Method of diagnosing reproductive function in men

FIELD: medicine.

SUBSTANCE: patient's blood serum is analysed by ELIZA, determining concentrations of antibodies, specific to components of male reproductive tissues. As components of male reproductive tissues, used is nuclear antigen of pachytene spermatocytes of rodents. If values of antibody concentration, expressed in units of optic density, equals or is higher than 0.37, presence of reproductive dysfunction in men is diagnosed.

EFFECT: invention makes it possible to obtain accurate evaluation of presence of reproductive dysfunction in men due to elaborated accurate quantitative diagnostic criterion.

10 ex

 

The invention relates to medicine, to methods of analysis of biological materials, and can be used for the diagnosis of reproductive disorders in men in urology, andrology, dermatology, reproductive medicine and surgery, Oncology, for monitoring the genetic consequences of natural and anthropogenic impacts, in veterinary medicine and animal husbandry.

Traditionally for assessment of reproductive function in particular of spermatogenesis know the use of the 4 approaches.

First the traditional approach long used in medicine, is a semiological analysis of the ejaculate, which practically characterize the concentration, motor activity and morphology of sperm, white blood cells and other somatic cells (see, for example SU 1330567, 15.07.1987). The disadvantage of this method is the inability when performing a semiological analysis of the ejaculate to get information about physiology or violation of the passage of reproductive cells through all the successive stages of spermatogenesis in General.

The second traditional approach for assessing different stages of spermatogenesis is a histological analysis of drugs of sliced biopsy of the testis with the definition of the characteristics of the cycle of the spermatogenic epithelium, the calculation of the index germ cells relative to the number of Sertoli cells (see for example the EP EN 2199117, 20.02.2003). The analysis is carried out after removal of a fragment of the testis (biopsy) and its fixation by dehydration piece of testicular biopsy in ascending alcohols concentration and xylene, fill in paraffin, making sections and staining with subsequent microcapillaries histological preparations of testicular biopsy. The disadvantage of this method is: injury of the testis when obtaining surgical biopsies, the development of an autoimmune process in the testis and impaired spermatogenesis and fertility; the cooking time of historiarum of testicular biopsy is 3-5 days; the results of the analysis of spermatogenesis get on the local site of the testis (biopsy)and cannot be extrapolated to all the eggs in whole or in both testicles, because they often run local processes. In addition, a biopsy of the testicle is allowed only for patients with azoospermia or oligozoospermia severe (who, 1982, 1999). Such patients are not more than 30% of the number of men seeking professionals about the violation of spermatogenesis and infertility. That is, about 70% of men do not have the ability to obtain qualified information about the causes and extent of disturbance of spermatogenesis up to its final stage - up of sperm.

The third approach for assessing different stages of spermatogenesis is cytological analysis of germ cells ejaculate on which all the successive stages of their development by determining the number of degenerating gametes (see for example EN 2328736, 10.07.2008).

The analysis is carried out after sampling germ and somatic cells of the sediment was centrifuged semen treated for one hour with a solution of 0.55-0,56% potassium chloride at a temperature 37,0 was 37.1°C; their subsequent fixation in a mixture of methanol alcohol and glacial acetic acid with the addition of 0.1-0.2 ml of chloroform. The product is dried and stained by the dye from the Institute, followed by microscopy. The disadvantage of this method is the complexity of the method, the quality of the preparation and interpretation of the results of the analysis depend on the skill of a technician, a small number of drugs that can analyze laboratory during the working day, the use of highly active poison (methyl alcohol), in the drying of the drug over the flame of a gas or alcohol burner easy cells to burn that will affect the analysis results.

The fourth approach - immunochemical associated with detection of antigens of sperm and sperm antibody associated with infertility. There are several antigens (AG) sperm antibodies which have agglutinins and immobilsarda sperm activity. They are all strong immunogenum, can cause the formation of antisperm antibodies (AST)is associated with clinical infertility, red eye reduction is affected fertility in experimental animals or influencing the process of fertilization in vitro. Suggest that immunological infertility is possible due to the combined effects of different antibodies to many antigens of sperm. Identifying and exploring private AG sperm serves the purpose of understanding the damage functions of sperm and mechanisms of fertilization, as well as towards the development of commercially available test kits for the detection of antigen-specific AST.

The method adopted by us for the prototype, characterized by obtaining the target antigen nuclear components of reproductive tissues from the tissue of the testes of young male albino rats, allocating kernel pachytene of spermatocyte, and sediment pachytene cores are extracted antigen. To isolate a target antigen suspension pachytene cores resuspending, are lysed hydralicious the chromatin, cleaned from coarse debris by centrifugation, and the supernatant subjected to ultracentrifugation in a stepwise sucrose gradient purification synaptonemal complexes, which are used as the target antigen, which covalently bind to the surface of the wells of the microplate, and then in a series of holes make a measured sample of biological fluid, which is a blood serum of a patient, and after the enzyme-linked immunosorbent assay for the spectrophotometric indices determine the optical tightly the TB samples (Gomberg M.A. Male infertility as a result of a breach of hepatolenticular barrier when HEE / Gomberg M.A., Anisova I.N., Dadashev, SJ // news of dermatology and venereology of the South Caucasus. - 2007. No. 1(4). - P.6-10).

The disadvantage of this method is the lack of accurate quantitative criteria for inclusion of patients in the group with impaired reproductive function. In addition, the applied method allows to diagnose disorders of reproductive function in a limited number of persons, namely in patients with urogenital chlamydial infection in history.

The technical objective of the proposed method of reproductive disorders in men is a further development of the methodology, which consists in producing accurate quantitative criteria for inclusion of patients in the group with impaired that allows you to develop on the basis of the way full automated assessment system, regardless of the qualifications of the laboratory; to expand the number of diagnosed diseases / conditions.

The solution of this problem is based on a fundamentally new approach to the method for the diagnosis of reproductive disorders-conduct typhoid and implemented by obtaining specific nuclear antigen from the testes of young male albino rats.

Detailed description of the invention.

1. Obtaining nuclear EN is Egan.

The target antigen receive, exposing enzymatic hydrolysis pachytene core spermatocyte from the testes of young male albino rats. Nuclear antigen spermatocyte rodents prepared as follows: allocate kernel pachytene of spermatocyte from the testes of young male albino rats weighing 50-70 g according to the standard technique (see, in particular, Horac GG, Safronov CENTURIES, Kolomiets O.L., Bogdanov Û.F. Biochemical and ultrastructural analyses synaptonemal complex in spermatocyte mammals. Cytology. - 1985. - T.XXVII. No. 12. - S-1352), from sediment pachytene cores are extracted antigen, resuspended in 10 ml of 0.1 mm MgCl2in 0.01 M Tris HCl (pH 7.4); then add 90 ml of a solution containing 5 mm EDTA and 0.01 M Tris-HCl (pH 8.5) and incubated for 5-10 min at room temperature, then add the DNA-ABC II (DNA II, 200-300 Kunitz units in 1 mg, Serva) of 85 units per 1 ml of suspension and incubated for 60 min at room temperature, periodically shaken; then the sample is centrifuged at 1000 rpm for 10 min at 4°C, 15 ml of the supernatant is applied on a step gradient of sucrose 1,5/1,8/2,0 M in 0.01 M solution of Tris-HCl (pH 7.4) and centrifuged at 80000 rpm for 90 min in the rotor SW-27.1; synaptonemal complexes are concentrated at the boundaries of layers 1,5/1,8 1,8/2,0 M Selected as described above, the target antigen is a definite the th and permanent set of proteins synaptonemal complex, who are able to experience to bind specific antibodies, and the antigen permanent biochemical composition can be obtained repeatedly. In addition, you can survey a large number of samples (up to 40 tests), and obtaining large quantities of prepared antigen and its long-term storage (up to 1 year) in a frozen state.

2. Application of antigen on the surface of the wells of the microplate.

Obtained as described above, the target antigen is covalently associated with the surface of the wells of the microplate according to the conventional methods (NGOs TT, Lenhoff, (as amended) Enzyme-linked immunosorbent assay. TRANS. eng. M.: Mir, 1988, p.172-192). To associate with the surface of the hole the target antigen diluted 1% solution of ammonium bicarbonate to a final protein concentration of 10 ng/ml Then the remaining binding sites score a 1% solution of bovine serum albumin. As linking (cross-linking) a bifunctional agent using 0.25% solution of glutaraldehyde. After the binding reaction, the microplate is washed and dried at 37°C for 3 hours. The solid-phase procedure immunofermentnogo analysis carried out in the traditional version (NGOs TT, Lenhoff, (as amended) Enzyme-linked immunosorbent assay. TRANS. eng. M.: Mir, 1988, s-209). As the study of the biological fluid can b the th used the serum. Serum get the standard method for doing this, for example, collect peripheral blood from a vein, followed by obtaining blood plasma and removal of fibrinogen by either natural plasma coagulation or precipitation of fibrinogen calcium ions.

For holding typhus were used secondary peroxidase labeled antibodies against mouse IgG (research Institute of epidemiology under them. Nofamily AMS) and normal sales set with replacement microplate prepared as above with carrying typhus according to the instructions. As an example, used a set of firm "Vectorvest" - "a Set of reagents for immunoassay detection of IgG class antibodies to Treponema pallidum.

Conducting immunoassay

All wells of the microplate, add 90 ál solution for dilution of sera (PC). 2 wells add 10 ál of positive control sample (+), 2 wells, and 10 µl of the negative control sample (K-), 1 hole - 10 ál of QC (control conjugate), in the remaining wells and 10 μl of whole of the tested sera, mix by pipetting. The microplate seal with tape and incubate for 30 min at 37°C. For 5-10 min until the end of the incubation to prepare a solution of conjugate working dilution. As a negative control sample ( -) used standard serum fertile patients.

<> After incubation the contents of the wells to collect in a container of disinfectant, rinse the wells of the microplate 5 times with washing solution. To each well add 100 ál of conjugate solution (individule antibodies conjugated with horseradish peroxidase in a working dilution. The microplate seal with tape and incubate for 30 min at 37°C. after incubation wash the wells 5 times with washing solution. To prepare a solution of tetramethylbenzidine (TMB) in your breeding. Then make each well 100 μl of TMB solution into the working dilution. The microplate placed on a 30 min protected from light place at (18-25)°C. Stop the reaction by adding to the wells, 100 μl of stop reagent and immediately measure the optical density (OD).

Registration and evaluation of results

The results of typhoid fever were recorded by a standard method using a spectrophotometer ("DOMBI PLATE", Russia), measuring the optical density (OD) in a two mode: main filter 450 nm, reference filter in the range of 620-650 nm. The removal of the spectrophotometer to zero ("form") is by air.

Analyzed the serum is considered positive if the OD value is equal to or greater than the magnitude of Opcrit, in which the result of the research was defined as 0,37.

Laboratory studies were about the Eden on the basis of the laboratory of cytogenetics, Institute of General genetics him. N.I. Vavilov RAS (Moscow) and in the Krasnodar regional center of family planning and reproduction (Krasnodar). This work was carried out on 56 serum samples infertile and fertile men. The fact is men have previously been estimated using: anamnestic data, semiological analysis, cytological (quantitative karyological analysis of the composition of immature germ cells in semen analysis and immunochemical determination of surface antisperm antibodies (AST) in the serum and spiroplasma.

Statistical processing of the obtained results was performed with the program STATISTICA v.6 (StatSoft, Inc. 2001). Determination of autoantibodies to nuclear components of the male reproductive tissues in high titer in blood samples infertile patients allowed us to identify reliable high OD value in the detection of ASAT in spiroplasma or AST in the serum (68% 71,43%, respectively, the difference being statistically significant (p=0,0004). Determination of autoantibodies to nuclear components of the male reproductive tissues in high titer in blood samples infertile and fertile men revealed that a significant increase in OD is to increase the number of unidentified germ cells, which was confirmed a high statistically significant correlation (r=0,8601 when p=0,019). Defined high way is almost significant correlation stages prelipceanu-zygotene with autoantibodies to nuclear components of the male reproductive tissues (r=1.00 and when p=0.005). Identified violations reflected on the functional and morphological state of Mature spermatozoa.

Examples of implementing the inventive method.

Example 1. Patient Abramov, 31. Marriage - 2; no children; complaint - infertility 6 years, intrauterine insemination - 5 attempts.

Moved urogenital infections: urogenital chlamydia

Semiological analysis: ejaculate volume 2.5 ml, color - grey, the term liquefaction 40 minutes; pH 7.2, the number of spermatozoa in 1 ml of ejaculate 203×106the total number of spermatozoa are 507, 5×106actively motile (a+b) - 70%, live spermatozoa - 77%, normal sperm - 77%, pathological forms - 23%, the cells of spermatogenesis - 1, leukocyte - 1×106. Conclusion: polytopia

Quantitative karyological analysis of the composition of immature germ cells in semen (ADC CDD): CDD Index - 20%; In prelature-zygotene 0%; pachytene 0%; Spermatocyte II + Spermatid - 91%; Narutolatina Spermatid 33%; Reidentify. sex cells - 9%; Cells - 2%; Leucocytes - 8%. Conclusion: violation of the mechanism of divergence of chromosomes in daughter cells during division of sex cells, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in the serum was not detected in spiroplasma is detected. The results of the laboratory is-waves using the claimed method - the optical density of autoantibodies to nuclear components of the male reproductive tissues to 0.39.

Example 2. Sick Gruskin, 31. Marriage - 1; no children; complaint - infertility 4.5 years, intrauterine insemination - 4 attempts.

Moved urogenital infections: urogenital chlamydia - 2 times.

Semiological analysis: ejaculate volume 3.0 ml, color - grayish-whitish, the term liquefaction 45 minutes; pH 7.3, the number of spermatozoa in 1 ml of ejaculate 5×106the total number of spermatozoa 15×106actively motile (a+b) - 44%, live spermatozoa - 97%, normal sperm - 40%, pathological forms - 60%, of the cells of spermatogenesis - 1,6, leukocytes to 0.2×106. Conclusion: Aligote

ADC CDD: Index CPP - 18%; In prelature-zygotene 0%; pachytene 0%; Spermatocyte II + Spermatid - 95%; Narutolatina Spermatid 23%; Reidentify. sex cells 5%; Cells - 1%; Leucocytes - 19%. Conclusion: violation of the mechanism of divergence of chromosomes in daughter cells during division of sex cells, desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in serum is detected, spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissue of 0.6.

Example 3. Sick of evco, 36 years. Marriage - 1; children - none; complaint - infertility 9 years, attempts: extrakorporale fertilization - 4 attempts, intrauterine insemination - 6 attempts.

Moved urogenital infections: genital herpes, ureaplasmosis.

Semiological analysis: ejaculate volume 3.0 ml, color - grayish-whitish, the term liquefaction 30 minutes; pH 7.5, the number of spermatozoa in 1 ml of ejaculate - 69×106the total number of sperm - 207×106actively motile (a+b) - 65%, live spermatozoa - 83%, normal sperm - 78%, pathological forms - 22%, the cells of spermatogenesis - 6, white blood cells and 2.5×106. Conclusion: leukospermia.

ADC CDD: Index CPP - 14%; In prelature-zygotene of 7.5%; pachytene 0%; Spermatocyte II+ Spermatid - 81,5%; Narutolatina Spermatid - 23,5%; Reidentify. sex cells - 11%; Cells - 5%; Leucocytes - 21%. Conclusion: violation of the mechanism of divergence of chromosomes in daughter cells during division of sex cells, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in serum is detected, spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissue - 0,57.

Example 4. Patient Ishchenko, 4 years. Married - 2 children - 1 from 1st marriage; complaint - infertility for 8 years, attempts: extrakorporale fertilization - 1 attempt, intrauterine insemination - 8 attempts.

Moved urogenital infections: genital herpes.

Semiological analysis: ejaculate volume 3.2 ml, color - grayish-white, the term liquefaction 20 minutes; pH 7.4, the number of spermatozoa in 1 ml of ejaculate 62×106the total number of spermatozoa 198,4×106actively motile (a+b) - 81%, live spermatozoa, 94%, and normal sperm - 83%, pathological forms - 17%, the cells of spermatogenesis - 2, leukocytes to 3.2×106. Conclusion: leukospermia.

ADC CDD: Index CPP - 14%; In prelature-zygotene 0%; pachytene 0%; Spermatocyte II+ Spermatid - 97%; Narutolatina Spermatid - 34%; Reidentify. sex cells - 3%; Cells - 14%; Leucocytes - 23%. Conclusion: violation of the mechanism of divergence of chromosomes in daughter cells during division of sex cells, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in serum is detected, spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues of 0.48.

Example 5. Sick Maroulakou, 34 years. Marriage - 1; children - no; complaints when the clicks the drop - infertility for 9 years.

Moved urogenital infections: urogenital chlamydia - 4 times, trichomoniasis.

Semiological analysis: volume of ejaculate 2.0 ml, color - grayish-white, the term liquefaction 20 minutes; pH 7.2, the number of spermatozoa in 1 ml of ejaculate 32×106the total number of spermatozoa 64×106actively motile (a+b) - 59%, live spermatozoa - 70%normal sperm - 82%, pathological forms - 18%, the cells of spermatogenesis - 2,4, leukocytes - 16×106. Conclusion: leukospermia.

ADC CDD: Index CPP - 5.6%, prelature-zygotene of 1.8%; pachytene - 0%; Spermatocyte II + spermacide is 98.2%; Narutolatina Spermatid of 5.5%; Reidentify. sex cells - 0%; Cells - 17%; Leucocytes - 31,5%. Conclusion: partial block of spermatogenesis in prophase of meiosis, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in serum is detected, spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues of 0.48.

Example 6. Patient Shugai, 46 years old. Married - 2 children - 1 from 1st marriage; complaint - infertility for 4 years.

Moved urogenital infections: urogenital chlamydia - 4 times, gonorrhea, trichomoniasis

Semiological analysis: the volume of the ejaculate 2.0 ml, color - grayish-white, the term liquefaction 45 minutes; pH 7.5, the number of spermatozoa in 1 ml of ejaculate 18×106the total number of spermatozoa 36×106actively motile (a+b) - 29%, live spermatozoa - 40%, normal sperm count for 75%of pathological forms - 25%, the cells of spermatogenesis - 2,4, leukocytes and 3.5×106. Conclusion: oligoasthenozoospermia, necrosphere, leukospermia.

ADC CDD: Index CPP - 19%; In prelature-zygotene - 5%; pachytene 0%; Spermatocyte II + Spermatid - 91%; Narutolatina Spermatid - 10%; Reidentify. sex cells - 9%; Cells - 12%; Leucocytes - 32%. Conclusion: partial block of spermatogenesis in prophase of meiosis, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in serum is detected, spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues of 0.62.

Example 7. Patient Savenko, 29 years. Married - 2 children - 1 from 1st marriage; complaint - infertility 3 years, intrauterine insemination - 4 attempts.

Moved urogenital infections: urogenital chlamydia - 1 times, gonorrhea, trichomoniasis.

Semiological analysis: volume of ejaculate 2.0 ml, color - grayish-white, the term liquefaction 20 minutes; pH 7.2, the number with which ermination in 1 ml of ejaculate 12×10 6the total number of spermatozoa 24×106actively motile (a+b) - 69%, live spermatozoa - 88%, normal sperm - 66%, pathological forms of 34%, the cells of spermatogenesis - 0,5, leukocytes to 0.8×106. Conclusion: oligozoospermia

ADC CDD Index: CPP - 4.3 per cent; In prelature-zygotene - 3%; pachytene 0%; Spermatocyte II + Spermatid - 89%; Narutolatina Spermatid - 7,3%; Reidentify, germ cells - 8%; Cells - 17%; Leucocytes - 38%. Conclusion: partial block of spermatogenesis in prophase of meiosis, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in the serum was not detected in spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues was 0.68.

Fertile men and patients whose barren marriage of female factor (control).

Example 8. Patient Barinov, 30 years. Marriage - 1; children - none; complaint - infertility 7 years attempt.

Moved urogenital infections: urogenital chlamydia.

Semiological analysis: ejaculate volume 4.0 ml, color - grayish-white, time dilution of 50 minutes; the pH to 7.4, the number of spermatozoa in 1 ml of ejaculate 106×106the total number of spermatozoa 424×106actively motile (a+b) - 53%, Evie sperm - 58%normal sperm - 83%, pathological forms - 17%, the cells of spermatogenesis - 1.0, leukocytes to 0.4×106. Conclusion: normozoospermia

ADC CDD: the CDD Index is 2.9%; In prelature-zygotene to 1.9%; In pachytene 0%; Spermatocyte II + Spermatid - 90,2%; Narutolatina Spermatid - 6,45%; Reidentify. sex cells - 7,76%; Cells - 28%; Leucocytes - 13,4%. Conclusion: no abnormalities, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in the serum was not detected in spiroplasma - detected.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues to 0.13.

Example 9. Patient Savchuk, 26 years. Marriage - 1; children - none; complaint - infertility 0.5 years.

Moved urogenital infections: urogenital chlamydiosis, mycoplasmosis

Semiological analysis: ejaculate volume 3.0 ml, color - grayish-white, the term liquefaction 40 minutes; pH 7.2, the number of spermatozoa in 1 ml of ejaculate 45×106the total number of spermatozoa 135×106actively motile (a+b) - 65%, live spermatozoa - 58%, normal sperm - 88%, pathological forms - 12%, the cells of spermatogenesis - 0.6 leukocytes to 0.9×106. Conclusion: normozoospermia

ADC CDD: the CDD Index is 2.7%; In prelature-zygotene - 0%; is pachytene 0%; The spermatocyte II+ Spermatid - 93%; Narutolatina Spermatid - 2,6%; Reidentify. sex cells - 7%; Cells - 11%; Leukocytes to 9.5%. Conclusion: no abnormalities, increased desquamation its germinative epithelium.

The presence of antisperm antibodies (AST)in the serum was not detected in spiroplasma - not found.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues to 0.13.

Example 10. Patient Rybalko, 26 years. Marriage - 1; children - 1 from 1st marriage; complaint - infertility 5 years.

Moved urogenital infections: a ureaplasmosis.

Semiological analysis: volume of ejaculate 2.0 ml, color - grayish-white, the term liquefaction 45 minutes; pH 7.3, the number of spermatozoa in 1 ml of ejaculate 42×106the total number of spermatozoa 84×106actively motile (a+b) - 58%, live spermatozoa - 82%, normal sperm - 61%, pathological forms - 39%, the cells of spermatogenesis 0,56, leukocytes to 0.2×106. Conclusion: normozoospermia

ADC CDD: Index CDD - 6%; In prelature-zygotene - 0%; pachytene 0%; Spermatocyte II + Spermatid - 67%; Narutolatina Spermatid - 17%; Reidentify. sex cells - 33%; Cells - 4%; Leucocytes - 9%. Conclusion: no abnormalities, increased desquamation its germinative epithelium.

Under the anti-spermatozoa antibodies (AST)in the serum not found in spiroplasma - not found.

Laboratory results using the inventive method is the optical density of autoantibodies to nuclear components of the male reproductive tissues of 0.25.

Thus, the proposed method for the diagnosis of disorders of reproductive function in men, consisting in the determination of autoantibodies to nuclear components of the male reproductive tissues, demonstrates a high degree of conformity with the data of cytological and semiological diagnosis. This method for the determination of autoantibodies to nuclear components of the male reproductive tissues in serum reveals violations of spermatogenesis, which is manifested in prophase I of meiosis and diagnosis of reproductive disorders in men, allows to obtain comparable quantitative indicators and does not require surgical intervention.

Use of the method of determination of autoantibodies against meiotic nuclear antigens allows you to give an objective assessment of the status of spermatogenesis, simple and fully automated, which allows for a large number of similar studies; to improve the accuracy and efficiency of diagnosis; requires low labor costs; does not depend on the human factor (qualified technician). Noninvasive method of IP which incorporates both the injury of the testis and risk of development of autoimmune complications.

A method for the diagnosis of reproductive disorders in men, including the study of biological fluid by conducting enzyme immunoassay, to determine the concentration of antibodies specific to the components of the male reproductive tissues, expressed in units of optical density (OD), which as a biological fluid investigate the serum of the patient, as components of male reproductive tissues using nuclear antigen pachytene of spermatocyte rodents and when the OD value equal to or exceeding 0,37, diagnose the presence of reproductive function in men.



 

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1 dwg, 7 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: offered is a method for selection of a contingent indicated for diphtheria prophylactic immunisation: vaccination or revaccination. Venous blood serum of a person being tested and a reference venous blood serum sample are examined for levels of specific IgG to diphtheria antitoxin, IgGTDA and IgGRDA respectively. Saliva of the person being tested and a reference saliva sample are examined for levels of specific secretory IgA to diphtheria dialysate antigen, slgATDDA and slgARDDA respectively. The derived values slgATDDA and slgARDDA are compared, and the produced values are also compared thereby. Provided IgGTDA≤IgGRDA and slgATDDA≤slgARDDA, diphtheria prophylactic immunisation is considered to be indicated.

EFFECT: method presents more reliable determination of indications for diphtheria prophylactic immunisation.

FIELD: medicine.

SUBSTANCE: admission laboratory blood plasma examination in the patients with acute calculous cholecystitis is added with evaluating a cholecystokinin level both preoperative one, and on the 9th day following cholecystectomy, and if the examination shows decreasing concentration more than in 2 times, function-type Oddi sphincter dyssynergia is predicted.

EFFECT: invention enables early prediction of developing function-type Oddi sphincter dyssynergia following cholecystectomy.

2 ex, 5 dwg

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

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