Method for predicting gestosis

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the present innovation deals with detecting the concentration of antibodies to neurospecific enolase (NSE) and gliofibrillo-acid protein (GFAP) in blood serum of pregnant women. The development of gestosis should be predicted at the level of anti-NSE-antibodies being above 0.6 mcg/ml or anti-GFAP-antibodies being above 0.9 mcg/ml. The innovation provides high information value and specificity of the method suggested to predict the development of gestosis.

EFFECT: higher efficiency.

3 ex, 1 tbl

 

The present invention relates to medicine, in particular to obstetrics.

The known method preclinical diagnosis of preeclampsia at the time of 13-14 weeks of pregnancy, based on the determination of antiphospholipid antibodies in the serum [patent RU 2135999 C1, G 01 N 33/53, issued in 1999]. This determines the level of Ig M and Ig G to cardiolipin, phosphatidylserine and phosphatidylcholine. When the level increases at least one of the antibodies is 1.5 times or more the norm diagnose the development of preeclampsia.

The disadvantage of this method is its low specificity, as increased levels of antiphospholipid antibodies is observed in other types of pathology.

Another known way to predict the development of preeclampsia based on the determination of the resistance of erythrocytes to peroxide hemolysis initiated by ferrous iron and autologous blood hemolysate [patent RU 2191384 C2, G 01 N 33/52 issued in 2002]. At the same time for 2-4 weeks prior to the onset of clinical signs of preeclampsia time stability of erythrocytes is less than 150; with more than 150 with preeclampsia in pregnant women does not develop. This method is also not precise enough, since the resistance of erythrocytes to hemolysis affect a number of other disorders.

Closest to the proposed method for predicting preeclampsia, starting from 20 weeks of pregnancy, is the definition retained the I-lymphocytes, bearing on its surface structure, responsive to USSR beta-glycoprotein [patent RU 2077722 C1, G 01 N 33/50, issued in 1997]. When the value of 18% or more is predicted the development of preeclampsia in pregnant women.

The disadvantage of this method is the relatively low accuracy associated with its low specificity.

The objective of the invention is to improve the accuracy of forecasting the development of preeclampsia in pregnant women due to the higher specificity of the method.

This object is achieved by determining the concentration of antibodies to neurospecific enolase (NSE) and lifebalance-acidic protein (GFAP) (anti-NSE antibodies and anti-GFAP antibodies) in the serum of pregnant women, and when the level of anti-NSE antibodies than 0.6 μg/ml or anti-GFAP antibodies higher than 0.9 µg/ml predict the development of preeclampsia.

The method is based on pathogenetic factor in the development of preeclampsia, which is caused by the development of acute endotheliosis due to the penetration of neurospecific proteins of the fetus in the maternal bloodstream, generating amino acid antibodies and formation of immune complexes that damage the endothelium.

The way is as follows.

A pregnant woman produces the fence peripheral venous blood in a volume of 2 ml, and then in serum to determine the level of the antibodies to NSE and GFAP by the method of enzyme-linked immunosorbent assay (ELISA).

When conducting ELISA in cell polystyrene tablets were made in 50 μl of solutions of NSE and GFAP in each well (at a concentration of 5 μg/ml)prepared in carbonate buffer with a pH of 9.6. The tablets were activated for 12 hours at 4°With (at this time there is adsorption of antigens on the surface of polystyrene solid phase of the bottom and walls of the cells). Then there was the washing of cells with PBS solution (1 l of solution: 8.0 g NaCl, 0.2 g KN2PO4, 2.9 g of Na2HPO4, 0.2 g KCl and 0.5 ml Tween-20, pH 7.4) - 3x for 3 minutes. To exclude nonspecific reactions in each cell was introduced into 200 μl of a solution of bovine serum albumin (1% BSA in PBS) and incubated at 37°With 1 hour. Then the cells were again washed as described above.

After washing, cells were introduced into 50 µl sample sera (diluted 1:10 in PBS); in individual rows of cells made of a standard sample with known concentration of monoclonal antibodies to NSE and GFAP (7-point calibration with step dilution in PBS). Incubation was performed at 37°With 1 hour. During this time, is the binding of the antibodies studied sera with adsorbed antigens. Unbound proteins were washed with the same wash buffer. In cells contributed reconstituted enzyme conjugate, 50 μl (conjugate to Ig G man was diluted with PBS in a ratio of 1:4000), incubated 1 hour at 37°s there was rinsed 5 times for 3 minutes. As a substrate for the manifestation of the enzymatic activity of peroxidase were used orthophenylphenol. The substrate for peroxidase was prepared directly before making and contributed to the wells at 50 μl (4 mg of orthophenylphenol, 10 ml substrate buffer, 4 μl of N2About2). The reaction was developed 10-15 minutes and was characterized by the appearance of yellow color of varying intensity in the wells containing the calibration and test samples. Then the reaction was stopped by adding to each well 50 µl of 10% sulfuric acid. Measurement of the optical density of the solutions in each cell of the tablets produced using a spot of reader TECAN, Austria) at a wavelength of 492 nm. Preparation of calibration graph and the final calculation of the concentrations of anti-NSE - and anti-GFAP antibodies produced by standard ELISA technology.

In physiological pregnancy (control group relative to the healthy pregnant) antibodies to NSE and GFAP is not specified or the level does not exceed 0.6 mg/ml and 0.9 mg/ml, respectively. The obtained data are presented in the table.

Table.

The concentration of AITI-NSE antibodies and anti-GFAP antibodies in the serum of pregnant women.
N (number of observations)Anti-NSE-Auditel, ug/ml Anti-GFAP antibodies, ug/ml
Women with physiological pregnancy300-0,6000-0,900
Pregnant, which was projected and developed preeclampsia290,610-2,7100,910-3,203
Pregnant, which was predicted and did not develop preeclampsia1of 0.6150,904

Example 1. Pregnant,, 35 years. This pregnancy 2nd. The first pregnancy ended in a timely deliveries in the period of 39 weeks and birth full-term baby weighing 4000 g, a growth of 53 cm, as assessed by the Apgar scale 8/8 points. In the first pregnancy in the third trimester diagnosed with preeclampsia, mild (nephropathy first degree). Conducted treatment. When this pregnancy is related to increased risk of developing preeclampsia. This pregnancy proceeds without complications. In the period of 23-24 weeks objective: BP - 110/70 mm Hg, swelling and protein in the urine are not available, weight gain 300 g per week. Biochemical analysis of blood within normal limits. Complaints during the inspection does not show.

According to the U.S.: pregnancy 23-24 weeks, the uterus in a normal tone, the fetus corresponds to the gestational age.

Diagnosis: pregnancy 23-24 weeks, the risk of developing the guestbook is for.

The study of peripheral blood in the period of 24 weeks for the presence of amino acid antibodies. The concentration of antibodies to neurospecific enolase was 2.2 μg/ml to lifebalance-acidic protein - 2.8 mg/ml, which allows to predict the development of preeclampsia.

When surveyed in the period 29-30 weeks pregnant, the observed increase in HELL to 130/90-140/90 mm Hg, swelling of the legs, abnormal weight gain to 400 g per 1 week, protein in the urine 0.66 g/l Pregnant, admitted to hospital with a diagnosis of preeclampsia mild. Was held pathogenetic therapy. The pregnancy ended in birth at 38 weeks and the birth of full-term ypotrofies girls by weight of 2700 grams and height of 50 cm, as assessed by the Apgar scale 7/8 points.

Example 2. Pregnant L., 31. Of somatic cells to a violation of fat metabolism II degree, neurocirculatory dystonia on hypertonic type. Gynecological anamnesis is not burdened. This first pregnancy. During the inspection no complaints.

Objectively in the period 22-23 weeks: HELL 120/80 mm Hg, protein in the urine no, no edema, weight gain per week is 250, According to ultrasound - pregnancy 22-23 weeks, the fetus corresponds to the gestational age.

Diagnosis: pregnancy 22-23 weeks, impaired fat metabolism II degree, neurotic the batery dystonia on hypertonic type, the risk for development of preeclampsia.

The concentration of antibodies to neurospecific enolase to 1.3 µg/ml, lifebalance-acidic protein and 1.4 µg/ml, which allows to predict the development of preeclampsia.

When surveyed in the period of 34 weeks pregnant L. objective: HELL 130/90, swelling of the legs, hands, protein in the urine - 0,99 g/l, abnormal weight gain - 370 g for 1 week.

Pregnant L. hospitalized with a diagnosis of preeclampsia mild. Conducted pathogenetic treatment. The pregnancy ended in birth in term of 40 weeks and the birth of full-term girls weighing 3200 g, height 50 cm, as assessed by Apgar 7/8 points.

Example 3. Pregnant U., 30 years. From somatic cells neurocirculatory dystonia on hypertonic type, hepatitis A. This pregnancy 2nd. The first pregnancy ended in a medical abortion at 8 weeks, with no complications.

Objectively in the period 21-22 weeks: HELL 100/60 mm Hg urine Analysis - normal. No edema. The weight gain is within the norm. According to ultrasound - pregnancy 21-22 weeks, the fetus corresponds to the gestational age.

Diagnosis: pregnancy 21-22 weeks, neurocirculatory dystonia of hypertensive type, the risk for development of preeclampsia.

The concentration of antibodies to neurospecific enolase - 0,66 mg/ml, to lifebalance-acidic protein - 0,98 g/ml, that of the criminal code which indicates the possibility of developing preeclampsia.

When surveyed in 30 weeks of pregnancy: HELL 120/90 mm Hg Swelling of the lower legs, hands, face, traces of protein in the urine, abnormal weight gain - 410 g per week, hypoproteinemia (total protein blood at 58.1 g/l), thrombocytopenia - 169·109/HP

Pregnant U. admitted to hospital with a diagnosis of preeclampsia mild. Conducted pathogenetic treatment. The pregnancy ended in an independent term births in the period 39-40 weeks and the birth of full-term girls weighing 3150 g, 52 cm, as assessed by Apgar 8/9 points.

The proposed method is vasocontraction and specific, clearly allows to predict the development of preeclampsia, timely measures to determine the tactics of a pregnant woman.

A method for predicting preeclampsia by analysis of blood serum of a pregnant, characterized in that the serum concentration is determined antibodies to neurospecific enolase and lifebalance-acid protein and the level of anti-NDE-antibodies than 0.6 μg/ml or anti-GFAP antibodies higher than 0.9 µg/ml predict the development of preeclampsia.



 

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