RU2549467C1 - Method of determination of atherogenicity of immune complexes - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention can be used for determination of atherogenicity of immune complexes containing multiply modified low density lipoproteins (IC-MMLDLP). For this purpose basic calcium superphosphate of IC-MMLDLP is prepared from human blood serum by treatment with the buffer containing 10% solution of polyethyleneglycol with a molecular weight 3350 (PEG-3350), in the ratio 1:2.5, then it is incubate within 10 minutes at a room temperature. IC-MMLDLP aggregates are pelleted, dissolved in the buffer without PEG-3350, analysed for the content of cholesterol in immune complexes (ChIK) and the level of guinea pig complement binding (EBC) by precipitated immune complexes. IC-MMLDLP atherogenicity is calculated as SSK to HIK ratio. If the value is below 24 units a high blood atherogenicity because of the reduced complement activating IgG function in IC-MMLDLP is stated.

EFFECT: method allows to assess IC-MMLDLP atherogenicity, diagnose atherosclerosis at a preclinical stage, and also to predict both the course of atherosclerotic process at individuals, and efficiency of the conducted therapy.

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Description

The invention relates to clinical immunology and can be used to determine the atherogenicity of immune complexes containing multi-modified low density lipoproteins (IR-mmLDL) in human serum.

Currently, in clinical laboratory practice, there are no methods available for routine research to determine the atherogenicity of IR-LDL. Given the important pathogenetic role of immune complexes containing multi-modified low-density lipoproteins in atherosclerosis, the development of simple methods for determining the atherogenicity of IR-LDLM [1, 3, 4] remains relevant.

A known method for determining the atherogenicity of blood serum by its ability to cause the accumulation of cholesterol in cultured cells [5]. The disadvantage of this method is the complexity, high cost, complexity and duration of the study.

As a prototype, a method for assessing the atherogenicity of immune complexes containing multiply modified low density lipoprotenes was used [2].

In the prototype, the precipitate of immune complexes containing multi-modified low-density lipoproteins is prepared from human blood serum by treatment with a buffer containing 8.3% PEG 3350 and 3.3% PVP 12600, in a ratio of 1: 1.2, incubated for 10 min at room temperature. The precipitate containing IR-mmLDPE is separated by centrifugation at 3100g for 10 min at 23 ° C, dissolved in a buffer without PEG and PVP, the cholesterol content in the immune complexes (CIC) is determined using an enzymatic kit, complement binding degree (CCK) of guinea pig , the atherogenicity of immune complexes containing mmLDL is calculated as the ratio of SSC to CIC.

The disadvantage of the method (prototype) is the study of the atherogenicity of IR-LDL only in patients with instrumentally confirmed atherosclerosis of the carotid arteries, which did not allow the authors to determine the reference values for the atherogenicity of these complexes.

The objective of the present invention is to develop a method for determining the atherogenicity of immune complexes containing LDL for routine studies with high accuracy and with a minimum investment of time.

This problem is solved in that the specific precipitation of circulating immune serum complexes containing mmLDPE is carried out by treating the serum with a buffer solution containing PEG-3350, incubated for 10 minutes at room temperature, then the formed aggregates of the immune complexes are precipitated by centrifugation, decanted, and the resulting precipitate is obtained dissolved in a buffer without PEG-3350, determine the cholesterol content in precipitated immune complexes using a standard enzymatic kit and Degree of guinea pig complement fixation, after which the calculated atherogenicity IR mmLPNP as a ratio to the CIU CCK. When the value of this indicator is below 24 IU, an increased atherogenicity of IR-mmLDPE is noted due to a reduced complement-activating function of IgG in IR-mmLDPE.

The method for determining the atherogenicity of IR-LDL includes:

1. Buffer-1 for precipitation of IR-LDL contains 10% polyethylene glycol with a molecular weight of 3350 (Sigma, USA) in 0.01 M Tris-HCl buffer with a pH of 7.4 containing 0.15 M NaCl;

2. Buffer-2 for dissolving the precipitate IR-LDL is 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl.

The method for determining the atherogenicity of IR-LDL is as follows. Fasting is carried out on an empty stomach from the human ulnar vein, serum is separated and cholesterol level and the degree of complement fixation of guinea pig in PEG precipitates are determined by treating blood serum with Buffer-1 in a ratio of 1: 2.5 to precipitate IR-mmLDPE by incubation for 10 min at room temperature, precipitation of the precipitate by centrifugation, decantation and subsequent dissolution of it in the initial volume in Buffer-2. The cholesterol in IR-mmLDPE is determined using the enzyme kit "Cholesterol". The complement-activating ability of immune complexes containing multi-modified LDL is studied using a hemolytic test using guinea pig complement, standardized (1.5 × 10 ⁸ cells / ml) sheep erythrocytes sensitized by rabbit antibodies (EA), in a veronal saline buffer containing 0 5 mM MgCl ₂ and 0.15 mM CaCl ₂ (VBS ²⁺ ).

The atherogenicity of IR-mmLDL in ED is calculated as the ratio of SSC to cholesterol in precipitated immune complexes containing mmLDL. When the atherogenicity of IR-mmLDL is less than 24 units, an increased blood atherogenicity is noted due to the reduced complement-activating function of IgG in IR-mmLDL.

Stage 1. Determination of cholesterol content in the immune complexes in the blood serum of donors and patients with atherosclerosis of carotid arteries. Preparation of the precipitate of immune complexes from serum: 125 μl of buffer-1 is added to 50 μl of blood serum and incubated for 10 min at room temperature. The resulting aggregates of immune complexes are precipitated by centrifugation at 3100g for 10 min at 23 ° C. The supernatant was carefully decanted and the pellet was dissolved in 50 μl of buffer-2. The cholesterol content is determined using a commercial set of "Cholesterol" company "ZAO Ekolab" (Russia). The results are presented in the table.

Stage 2. Determination of the degree of binding of guinea pig complement to immune complexes containing mmLDL. To 10 μl of precipitate solutions diluted 1:99 with VBS ²⁺ buffer, 12 μl of a diluted 1:19 guinea pig complement solution was added. The total volume was adjusted to 0.3 ml with VBS ²⁺ buffer and incubated for 20 min at 37 ° C. After incubation, add 200 μl of EA and re-incubate for 30 min at 37 ° C. After incubation, the reaction is stopped by adding 2.5 ml of a cold solution of 0.15 M NaCl, centrifuged and the red blood cell lysis value is determined by the release of hemoglobin into the supernatant. The control sample does not contain precipitate of immune complexes. Reduced hemolysis in experimental samples compared with the control indicates the presence of complement binding. The degree of lysis of red blood cells (U) is determined by the formula:

Y (%) = [(X-R) / (H-R) × 100],

where H, R and X are the optical density values A ₄₀₅ in the hemolytic systems of the control sample, in the control of EA spontaneous lysis and in the experimental sample, respectively. The degree of complement fixation (CCK) is determined by the formula:

SSK (%) = 100-U.

The results are presented in the table.

Stage 3. Determination of the atherogenicity of immune complexes containing mmLDL (AIC-mmLDL). AIC-mmLDPE is calculated in the blood serum of the examined individuals as the ratio of the degree of complement binding to cholesterol of precipitated immune complexes containing mmLDPE (CIC). The data obtained are presented in the table.

As can be seen from the data presented in the table, the HIC content in the blood serum of healthy donors in 100% of cases was below 10 mg / dl (scatter from 2.3 to 9.2 mg / dl), while the HEC in the blood serum was neurological patients with instrumentally confirmed atherosclerosis of the carotid arteries was on average 2.5 times higher than normal values and ranged from 13.7 to 44.6 mg / dl. The degree of binding of guinea pig complement by PEG precipitate in the group of healthy donors was statistically significantly higher (p = 0.028; t - Student's test) than in the group of patients (average 62% and 42%, respectively).

The obtained results on the assessment of the atherogenicity of immune complexes containing LDL-M indicate that the immune complexes of patients with atherosclerosis of carotid arteries have a reduced complement-activating ability, in this case a heterogeneous complement of guinea pig. And, as a result, an increased level of immune complexes containing mmLDPE in the blood of patients with atherosclerosis of the carotid arteries due to impaired solubilization and elimination with the participation of an autologous complement system. The reduced complement activating ability of IgG in the immune complexes of patients with atherosclerosis also leads to incomplete phagocytosis and, as a result, the formation of “foamy” cells in this pathology.

Thus, the proposed method allows a functional assessment of the atherogenicity of immune complexes containing MMLPD with a minimum expenditure of time and money, as well as conducting extensive screening examinations, diagnosing atherosclerosis at the preclinical stage, and predicting the course of the atherosclerotic process in subjects. Identification of elevated levels of CIC with extensive screening tests can be a predictor of atherosclerosis in the preclinical stage. The availability of reagents and the simplicity of the method for determining CIC in laboratory practice allow an in-depth study of the pathogenesis of atherosclerotic diseases. On the other hand, the introduction of a method for determining the atherogenicity of immune complexes containing mmLDPE into laboratory practice will allow the development of new methods for treating atherosclerosis and monitoring the effectiveness of the treatment.

Literature

1. Sobenin I.A., Karagodin V.P., Melnichenko A.A., Orekhov A.N. Cholesterol of immune complexes as an indicator of atherosclerosis // Pathological physiology and experimental therapy. - 2012. - No. 3. - S.99-103.

2. Shoybonov B.B., Kravchenko M.A., Shabalina A.A., Kostyreva M.V., Baronets V.Yu., Panchenko L.F. Assessment of the atherogenicity of immune complexes containing multi-modified low-density lipoproteins // Materials of the All-Russian Scientific and Practical Conference "Non-Infectious Diseases and the Health of the Population of Russia" May 16-17, 2013 Moscow. Thes. Reports. // Preventive medicine. - 2013. - T. 16, No. 2 (issue 2). - S. 150.

3. Burnt D.F.P., Karim Y., Ferns G.A.A. The Role of Immune Complexes in Atherosclerosis // Angiology. - 2010. - V.61, No. 7. - P.679-689.

4. Lopes-Virella M.F., Brent McHenry M., Lipsitz S., et al. Immune complexes containing modified lipoproteins are related to the progression of internal carotid intima-media thickness in patients with type 1 diabetes // Atherosclerosis. - 2007. - V.190. - P. 359-369.

5. Tertov V.V., Orekhov A.N., Nikitina N.A. et al. Peritoneal macrophages: model for detecting atherogenic potential in patiens' blood serum Ann. Med. - 1989 .-- V.21 (6). - P.455-459.

The cholesterol content of immune complexes (CIC), the degree of complement binding (CCK) and the atherogenicity of immune complexes containing mmLDL (AIC-mmLNLP) in PEG precipitates of blood serum from donors and neurological patients Donors HIK, mg / dl SSK,% AIK-mmLDPN Sick HIK, mg / dl SSK,% AIK-mmLDPN one 5.2 56 108 one 30,0 43 fourteen 2 6.4 70 109 2 26,4 35 13 3 2,3 27 117 3 21.1 33 16 four 4,5 80 178 four 16.9 39 23 5 9.2 78 85 5 44.6 72 16 6 5,0 41 82 6 27.0 51 19 7 5.3 53 one hundred 7 29.1 3 one 8 5.7 82 144 8 16.6 41 3 9 6.8 73 107 9 14.8 35 24 10 7.8 69 89 10 40,0 77 19 eleven 8.4 49 58 eleven 13.7 33 24 M ± m 6.1 ± 2.0 62 ± 18 107 ± 32 M ± m 25.5 ± 10.2 42 ± 20 16 ± 8

Claims (1)

A method for determining the atherogenicity of immune complexes containing multi-modified lipoproteins (IR-mmLDPE) in human blood serum by treating it for 10 min with a buffer containing polyethylene glycol (PEG), precipitation of IR-mmLDPE by centrifugation, determination of cholesterol in immune complexes and the degree of complement fixation characterized in that the serum is treated with a 10% solution of PEG-3350 in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, with a volume ratio of serum: PEG-3350 (1: 2.5) calculate the atherogenicity of IR-m mLDPN as a ratio of the degree of complement fixation to the cholesterol content in the immune complexes and at a value of less than 24 PIECES indicate an increased blood atherogenicity due to a decreased complement-activating function of IgG in IR-LDL.