RU2303267C2 - Method for quantitative determination of antibodies concentration specific to human endometrial tissue stromal cells antigen and in human biological fluid containing specific antibodies - Google Patents

Abstract

FIELD: medicine, immunology, clinical laboratory diagnosis.

SUBSTANCE: method involves using the usual solid-phase enzyme immunoassay based on chemical binding antigen with microplate surface followed by placing antibodies-containing biological fluid on microplate, incubation, carrying out the color reaction and spectrophotometric evaluation of the color reaction indices. The end antigen is prepared by enzymatic hydrolysis of human normal tissue samples containing stromal cells of endometrial tissue. The enzymatic hydrolysis reaction is terminated by addition of chelating agent. Prepared mixed suspension of cells is centrifuged in continuous density gradient resulting isolation of stromal cells. Microsomal fraction of stromal cells is used as the end antigen that is diluted and bound covalently with microplate well surface. Then calibrating material prepared on basis of standard preparation antibodies specific to the end antigen is added into wells set, and biological fluid samples for analysis are added into another wells set. After carrying out the usual solid-phase enzyme immunoassay and using spectophotometry data a calibrating curve is plotted and the concentration of specific antibodies is determined using this curve. Invention provides the development of method for assay of antibodies amount specific to endometrial tissue stromal cells exceptionally that gives possibility for clinical diagnosis of autoimmune syndrome. Invention can be used in gynecology and obstetrics for diagnosis of autoimmune syndrome accompanying to clinical picture of endometriosis and other diseases in female reproductive system.

EFFECT: improved method of quantitative assay.

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Description

The invention relates to the field of clinical laboratory diagnostics and may find application in gynecology and obstetrics for the diagnosis of autoimmune syndrome, the concomitant clinical picture of endometriosis and other diseases of the female reproductive system.

A known laboratory method for the determination of autoantibodies to endometrial antigen is described in the article "The prevalence of endometrial immunoglobulin G antibodies in patients with endometriosis". OAOdukoya, N. Whitecroft, APWeetman, IDCooke // Human Reproduction, Oxford University press, Oxford, England, 1995, vol. 10, No. 5, pp. 1214-1219 ("Prevalence of endometrial antibodies of type immunoglobulins G in patients with endometriosis. "OA Odukoya, N. Whitcroft, AP Whitman, ID Cook // Human Reproduction, Oxford University Press, Oxford, England, 1995, Volume 10, No. 5, p. .1214-1219 - see Appendix 1). The known method is enzyme-linked immunosorbent assay (ELISA). In this work, a homogenate of endometrial tissue samples containing both stromal and epithelial cells was used as an antigen. As described in the implementation of this method, the samples were homogenized at + 4 ° C in the presence of proteinase inhibitors. The heavy fractions were precipitated by centrifugation, IgG was removed by incubation with Protein A Sepharose, and the resulting suspension of cellular components was used as antigen.

Obtained by the described method, the antigen includes components of epithelial and stromal cells and does not allow the determination of antibodies specific for the antigens of cells of each type.

As the closest analogue, we consider a laboratory method for more accurately determining antibodies to the endometrial antigen (target antigen) by conducting an enzyme-linked immunosorbent assay (TIFA), described in the article "Antiendometrial antibodies in women measured by an enzyme-linked immunosorbent assay". S. Femandez-Shaw, S. H. Kennedy, B. R. Hicks, K. Edmonds, P. M. Starkey, B. H. Barlow // Human reproduction, Oxford University press, Oxford, England, 1996, vol. 11, No. 6, pp. 1180-1184. (“Anti-endometrial antibodies in women, measured by enzyme-linked immunosorbent assay (ELISA).” K. Fernandez-Shaw, C.H. Kennedy, B.R. Hicks, C. Edmonds, P.M. Starki, B.Kh. Barlow // Human Reproduction, Oxford University Press, Oxford, England, 1996, Volume 11, No. 6, pp. 1180-1184 - see Appendix 2). The authors proposed three options for the preparation of antigen: the first - directly from samples of endometrial tissue of patients, the second - using the isolation of endometrial epithelial cells of patients and the third - using a laboratory culture of endometrial epithelial cells as a source of antigen. Endometrial epithelial cells were isolated by immunomagnetic separation, the cell components were separated by repeated centrifugation, and the various suspensions of the cell components obtained were used as the target antigen for enzyme immunoassay.

Obtained by the described method, the antigen includes components of epithelial cells and does not allow the determination of antibodies specific for stromal cell antigens.

According to the authors, endometrial stromal cells by their immunochemical properties have a high degree of specificity and antigenic activity. This is confirmed by statistically reliable results of both clinical and laboratory diagnosis of endometriosis. It follows that the use of stromal cells as a source of antigen provides an opportunity for a more accurate diagnosis compared to the use of epithelial cells. The microsomal fraction of cells of various tissues is traditionally used as antigens in immunochemical diagnostics when performing enzyme immunoassay. A fraction in its composition is an accumulation of internal cell membranes with which enzyme complexes are associated. Almost always, the microsomal fraction has a high affinity (degree of binding) for autoantibodies circulating in the blood of patients. Accordingly, this cell fraction can be considered as the most specific and active antigen for enzyme-linked immunosorbent assay.

The invention is aimed at obtaining diagnostic indicators, allowing with a high degree of reliability to testify about a disease (pathology) of an autoimmune nature. The proposed method allows to identify and diagnose autoimmune pathologies in case of impaired reproductive apparatus in women, as well as for systemic diseases in women. When using this antigen, it was possible to minimize the phenomenon of "immunological crossover", in which there is a concomitant reaction with antigens of other tissues. Accordingly, the risk of an erroneous diagnosis of autoimmune syndrome is reduced due to the simultaneous detection of autoantibodies to tissues of other organs.

The solution of this problem is based on traditional TIFA and is implemented due to a fundamentally new approach to the method of producing antigen based on the isolation of isolated stromal cells from samples of normal human endometrial tissues and the subsequent receipt of a microsomal fraction from them. This approach allows you to determine the number of antibodies specific only to the antigen of stromal cells of the endometrium, which makes it possible to clinical laboratory diagnosis of autoimmune syndrome.

The target antigen isolated in the manner described above is a microsomal fraction of stromal cells, to which the vast majority of autoantibodies are specific. The interaction of autoantibodies of this specificity with the antigen of the microsomal fraction of stromal cells is one of the causes of the autoimmune syndrome in endometriosis. The presence of these autoantibodies is an important diagnostic feature. The use of the antigen obtained by the described method can reduce the level of non-specific reactions.

Conventional enzyme-linked immunosorbent assay (TIFA) involves the chemical binding of an antigen to the surface of a microplate, followed by placement of a biological fluid containing antibodies on a microplate, incubation, color reaction and spectrophotometric evaluation of color reaction parameters. In this case, the target antigen is obtained by enzymatically hydrolyzing samples of normal human tissues containing endometrial stromal cells. The enzymatic hydrolysis reaction is stopped by the addition of a chelating agent. The resulting mixed cell suspension is centrifuged in a continuous density gradient, whereby stromal cells are isolated. The microsomal fraction of stromal cells is used as the target antigen, which is covalently linked to the surface of the wells of the microplate in the selected dilutions, then calibration material prepared on the basis of the standard preparation of antibodies specific for the target antigen is added to the series of wells in another series of wells - the test samples biological fluid, and after carrying out traditional TIFA, a calibration curve is constructed from spectrophotometric indicators and the concentration is determined from it specific antibodies.

This method allows you to establish a "basic" (normal) level of autoantibodies specific for antigens of stromal cells of the endometrium, as well as to quantify in arbitrary units (E / ml) the level of concentrations of natural (natural) autoantibodies to endometrial antigen in women. The level of such antibodies above the "baseline" indicates an autoimmune disease (pathology). The establishment of a high level of specific autoantibodies (concentration of autoantibodies above 200-250 U / ml) is the basis for the diagnosis of autoimmune disease, usually associated with endometriosis.

The target antigen isolated and immobilized in the manner described above is a microsomal fraction of endometrial stromal cells (MFSC) adsorbed in the wells of a microplate, which are capable of binding specific antibodies in an experiment, and an antigen of constant chemical composition and molecular structure can be obtained repeatedly. In addition, it becomes possible to simultaneously examine a large number of samples (up to 40 tests), as well as obtain relatively large (up to 600 mg) quantities of the finished antigen and its long-term storage in the frozen state.

Figure 1 presents a diagram of the application of calibration material in the wells of the microplate. Figure 2 presents an example of constructing a calibration curve.

The method is as follows.

Samples of normal (without visible anatomical and histological pathology) human endometrial tissues obtained during diagnostic examinations, and frozen are subjected to thawing and then enzymatic hydrolysis. Hydrolysis is carried out as follows: tissue samples are incubated with collagenase (purified bacterial collagenase is a commercial preparation “collalysin”, pr-research institute of vaccines and serums, St. Petersburg, Russia). Incubation is carried out in a thermostat at 37 ° C for 60-80 minutes until visual signs of maceration of the samples appear. After the appearance of signs of maceration, enzymatic hydrolysis is continued for another 10 minutes, after which the reaction is stopped by the addition of ethylene diamine tetraacetate - disodium salt (EDTA-2Na) to a final concentration of 1%. Macerate with a solution of EDTA-2 Na is incubated for 20 minutes at room temperature with constant stirring, after which the macerate is washed with standard buffered saline (PBS). The mixed cell suspension is forced through a 220 μm diameter nylon filter and concentrated by centrifugation for at least 10 minutes at 100 g. To separate the epithelial and stromal cells of endometrial tissues, a continuous density gradient is prepared on the basis of stock solutions of ficoll: A and B with a concentration of 10 and 15% ficoll, respectively, on standard PBS. Lamb serum is added to each mother liquor to a final concentration of 5%. Using a mixer "GM" create a solution of ficoll with a continuous distribution of the density gradient. The gradient is stabilized by centrifugation for one hour at 5000 g. The mixed cell suspension is applied to the surface of the ficoll solution and the cells are separated by centrifugation for 2.5 hours at 10,000 g. During centrifugation, epithelial cells are concentrated mainly in the upper layer of the gradient due to lower density, and stromal (more dense) cells are concentrated in the lower one. After centrifugation, approximately 1/5 of the volume is taken from the bottom of the “column” of the density gradient, the selected material is washed three times with PBS with the addition of five percent lamb serum. The resulting suspension of stromal cells is then subjected to destruction by repeating the procedure "freezing-thawing", which is carried out by alternately immersing the capsule with a suspension of cells in liquid nitrogen and water at room temperature at least ten times. The resulting suspension of cell components is further centrifuged. In this case, the microsomal fraction of stromal cells of the endometrial tissue remaining in the supernatant is separated, which is used as the target antigen in the determination of autoantibodies specific to endometrial tissue.

The solution of the target antigen is fixed by adding formaldehyde to a final concentration of 2.5% and then, if necessary, stored in a 50% glycerol solution on PBS at a temperature of 20 ° C.

Obtained as described above, the target antigen is covalently bound to the surface of the wells of a microplate according to generally accepted methods [Ngo TT, Lenhoff G. (ed.) Enzyme-linked immunosorbent assay. Per. English, Moscow, Mir, 1998, pp. 172-192. [one]]. To immobilize the obtained antigen on the solid phase, the microsomal stromal cell fraction (MFSC) used as antigen is diluted with PBS to a final concentration of 1 μg / ml total protein and 100 μl of MFSC are added to the wells of the TIFA microplate. The solution is left for two hours in the refrigerator at a temperature of + 4 ° C, then glutaraldehyde is added to the wells to a final concentration of 5%. After that, the microplate with immobilized antigen is left at a temperature of + 4 ° C overnight (10-12 hours) for complete binding. The enzyme-linked immunosorbent assay is carried out in the traditional way [[1], pp. 193-209]. As the studied biological fluid, in addition to blood serum, amniotic fluid, cerebrospinal fluid, etc. can be used.

Reagents and consumables used during TIFA:

1. Microplate for conducting an enzyme-linked immunosorbent reaction with applied antigen, the preparation of which is described above.

2. A standard solution for washing the microplate (PR), which is prepared as follows: to PBS add a nonionic detergent tween-20 to a final concentration of 0.05%.

3. A standard antibody preparation is prepared from pre-selected blood sera of patients who have antibodies that are specific for the target antigen, exhibiting an optical density of at least "0.9" in TIFA. Selected samples of human blood serum are poured together (make a "pool") and the immunoglobulin fraction is isolated by ion exchange chromatography according to the generally accepted methodology [X. Fremel (ed.) "Immunological methods". Per. German, M., Medicine, 1987]. Then, the method of successive dilution [[1], pp. 222-224] determines the minimum number of specific antibodies that give a reaction in TIFA with 10 ng of the target antigen bound to the surface of the microplate. The antibody amount thus determined is designated as the unit “E” of the standard antibody preparation. The immunoglobulin fraction is adjusted to a concentration of 25,000 U / ml and used as a standard preparation of antibodies specific for the target antigen.

4. Enzyme-linked conjugate (IFC) - monoclonal antibodies labeled with horseradish peroxidase with specificity for immunoglobulins of the corresponding class (IgG, IgM, IgA) or all classes (specificity for immunoglobulin kappa and lambda) are used as an enzyme-linked conjugate. The analysis uses monoclonal antibodies produced by the biotechnology laboratory of monoclonal antibodies of the Central Radiological Institute (TsNIRRI, St. Petersburg).

5. The diluent is a solvent (DL) for the dilution of calibration material prepared on the basis of a standard preparation of antibodies, samples of the studied biological fluids and enzyme immunoassay. DL is prepared by adding sterile horse serum to PR to a final concentration of 1%.

6. Substrate mixture - any chemical substrate for the manifestation of an enzymatic peroxidase reaction, for example, the commercial preparation tetra-methyl-benzidine (TMB) in solution.

Preparation of reagents and test samples for work:

1. Preparation of calibration material: a standard antibody preparation with specificity for IFSCs is diluted 25 times (for example, using a single microplate, 10 μl of a standard antibody preparation is added to 250 ml of DL) and then, by successive dilutions, two times respectively receive samples for calibration of 1000 ; 500; 250; 125; 62.5; 31.5 and 15.6 U / ml.

2. The test samples are diluted 25-100 times depending on the concentration of antibodies in specific biological fluids, for example, blood serum samples are diluted 50 times, that is, 5 μl of serum is diluted in 250 μl of DL.

3. Preparation of the IFC working solution: the initial commercial IFC solution is diluted with DL according to the manufacturer's recommendations. For example, the used CPI from ZNIRRI are diluted 150 times; when using one microplate in an experiment, 10500 μl of DL are added to 70 μl of a commercial solution.

4. A working solution of a substrate mixture for the manifestation of a color enzymatic reaction is prepared immediately before use by mixing a solution of tetra-methyl-benzidine (TMB) and a solution of hydrogen peroxide (for example, when using reagents manufactured by Protein circuit JSC (St. Petersburg), equal volumes are mixed Reagent and Substrate solutions).

5. As a reagent that stops the color reaction (stop solution), use a 9% solution of hydrochloric acid.

Carrying out TIFA:

1. 100 μl of calibration material are added to the wells of the microplate of vertical rows 1 and 2 in an increasing order of concentrations: 0 (blank sample - pure DL); 15.6; 31.5; 62.5; 125; 250; 500 and 1000 U / ml (see figure 1). Moreover, the material of each concentration is introduced in parallel into two adjacent wells of the same vertical row, for example, calibration material 15.6 U / ml is introduced into the wells "C" and "D" of row "1". In the wells of the vertical rows 3 to 12, 100 μl of diluted samples of the studied biological fluids are also added in pairs in parallel, for example, one sample is introduced into the wells “A” and “B” of the vertical row “3”.

2. The microplate with the introduced calibration material and the test samples is covered and incubated in a thermostat at 45 ° C for one hour.

3. The contents of the microplate wells are removed by sharp shaking and the wells are washed six times with a solution of PR.

4. Into all wells of the microplate add 100 μl of a freshly prepared working solution of IFC, cover the microplate and incubate in an incubator at 45 ° C for 40 minutes.

5. The wells of the microplate are washed similarly to paragraph 3.

6. In all wells of the microplate contribute 100 μl of the finished working solution of the substrate mixture for the manifestation of a color reaction.

7. The microplate is placed in a dark place and incubated at room temperature for approximately 10-15 minutes until the visible visual manifestation of "stepwise" color in the wells with calibration material. The remaining wells show color depending on the amount of specific antibodies contained in the samples.

8. The color reaction is stopped by adding 50 μl of stop solution to each well.

9. The results of the color reaction are quantified using a vertical optical absorber (for example, "Uniplan", JSC "Picon", Russia).

Calculation of results: the concentration of specific antibodies in arbitrary units (E / ml) is calculated using a FIA-CALC type software package (Wallac, Finland) in the mode of constructing a calibration curve in double logarithmic coordinates (Log / Log) with curve alignment according to the spline interpolation method. The results can also be calculated by manually plotting the calibration curve on a double logarithmic scale, followed by finding the desired results on the graph (see figure 2) [Chard T. Radio-immune methods, M., Mir, 1981].

Laboratory studies conducted on the basis of the Research Institute of Obstetrics and Gynecology. DO Otta RAMS (St. Petersburg) showed that the “baseline” level of normal autoantibodies to the target antigen in healthy women is 200-250 U / ml, and in men, 100-150 U / ml, respectively.

Example 1

Patient Polyakova AB, 40 years old, case history of NPO NIIAG named after D.O. Ott RAMS 5840/02. Clinical diagnosis: secondary infertility, external genital endometriosis 1, uterine fibroids, autoimmune thyroiditis (AIT), ovarian failure (NLF), autoimmune endometritis. The level of autoantibodies to the endometrial antigen is 450 U / ml. After complex therapy: coagulation of foci of endometriosis and conservative myomectomy. Gonadoliberin agonist therapy (Buserilin for 4 months), AIT and NLF replacement therapy, intrauterine laser therapy. The level of antibodies to the target antigen decreased: 255 U / ml.

Example 2

Filippova E.N., 25 years old, medical history NPO NIIAG named after D.O. Ott RAMS 2510/04. Clinical diagnosis: primary infertility, external genital endometriosis 3. The level of autoantibodies to the target antigen is 915 U / ml. After complex therapy: coagulation of foci of endometriosis and therapy with gonadoliberin agonists (Zoladex for 3 months), intrauterine laser therapy. The level of antibodies to the target antigen decreased: 195 U / ml.

Claims (2)

1. The method of quantitative determination of the concentration of antibodies specific for the antigen of stromal cells of human endometrial tissue in human biological fluids containing specific antibodies by conducting an enzyme-linked immunosorbent assay, including applying the target antigen to a microplate, followed by placing the test biological fluid on a tablet, incubating, conducting color reaction and spectrophotometric evaluation of indicators of color reaction, characterized in that as the initial samples of normal human endometrial tissues are taken on the material for producing the target antigen, which are subjected to enzymatic hydrolysis until visible signs of maceration are observed followed by treatment with a chelating agent, then stromal cells are isolated from the mixed cell suspension by continuous density gradient and subjected to destruction by repeating the procedure " freezing-thawing "at least 10 times, from the suspension of cellular components obtained as a result of destruction By centrifugation, the microsomal fraction of stromal cells of the endometrial tissue is separated, which is used as the target antigen, which is covalently linked to the surface of the wells of the microplate in the selected dilutions, then, during the enzyme-linked immunosorbent assay, calibration material prepared on the basis of a standard preparation of specific to the target antigen of antibodies, in another series of wells - the studied samples of biological fluid, microplate with They are incubated with the calibration material and the test samples, followed by a color reaction and spectrophotometric indices are used to construct a calibration curve by which the concentration of specific antibodies is determined. 2. The method for quantitatively determining the concentration of antibodies according to claim 1, characterized in that a continuous density gradient is prepared on the basis of two stock solutions of ficoll-400 with a concentration of 10% and 15%, respectively, and the gradient is stabilized by centrifugation for one hour at 5000 g.

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