RU2549466C1 - Instant method for assessing atherogenicity of immune complexes of human blood serum - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for the instant assessment of atherogenicity of the immune complexes (IC) of human blood serum. Precipitated ICs from human blood serum are prepared by treating with a buffer containing 10% polyethylene glycol of molecular weight 3,350 (PEG-3350) in ratio 1:3.5, incubating for 10 min at room temperature. Aggregated ICs are deposited by centrifuging and dissolved in PEG-3350 free buffer; the immune complex cholesterol (ICC) is measured, and a guinea pig's complement fixation (CF) by the precipitated immune complexes is determined. The IC atherogenicity is calculated as CF/ICC relation, and if the derived value is less than 4 units, the high blood atherogenicity is stated.

EFFECT: higher assessment accuracy.

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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 the ratio 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 mLDPL is calculated as the ratio of SSC to HIC.

The objective of the present invention is to develop a method for the rapid determination of the atherogenicity of human serum immune complexes 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 IR is dissolved in a buffer without PEG-3350, the cholesterol content of precipitated immune complexes (CIC) is determined using the Cholester commercial kit n 'and degree of binding complement of the guinea pig, after which atherogenicity calculated as the ratio of IR to CCK CIU. When the value of this indicator is below 4 IU, an increased atherogenicity of IR-mmLDPN is noted.

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

1. Buffer-1 for IR precipitation 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 IR precipitate is 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl.

The method of rapid determination of atherogenicity of IR blood serum 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: 3.5 to precipitate IR, 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. IR cholesterol is determined using the Cholesterol commercial kit. The complement-binding ability of immune complexes containing multi-modified LDL is examined using a hemolytic test using guinea pig complement, standardized (1.5 × 10 ⁸ cells / ml) sheep erythrocytes sensitized by rabbit antibodies (EA), in veronal saline buffer, containing 0.5 mm MgCl ₂ and 0.15 mm CaCl ₂ (VBS ²⁺ ).

The atherogenicity of IR in ED is calculated as the ratio of SSC to cholesterol in precipitated immune complexes (CIC). When the SSC / HEC value is less than 4 units, an increased atherogenicity of human serum immune complexes is noted.

Stage 1. Determination of cholesterol content in the immune complexes in the blood serum of donors and patients with coronary artery atherosclerosis. 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 3000 g 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⁺ , add 12 μl diluted 1:19 guinea pig complement solution. 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 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 of cardiac patients with instrumentally confirmed coronary artery atherosclerosis, was on average 4.7 times higher than normal values and ranged from 13.8 to 67.5 mg / dl. The degree of binding of guinea pig complement by PEG precipitate in the group of healthy donors was significantly higher than in the group of patients (on average 62% and 47%, respectively).

The results obtained in assessing the atherogenicity of immune complexes clearly indicate that the immune complexes of patients with coronary artery atherosclerosis have a reduced complement-activating ability, in this case, a heterogeneous complement of guinea pig. And as a result, there is 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 immunoglobulins 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 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 patients in dynamics. 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 allows 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. Burut 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: a model for detecting atherogenic potential in pattens ′ blood serum // Ann. Med. - 1989 .-- V.21 (6). - P.455-459

Table 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 donors and cardiac patients Donors HIK, mg / dl SSK,% AIK-mmLDPN Sick HIK, mg / dl SSK,% AIK-mmLDPN one 5.2 56 108 one 16.6 43 2.6 2 6.4 70 109 2 15.6 52 3.3 3 2,3 27 117 3 33,4 74 2.2 four 4,5 80 178 four 13.8 eighteen 3,5 5 9.2 78 85 5 44.5 8 0.2 6 5,0 41 82 6 67.5 22 0.3 7 5.3 53 one hundred 7 34.2 19 0.6 8 5.7 82 144 8 17.5 45 2.6 9 6.8 73 107 9 27.7 89 3.2 10 7.8 69 89 10 24.9 78 3,1 eleven 8.4 49 58 eleven 18.6 74 4.0 M ± m 6.1 ± 2.0 62 ± 18 107 ± 32 M ± m 28.6 ± 16.1 47 ± 28 2.3 ± 1.4

Claims (1)

An express method for determining the atherogenicity of immune complexes (IR) of human blood serum by treating it for 10 min with a buffer containing polyethylene glycol (PEG), precipitation by IR centrifugation, determination of cholesterol in immune complexes (CIC) and the degree of complement fixation (CCK), characterized in that the processing of serum is carried out 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: 3.5), atherogenicity is calculated IR as the ratio of SSC to CIC and at a value of less than 4 PIECES iruyut increased blood atherogenicity.