RU2240561C1 - Method for determination of circulating autoantibody level in biological fluids - Google Patents

Abstract

FIELD: medicine, immunology.

SUBSTANCE: invention can be used for diagnosis of autoimmune process. Method involves assay of the level of circulating autoantibodies in biological fluids by detection of interaction of autoantigens with autoantibodies, the following identification and determination of the level of circulating autoantibodies using a labeled conjugate. Anti-IgE antibodies that are complementary to epsilon-chains of immunoglobulin E conjugated with a label are used as a conjugate with the following identification. Invention provides the development of method providing high sensitivity and specificity.

EFFECT: improved assay method.

1 tbl, 3 ex

Description

The invention relates to medicine, in particular to immunology, and can be used to diagnose an autoimmune process and the subsequent effect on this link of pathogenesis due to the possibility of determining the presence or absence of an atopic mechanism among other autoaggression mechanisms aimed at an organ target.

From such different approaches, therapeutic tactics also change. If we are talking about foreign allergens, that is, classical allergies, then one of the regular methods of the attending physician is the elimination of antigens, prevention of the patient’s contact with them, their destruction, and vice versa, if it is an autoimmune process, then the antigen’s goal is to protect the antigen, so how it is a necessary body.

Currently, for the determination of autoantibodies, methods of different sensitivity and specificity are used that do not allow verification of the atopic mechanism from other autoaggression mechanisms (see RU 2173464 C1, 09/10/2001).

The closest analogue to the claimed method is a method for determining the level of autoantibodies in which the interaction of autoantigens with autoantibodies is detected, the identification and determination of the level of circulating autoantibodies is carried out using labeled Amersham conjugate (biotinylated human anti-IgG and avidin peroxidase) by solid-phase enzyme analysis see Sidorik LL et al. Immune response to some intracellular antigens in patients with dilated cardiomyopathy (http://www.rql.kiev.ua/cardio j / 2000/3 / ry abenko.htm. 1999).

The method allows to solve the problem of determining autoantibodies, where conjugates to conjugates to antibodies of classes considered to be classical for autoimmune syndromes were used, but it is not possible to verify the atopic link of the pathogenesis of autoaggression to the affected target organ by determining the level of antigen-specific IgE.

The aim of the present invention is to develop a non-invasive method that allows you to verify the atopic component of autoaggression on the affected target organ. The developed method is implemented by determining the level of antigen-specific IgE and allows you to really influence the course of the autoimmune process. This is achieved by the fact that to determine the level of circulating autoantibodies in biological fluids, the interaction of autoantigens with autoantibodies is detected using a conjugate, in the quality of which antibodies labeled for subsequent identification are used that are complementary to immunoglobulin E epsilon chains manufactured by TsNIRRI, St. Petersburg, Russia. The method for producing antibodies is given in the publication Bohn A, Konig W. Generation of monoclonal murine anti-DNP-IgE, IgM and IgG1 antibodies: biochemical and biological characterization // Immunology. 1982 Oct; 47 (2): 297-3H.

The method is as follows.

IgE class circulating autoantibodies are determined in biological fluids (blood, lymph, saliva, cerebrospinal fluid, semen, bile, urine, exudate, edematous fluid, ophthalmic fluid, etc.) using pre-labeled antibodies for complementary identification of IgE epsilon chains . For this, polyclonal and monoclonal anti-IgE antibodies are used, which are included in the conjugate, in which a dye fluorescent in ultraviolet light, an isotope detected by radiometry, etc., can be used as a label, depending on the method used to determine the level of autoantibodies.

At the first stage of the study, specific binding of autoantigens (proteins of target organs of the autoimmune process) to IgE class autoantibodies circulating in the studied biological fluid occurs. Antigen-antibody complexes arise, which are then detected using the aforementioned identifiable conjugate affinely interacting with IgE class antibodies, acting in this case as an antigen. The result of the reaction is measured using highly sensitive instruments that allow the quantitative identification of the labeled conjugate and, therefore, the desired IgE autoantibodies of detectable antigen specificity.

Examples of specific performance of the method

Example 1

1. The patient is 72 years old. Hospitalized for the period from 19.11. on January 9, 02 with a diagnosis of Chronic viral hepatitis B, HBeAv (+), HB coh (+). Aggravation. IHD. General atherosclerosis. Diffuse non-toxic goiter. Euthyroidism. During curation, a phase was observed both in the dynamics of the patient’s well-being and in the nature of the course of the main hepatological syndromes. So, the patient’s well-being gradually worsened until 03.12, after which the tendency reversed. The main biochemical marker of the cytolysis syndrome, the activity of the AlAT enzyme, initially systematically increased from 1102 e / l 20.11 to 1111 e / l 23.11, then it also gradually decreased to 397 e / l 04.12 (normal - 11-40 e / l).

Bilirubinemia, a biochemical marker of jaundice syndrome in the dynamics of the first phase, showed periods of increase with a slight decrease in the concentration of bilirubin (also 23-24.11). In general, however, the indicator increased from 20.11 (376 μmol / L) to 3.12 (510 μmol / L), and then gradually decreased to 199 μmol / L 04.01.

It is difficult for us to indicate the factor that influenced the trend reversal in the dynamics of cytolysis and the transient decline in bilirubin concentration 23-24.11. The nature of the phase change phenomenon in the dynamics of well-being and jaundice syndrome is obvious: it was on 03.12 the patient underwent a repeated plasmapheresis procedure. Such a relief effect of the indicated therapy on the patient’s condition indirectly confirms the antibody-dependent mechanism of development of her disease. We find confirmation of this in the data of immunological studies.

Blood was withdrawn for them on November 21 and 5.01, i.e. accordingly, in the phase of rise and fall in the activity of the main hepatological syndromes, as well as against the background of deterioration and improvement of the patient's subjective well-being. The level of circulating autoantibodies was determined by enzyme-linked immunosorbent assay using a conjugate that contains antibodies complementary to the epsilon chains of immunoglobulin E. The obtained data showed that the level of autoantibodies is significantly different in the above phases of the pathological process. For general antibodies, it is in the optical density of 0.777 and 1.129 for antibodies of the IgE class - 1.483; 0.913, respectively. This indicates that this patient has an autoimmune process in the pathogenesis of chronic viral liver disease, mainly mediated not by classes of antibodies generally accepted for such processes, but by antigen-specific IgE antibodies.

Example 2

The patient is 43 years old. She was hospitalized for the period from May 20 to June 10 with the diagnosis: “Chronic viral hepatitis B, HBsAg +, cirrhotic stage, decompensation. Portal hypertension, edematous ascites syndrome. Hemorrhagic syndrome. Chronic cholecystitis, exacerbation. " According to the district infectious disease specialist (attending physician), this hospitalization is associated with ascites, weakness, and shortness of breath that increase during the week.

Subsequently, during hospitalization, the state of moderate severity was observed in the subject until 26.05 inclusive; from May 27 until the day of discharge (June 10), it was assessed as satisfactory or relatively satisfactory.

Giving a general assessment of the patient's condition and well-being during the hospital observation, it should be noted that the severity of her condition was mainly determined by the manifestations of portal hypertension syndrome.

Indeed, bilirubinemia from the moment the patient arrived at the hospital observation did not exceed the upper boundary of the moderate course area (classification of the Riga Hepatology Center, 1984), and then steadily decreased. This dynamics does not fully reflect the dynamics of the severity of the patient's condition.

To a lesser degree, the dynamics of severity was reflected in the biochemistry of cytolysis but ALT syndrome. This parameter fluctuated around 60 units / l with wave-like dynamics of the patient's well-being.

Meanwhile, the dynamics of the manifestations of the clinical syndrome of portal hypertension (weakness, ascites, dyspnea) served as a reason for hospitalization and discharge of the patient: due to the disappearance of weakness and shortness of breath, decreased ascites, the patient’s further hospital stay was “considered inappropriate (9.06, examination of the manager department). ” The patient was discharged 10.06. The district infectious disease specialist who observed the patient before hospitalization also notes a general improvement in her condition. According to ultrasound of the abdominal organs from 3.06, the formation of micronodular cirrhosis of the liver with portal hypertension IV takes place.

Thus, in contrast to the previous case, the hepatological autoimmune process can mainly affect the severity of the portal hypertension syndrome. The level of circulating autoantibodies was determined by the radioimmune method using a conjugate that contains antibodies complementary to the epsilon chains of immunuloglobulin E. Moreover, the levels of antibodies to the f-protein of both general and IgE class are significantly different and amount to 1,025; 0.94 and 0.185; 0.09 respectively. The data obtained indicate that this patient in the pathogenesis of chronic viral liver disease has an autoimmune process along with classes of antibodies generally accepted for such processes, mediated by antigen-specific antibodies of the IgE class.

Example 3

The patient is 19 years old. The outpatient was examined and treated. During October 2002 he was observed at the outpatient department of the City Hepatology Center with the main diagnosis: “Chronic viral hepatitis B. HBsAg +. HBeAg +. PCR HBV +. ”Blood for immunological studies was withdrawn on 10/11/02 and 10/23/02. The patient had a history (December 2001) of hospital treatment in the hepatology center for the first detected viral hepatitis B, followed by observation by the district infectious disease specialist at the place of residence and in the outpatient department of the hepatology center. Until August 2002, the patient maintained cytolytic activity with ALAT numbers 3-4 times higher than normal. At the time of this examination, the patient had mild hepatomegaly (+1 cm) palpation.

Analyzing the data of biochemical examination, we came to the obvious conclusion that at the time of observation the patient had a uniform increase in the pathological activity of indicators marking the activity of the main hepatological syndromes, such as jaundice syndrome and cytolytic syndrome. The indicated uniform increase in biochemical parameters also corresponds to a mild clinical activity, manifested by mild hepatomegaly. That is, according to clinical and laboratory data, there is reason to talk about an exacerbation of chronic viral liver disease. An immunological study of the biological fluid of bile by the claimed method showed that this exacerbation corresponds to and, apparently, to a large extent determines its growth in the intensity of autoimmune hepatological syndrome, manifested by a corresponding fluctuation in the level of antibodies to the f-protein of common and circulating autoantibodies of the IgE class, while the optical density is 0.355: 0.525 and 0.155: 0.365, respectively. The data obtained indicate that this patient in the pathogenesis of chronic viral liver disease has an autoimmune process along with classes of antibodies generally accepted for such processes, mediated by antigen-specific antibodies of the IgE class.

In general, from clinical cases No. 1-3, the relevance of studying the level of circulating autoantibodies in blood serum at different stages and with different leading syndromes of the studied disease in different statistical groups of patients (hospitalized and outpatients, patients with a newly diagnosed and with an exacerbation of a previously diagnosed diseases, etc.).

The given examples indicate that this method of determining the level of circulating autoantibodies in biological fluids (in particular, in the blood and bile of patients) can be used in the diagnosis of the autoimmune process and, in addition, really influence this pathogenesis link. Scientific and practical medicine has been dealing with IgE-induced anaphylaxis effectors for decades. Transferring the methods developed to combat them to the field of treatment of the autoimmune components of pathogenesis in diseases of various etiologies can be of significant help in their effective therapy.

For a wider testing of the method, 10 samples of another biological fluid, specifically the edematous fluid of patients who died of chronic viral hepatitis B, were also studied (see table).

The data obtained indicate that the determination of the level of circulating autoantibodies in biological fluids allows one to bind the level of IgE antibodies to the patient’s pathological process and isolate the atopic mechanism in the pathogenesis of this process.

Claims (1)

A method for determining the level of circulating autoantibodies in biological fluids, comprising detecting the interaction of autoantigens with autoantibodies and subsequent identification and determination of the level of circulating autoantibodies using a labeled conjugate, characterized in that anti-IgE antibodies complementary to the epsilon immunoglobin epsilon chains are used as conjugate tagged for later identification.