RU2216742C2 - Method for quantitative detecting concentration of antibodies specific to antigens of human steroid-producing cells in human biological liquids containing specific antibodies - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: one should carry out a solid-phase immuno-enzymatic assay (sIEA). Corresponding samples of normal human tissues containing steroid- producing cells undergo fermentative hydrolysis in standard mode till availability of maceration signs. Then tissues should be treated with chelating agent and steroid- producing cells should be isolated by centrifuging in discontinuous density gradient followed by destruction due to repeated procedure of "freezing-thawing out" for not less than 4 times and "thawing out" procedure is combined with ultrasound treatment. By centrifuging one should separate microsomal fraction of steroid-producing cells to be applied as a target antigen. The latter in chosen dilutions is covalently bound with the surface of microplate's holes. Some holes are introduced with calibrating material, other - with samples of biological liquid under testing. The mentioned sIEA is carried out and by spectrophotometric values obtained it is possible to make a calibrating curve to detect concentration of specific antibodies. EFFECT: higher efficiency. 2 dwg, 3 ex

Description

 The invention relates to the field of clinical laboratory diagnostics and may find application in gynecology, andrology and endocrinology for the diagnosis of diseases of an autoimmune nature, or with systemic pathology.

A known laboratory method for diagnosing autoimmune pathologies in women is described in the article "Circulating antiovarian antibodies in premature ovarian failure", Damewood MD, Zacur H., Hoffmann GJ, Rock JA // Obstetrics and Gynecology, New York, Elsevier, 1986, v. 68, 6, Dec., p. 850-854. (Circulating anti-varial antibodies in case of premature ovarian failure, Daimwood M.V., Zakur X., Hoffman G.J., Rock J.A. // Obstetrics and Gynecology, New York. Elsevir, 1986, v. 68, 6, December, pp. 850-854). This method is an indirect immunofluorescence. When implementing this method, the tissue of normal ovaries of women was obtained by laparoscopy. The material was quickly frozen in 100% alcohol with dry ice and sections were made in a cryostat. Slices with a thickness of 6 μm were placed on slides pretreated with 0.5% gelatin. In this case, the sections performing the antigen function were dried for 1-2 minutes and placed for 10 minutes in a glycine buffer solution containing 0.1% gelatin, and then washed with distilled water. Diluted (from 1/4 to 1/32) buffer solution, samples of the studied blood serum containing circulating anti-varial antibodies were applied to a glass slide of 50 μl, incubated for 30 minutes at 37 ^° C in a humid chamber, and then washed 5 times with glycine buffer solution . Next, affinity-purified polyvalent goat antibodies against human immunoglobulins labeled with 1/16 fluorescein-isothiocyanate labeled were added and incubated for 30 minutes, and then washed. Then, a drop of 50% glycerol in a phosphate-buffered saline solution was placed on a glass slide and the drop was covered with a coverslip. The reaction was evaluated visually under a microscope according to the intensity of luminescence in scores from 1+ (weak) and 2+ (moderate) to 4+ (bright), which corresponded to low, medium and high content of specific antibodies. The absence of luminescence was regarded as the absence of antibodies. As a result of the assessment, it was possible to ascertain the presence or absence of anti-ovarian antibodies in blood serum.

 A laboratory method for diagnosing autoimmune pathologies in women is also known, described in the article "Ovarian antibodies detected by immobilized antigen immunoassay in patients with premature ovarian failure" Luborsky JL, Visitin I., Boyers S., Asari T., Caldwell B., DeCherney A .// Joural of Clinical Endocrinology and Metabolism, 1990, vol. 70, 1, p. 69-75, Lippincot, Philadelphia, Pennsilvania, USA. ("Determination of ovarian antibodies in patients with premature ovarian failure by immunoassay with immobilized antigen." Luborski, J.L., Vizitin I., Boyers S., Asari T., Caldwell B., De-Chirni A. // Journal of Clinical Endocrinology and Metabolism, 1990, v. 70, 1, pp. 69-75, Lipincot, Philadelphia, USA). This article describes a method for the study of blood serum of 45 women by conducting an enzyme-linked immunosorbent assay (TIFA). We examined women (normal karyotype, age less than 40 years) with clinical manifestations of ovarian failure (autoimmune pathology), in which menstrual irregularities and / or hormonal deviations were observed. At the same time, blood serum (biological fluid containing specific antibodies to the ovarian antigen) of 19 healthy female volunteers with a normal menstrual cycle, which made up the control group, was examined. Normal ovaries of women (containing ovarian antigen) were obtained during the operation of a hysterotomy.

Reagents and consumables used during TIFA:
1. Microplate for TIFA with antigen adsorbed on the surface of the wells.

 2. Bovine serum albumin (BSA) to block non-specific binding sites.

 3. Standard phosphate-saline buffer solution (FSB), as well as FSB with the addition of 0.05% detergent "Triton X-100" (FSB-T).

 4. Goat antibodies against light and heavy chains of human IgG class immunoglobulins labeled with alkaline phosphatase - enzyme-linked immunosorbent enzyme conjugate (IPA).

 The antigen was prepared as follows.

The ovaries were collected in the FSB, cut into pieces 3-4 mm in size and quickly frozen on dry ice (solid carbon dioxide). In an acetone bath with dry ice, ovarian homogenate was prepared by grinding pieces of tissue to a powder state. The obtained ovarian tissue homogenate was washed twice with a FSB solution, and large aggregates of cellular components were removed by centrifugation. For further use, a suspension of ovarian tissue homogenate was diluted to a final concentration of 0.1 mg / ml total protein. Antigen was adsorbed onto the surface of the microplate wells. Used 96-well microplate for typhoid. After antigen adsorption in the wells, excess ovarian homogenate solution was removed by vigorous shaking of the microplate, and nonspecific binding sites were blocked with a 3% BSA solution in wash buffer (0.15 ml, 16 hours; 4 ^° C).

Carrying out TIFA:
1. 0.1 ml of control blood serum samples were added to a portion of the microplate wells, and blood serum samples of patients with ovarian insufficiency were added to the remaining wells.

2. Samples were incubated for 0.5-2 hours at 22 ^o C.

 3. The contents of the microplate wells were removed by sharp shaking and the wells were washed twice with PBS-T and four times with distilled water.

4. In all wells of the microplate were added 50 μl of IFC and incubated at 22 ^o C for 2 hours.

 5. The contents of the wells were removed and the wells of the microplate were washed in the same manner as in claim 3.

 6. A color reaction was carried out by adding a specific substrate of alkaline phosphatases: 1 mg / ml p-nitrophenyl phosphate in a 1 M solution of diethanolamine with the addition of 0.24 mm magnesium chloride at a pH adjusted to 9.8.

 7. The results of the color reaction were measured using a vertical optical absorber at a wavelength of 405 nm, the indicators of color reaction were expressed in units of optical density (OD).

 Evaluation of the results was carried out, taking the OD of the samples of blood serum of healthy women for the value of "cut off", that is, samples having an OD index below this value were taken as negative and denoted by "-"; whereas samples in which this indicator exceeded “cut off” were taken as positive and denoted as “+”. The final results were presented in alternative form, that is, were designated as positive or negative (respectively + or -). The OD value = 0.27, according to the authors, should be considered "borderline" and samples with higher OD values were considered positive, that is, the presence of specific antibodies was recorded in such samples. The described method, adopted for the closest analogue, as well as the analogue, makes it possible to ascertain the presence or absence of specific antibodies in the blood serum, but not to measure their concentration.

 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 makes it possible to identify and diagnose autoimmune pathologies in cases of impaired development of the sexual and endocrine spheres not only in women, but also in men, as well as systemic diseases in people of both sexes.

 The solution to this problem is based on traditional TIFA and is implemented through a fundamentally new approach to the method of producing antigen, based on the isolation of steroid-producing cells responsible for immune reactions from tissue samples, followed by additional purification of subcellular fractions, as well as by applying the construction principle calibration curve by serial dilution method.

 Traditional ELISA involves chemically binding the antigen to the surface of the microplate, followed by placing the biological fluid containing the antibodies on the microplate, incubating, performing a color reaction and spectrophotometric evaluation of the color reaction. In this case, the target antigen is obtained by enzymatically hydrolyzing the corresponding samples of normal human tissues containing steroid-producing cells: the ovarian antigen is obtained from the ovaries of women, the testicular antigen from the male glands, the adrenal antigen from the adrenal cortex. The enzymatic hydrolysis reaction is stopped by the addition of a chelating agent. The obtained mixed cell suspension is centrifuged in a stepwise density gradient, as a result of which steroid-producing cells are isolated, which are then subjected to destruction by repeating the freeze-thaw procedure at least 4 times and the thawing procedure is combined with ultrasound treatment. The suspension of cellular components obtained after destruction is centrifuged to precipitate ballast cell fractions, and the microsomal fraction contained in the supernatant is used as the target antigen, which is covalently linked to the surface of the wells of the microplate in the selected dilutions, and then calibration material prepared for the basis of the standard preparation of antibodies specific for the corresponding target antigen; in another series of wells, the studied samples of biological After carrying out traditional TIFA, a calibration curve is constructed from spectrophotometric indicators and the concentration of specific antibodies is determined from it.

 This method allows you to establish a "basic" (normal) level of autoantibodies specific for antigens of steroid-producing human cells, as well as to quantify in arbitrary units (E / ml) the level of concentrations of natural (natural) autoantibodies to antigens of ovarian follicular cells in women ( ovarian antigen), Leydig cells in men (testicular antigen), and steroid-producing adrenal cells (adrenal antigen) in patients of both sexes. The level of such antibodies above the "base" indicates an autoimmune disease (pathology). The establishment of a high level of specific autoantibodies serves as the basis for the diagnosis of autoimmune oophoritis in women, respectively (the concentration of antibodies to the ovarian antigen is higher than 260 U / ml), autoimmune orchitis in men (the concentration of antibodies to the testicular antigen is higher than 310 U / ml), and the systemic disease is polyglandular syndrome in patients of both sexes (the concentration of antibodies to the adrenal antigen is higher than 350 U / ml).

 The target antigen isolated in the manner described above is a definite and constant set of proteins and glycoproteins that are capable of binding specific antibodies in an experiment, and an antigen of a constant biochemical composition 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 400 mg) quantities of the finished antigen and its long-term storage (up to 1 year) in a frozen state; while the method chosen as the closest analogue requires “piece-wise” production of antigen during each series of experiments due to the immunochemical heterogeneity of the starting material (adrenal tissue, female and male gonads).

 In FIG. 1 is a diagram of the application of calibration material to the wells of a microplate. Figure 2 presents an example of constructing a calibration curve.

 The method is as follows.

Samples of normal (without pathology) tissues containing steroid-producing cells are obtained as a result of surgery - the ovaries of women - by laparoscopy, the male glands of a surgical operation to change sex, and samples of the cortical zone of the adrenal glands - during partial adrenalectomy. Tissue samples are subjected to enzymatic hydrolysis at a standard temperature for this enzyme (37 ^° C for bacterial collagenase) until visible signs of maceration appear (20-35 minutes for samples of female and male gonads, 30-40 minutes for samples of the adrenal glands), after which the reaction is continued 10 minutes more. As an enzyme, purified bacterial collagenase is used (the commercial drug "collalysin" manufactured by the Research Institute of Vaccines and Serums, St. Petersburg, RF). The enzymatic hydrolysis reaction is stopped by the addition of a chelating agent and incubated for 30 minutes with constant stirring. As a chelating agent, ethylene diamine tetraacetate disodium salt (EDTA-2Na) is used in a concentration of 0.5%. The resulting mixed cell suspension is forced through a nylon sieve, concentrated by centrifugation, and then centrifuged in a stepwise gradient of medium density, whereby steroid-producing cells are isolated. Isolated steroid-producing cells are destroyed by repeating the freeze-thaw procedure at least 4 times and the thawing procedure is combined with ultrasound treatment. For an optimal yield of the target antigen, repeating the above procedure 10 times is sufficient. After destruction, the ballast cell fractions are precipitated by centrifugation, and the microsomal fraction in the supernatant is used as the target antigen.

 For the formation of a stepwise density gradient, solutions of ficoll-400 of various concentrations based on FSB with the addition of horse serum to a final concentration of 1% are used (it is also possible to use percoll or another similar polymer reagent). The procedure for isolating the cells of the granulosa layer of the female follicles is carried out at 5000g for 2 hours in a 14% solution of ficoll-400, isolation of Leydig cells of the male gonads at 5,000g for 2 hours in a 13% solution of ficoll-400, and the steroid is isolated -producing cells of the human adrenal gland at - 4500g 2.5 hours in a 15% solution of ficoll-400. These centrifugation modes provide the most effective isolation of cells of the corresponding type.

Obtained as described above, the target antigen is covalently bound to the surface of the wells of the microplate according to generally accepted methods [Ngo TT, Lenhoff G. (ed.) Enzyme-linked immunosorbent assay. Per. English., M., "World", 1988, p. 172-192, / 1 /]. For binding to the surface of the wells, the target antigen is diluted with 1% ammonium bicarbonate solution to a final concentration of total protein: ovarian antigen - up to 1 μg / ml, testicular antigen - up to 2 μg / ml and adrenal antigen - up to 500 ng / ml. A 0.25% glutaraldehyde solution is used as a binding (crosslinking) bifunctional agent. After completion of the binding reaction, the microplate is washed and dried at 37 ^{° C.} for 3 hours. The enzyme-linked immunosorbent assay is carried out in the traditional way [/ 1 /, p. 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 a coated antigen, the preparation of which is described above;
2. A standard solution for washing microplates (PR), which is prepared as follows: to a standard solution of FSB 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 detected antibodies specific for the corresponding antigen (ovarian, testicular or human adrenal antigen), showing 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 method [X. Fremel (ed.). "Immunological methods." Per. German, M., "Medicine", 1987]. Then the method of successive dilutions [/ 1 /, p. 222-224. ] determine the minimum number of specific antibodies that gives a reaction in TIFA with 10 ng associated with the surface of the microplate of the corresponding antigen. 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 of appropriate specificity.

 4. Enzyme-linked immunosorbent conjugate (IFC) - monoclonal antibodies labeled with horseradish peroxidase with specificity for immunoglobulins of the corresponding class (IgG, IgM, IgA) or all classes (specificity for kappa and lambda-chains of immunoglobulins) are used as an enzyme-linked conjugate. The analysis used monoclonal antibodies produced by the Biotechnology Laboratory of Monoclonal Antibodies of the Central X-ray and Radiological Institute (TsNIRRI, St. Petersburg, RF).

 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 of ortho-phenylene-diamine (CRF) in 5 mg tablets.

Preparation of reagents and test samples for work:
1. Preparation of calibration material: a standard preparation of antibodies of appropriate specificity is diluted 25 times (for example, when using one microplate, 10 μl of a standard preparation of antibodies is added to 250 μl of DL) and then samples for calibration of 1000 are obtained by successive dilutions of 2 times; 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 blood 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; the used IFCs from TsNIRRI are diluted 150 times, for example, when using 1 microplate in the experiment, 10500 μl of DL are added to 70 μl of the commercial IFC solution.

 4. Preparation of the substrate mixture: the substrate mixture is prepared according to the manufacturer's recommendations, for example, when using OFD to prepare the working solution, one tablet of OFD in 5 mg is dissolved in 10 ml of 0.1 M citrate buffer solution (pH 5.0) and 70 μl of standard 5% perhydrol solution.

Carrying out TIFA:
1. In the wells of the microplate of vertical rows 1 and 2, 100 μl of calibration material are added in an increasing order of concentrations: 0 (blank sample is pure DL); 15.6; 31.5; 62.5; 125; 250; 500 and 1000 U / ml (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 wells “C” and “D” of row “1”, etc. In the wells of the vertical rows 3 through 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” (Fig. 1).

2. The microplate with the introduced calibration material and the test samples is covered and incubated in a thermostat at 45 ^o C for 40 minutes.

 3. The contents of the microplate wells are removed by sharp shaking and the wells are washed 6 times with PR.

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

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

 6. 100 μl of a freshly prepared (immediately before use) working solution of a substrate mixture is added to all wells of a microplate to display 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 9% sulfuric acid to each well.

 9. The results of the color reaction are measured quantitatively using a vertical optical absorptiometer (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 (figure 2) [Chard T. Radio-immune methods, M., Mir, 1981].

 Laboratory studies conducted at the Research Institute of Obstetrics and Gynecology, Russian Academy of Medical Sciences D.O. Otta (St. Petersburg) and the Medical Academy of Postgraduate Education (St. Petersburg), showed that the "basic" level of normal autoantibodies in healthy people is 260 U / ml for ovarian antigen, 310 E / for testicular antigen ml and for adrenal antigen 350 U / ml.

 Example 1. Patient K., an anamnesis - from the age of 15 was observed, examined and treated by an endocrinologist, a preliminary diagnosis was delayed sexual and physical development, and from 16 years old he received chorionic gonadotropin as a therapy. The results of laboratory studies of hormonal levels within normal limits. Functional test with chorionic gonadotropin is negative (there was no increase in testosterone per test). The content of testicular antibodies is 1398 U / ml. The final diagnosis: autoimmune orchitis against the background of normogonadotropic hypogonadism.

 Example 2. Patient W., age 22 years; complaints during treatment - menstrual irregularities.

 The results of laboratory tests 06/20/2001: the concentration of ovarian antibodies is 324 U / ml. Dexamethasone therapy has been prescribed. Repeated laboratory study 09/20/2001 - 230 U / ml, menstruation with severe pain, the patient is directed to an ultrasound examination of the pelvic organs. Ultrasound findings: polycystic ovary syndrome, probably of an autoimmune nature.

 Example 3. Patient P., 31 years old. Complaints during treatment: discharge - from the mammary glands, pain in the pelvic area. Gynecological diseases: chlamydia, thrush, amenorrhea. The results of a laboratory examination of thyroid function showed a slight excess of thyroglobulin. The patient came on 6/06/2001 for examination. A laboratory examination of 06/26/2001 revealed a high content of ovarian antibodies of 720 U / ml. Assigned therapy with the drug "Wobenzym", the results of repeated laboratory examination for ovarian antibodies - 389 U / ml. The patient is directed to laparoscopy, the final diagnosis on the basis of laparoscopic examination: ovarian failure on the background of autoimmune oophoritis.

 Thus, the proposed method for determining autoantibodies demonstrates a high degree of compliance with the data of clinical and histological diagnostics. In addition, the method allows to obtain comparable quantitative indicators and does not require surgical intervention.

Claims (1)

 A method for quantitatively determining the concentration of antibodies specific for antigens of steroid-producing human cells in human biological fluids containing specific antibodies, including carrying out an enzyme-linked immunosorbent assay by applying antigen to a microplate, followed by placing the test biological fluid on a plate, incubating, carrying out a color reaction and spectrophotometric assessment of the color reaction, characterized in that the target antigen of steroid-producing cells human ok is obtained by subjecting appropriate samples of normal human tissues containing steroid-producing cells to enzymatic hydrolysis in a standard mode until visible signs of maceration appear, followed by treating the tissues with a chelating agent, steroid-producing cells are isolated from the obtained mixed cell suspension by centrifugation in a stepwise density gradient which are subjected to destruction by repeating the procedure of "freezing-thawing" at least four times and the procedure of "thawing" with sonicated, the microsomal fraction of steroid-producing cells is separated from the suspension of cell components by centrifugation, which is used as the target antigen, which is covalently linked to the surface of the microplate wells in the selected dilutions, then the calibration material is introduced into the series of wells in the specified dilutions , prepared on the basis of a standard preparation of antibodies of the corresponding specificity, into another series of holes - the studied samples are biologically th liquid, and then carrying out an ELISA calibration curve and the concentration of specific antibodies on it by spectrophotometric indicators.

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