RU2478971C1 - Diagnostic technique for autoimmune disorder of peripheral parasympathetic vegetative nervous system (version) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: in the first case, blood serum is examined for the presence of α3-ACR antibodies, with the α3-ACR antibodies detected by rapid western blotting in blood serum by the reaction with an extracellular site of an α3 subunit of neuronal ACR, while the presence of the α3-ACR antibodies is stated by staining with precipitant tetramethylbenzidine used as a chromogen. In the second case, blood serum is examined for the α3-ACR antibodies by EIA by by the reaction with an extracellular site of a α3 subunit of neuronal ACR; the level of the α3-ACR antibodies is stated by staining with tetramethylbenzidine used as a chromogen; optical light absorption is measured by means of an EIA analyser at wave length 450 nm in optical density units (OD), with increasing the level of the α3-ACR antibodies more than 0.24 OD_450nm.

EFFECT: invention is cheaper and safer; with the same specificity and more simple and efficient procedures, the technique enable diagnosing an autoimmune disorder of peripheral VNS accompanying neural and neuromuscular diseases.

2 cl, 2 dwg

Description

The invention relates to medicine, namely to neurology, and can be used to detect autoimmune damage to the parasympathetic department of the autonomic nervous system at the ganglion level.

Known clinical (dermographism, pupillometry, pilomotor reflex, dynamics of blood pressure and heart rate in ortho-, clinostatic and reflex tests with the calculation of various coefficients, etc.) and electrophysiological (measurement of the RR interval at various loads, evaluation of the skin-galvanic reflex etc.) research methods allow us to establish the predominance of the sympathetic or parasympathetic department of the autonomic nervous system (ANS) in various pathological conditions. Methods for assessing the state of the ANS with pharmacological tests (adrenergic and anticholinergic drugs) reveal the presence of autonomic dysfunction and in some cases localize the level of its damage, but the introduction of these (mainly potent group A) drugs is associated with a risk of severe side effects, especially in patients with organic damage to the vital structures of the peripheral ANS (Methods for the study of the autonomic nervous system. In the book. Vegetative disorders. Clinic, diagnosis, treatment. Ed. A.M. Vejna. - M .: Medical Information Agency LLC, 2003, pp. 44-102). However, these methods do not allow to establish the cause and level of affection of the ANS.

Closest to the invention is a method for diagnosing autoimmune lesions of the peripheral ANS by detecting antibodies to the ganglionic subunit of the α3-acetylcholine receptor (α3-AChR) by radioimmunoprecipitation, where a receptor isolated from a neuroblastoma cell culture and bound to epibatidine labeled with isotope 1 was used as an antigen -125 (Vemino S., Low PA, Fealey RD, Stewart JD, Farrugia G., Lennon VA. Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies., N. Engl. J. Med., 2000, 343, 847-855 )

This method allows the determination of antibodies in blood serum, to establish the level of affection of the ANS, however, along with high specificity, it is laborious to perform, since it involves working with radioactive isotopes, requires special equipment and facilities, is more expensive.

Our proposed variants of the methods are cheaper and safer for determining antibodies in blood serum by interaction with the extracellular domain of α3-AChR by enzyme-linked immunosorbent assay and Western Blotting. In addition, these methods allow for the same specificity in a simpler and more economical way (in particular, not requiring radiation protection measures) to diagnose autoimmune lesions of the peripheral ANS in nervous and neuromuscular diseases.

This, in turn, allows prescribing adequate therapy to patients not only in neurological, but also in cardiology, gastroenterology, urology and ophthalmology.

The specified technical result is achieved by the fact that in the first case, the presence of antibodies to α3-AChR in the blood serum is determined in order to diagnose autoimmune damage to the peripheral VNS, and antibodies to α3-AChR are determined by the express method of Western Blotting in blood serum by interaction with extracellular plot of the α3-subunit of neuronal AChR, and the presence of antibodies to α3-AChR is determined by staining using tetramethylbenzidine precipitating as a chromogen. And in the second case, to diagnose an autoimmune lesion of the peripheral parasympathetic autonomic nervous system, antibodies to α3-AChR are determined in blood serum, and antibodies to α3-AChR are determined by ELISA in a serum sample by interaction with the extracellular region of the α3 subunit of neuronal AChR, the level of antibodies to α3-AChR is determined by staining using tetramethylbenzidine as a chromogen, the optical absorption of light is measured on an ELISA analyzer at a wavelength of 450 nm in optical units density (OD) and with an increase in the level of antibodies to α3-AChR more than 0.24 OD _450HM , an autoimmune lesion of the peripheral ANS at the level of parasympathetic ganglia is diagnosed with respect to control.

The method is as follows.

For the express method of Western Blotting and TIFA, the patient is sampled from the cubital vein, then the serum is separated from the blood clot by centrifugation for 15 minutes at 3000 rpm. Serum was tested for the presence of antibodies to α3-AChR in ELISA.

The extracellular region of the α3 subunit of neuronal AChR obtained in the laboratory of finished dosage forms of the Center for Bioengineering of the Russian Academy of Sciences was used as an antigen for the determination of autoantibodies by ELISA.

Western bloting revealed the presence of antibodies in the blood serum of a patient with autoimmune autonomic gangliopathy by interacting with the extracellular domain of the α3 subunit of neuronal AChR. As a control, the blood serum of a healthy donor was investigated. Western blotting of the blood serum of a patient and a healthy donor was carried out according to the following scheme: the nitrocellulose membrane after electrophoresis and electrotransfer, containing the extracellular region of the α3 subunit of AChR, was incubated with blood serum diluted (1: 100) for 60 minutes at 37 ° C, followed by 3 washing three times (0.2% BSA in PBS / Tween-20) for 3 minutes. The membrane was then incubated with conjugate of peroxidase with anti-human IgG (Sigma) at a dilution of 1: 1000 for 60 minutes at 37 ° C, after which 3-fold washing was performed (0.2% BSA in PBS / Tween-20) for 3 minutes. For staining, a single component tetramethylenebenzidine precipitating substrate (USA) was used as a chromogen.

The results of the analysis of the express method of Western Blotting are presented in Figs. 1a and 1b.

In Fig.1a - Patient P., 54, on 2 and 3 paths, the presence of antibodies was detected, the diagnosis: Autoimmune autonomous gangliopathy.

In Fig.1b - Control (serum of a healthy donor) antibodies on tracks 2 and 3 are absent.

In the figures: 1-track — extracellular domain of α3-AChR

2-track - extracellular domain of α3-AChR

3-track - protein markers.

To quantify the level of autoantibodies to α3-AChR, the following study was performed using ELISA.

The costar 96-well plates sorbed the extracellular domain of the α3 subunit of neuronal AChR at a concentration of 2 μg / well and incubated for 60 minutes at 37 ° C. The first row of wells was left free for conjugate control. To block nonspecific binding, a 2% solution of BSA (Sigma) in PBS / TBHH-20 at 200 μl / well was added to the wells and incubated for 60 minutes at 37 ° C. After incubation, serums of control donors were added to the second row, and the serums of patients with autoimmune autonomic gangliopathy, myasthenia and myasthenia with thymoma diluted 1: 100 were added to all subsequent rows, and incubated for 60 min at 37 ° C followed by 3-fold washing (0.2 % BSA in PBS / TBHH-20 at 200 μl / well) for 3 minutes. The presence of antibodies was determined by staining using a conjugate of peroxidase with human anti-IgG (Sigma), which was added at 100 μl / well at a dilution of 1: 1000 and incubated for 60 minutes at 37 ° C, after which 3-fold washing was performed (0.2% BSA in PBS / Tween-20 at 200 μl / well) for 3 minutes. Tetramethylenebenzidine (Sigma) was used as a chromogen: 10 mg was dissolved in 5 ml of nitrate buffer (pH 4.5) and 15 μl of hydrogen peroxide and 100 μl / well were added. Staining developed within 20 minutes. Inhibition of the reaction was carried out with 0.25 M sulfuric acid solution - 100 μl / well. The measurement of optical light absorption at a wavelength of 450 nm was carried out on an ELISA analyzer.

The results were expressed in units of optical density (OD). The average value of the antibody content in the control group was 0.2 ± 0.04 OD as the normal amount of antibodies to α3-AChR.

The proposed method was examined 3 patients with a generalized form of myasthenia gravis, 3 - myasthenia gravis with thymoma, 4 autoimmune autonomic gangliopathy and 10 control healthy donors. In all cases, the level of antibodies corresponded to the condition of patients and healthy donors.

Thus, the proposed methods have a high specificity for detecting the presence of antibodies to α3-AChR in Western Blotting and TIFA and make it possible to detect with high accuracy both the presence of antibodies (the appearance of a dark colored band) and the quantitative level of antibodies in which autoimmune damage to the peripheral region is diagnosed ANS.

This provides timely adequate treatment aimed at suppressing autoimmune lesions of the ANS and improving the quality of life of patients with peripheral autonomic insufficiency (PVN).

Thus, the methods offered by us differ from the prior art in the following:

1) allow to evaluate the ANS by examining the blood of patients, developed by us by the method of ELISA in blood serum by interaction with the extracellular region of the α3 subunit of neuronal AChR;

2) allow you to assess the level of damage to the ANS;

3) allow you to abandon the use of radioactive isotopes as reagents when performing the methodology;

4) allow differential diagnosis, objectify the dynamics of the pathological process, and also facilitate the selection of adequate therapy in patients with signs of PVN;

5) are more economical and no less sensitive than other methods;

6) do not require the introduction into the body of patients of potent drugs that are specially registered;

7) one of the methods allows to quantify the level of antibodies to α3-AChR, which can characterize to some extent the severity of autoimmune lesions of the peripheral ANS at the level of parasympathetic ganglia.

Examples of the method.

Example 1. Patient P., 54 years old, was observed in the clinic of nervous diseases named after Kozhevnikov I MGMU them. Sechenova I. with signs of peripheral autonomic insufficiency, progressing over 8 years: Horner's syndrome on the right, impaired accommodation with a distorted reaction of the pupils in the sample with adrenaline, the opposite reaction of blood pressure in the clinostatic and orthostatic sample (increase in the supine position and a significant decrease - in a standing position), lack of dynamics of heart rate when changing posture, change of constipation with frequent loose stools (up to 12-15 times per night), weakness in a standing and sitting position, inability to move to be played without dragging clothes, fatigue, dry skin and mucous membranes with proximal hyperhidrosis. She received symptomatic therapy (enhanced water regimen, mineralocorticoids, skin-tightening clothes) with an insignificant temporary effect against the background of disease progression. A study of the patient's blood serum by the proposed quantitative ELISA method revealed a significant increase in the level of autoantibodies to α3-AChR: 1.78 OD _{450 nm} .

Based on the analysis, a diagnosis of autoimmune autonomic vegetative gangliopathy was made, mineralocorticoids were replaced with glucocorticoids, the patient's condition gradually improved with immunosuppressive therapy.

Example 2. Patient D., 37 years old, is observed in the Neurology Scientific Center of the Russian Academy of Medical Sciences with a diagnosis of myasthenia gravis with thymoma (teratoma with signs of a dermoid cyst - according to MRI). The clinical picture was dominated by symptoms of severe myasthenia gravis: asymmetric dynamic ptosis, external ophthalmoparesis with diplopia, severe bulbar syndrome (to the point of inability to swallow and aphonia), fatigue when chewing, weakness and fatigue of the muscles of the face, neck and limbs, difficulty breathing due to weakness of the respiratory muscles. There are no clinical signs of autonomic nervous system dysfunction. In such cases, first-line drugs for the treatment of myasthenia gravis are glucocorticosteroids. At the same time, a blood serum test using the ELISA method revealed the presence of autoantibodies to α3-AChR up to 0.45 OD _{450 nm} , which allowed us to prescribe adequate immunocorrective therapy and avoid the expressed side sympathomimetic effects of glucocorticoids (psychomotor agitation, tachycardia, increased blood pressure, impaired gastrointestinal motility intestinal tract, sweating disorders, etc.). The patient underwent a course of plasmapheresis followed by the appointment of cytostatics, with intercurrent infection - a course of iv normal human immunoglobulin with a positive effect.

Example 3

Patient I., 46 years old, is observed in the SC Neurology RAMS with a diagnosis of myasthenia gravis, generalized form. The patient showed symptoms of autonomic dysfunction in the form of attacks of tachycardia, sweating with dry mucous membranes, visual impairment (in addition to diplopia associated with myasthenic external ophthalmoparesis, periodically “defocusing” of the contours of objects at an average distance), in addition to weakness and fatigue of voluntary muscles, general weakness fatigue, not responding to AChE inhibitors. The study of blood serum by the proposed method did not reveal autoantibodies in the blood serum, which allowed us to attribute these disorders to suprasegmental level of VIS dysfunction against the background of dishormonal disorders (4 years ago it was operated on for thyroid cancer, there are menstrual irregularities with alternating uterine bleeding and long periods of amenorrhea ) and allowed to adjust the ongoing therapy.

Thus, the determination of AT to α3-AChR by the express method of Western Blotting of TIFA in blood serum by interaction with the extracellular region of the α3 subunit of neuronal AChR reflects the ganglion level of lesion of the parasympathetic division of the ANS. The analysis allows you to establish a diagnosis of autoimmune autonomic gangliopathy, to identify the presence of autonomic insufficiency in a patient with myasthenia gravis and thymoma, and also to exclude damage to the peripheral ANS in a patient with myasthenia gravis and autonomic dysfunction.

The inventive express method of Western Blotting and TIFA can improve the diagnostic process in patients with signs of peripheral autonomic insufficiency in a short time, track the dynamics of the pathological process and choose the right treatment tactics. With high specificity, the methods are simpler and more economical and provide the possibility of diagnosing autoimmune lesions of the peripheral ANS in nervous and neuromuscular diseases.

Claims (2)

1. A method for diagnosing an autoimmune lesion of the peripheral parasympathetic autonomic nervous system, comprising determining the presence of antibodies to α3-AChR in the blood serum, characterized in that antibodies to α3-AChR are determined by the express method of Western Blotting in blood serum by interaction with the extracellular portion of α3 -subunits of neuronal AChR, and the presence of antibodies to α3-AChR is determined by staining, using precipitating tetramethylbenzidine as a chromogen. 2. A method for diagnosing an autoimmune lesion of the peripheral parasympathetic part of the autonomic nervous system, comprising determining antibodies to α3-AChP in blood serum, characterized in that antibodies to α3-ACP are determined by ELISA in a blood serum sample by interacting with the extracellular region of the α3 subunit of neuronal ACP , the level of antibodies to α3-AChR is determined by staining using tetramethylbenzidine as a chromogen, the optical absorption of light is measured on an ELISA analyzer at a wavelength of 450 nm in units of opt specific density (OD) and with an increase in the level of antibodies to α3-AChR more than 0.24 OD _450HM , an autoimmune lesion of the peripheral part of the ANS at the level of parasympathetic ganglia is diagnosed with respect to control.

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