RU2415430C1 - Method for detecting circulating immune complexes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: differentiated detection large and small circulating immune complexes in blood serum is ensured by a follow-on examination of blood serum clarified by short centrifugation with a method of circulating immune complexes precipitation PEG -6000 of the end concentration 4 % and 6 %. Thereafter, the results are recorded by a turbidimetric method with using a microplate spectrophotometer in a two-wave mode: basic - 340 nm, reference filter - 620 nm. Duration of an incubation step at temperature +18-25°C is 15 minutes. Using the method enables higher effectiveness and reliability of determining the level of large circulating immune complexes, results reproducibility, as well as differential measurement of the level of small CIC which are a diagnostically significant indicator of human body immune responsiveness in many types of a pathology, decreased volume of analysed serums to 0.06 ml, and incubation duration to 15 minutes.

EFFECT: method is suitable for clinical screenings.

1 dwg, 1 tbl, 1 ex

Description

The invention relates to medicine, namely immunological and biochemical methods for studying the parameters of biological substrates, and can be used in clinical laboratory diagnostics and research to determine circulating immune complexes (CEC) in human serum.

The formation of antigen-antibody complexes (immune complexes) is a natural immunological reaction of a healthy organism, aimed at eliminating a foreign antigen and maintaining homeostasis. With rheumatological, autoimmune, allergic diseases, as well as infections of various etiologies and neoplasms, an increase in the content of the CEC is noted [Konstantinova N.A. Immune complexes and tissue damage. - M .: Medicine, 1996. - 256 s].

The following methods are known for determining CEC in biological fluids [Kasali R. et al. Immune complexes and tissue damage: in the book. "Immunological aspects of infectious diseases." / Ed. J. Dick, per. from English A.S. Apta, V.I. Litvinova. - M: Medicine, 1982. - S.334-384]:

Type 1 - antigen-specific, based on the identification of CECs with a known antigen due to differences between the free and antibody-associated antigens. Currently, they have not found widespread use, since in most cases the antigen that is part of the CEC is unknown or difficult to determine.

Type 2 - antigen-specific methods. Found the greatest distribution in clinical trials and are divided into several main groups:

1. Based on the biological properties of immune complexes.

1.1. Determination of CEC during interaction with complement, in particular, general complement binding activity (KSA), reactions with Clq or C3b subcomponents (classic or alternative way of complement activation, respectively) [Konstantinova N.A., Lavrentiev V.V., Kovalchuk L. V., Petrov R.V. Determination of circulating immune complexes in humans. // Laboratory work. - 1982. - No. 11. - S.673-675; Patent No. 2343484, G01N 33/50 according to Application No. 2005129801/15 dated September 28, 2005, published on January 10, 2009, “Method for the determination of circulating immune complexes”; Patent No. 2006125176, G01N 33/53, G01N 33/564 according to the application No. 2006125176/15 dated 07/13/2006, published July 10, 2008, "Method and kit for enzyme-linked immunosorbent assay for complement-binding circulating immune complexes"]. Dissolution of the CEC under the action of complement is a very important process of removing them from the body. However, the determining factor in the measurement of KSA is the size of the CEC; small CECs are not solubilized under the action of complement, even with a significant excess in the solution. The presence of C3-IgG complexes in the blood can be considered as a complement activation product, but not as evidence of the presence of CEC in the blood. IgA and IgE do not have effector centers for C1q. A drawback of the tests developed so far, including ELISA, is the possibility of cross-reaction with DNA, endotoxin, rheumatoid factor, C-reactive protein, various polysaccharides, which significantly reduces their diagnostic specificity and sensitivity. Along with this, their use is limited by the fact that the diagnostic value of determining C1q or C3b for a certain pathology is different. So, in the vast majority of cases, in patients with rheumatoid arthritis, CECs contain C1q, but not C3b, but vice versa with systemic lupus erythematosus. In addition, the activation of the complement system is not always necessary for the development of damage associated with the CEC, in particular with glomerulonephritis. And the development of immunocomplex pathology may be the result of a deficiency or decrease in the activity of various components of complement.

1.2. Determination of CEC using anti-immunoglobulin antibodies, in particular rheumatoid factor (RF), allows to detect IgG-containing CEC in a fairly wide range of molecular weight complexes. Their main disadvantage is the receipt of false positive results in the presence of aggregated IgG or RF of their own in the test sample.

1.3. Determination of CEC by interaction with receptors of various cells. The most well-known method is the use of lymphoblastic cell line (Raji cells), which allows one to determine small CECs carrying C3b and is highly sensitive and specific, however, the complexity and complexity of cell culture technology limit the use of this group of methods.

2. Methods based on the physicochemical properties of the CEC. The largest number of CEC determination methods was developed based on the use of a linear uncharged polymer of polyethylene glycol (PEG) with a molecular weight of 6000, which allows the determination of complexes of various molecular weights and sizes (directly proportional to the concentration of PEG), both complement fixing and non-complement binding [Immunological methods. / Ed. G. Fremel, per. with him. A.P. Tarasova. - M .: Medicine, 1987. - 472 s; Clinical laboratory analytics. Private analytical technology in a clinical laboratory. / Ed. prof. V.V. Menshikova. - M .: Labinform - RAMLD, 1999 .-- 352 s; Clinical immunology: a textbook for students of honey. Universities / Ed. A.V.Karaulova. - M .: Medical inform. Agency, 1999. - 604 s; Patent No. 2107299, G01N 33/539 according to application No. 94043266/14 dated 12/07/1994, published on 03.20.1998 "Method for the determination of circulating immune complexes in blood serum"]. It was shown that an increase in the content of CIC in the PEG precipitate correlates with some clinical conditions.

Closest to the proposed invention (prototype) is a method of precipitation from blood serum (0.3 ml) of antigen-antibody complexes in a 3.75% PEG-6000 solution prepared using 0.1 M borate buffer (pH 8.4), at room temperature followed by (after 60 min) photometric determination of the precipitate density at a wavelength of 450 nm [Grinevich Yu.A., Alferov AN Determination of immune complexes in the blood of cancer patients. // Laboratory work. - 1981. - No. 8. - p. 493-496].

The disadvantage of this method is the ability to determine the concentration of CECs of predominantly large size, while small complexes have the greatest pathogenic effect. In addition, a sufficiently large volume of serum is required for the study, which may be of particular importance when examining children. And the duration and technical support (spectrophotometer with a cuvette compartment) of the method reduces the possibility of simultaneous research to single samples.

The aim of the invention is to improve the methods of PEG precipitation to develop a screening test for determination of serum CEC levels of both large and small sizes, characterized by reliability, simplicity, reproducibility and the possibility of widespread use in clinical laboratory practice.

The essence of the invention lies in the fact that blood serum is further clarified by brief centrifugation, and the measurement of the increase in turbidity of the sample in the presence of PEG with a final concentration of 4% to determine large CECs and 6% for small CECs is carried out using a microplate spectrophotometer in a two-wave mode: the main one is 340 nm, the reference filter is 620 nm, while the incubation time is reduced to 15 minutes.

The technical result consists in the fact that the implementation of the invention allows, on the one hand, to increase the efficiency and reliability of determining the level of large circulating immune complexes and reproducibility of results, on the other hand, to differentially measure the level of small-size CECs, which are a diagnostically significant indicator of the immunological reactivity of an organism human with many types of pathology. Moreover, the use of the proposed method makes it possible to reduce the volume of the analyzed samples to 0.06 ml, and the incubation time to 15 minutes. The proposed method is suitable for mass clinical trials as a screening test for identifying individuals with immunocomplex pathology, as well as monitoring changes in the level of CEC during the development of the disease and the effectiveness of the treatment.

The method is as follows:

1. The preparatory phase.

Prepare working solutions: solution 1 - buffered saline solution (pH 7.4); a solution of 2-8% PEG solution (8.0 g of PEG - 6000 + 100.0 ml of solution 1); solution of 3-12% PEG solution (12.0 g of PEG - 6000 + 100.0 ml of solution 1).

The material for the study is human blood serum, obtained as a standard from capillary or venous blood. Additionally, serum is clarified by centrifugation at 10,000 rpm for 2 minutes. For further studies, a supernatant is used. The serum is examined on the day of blood sampling, or immediately frozen and stored at a temperature not exceeding -16 ° C for up to 3 months. A single freezing-thawing of samples is allowed. Serum is thawed quickly at a temperature not exceeding 30 ° C. An analysis requires 0.06 ml of serum.

Before the study, all working solutions and serum samples are kept at a temperature of + 18-25 ° C for 30 minutes. Samples are thoroughly mixed on a vortex until a homogeneous consistency.

The observance of these conditions determines the accuracy and reproducibility of the research results.

2. The course of analysis.

Blood serum (30 μl) is pre-diluted 10 times in solution 1 (270 μl) using a separate microplate (strips) for immunological studies with a well volume of at least 300 μl, each sample being duplicated.

100 μl of working solutions are added to the wells of a standard 96-well plate (strips) for immunological studies: in all wells of strip 1 — solution 1; strip 2 - solution 2; strip 3 - solution 3; if necessary, fill in the following strips in the same sequence.

Using an eight-channel dispenser, 100 μl of previously diluted sera are added to each strip (1, 2, 3, respectively), as shown in Fig. 1, while the final dilution of the sample is 20 times. Pipette the contents of the wells 3-4 times in the direction from solution 1 to solution 3, preventing the formation of air bubbles, and immediately notice the incubation time of 15 minutes. Leave the tablet covered with a lid at a temperature of + 18-25 ° C.

3. Registration and evaluation of results.

Data is recorded using a microplate spectrophotometer, measuring the optical density (OD) in a two-wave mode: main - 340 nm, reference filter - 620 nm.

Then, on the basis of the obtained OP data, the level of large and small circulating immune complexes (to [CEC] and m [CEC], respectively) is calculated, in conv. units:

to [CEC] = (OD cf. p-r 2 - OP cf. p-1) × 1000;

m [CEC] = (OD cf. solution 3 - OP cf. solution 1) × 1000,

where OP cf. is the arithmetic mean value of the optical density of the sample.

The level of PIK is estimated differentially for large and small sizes, comparing with the normative indicators obtained in the study of the proposed method for the serum of primary blood donors (n≥30).

An example of a specific implementation of the method:

Blood donor ("A"), patients with chronic hepatitis C (CHC) ("B"), autoimmune type 2 hepatitis (AIH-2) ("C") and systemic lupus erythematosus (SLE) ("G") produced venous blood sampling in a volume of at least 0.5 ml, from which serum was obtained by the conventional method. The samples were further clarified by centrifugation at 10,000 rpm for 2 minutes. The obtained supernatant was used for research on the same day.

Working solutions 1, 2 and 3 were kept at a temperature of + 18-25 ° C for 30 minutes. Samples were thoroughly mixed using vortex.

270 μl of solution 1 was added to all wells of the strip for preliminary dilution of the sera. Then, 30 μl of the serum sample “A” was added to the first two wells; in the next two holes - sample "B", then "C" and after "G".

100 μl of solution 1, strip 2 of solution 2, strip 3 of solution 3 were added to all wells of working strip 1; 100 μl of previously diluted sera were transferred to each strip using an eight-channel dispenser and pipetted 3-4 times, avoiding the formation of bubbles air, in the direction from strip 1 to strip 3. Left for 15 min at a temperature of + 18-25 ° C, covered with a lid.

The results were recorded on a microplate spectrophotometer in a two-wave mode: base - 340 nm, reference filter - 620 nm.

Received the following values of optical density (OD):

Serum sample Row Solution (strip) one 2 3 "BUT" BUT 0.045 0.067 0.177 AT 0.048 0.070 0.174 "B" FROM 0.070 0.079 0.276 D 0.069 0.077 0.268 "AT" E 0.050 0.094 0.280 F 0.054 0.091 0.283 "G" G 0.113 0.180 0.399 N 0.118 0.191 0.378

Calculated the level of circulating immune complexes, in conventional units:

Sample "A": k [CEC] = (0.069-0.047) * 1000 = 22; m [CEC] = (0.176-0.047) * 1000 = 129. Sample "B": k [CEC] = (0.078-0.070) * 1000 = 8; m [CEC] = (0.272-0.070) * 1000 = 202. Sample "B": k [CEC] = (0.093-0.052) * 1000 = 41; m [CEC] = (0.282-0.052) * 1000 = 230. Sample "G": k [CEC] = (0.186-0.116) * 1000 = 70; m [CEC] = (0.389-0.116) * 1000 = 273.

According to our own research, the proposed method for the serum of primary blood donors (n = 31) the upper allowable limit of normative indicators of the level of circulating immune complexes amounted to 30 standard units for m [CEC], 160 standard units for m [CEC].

As a result of the study, in the blood serum samples of patients with AIH-2 ("B") and SLE ("G"), compared with standard values, an increased content was found along with large and small circulating immune complexes. And in the blood serum of a patient with chronic hepatitis C ("B"), the content of circulating immune complexes according to the prototype did not exceed the standard indicator, while the level of CEC of small sizes was significantly high, which was combined with a severe clinical course of the infection and extrahepatic manifestations.

Thus, additional clarification of the studied blood serum by brief centrifugation and a final dilution of 20 times, along with the use of a two-wave measurement mode, provide optimal sensitivity of the method, accuracy and reproducibility of the research results. The choice in favor of using PEG-6000 with a final concentration of 4% for the detection of high molecular weight CECs (large sizes) and 6% for the most complete precipitation of medium and low molecular weight CECs (small sizes), but, importantly, not yet free IgG, is justified by the data own studies of the molecular weight distribution obtained at various concentrations of PEG CEC precipitates by high performance liquid chromatography. In addition, we used the kinetic type of measurements of the increase in the turbidity of the samples in the process of PEG precipitation, which made it possible to determine that determining their level based on data obtained by measuring at the "end point", according to the prototype after 60 minutes, is methodologically inappropriate. The kinetic curves of the process of PEG precipitation of the CEC in the blood serum in normal and pathological conditions, shown in Fig. 2, indicate that, on the one hand, as a rule, the OD values reach maximum values and reach a “plateau” no later than 15 min from the moment the sample is introduced into the PEG solution. On the other hand, in individual samples over an interval of more than 15 min, regardless of the concentration of PEG, spontaneous self-aggregation of precipitated CECs can be observed, which is accompanied by enlightenment of the medium, as occurs in sample “B” (patient AIH-2), and results to register inadequate understated results. This phenomenon can be caused either initially by the peculiarities of the composition and properties of the CEC for a certain type of pathology, or by a modification of the physicochemical characteristics of the CEC in the PEG precipitate. Therefore, the incubation time, not exceeding 15 minutes, is optimal for obtaining methodologically correct data for the determination of CEC by the method of PEG precipitation.

The availability of the application of the method is ensured by the widespread use in standard healthcare facilities of standard equipment for ELISA studies.

The method was developed and tested in the laboratory of immunochemistry of the Federal State Institution of Health Research Institute named after Academician I.N. Blokhina of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare.

Claims (1)

A method for determining circulating immune complexes in human serum by precipitating them with a solution of polyethylene glycol with a molecular weight of 6000 (PEG-6000), followed by recording the results by turbidimetry, characterized in that the blood serum is further clarified by brief centrifugation, and measuring the increase in turbidity of the sample in the presence of a PEG solution with a final concentration of 4% for the determination of large CECs and 6% for small CECs, a double-wavelength microplate spectrophotometer is used ovom mode (basic - 340 nm, reference filter - 620 nm), the duration of incubation is 15 min.

Images