RU2107913C1 - Method for performing screening examination of women of productive age by means of eli-p test system to predict embryo/fetus development and birth of healthy or anomalous baby - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves taking blood samples in early pregnancy period or before pregnancy. Blood serum is produced, ELI-P test system (cleaned antigens) is obtained and enzyme immunoassay (solid phase) is carried out to determine immune reactivity of serum auto-antibodies of female organism exerting essential influence upon normal embryo/fetogenesis and directed to antigens participating in processes of fetal tissues and organs development, for instance to protein S 100 as basic myelin protein, stable anion-binding protein of chromatin ABX 14-18, membrane protein MP65. Data on tested sera immune reactivity deviation from normal values level to any of them being obtained, fetus development disorder risk level is determined from the marked deviation degree. EFFECT: availability for mass-scale examination. 9 tbl

Description

 Field of technology: medicine, obstetrics and gynecology, immunology, biotechnology.

 There is growing evidence that the immune system, through organ-specific immune responses (humoral and cellular type), is directly involved in organogenesis at the stages of early embryonic development (V. Abramov, Interaction of the nervous and immune systems. Novosibirsk: Nauka, 1988 ) Accordingly, it can be expected that failures in the mechanisms of immunoregulation caused by various external or internal causes that occur during pregnancy or shortly before pregnancy can be accompanied by impaired formation of individual organs and tissues and the embryo as a whole. As a result of such malfunctions, one can expect the birth of a newborn with one or another congenital anomaly.

 A distinctive feature of the immune system is the ability of immunocompetent cells to synthesize and secrete antibodies to foreign antibodies (e.g., viral, bacterial) into the general bloodstream. In addition, the immune system of each organism produces "natural" regulatory antibodies (autoantibodies) to any antigenic components of its own body. The synthesis of the latter in a healthy organism is maintained within certain limits necessary for the fulfillment of certain regulatory functions by "natural" antibodies, and their hyperproduction (as well as hypoproduction) can lead to the development of certain pathological conditions (AB Poletaev, Autoantibodies as an universal modulators of biological functions. In: Bio-medicine 2000 - Proc. 1th Int. Workshop, Moscow, 1993, 42-48). The foregoing fully applies to the development of embryos and fruits. For example, if mothers shortly before pregnancy or during pregnancy had elevated titers of “natural” antibodies to the protein of nerve cells S100, then their offspring had various kinds of disorders in the development of the nervous system (AB Poletaev, OP Selifanova, Transfer of elevated anti-S100 autoimmunity from mother to their offsprings in rats. Life Sci., 1994, v. 54, No. 18, 1377-1381).

 Purely genetic developmental defects (that is, equally dependent on the mother and father) are the cause of no more than 2-3% of congenital malformations. The remaining 97-98% of congenital pathology does not depend on the state of the father and are determined by various kinds of epigenetic factors that adversely affect the state of the mother-fetus system. Etiological factors here may include infectious diseases of women, severe stresses, and harmful chemical and physical effects such as ionizing radiation and many other reasons. It is important to note that the immune system as a whole of the body reacts most early to the noted potentially harmful effects. It is possible that the increased frequency of birth of children in various congenital malformations in regions belonging to ecologically unfavorable zones is largely caused by environmental-induced disturbances in the immunoregulatory mechanisms of their mothers, since it is the woman who is the connecting and mediating link between the fetus and the environment , and any external influences are primarily reflected in her body. Including, on the amounts of regulatory antibody produced by a woman molecules, which are important for differentiation and morphogenesis of organs and tissues of a developing embryo and fetus.

 As additional factual justifications of the invention, publications may be made which provide data on the availability of internal organs of the forming embryo and fetus for maternal antibodies (Fabian RH, Welch AL, Chen YC, Hulsebosch CE Maternal antibodies access the neonatal CNS. In: Soc. Neurosci. Abstr., 1993, v. 19, pt. 2, p. 1463); on the need for a series of blocking antibodies in maternal serum for successful fetal development (Takeuchi S. Is production of blocking antibodies in successful human pregnancy an epiphenomenon? Amer. J. Reprod. Immunol., 1990, v.24, N 4, p. 108- 119); on growth retardation and fetal growth retardation under the influence of maternal cytotoxic antibodies (Hasegawa I., Takakuwa K., Adachi S., Kahazawa k. Cytotoxic antibody against trophoblast and Lymphocytes present in pregnancy with intrauterine fetal growth retardation and its relation to anti-phospholipid antibody. J. Reprod. Immunol., 1990, v. 17, N2, p. 127-139).

 The above was the basis for the development of the ELI-P test system (from ELISA-detected Probability of Pathology), suitable for the prognostic assessment of the levels of a number of “natural” regulatory autoantibodies of a woman’s body that are important for normal embryo / fetogenesis directed to antibodies directly involved in the processes development of organs and tissues of the fetus.

 No direct analogues of the invention have been identified. As a distant (indirect) analogue, one can consider a method for diagnosing fetal development disorders, performed on the basis of an enzyme-linked immunosorbent assay for alpha-fetoprotein (AFP) in amniotic fluid. Among laboratory methods for diagnosing possible fetal development disorders based on the detection of humoral (molecular) changes, test systems used to determine the content of AFP immunoregulatory protein in the amniotic fluid of a pregnant woman (Pecchio F., Rapellino M., Torre GC, eds) are widely used. ., International workshop on AFP: new trends and perspectives., J. Nucl. Med. Allied Sci., 1989, 33, suppl. 1-137). It has been established that defects in the development of the neural tube of the fetus are usually accompanied by a slight increase in the AFP content in the amniotic fluid. This allows you to use this method as a diagnostic test in obstetrics.

At the same time, the considered method has a number of significant disadvantages:
firstly, the method of determining the level of AFP requires obtaining a sample of amniotic fluid, which is a rather complicated invasive procedure, potentially dangerous for a developing fetus;
secondly, the method is applicable only starting from the stages of the appearance of a sufficiently developed neural tube of the fetus, i.e. quite late (from 15-18 weeks of pregnancy);
thirdly, there are many reasons caused by changes in the body of women, leading to a sharp increase in the content of AFP (primarily various liver diseases - Taketa K., alfa-Fetoprotein: reevaluation in hepatholigy. Hepathology, 1990, 12, 6, 1420-1432 ), not associated with impaired development of the neural tube of the fetus: this can lead to erroneous overdiagnosis of developmental pathology.

 In addition, the method based on the detection of the AFP content does not allow to diagnose other (not associated with impaired neural tube development) forms of pathology.

In contrast to the above method for detecting fetal development disorders, which can be considered as a distant analogue or prototype of the invention, the proposed method allows you to:
use for diagnosis 0.01 ml of a woman’s blood (from a fingertip), instead of a sample of amniotic fluid;
to examine women both in the initial stages of pregnancy (already in the first weeks), and before pregnancy;
suitable for predicting the risk of various forms of pathology of the unborn child, including those not associated with impaired neural tube formation.

 The aim of the proposed method is to increase the reliability of the prognosis of normal or abnormal development of the embryo and fetus based on an assessment of the content in the blood serum (in pregnant women or women examined before pregnancy) of regulatory autoantibodies directed to the components of the ELI-P test system (proteins involved in mechanisms early development), the content of which is critical for the normal development of the embryo and fetus.

The goals are achieved by implementing the essential features of the proposed method, namely as a result of sorption on standard 96-well plates for ELISA a) protein OBM (AG1), b) protein S100 (AG2), c) anionic chromatin proteins ABX 14-18 (AG3 ), d) membrane protein MB 65, applying to them test samples of sera and reference reference serum obtained from healthy individuals, detecting the resulting antigen-antibody complexes using conjugates of secondary (antispecies) immunoglobulins labeled with horseradish peroxidase, the manifestation of the reaction in nkah tablet o-phenylenediamine / H ₂ O ₂ and comparing the level of intensity in the reaction wells containing the test sera to the wells containing the reference serum. For the ELI-P analysis, it is required to obtain 0.01-0.02 ml of a woman's blood serum (for example, from a fingertip).

Antigens used in the ELI-P test system
1. Obtaining the main protein of myelin (antigen 1). Various methods can be used to obtain this component of the test system (for example, described by Deibler et al. - J. Neurochem., 1986, 47, 4, 1219-1225) or ready-made commercially available preparations of the myelin basic protein (for example, a product from CALBIOCHEM) .

 2. The method of obtaining protein S100 (antibodies 2). Various methods can be used to obtain this component of the test system (for example, described by A. B. Poletaev (Doc. Diss., Moscow, 1988), or a commercial protein preparation S100 (for example, CALBIOCHEM company) was used.

 3. Obtained tightly bound chromatin anion proteins (ABC 14-18; antigen 3) 3.1 A fraction of cell nuclei was isolated from fresh cattle brain (for example, the brain is homogenized in 0.15 M NaCl with 0.01 M Tris-HCl pH 7.5 ; centrifuged for 5 min at 1000 g; the resulting precipitate was resuspended in 0.32 M sucrose; centrifuged again for 5 min at 1000 g; resuspension in sucrose / centrifugation solution was repeated 5-7 times).

 3.2. The resulting nuclei were homogenized in 0.2 M NaCl, centrifuged, and the extract containing nucleoplasmic proteins and weakly bound chromatin proteins was discarded. The washed precipitate was homogenized in 0.01 M Tris-HCl buffer pH 8, with 2M NaCl, 2M urea and 0.1% X-100 Triton. It was centrifuged, the supernatant was collected, dialyzed against the same buffer without NaCl, and applied to a DEAE Sepharose column equilibrated with the same buffer. Stepwise protein elution was performed using NaCl gradients on the same buffer. Stepwise protein elution was performed using NaCl gradients on the same buffer. A fraction eluted in the range of 0.3-0.7 M NaCl was selected. After dialysis against the same buffer without NaCl, the proteins were loaded onto the anion exchanger column and eluted in a linear gradient of 0.1 M - 1.0 M KSCN with UV detection at 280 nm. We collected the last peak.

 3.3. According to the SDS-Na gradient (8-20%) polyacrylamide gel electrophoresis, the resulting material gave three discrete intensely stained zone zones characterized by molar masses of 14, 16 and 18 kD. The resulting material was used in the work.

4. Obtaining membrane-bound protein (MB 65; antigen 4)
4.1. A fraction of cell membranes is isolated from fresh cattle brain (for example, the brain is homogenized in 0.15 M NaCl with 0.01 M Tris-HCl pH 7.5; centrifuged for 5 min at 1000 g; the resulting pellet is discarded. Sucrose is added to the supernatant to 0.32 M; centrifuged again for 10 min at 3000 g; the precipitate was discarded; an equal volume of 0.32 M sucrose was added to the supernatant. The procedure was repeated 2-3 times, then the supernatant was diluted 2 times with water and centrifuged for 60 min at 20,000 g. cell membrane precipitate.

 4.2. The resulting membranes were homogenized in 0.01 M Tris-HCl buffer pH 7, with 0.2 M NaCl. It was centrifuged, the precipitate was collected, and washed with 0.01 M Tris-HCl buffer pH 7.5. Homogenize the pellet in 0.01 M Tris-HCl buffer, pH 7.5 with 0.1% Triton X-100 2M urea. Centrifuged for 60 min at 20,000 g. The supernatant was applied to an anion exchanger column equilibrated with the same buffer. Stepwise protein elution was performed using NaCl gradients on the same buffer. A fraction eluted in the range of 0.1-0.3 M NaCl was selected. After dialysis against 0.01% Triton X-100 with 1 M urea in Tris-HCl buffer pH 7.5, the proteins were applied to a Mono-Q column and eluted in a linear gradient of 0.01 M - 0.4 M KSCN c 0, 01% Triton X-100 c with UV detection at 280 nm. We collected the last peak.

 3.3 According to the data of electrophoresis in a gradient (8-20%) polyacrylamide gel with SDS-Na, the resulting material gave one main zone (> 80% protein), characterized by a mol. weighing 65 kD. The resulting material was used in the work.

Indirect ELISA technique
1. For sorption on standard 96-well flat-bottomed polystyrene plates for enzyme immunoassay, solutions of each of the antigen proteins are prepared on carbonate buffer. The prepared solutions are introduced into individual wells of the tablet. Coated plates incubate overnight in the refrigerator at + 2-4 ^o C.

2. Remove (shake) protein solutions from the wells and (without additional washing) fill in blocking buffer (for example, 0.5% gelatin solution in 0.15 M NaCl. Incubate for 1 h at 37 ^o C.

 3. Wash the plates with wash buffer (0.15 M NaCl / 0.05% Tween-20).

4. Application of test sera:
a) In separate bottles or trays for ELISA, prepare dilutions of 1: 200 sera.

 b) add diluted serum to the wells.

c) the covered covers of the plates incubate overnight at + 4 ^o C.

 In our work, we used the following scheme for pouring antigens and test sera into the wells of a plate (Art. Table 1).

 5. Wash the plates with wash buffer.

6. Manifestation of the reaction:
a) the initial solution of horseradish peroxidase conjugate (or another enzyme) with antibodies to human IgG immunoglobulins is diluted to the required number of times (depending on its reactivity) with washing buffer;
b) the conjugate solution is added to all wells of the tablet;
c) the coated plates are incubated for 60-90 minutes at 37 ^° C, or 12-16 hours at + 2-4 ^° C. The conjugate is removed and the tablets washed with washing buffer;
d) immediately after this, a substrate solution (0.01-0.05% o-phenylenediamine in 0.001-0.001% hydrogen peroxide in 0.01-0.05 M citrate-phosphate buffer in the case of peroxidase conjugates) is added to the wells. Incubate the tablets in the dark for 15-20 minutes at room temperature.

 9. Upon reaching the optimal level of staining (after 10-20 minutes of incubation in the dark), the reaction is stopped by adding 0.2-0.4 M sulfuric acid to each well.

 10. Registration of the results of the reaction: the intensity of the staining is evaluated using an ELISA reader of any model.

11. Processing of received data:
a) from several (usually three) duplicating values of the results of the reaction of the reference and test sera, the average is selected (in the case if of 3 takes one is very different from the other two, i.e. it is obviously an artifact, its value is not taken into account) ;
b) the obtained values of the optical density of the contents of the wells calculate the relative intensity of the reaction of the tested sera with proteins (1), (2), (3) and (4) with respect to the standard (in percent).

12. Interpretation of the received data:
If the intensity of the reaction of the test serum with any of the four protein antigens does not go beyond -15% to + 40% with respect to the reaction of the reference serum (reference), this serum is assigned to the classification group K1 (normal).

 If the intensity of the reaction of the test serum with any of the four protein antigens goes beyond the K1 group, but is not less than -40% and not more than + 65% with respect to the reaction of the standard serum, this serum to the classification group K2 (weak deviations).

 If the intensity of the reaction of the test serum with any of the four protein antigens goes beyond the groups K1 and K2, but is not less than -60% and not more than + 100% with respect to the reaction of the reference serum, this serum is assigned to the classification group K3 (moderate abnormalities).

 If the intensity of the reaction of the test serum with any of the four protein antigens goes beyond the groups K1, K2 and K3, but is not less than -70% and not more than + 150% with respect to the reaction of the reference serum, this serum is referred to classification group K4 (significant deviations).

 If the intensity of the reaction of the test serum with any of the four protein antigens goes beyond the K4 group by negative values below -70%, and by positive values it is in the range from + 150% to + 200% with respect to the reaction of the reference serum , this serum belongs to the classification group K5 (sharp deviations).

 If the intensity of the reaction of the test serum with any of the four protein antigens goes beyond the groups K1, K2, K3, K4 and K5, this serum is assigned to the classification group K6 (very sharp, excisational deviations).

 The empirical data obtained by analyzing the results of childbirth of women previously examined using the ELI-P method revealed the patterns presented in table. 2.

 Thus, the probability of a favorable pregnancy outcome directly depends on the level of determined regulatory antibodies in the blood serum of women, and the frequency of birth of abnormal newborns directly correlates with the level of their deviations. So, if the relevant parameters did not go beyond -15% to + 40% of the standard (group k1), the probability of having a clinically healthy baby was more than 90%, and there were no cases of large anomalies or stillbirths. On the contrary, the more pronounced the deviations, the higher the risk of congenital anomalies and stillbirths, up to group k6, in which we did not observe a single case of the birth of healthy children. In the intermediate groups (K2-K5), the risk of serious pathology / stillbirths progressively increased (from 15% in women of the K2 group to 75% in the K6 group).

 The positive effect of the invention is to obtain a test system (diagnosticum) ELI-P, with which you can identify women whose sera contain abnormal levels of regulatory autoantibodies, which is a universal sign of the presence (or onset of development) of various forms of developmental disorders of the embryo and fetus . The data obtained as a result of using the ELY-P test system make it possible to quickly and objectively, based on instrumental enzyme-linked immunosorbent assay, already at the beginning of the first trimester of pregnancy, or before pregnancy, identify individuals belonging to a high risk group for fetal and fetal abnormalities.

 It should be noted that the analysis is performed in vitro without any harm to the subject.

 Example 1

 Examined A.M., 24 years old. No kids. The second pregnancy is 8 weeks. Heredity is not burdened.

 The results of the diagnostic test ELI-P are given in table. 3.

 Delivery results: Urgent delivery, healthy girl 52 cm tall, 3,800 weight, Apgar score 9.

 Example 2

 Examined OS, 22 years old. No kids. First pregnancy 5 weeks. Heredity is not burdened.

 The results of the diagnostic test ELI-P are given in table. 4.

 Delivery results: Urgent delivery, healthy boy, height 51 cm, weight 3100, Apgar score 9.

 Example 3

 Surveyed M.R., 36 years old. Fifth pregnancy, 15 weeks. Two children are healthy. Heredity is not burdened.

The results of the diagnostic test ELI-P are given in table. 5
Delivery results: urgent delivery, signs of prematurity 2 tbsp., Girl 42 cm tall, weight 1900, Apgar score 6-7.

 Example 4. Surveyed M.O., 26 years old. Second pregnancy, 5 weeks. Two children are healthy. Heredity is not burdened.

 The results of the diagnostic test ELI-P are given in table. 6.

 Delivery results: urgent delivery, a girl 50 cm tall, 2400 weight, Apgar 7 score, increased irritability, impaired intestinal motility.

 Example 5. Surveyed N.E., 29 years old. Third pregnancy, 12 weeks. No kids. Heredity is not burdened.

 The results of the diagnostic test ELI-P are given in table. 7.

 Delivery results: urgent delivery, a girl height of 45 cm, weight 1600, a score on the Apgar scale of 6-7, malnutrition, hypertonicity, increased intracranial pressure, early fontanel infection, neurological changes. The girl died at the age of 4 months. Pathomorphological diagnosis: immaturity of the brain; microcephaly.

 Example 6

 Examined A.G., 39 years old. Third pregnancy 9 weeks., Two miscarriages. Heredity is not burdened.

 The results of the diagnostic test ELI-P are given in table. eight.

 According to an ultrasound scan for 22 weeks. pregnancy - developmental abnormalities.

 Results of childbirth: pregnancy is interrupted (premature artificial birth for a period of 23 weeks), with autopsy - multiple malformations.

 Example 7. Surveyed T. T., 33 years old. The fourth pregnancy is 11 weeks. One child diagnosed with oligophrenia.

 The results of the diagnostic test ELI-P are given in table. nine.

 Delivery results: Premature artificial birth for a period of 20 weeks; multiple malformations according to pathomorphological research.

Techno-economic and social significance
1. The use of the ELI-P diagnosticum for examining pregnant women in the early stages of embryo and fetal development in order to identify individuals who are at increased risk of teratogenesis will significantly reduce the risk of giving birth to mentally and physically unhealthy children. The study is carried out on the basis of an enzyme-linked immunosorbent assay in vitro, requires 10 μl of blood from the test person and is safe for women and unborn children.

 2. Obtaining reagents does not require complex and expensive equipment and reagents, and analysis procedures can be carried out by a laboratory operator.

 3. The feasibility, social and medical significance of new objective methods for early prediction of deviations in the development of the embryo and fetus is obvious, since the incidence of congenital malformations (up to severe malformations) is up to 10% of the total number of newborns and is especially high in ecologically unfavorable regions. At the same time, reliable and objective methods of early diagnosis (earlier than 18-20 weeks of pregnancy) are currently lacking.

Claims (1)

 A method for screening women of childbearing age with early pregnancy or before pregnancy, including blood sampling, obtaining serum, obtaining the ELI-P test system (purified test antigens) and conducting an enzyme-linked immunosorbent assay, characterized in that the immunoreactivity of the serum autoantibodies of a woman’s body is determined, having a significant effect on normal embryo / fetogenesis, and directed to antigens involved in the development of organs and tissues of the fetus, for example, to the main myelin protein, protein S1 00, anionic tightly bound chromatin protein ABX 14-18, MP65 membrane protein and upon receipt of data on the deviation of the immunoreactivity of the tested sera from the level of normal values to any of them, the degree of severity of the deviations determines the level of risk of fetal development disorders, based on the data of the “Forecast Table” based on the analysis of accumulated data banks.

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