _ULA (less than 1 unit);

Group 2 - 22 samples with an average value of K _ULA (from 1.0 UNITS to 2.0 UNITS);

Group 3 - 10 samples with K _ULA (more than 2.0 units).

An analysis of the magnitudes of the coefficients in the mmLNP and IR-mmLNP pairs indicates multidirectional shifts, i.e. in some samples, an increase in K _ALA is observed, in others, on the contrary, a decrease in this coefficient. In the third group, K _ULA remains constant.

Thus, to assess the lytic activity of immune complexes containing mmLNP, the proposed test analyzes in paired samples of K _ULA (mmLNP and IR mmLNP). The results of a pair analysis indicate:

1. In the case of a decrease in the K _ULA IR-mmLNP compared with K _ULA mmLNP in paired samples, the nature of the autoimmune reaction of the body to mmLNP is evaluated as protective (favorable). The effect of neutralizing lytic activity in the interaction of autoantibodies with mmLNP is probably associated either with the neutralization of lipoprotein-associated phospholipase A2, or the neutralization of lysophosphatidylcholine in the mmLNP molecule in immune complexes, or the binding of autoantibodies to mmLNP causes conformational changes in the pathogen test (see table 5);

2. In the case of an increase in K _ALA in paired samples of mmLDL and IR-mmLNP, the nature of the autoimmune response of the body to mmLDL is assessed as a pro-inflammatory (pathogenic) response to mmLDL. It is possible that an increase in the pathogenicity (lytic activity) of LDL in the composition of immune complexes is associated with conformational changes in the lipoprotein molecule. This fact is a completely new effect for immune responses, (see table 6);

3. In the case of equal values of K _ULA in the pairs of mmLDL and IR-mmLNLP, on the one hand, the value of this coefficient is important for assessing pathogenicity, on the other hand, this fact indicates that autoantibodies, binding to mmLNP in immune complexes do not affect the lytic activity of these complexes. In this situation, the pathogenic effect of mmLNP and IR-mmLNP depends only on their concentration in the blood of the examined individual (table 7).

Thus, the method for assessing the lytic activity of IR-mmLNP in the human body allows the diagnosis of the course of the atherosclerotic process, expands the possibilities for correcting therapy under the control of the coefficient of specific lytic activity mmLNP and IR-mmLNP, and opens up a new approach to assessing the atherogenicity of mmLNP and IR-mmLNP " from quantitative ”indicators to“ qualitative ”(functional) indicators. That is, at a normal level of mmLDL and IR-mmLDL, the pathological process caused by these complexes can proceed at different speeds, depending on the specific lytic activity coefficient, i.e. from the pathogenicity of these complexes.

The proposed method is simple to perform, does not require expensive reagents and equipment. The stage of centrifugation is not required for registration of erythrocyte lysis. The use of a simple and informative method for assessing the nature of the body's autoimmune reaction to a natural metabolite, IR-mmLNP, allows screening and identification of people with an acute course of the pathological, in particular, atherosclerotic process, both in preclinical stages of the disease, and in clinical atherosclerosis.

Claims (1)

A method for assessing the lytic activity of multi-modified low-density lipoproteins, including the preparation of precipitate from human blood serum by incubating the supernatant obtained after pre-precipitation of the multi-modified low-density lipoproteins (mmLNP) with a buffer containing 20% polyvinylpyrrolidone with a molecular weight of 35,000 (PVP-35000), for 30 min at 4 ° C, then the formed aggregates of immune complexes containing mmLNP (IR-mmLNP) are precipitated by centrifugation at 4 ° C for 10 min at 31000 g, decanted, the precipitate obtained is dissolved in a buffer without PVP-35000, the cholesterol content in the immune complexes is determined, characterized in that standardized sheep erythrocytes are added to mmLNP and IR-mmLNP samples, incubated for 24 hours at 37 ° C is calculated specific lytic activity coefficient (K _ULA) and IR mmLNP mmLNP as the ratio of the degree of lysis to cholesterol in these complexes, the value of K is compared _ULA mmLNP and IR mmLNP in human subject, in case of decrease of this ratio IR mmLNP compared with By _ULA mmLNP estimated as neutralization lytic activity, with the opposite effect, with a tendency to an increase of this ratio IR mmLNP is measured as an increase in the lytic activity of equal magnitude K _ULA (mmLNP) and K _ULA (IR mmLNP) indicate that autoantibodies By binding to mNLP in immune complexes, they do not affect lytic activity.

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