Method for detecting circulating immune complexes

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

 

The invention relates to medicine, namely immunological and biochemical methods for study of biological substrates and can be used in the clinical laboratory and research work for the determination of circulating immune complexes (CIC) in the serum of a person.

The formation of complexes of antigen-antibody (immune complexes) is a natural immunological reaction of a healthy body, aimed at the elimination of foreign antigen and maintaining homeostasis. When rheumatologic, autoimmune, allergic diseases, and infections of various etiologies and tumors observed increase in the content of the CEC [Konstantinova N.A. Immune complexes and tissue damage. - M.: Medicine, 1996. - 256].

Known methods of determining the CEC in biological fluids [Casali R. and other Immune complexes and tissue damage: in the book. "Immunological aspects of infectious diseases". Ed. Jdike, Per. s angl. Asepta, Wealthview. - M.: Medicine, 1982. - S-384]:

1 type - antigen-specific, based on the detection of the CEC with a known antigen due to differences between the free and bound with antibodies to the antigen. Currently, they are not widely used, because in most cases the antigen included in SOS is AB CEC, unknown or difficult to identify.

2 type - antigenspecific methods. Found most prevalent in clinical studies and are divided into several major groups:

1. Based on the biological properties of immune complexes.

1.1. Determination of CEC in the interaction with complement, in particular the total complimentative activity (KSA), reaction with Clq - or C3b-subcomponents (classical or alternative pathway activation of complement, respectively) [N.A. Konstantinova, Lavrent'ev V.V., Kovalchuk L.V., Petrov R.V. detection of circulating immune complexes in humans. // Laboratory work. - 1982. No. 11. - S-675; Patent No. 2343484, G01N 33/50 on Application No. 2005129801/15 from 28.09.2005, published 10.01.2009 "method for the determination of circulating immune complexes"; Patent No. 2006125176, G01N 33/53, G01N 33/564 on application No. 2006125176/15 from 13.07.2006 published 10.07.2008 "Method and kit immunoassay determination complimentative circulating immune complexes]. The 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 CEC not solubilizers under the action of complement, even when a large excess in the solution. The presence in the blood of complexes C3-IgG can be considered as the product of the activation of the complement is, but not as proof of the CEC in blood. IgA and IgE do not have effector centers for C1q. The lack of developed up to the present time tests, 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 restricts their use and the fact that the diagnostic value of determination of C1q or C3b at a certain pathology is different. So, in the vast majority of cases, patients with rheumatoid arthritis CEC contain C1q, but not C3b, and systemic lupus erythematosus Vice versa. In addition, it is not always the activation of the complement system necessary for the development of damage associated with the CEC, in particular in glomerulonephritis. And the development of immune pathology may result from deficiency or reduced activity of the various components of complement.

1.2. Determination of CEC using antiimmunoglobulin 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 false-positive results in the presence in the sample of the aggregated IgG or native of Russia.

13. Determination of CEC when interacting with receptors of various cells. The most well-known method using the cell lymphoblast lines (Raji cells), which allows the detection of small CEC bearing b and has a high sensitivity and specificity, however, the complexity and the complexity of technology of cultivation of cells restrict the use of this group of methods.

2. Methods based on physico-chemical properties of the CEC. The greatest number of methods for the determination of the CEC developed based on the use of linear uncharged polymer of polyethylene glycol (PEG) with molecular weight of 6000, which allow you to define different molecular weight and size of the complexes (directly proportional to the concentration of PEG), as complementfixing and does not bind complement [Immunological methods. Ed. Grimes, Tr. Aphtharsia. - M.: Medicine, 1987. - 472 p; Clinical laboratory analyst. Private analytical technologies in the clinical laboratory. / Under the editorship of Prof. Riv. - M.: Labelform - RAMLA, 1999. - 352 p; Clinical immunology: a textbook for students of honey. universities / edited Averella. - M: Medical info. Agency, 1999. - 604 s; Patent No. 2107299, G01N 33/539 on application No. 94043266/14 from 07.12.1994, published 20.03.1998 "Method for determination of circulating immune complexes in serum CROs and"]. It is shown that the increase of the content of the CEC in the PEG-precipitate correlated with some clinical conditions.

Closest to the proposed invention (the prototype) is the method of precipitation of serum (0.3 ml) of complexes of antigen-antibody 3.75% solution of PEG-6000, prepared using 0.1 M borate buffer (pH 8,4), at room temperature, followed (after 60 min) photometric determination of the density of the precipitate at a wavelength of 450 nm [Grinevich Y.A., Alferov A.N. Determination of immune complexes in the blood of cancer patients. // Laboratory work. - 1981. No. 8. - s-496].

The disadvantage of this method is the possibility of determining the concentration of the CEC mostly of large size, while most pathogenic effect have small complexes. In addition, for research requires a fairly large amount of serum that can be important in particular in the examination of children. And the duration and technical support (spectrophotometer with sample compartment) method reduces the possibility of simultaneous studies to single samples.

The aim of the invention is the improvement of methods of PEG-precipitation to develop a screening test to determine the serum level of the CEC both large and small sizes, which are characterized by what hadinoto, simplicity, reproducibility, and the possibility of widespread use in clinical laboratory practice.

The essence of the invention lies in the fact that additionally lighten the serum by brief centrifugation, and measuring the increase in turbidity of the sample in the presence of PEG with a final concentration of 4% to determine large the CEC and 6% for the CEC of small dimensions, make use of a microplate spectrophotometer in a two mode: main - 340 nm, reference filter of 620 nm, and the incubation duration reduced to 15 minutes.

The technical result consists in the fact that the implementation of the present invention allows, on the one hand, to increase the efficiency and reliability of determination of the level of the large circulating immune complexes and the reproducibility of the results, on the other hand, is differentially measure the level of the CEC of small sizes, which are diagnostically significant indicator of immunological reactivity of the organism in many pathologies. The use of the proposed method allows to reduce the volume of samples to 0.06 ml, and incubation duration to 15 minutes. The proposed method is suitable for mass-clinical studies as a screening test in the detection of individuals with immunocomplex the Oh pathology, as well as monitoring changes in the level of the CEC in the process of development of the disease and effectiveness of treatment.

The method is as follows:

1. The preparatory phase.

Prepare working solutions: solution 1 - buffered saline (pH 7,4); solution of 2-8% solution of PEG (8.0 g PEG - 6000+to 100.0 ml solution 1); solution 3-12% solution of PEG (12.0 g PEG - 6000+to 100.0 ml solution 1).

The research material is serum human blood, obtained standard from capillary or venous blood. Additionally, serum lighten by centrifugation at 10,000 rpm./min for 2 min For further research using the supernatant. Serum examined on the day of blood sampling, or immediately frozen and stored at a temperature not above -16°C up to 3 months. Pets single freeze-thaw samples. Thawed serum rapidly at a temperature not exceeding 30°C. For analysis requires 0.06 ml of serum.

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

Observance of these conditions depends on the accuracy and reproducibility of research results.

2. The course of analysis.

Serum (30 ml) previously diluted 10-fold in a solution of 1 (270 μl), use the I separate microplate (strips) for immunological studies with the volume of the hole is not less than 300 ál, each duplicate sample.

In the wells of a standard 96-well tablet (strips) for immunological studies contribute 100 ál of working solutions to all wells of the strip 1 - solution 1; strip 2 - solution 2; strip 3 - solution 3; if necessary, fill out the following strips in the same sequence.

Using an eight-channel dispenser, each strip (1, 2, 3, respectively) add 100 ál prediluted serum, as shown in figure 1, and the final dilution of the sample is 20 times. Will pipeinput 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 is 15 minutes Leave the tablet, covered with lid, at a temperature of 18-25°C.

3. Registration and evaluation of results.

Record data using a microplate spectrophotometer by measuring the optical density (OD) in a two mode: main - 340 nm, reference filter of 620 nm.

Then, on the basis of the received data OP expect the level of large and small circulating immune complexes ([CEC] and m[CEC], respectively), usl. units:

[CEC]=(OP cf. R-R 2 OP cf. R-R 1)×1000;

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

where OP AVG - average value of the optical density of the sample.

Level PEAK assess differential is new for large and small sizes comparing with the standard indexes obtained in the study of the proposed method, the primary sera of blood donors (n≥30).

An example of a specific implementation of the method:

The blood donor (A), patients with chronic hepatitis C (CHC) (B), autoimmune hepatitis type-2 (AGG-2) ("In") and systemic lupus erythematosus (SLE) ("G") has made the sampling of venous blood in the amount of not less than 0.5 ml, from which the accepted way received the serum. Next, the sample additionally gave an introduction by centrifugation at 10,000 rpm./min for 2 min Obtained supernatant was used for studies on the same day.

Working solutions 1, 2 and 3 passed at a temperature of 18-25°C for 30 minutes, the Sample was thoroughly mixed using a vortex.

All wells strip for pre-dilution of the sera were made in 270 μl of solution 1. Later in the first two wells were added to 30 μl of serum sample "a"; in the following two holes - sample "B", then "In" and after "G".

All wells of the working strip 1 made of 100 μl of solution 1, strip 2 - solution 2, strip 3 solution 3. Each strip using eight-channel dispenser has transferred 100 ál prediluted serum and pietravalle 3-4 times, avoiding the formation of air bubbles, in the direction of the strip 1 to strip 3. Left for 15 min at a temperature of 18-25°is, cover with the lid.

Recorded results on a microplate spectrophotometer in a two mode: 340 nm, reference filter of 620 nm.

Got the following values of optical density (OD):

The serum sampleA numberSolution (strip)
123
"A"And0.0450.0670.177
In0.0480.0700.174
"B"0.0700.0790.276
D0.0690.0770.268
"B"E0.0500.0940.280
F0.0540.0910.283
"G"G0.1130.1800.399
N0.1180.1910.378

Calculated levels of circulating immune complexes in used:

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

According to our own studies offered by way of primary sera of blood donors (n=31) of the upper forth the permissible limit of normative indicators of the level of circulating immune complexes accounted for to the[CEC] - 30 usled, m[CEC] - 160 usled

The study in samples of blood sera of patients AGG-2 ("In") and SLE (G) compared with normative values revealed a high content along with large and small circulating immune complexes. And in the serum of the patient CHC (B) content of circulating immune complexes on the prototype did not exceed the standard rate, while the level of the CEC of small sizes were significantly high, which combined with a severe clinical course of infection and extrahepatic manifestations.

Thus, further clarification of the analyzed serum samples by brief centrifugation and the final dilution in 20 times along with the use of the two-wavelength measurement mode provides optimum sensitivity, accuracy and reproducibility of research results. The choice in favor of the use of PEG-6000 with a final concentration of 4% for the detection of high-molecular CEC (large size) and 6% for the most complete precipitation of the medium - and low CEC (small size), but, importantly, not yet free of IgG, as well as data of its own investigation of the molecular mass distribution obtained at various concentrations of PEG precipitates CEC by high performance liquid chromatography. In addition, we use the Yong kinetic measurement of the increase in turbidity of the sample as PEG-precipitation, which has allowed to establish that the determination of their level on the basis of data obtained by measurement on the "end point", according to the prototype after 60 minutes, is methodologically inappropriate. Presented in figure 2 kinetic curves of the process PEG-precipitation of the CEC in the serum in normal and pathological conditions, indicate that, on the one hand, as a rule, the value of OP is maximized and go out on a "plateau" no later than 15 minutes from the time of placing the sample in a solution of PEG. On the other hand, in individual samples in the interval more than 15 minutes, regardless of the concentration of PEG, may be spontaneous semiregular precipitiously CEC that accompanied the enlightenment environment, as it occurs in the sample (patient AGG-2 type), and eventually leads to inadequate registration low results. This phenomenon can be due to either the original features of the composition and properties of the CEC at a certain pathology or modification of the physico-chemical characteristics of the CEC in the PEG-precipitate. Therefore, incubation period, not exceeding 15 minutes is optimal for obtaining methodically the correct definition data of the CEC method PEG-precipitation.

Accessibility of the application of the method is provided by a wide spread in institutions practical what about the health standard equipment for ELISA studies.

The method developed and tested in the laboratory of immunochemistry fsri NIIEM imagedata Innlogging of Rospotrebnadzor.

The method of determination of circulating immune complexes in the serum of a person by precipitation them with a solution of polyethylene glycol with a molecular weight of 6000 (PEG-6000) with the subsequent registration of the results of the method turbidimetry, characterized in that it further serum lighten brief centrifugation, and measuring the increase in turbidity of the sample in the presence of PEG solution with a final concentration of 4% to determine large the CEC and 6% for the CEC of small dimensions, make use of a microplate spectrophotometer in a two-mode (main - 340 nm, reference filter of 620 nm), and the incubation duration is 15 minutes



 

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EFFECT: high accuracy and specificity of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves pouring venous blood treated with heparin into five conic test-tubes in the amount of 0.1 ml. The first three of them contain 0.1 ml of non-colored latex suspension with particle size of 1.5 mcm, the fourth one contains 0.1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue, the fifth one contains .1 ml of latex suspension and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue. The first test-tube is incubated in thermostat for 5 min at37°C, the second one for 30 min, the third one for 1 h, the fourth and the fifth one for 40 min. Smears are prepared from 0.2 ml of incubation mixture on glasses and dried at 37°C, fixed in burner flame, stained with 0.1% aqueous solution of tetrazole nitro blue, repeatedly dried and studied with microscope under immersion with magnification of 90x10. Test results are evaluated from absorption activity in phagocytosis reactions in determining the number of phagocytes, phagocytic number, phagocytic integral index and phagocytosis rate values. Tetrazole nitro blue test response is determined by counting formazan-positive cell number, calculating cytochemical activity index and tetrazole nitro blue test stimulation index.

EFFECT: accelerated test; high accuracy and low cost of examination.

1 dwg, 3 tbl

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