RU2285263C1 - Method for predicting tuberculosis - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: it is necessary to study saliva due to carrying out immunoenzymatic assay by applying mycobacterial antigen specifically binding with human secretory immunoglobulin A only, and specific monoclonal antibodies directed against human secretory immunoglobulin A. The present method is considered to be of high efficiency, noninvasive, simple in application, of high information value, specificity and sensitivity to tuberculosis.

EFFECT: higher accuracy of diagnostics.

3 ex, 2 tbl

Description

The invention relates to medicine, namely phthisiology, and can be used in the initial diagnosis of tuberculosis by determining specific anti-tuberculosis antibodies to adjust treatment tactics for patients with tuberculosis, as well as to study the pathogenetic mechanisms of the development of this disease.

Tuberculosis remains a grave, deadly chronic disease. Every year, 3 million people die from tuberculosis in the world, and in developing countries, one in every five deaths is associated with tuberculosis [Barry R. Bloom, Tuberculosis. Pathogenesis, protection, and control. 2002, ASM Press, Washington DC]. About a third of the world's population is infected with mycobacterium tuberculosis and is at risk of developing this disease. In the Russian Federation, the incidence of tuberculosis is increasing every year and in 2002 reached 95.2 cases per 100 thousand people [Scientific works on the 75th anniversary of the leading TB institution in Moscow. Edited by Academician RAMS V.I. Litvinova. Moscow. 2002]. Timely identification of persons infected with M. tuberculosis is crucial since 10% of them develop active contagious forms of tuberculosis throughout their lives, with clear clinical signs appearing late [Stead, W.W. 1992. Genetics and resistance to tuberculosis. Ann. Int. Med. 116: 937-94].

A known method for the diagnosis of tuberculosis based on a delayed-type skin hypersensitivity reaction is the so-called tuberculin test or Mantoux test [Palmer, C.E., and L. B. Ddwarsd, 1967. Tuberculin test in restospect. Arch. Environ. Health 15: 792-808]. The antigen is injected subcutaneously into the patient's body by an injection or scratch. Complex antigens (usually tuberculin or PPD - protein determined derivate) are used as antigens [Koch, R. 1891]. The response skin reaction develops within 24-48 hours and may persist for several days. It is considered positive when hyperemia, infiltration or blistering occurs. The patient is referred for further examination for the presence of a tuberculosis process.

However, the sensitivity and specificity of tuberculin samples is very limited, since among patients with tuberculosis a positive sample is observed only in 50% of cases, and due to the inability to distinguish patients with active forms of tuberculosis from previously infected, vaccinated or infected mycobacteria of other species.

The disadvantage of this method is its lack of information in a significant number of HIV-infected, when it is important to identify not only the presence of a disease caused by mycobacteria, but also determine the type of mycobacteria, as well as obtain information for effective treatment tactics. It is also difficult to diagnose with severe tuberculosis, when the immunological defense system of the body under the influence of mycobacteria is in a depressed state and cannot function effectively. [Huebner, R.E., M.F. Schein, and J. B. Bass, Jr. 1993 b. The tuberculin skin test. Clin. Infect. Dis. 17: 968-975].

Nevertheless, despite all the described limitations, a tuberculin skin test remains the main officially approved test and the risk groups for the occurrence of infection are determined by its results.

A known method for the diagnosis of tuberculosis by detecting the causative agent of tuberculosis in biological material taken from a patient. [Kent, B. D., and G.P. Kubica. 1985 Public health mycobacteriology a guide for the level III laboratory, 207 p. U.S. Department of Health and Human Services. Centers for Disease Control, Atlanta]. Standard procedures begin with microscopic examination of sputum smears and pleural exudate of patients with preliminary staining (AFB-method) for the presence of acid-resistant mycobacteria, followed by inoculation of material and biochemical testing of microorganisms grown on selective media in order to identify the type of mycobacteria found. The presence of the causative agent of tuberculosis is diagnosed with the presence of a tuberculosis process.

The disadvantage of this method is that all these procedures require a significant time - from 4 to 6 weeks from the moment of collection of the material, due to the slow growth of mycobacteria. In addition, cytological identification is not sensitive enough, is fraught with errors and depends on the qualifications of the staff. The use of the test in the laboratories of developing countries is detected only 20-40% of patients with tuberculosis, in developed countries ~ 40-60%.

More modern methods for the diagnosis of tuberculosis are also known, based on the polymerase chain reaction (PCR, or PCR) method. The principle of the method is to increase 10 ⁶ -10 ⁸ times copies of a specific DNA fragment of a microorganism activated by DNA polymerase in an automatic mode. This is a highly specific and extremely sensitive test, with the help of which it is fundamentally possible to identify in the analyzed sample the presence of even one single DNA molecule of the pathogen (sensitivity is about ^10-15 M) [Reischl U., Naumann L. / [Molecular biology methods in detection of mycobacteria. Improved and newly developed test systems for the diagnosis of mycobacteria] / Fortschr.Med. - 1996. - Vol. 114. - P.237-241]. Ha PCR method has high expectations, it is constantly being improved and is currently used in the diagnosis of tuberculosis.

The disadvantage of this method is its high cost, the complexity of execution, expensive tool and instrumentation. The result depends on the skill level of the staff and compliance with the special requirements for the laboratory facilities in which the analysis is carried out. Minimal contamination of the analyzed samples, instruments and air in the room with nucleic acid of mycobacteria can give a false positive result. In addition, due to its ultra-high sensitivity, this test often determines an inactive form of tuberculosis. For these reasons, the use of this method in laboratory diagnostics is still quite limited.

As the closest analogue, a method for diagnosing tuberculosis by determining specific anti-tuberculosis antibodies by enzyme-linked immunosorbent assay (ELISA) [Engvall E., Perlmann R. (1971). Enzyme-linked immunosorbent assay (ELISA), Quntitive assay of immunoglobulin G. Immunochem., 8, 871-874]. The method is as follows. Blood samples are taken from patients; serum samples prepared from it are examined. An enzyme-linked immunosorbent reaction is carried out, for which a mycobacterial antigen is introduced into the wells of the tablet, washed with a buffer, and a solution of bovine serum albumin (BSA) is introduced into each well. Serum samples are diluted in a BSA solution and added to the wells of the plate. In addition to the test samples, control samples were added to three wells. The tablet is incubated, washed. Then, a solution of specific anti-tuberculosis antibodies labeled with horseradish peroxidase in a BSA solution is added to the wells, incubated and washed. After that, a color reaction is carried out by introducing a color reaction substrate into the wells of the plate. The spectrophotometer determines the degree of staining by the absorption spectrum, which determines the concentration of specific anti-tuberculosis antibodies, expressed in units of optical density. The critical value of the optical density is calculated, and the optical density of the studied samples is compared with it. With a value of this value equal to or greater than the critical value of optical density, the reaction is considered positive and preliminary diagnosed with the presence of tuberculosis, and with a lower value - negative.

The known method has high sensitivity (determines up to 0.05 ng / ml of substance) and expressivity, which contributed to the widespread introduction of ELISA in laboratory practice for the diagnosis of many infectious diseases.

However, the sensitivity of the method does not always satisfy practitioners. This is due to the fact that antibodies to mycobacteria in one quantity or another are present in the blood not only of patients, but also of healthy “contact” people. In the blood serum of patients, antibodies can sometimes reach very large values, which is not always associated with the duration of infection or the severity of the disease. To a greater extent, this is determined by the characteristics of the course of the disease and the state of the patient's immune system. The antibody content in people with inactive tuberculosis is usually still significantly lower than in patients, and this makes it possible, subject to certain rules, to derive a certain discriminatory level, overcoming of which we can speak of a high probability of the presence of active tuberculosis.

In addition, the method is invasive; blood sampling by trained medical personnel in a room specially equipped for this purpose is necessary for its implementation. In this regard, this method cannot be used for large-scale research, as well as in the field.

The objective of the invention is to provide a highly effective, faster, simpler, more accurate and non-invasive method for the diagnosis of tuberculosis.

The essence of the invention lies in the fact that the proposed method for the diagnosis of tuberculosis, including the study of biological fluid by conducting an enzyme-linked immunosorbent assay, for which they add mycobacterial antigen to the wells of the tablet, wash them with buffer, add bovine serum albumin solution to each well, and test samples are diluted in serum albumin solution , add them and control samples to the wells of the tablet, incubate, wash, then add a solution of specific anti-tuberculosis antibodies labeled with horseradish peroxidase in a solution of serum bovine albumin is incubated and washed, then a color reaction is carried out and the concentration of specific anti-tuberculosis antibodies, expressed in units of optical density, is determined on a spectrophotometer, the critical optical density is calculated, and if the optical density of the test samples is equal to or greater than critical value of optical density, the reaction is considered positive and diagnosed with the presence of tuberculosis infection, and with a lower value negative, saliva is examined as biological fluid, which is preserved in a volume of 3-4 ml by adding 30 μl of sodium azide solution in a concentration of 0.002%, while mycobacterial antigen is used to carry out an enzyme-linked immunosorbent assay, which binds with a high degree of specificity for secretory IgA, and specific monoclonal antibodies directed against human secretory IgA.

Using the invention allows to obtain the following technical result.

For the first time, a method was developed for the determination of anti-IgA anti-tuberculosis secretory antibodies in patients' saliva by enzyme-linked immunosorbent assay for the diagnosis of tuberculosis.

The method has several advantages compared with the known ones, the most important of which is its non-invasiveness, which is especially important in young children during mass studies in institutions and in the field. When conducting extensive studies in adults, it is also much simpler, faster, and more economical to collect diagnostic samples of saliva rather than blood. In this case, the presence of special medical personnel, tools for blood sampling and specially equipped rooms is not required.

The method is simple to execute. The entire diagnostic process, including the collection of biological material, takes no more than 3 hours.

The method has high information content, specificity and sensitivity in relation to tuberculosis. It can be used in any clinical diagnostic laboratory where the enzyme-linked immunosorbent assay is used.

The method can be recommended for large-scale diagnostic studies in any conditions, for any population. This will allow early diagnosis of tuberculosis in a short time, prescribe timely and adequate treatment, reduce its time and material costs, and thus stop the growth of the disease due to transmission of the infection from patients with an open process to healthy individuals.

The technical result is achieved due to the fact that the authors first proposed to conduct a study of human saliva for the diagnosis of tuberculosis. An informative bloodless method has been developed, which is based on the detection of anti-TB secretory IgA in tuberculosis patients.

It is shown for the first time that the level of anti-tuberculosis secretory IgA in saliva in comparison with unchanged rates in healthy donors can be used to assess the presence of an infectious process in this disease.

The authors took the fact that the saliva of a person contains IgA and the saliva of tuberculosis patients contains anti-tuberculosis IgA antibodies that react with a specific antigen of mycobacterium tuberculosis. The main objective of the authors was to obtain a mycobacterial antigen that binds to secretory IgA with a high degree of specificity. For these purposes, a standard chromatographic method was used, by which, by separation and purification, a highly purified mycobacterial antigen consisting of fragments of the bacterial cell wall devoid of lipid components was obtained. For this, various fractions of mycobacteria were obtained, investigated, the specificity of bacterial antigens with respect to secretory IgA was determined, and the most effective ones were selected for further work. Another component of the scientific search was the production of highly specific MAT-4E7 monoclonal antibodies directed against human saliva IgA, which were obtained using the standard periodic method.

An essential feature of the method is also the conservation of saliva, because it allows you to save its native properties to obtain reliable results. In other conditions, in the presence of mycobacteria in the saliva, the latter can multiply rapidly, and in this case, the diagnostic result will not reflect the initial state of the studied samples.

The method is as follows.

The patient is sampled saliva, 20 minutes before which he rinses the oral cavity with mineral alkaline bicarbonate water such as "Narzan" or "Borjomi". Saliva is taken into a plastic container in a volume of 3-4 ml and preserved by adding 30 μl of sodium azide solution at a concentration of 0.002%. Samples are stored in a refrigerator at 4-6 ° C. If necessary, long-term storage, more than a day, samples are frozen at -20 ° C. The obtained samples of saliva are examined by enzyme immunoassay.

For carrying out the enzyme immunoassay, 96-well polystyrene plates with high binding ability, type 1 (Costar, USA, cat. No. 9018) are used. 50 μl of sodium phosphate buffer (PBS, pH 7.2) containing specific mycobacterial antigen at a concentration of 1 μg / ml was added to each well of the plate and left in the refrigerator at -4-6 ° C overnight. Mycobacterial antigen is obtained by standard chromatographic methods (Avdienko V.G., Cosmiadi G.A., Baensky A.V. et al. Immunodominant antigens. J. Problems of Tuberculosis, 2002, No. 2, pp. 30-33).

Then the wells of the plate are washed 4 times, adding 200 μl of sodium phosphate buffer (pH 7.2) containing 0.1% Tween-20 (detergent) to each well, after which the remnants of the wash buffer are completely removed from the wells of the plate by tapping the plate on folded in several layers of filter paper placed in the tray. Then, 100 μl of 1% BSA solution in sodium phosphate buffer medium is added to each well.

Samples of saliva are diluted in a 1% BSA solution (in a 1: 1 ratio) and 50 μl are added to the wells. In addition to the test samples, 50 μl of control samples, which are a pool (mixture) collected from 200 healthy people, are added to three wells of the tablet. The plate is covered and incubated for one hour at 37 ° C. Then the tablet is washed three times with sodium phosphate buffer (pH 7.2).

50 μl of a solution of specific monoclonal antibodies directed against human secretory IgA labeled with horseradish peroxidase in a 1% BSA solution at a concentration of 1 μg / ml was added to each well. These monoclinal antibodies against human IgA are obtained by the standard periodic method (Avdienko V.G., Kondrashev S.Yu., Kulikovskaya N.V. et al. J. Clin. Laboratory diag. 2000, No. 6, p. 22-35) .

The tablet is covered and incubated for one hour at 37 ° C. The plate is then washed 5 times with sodium phosphate buffer (pH 7.2)

After this, a color reaction is carried out, for which 50 μl of a color reaction substrate containing 0.04% o-phenylenediamin (Sigma, USA) and 0.012% hydrogen peroxide in citrate-phosphate buffer containing 0.05 M sodium citrate are added to each well. -NOOSS (OH) (CH ₂ COONa) ₂ and 0.1 M potassium phosphate K ₂ NRA (pH 5.0). Then the tablet is covered with a lid and incubated for 30 min at 37 ° C. After incubation, the reaction by adding 10 μl of 1 M sulfuric acid to each well of the plate.

Analysis results are taken into account within 10-15 minutes after the reaction is stopped. The reaction results are recorded using an ELx800 automatic spectrophotometer (Bioline, USA) or devices of a similar type. The degree of staining is determined by recording the absorption spectrum, and the absorbance values are obtained, which determine the concentration of specific anti-tuberculosis antibodies. Depending on the concentration of specific anti-tuberculosis antibodies, the test and control samples are stained, which is recorded by the device and is expressed in units of optical density (OD) at a wavelength of 492 nm.

To assess the results of the reaction, the critical optical density (ODcrit) is calculated by the formula:

OPcrit = OD (K) +0.2,

Where OD (K) is the arithmetic mean of the OD values obtained when setting control samples in 3 wells.

The optical density value of the test samples is compared with the critical optical density value. With a value of this value equal to or greater than the critical value of the optical density, the reaction is considered positive and previously diagnosed with the presence of the tuberculosis process, and with a lower value - negative.

The method was tested on the basis of DTS-BIO LLC and the Central Research Institute of Tuberculosis RAMS for 56 patients with pulmonary tuberculosis (23 women and 33 men) aged 12-65 years and 56 healthy plasma donors aged 25-53 years (tables 1 and 2 ) All patients with tuberculosis had a radiologically and microbiologically confirmed diagnosis with a process duration of at least 3 months.

Example 1. Patient X, the diagnosis of infiltrative tuberculosis S1-S2, suffers from the disease for about six months. Under the supervision of a doctor, she rinsed her mouth with mineral water “Narzan”, and after 20 minutes, her saliva was collected in a volume of 3.5 ml in a special container. Preserved samples by adding 35 μl of sodium azide with a concentration of 0.002%. The sample was frozen at -20 ° C and stored for 2 weeks.

For analysis, a previously thawed saliva sample of patient X was diluted in a BSA solution at a 1:10 dilution. Next, the resulting dilution was added 50 μl to a 96-well plate in duplicates, which were washed in advance with buffer. After one hour incubation at 37 ° C, the plate was washed three times with buffer and 50 μl of the “second” antibodies labeled with horseradish peroxidase directed against human IgA were added. The plate was incubated for one hour at 37 ° C. After washing five times with buffer, 50 μl of substrate was added to the plate and after 30 minutes the reaction was stopped by adding 10 μl of 1 M sulfuric acid. The reaction level was measured at a wavelength of 492 nm on an ELx800 automatic vertical-beam spectrophotometer / reader (Bioline, USA). For the test sample, the desired value was 2.036 ± 0.018 units. opt. density. By comparing the obtained value of the optical density of the test sample with OD crit (0.946 + 0.2), it was determined that this sample of saliva belongs to a patient with tuberculosis and the intervention of a TB specialist is required.

Example 2. Patient P, 17 years old, for 2 months has a low-grade fever, Mantoux 12 mm, cough, difficulty breathing. Saliva was collected according to the above procedure. The sample was immediately examined in ELISA according to the above procedure. For the test sample, the desired value was 1.547 ± 0.005 units. opt. density. Comparing the obtained value with a control sample, the average optical density of which was 1,020, we determined that this sample of saliva contains the number of secretory anti-tuberculosis IgA antibodies. The patient was examined by a TB doctor, and after additional research methods, including X-ray, microscopic examination of sputum smears, PCR was hospitalized with a diagnosis of infiltrative tuberculosis.

Example 3. A sample of saliva was obtained from patient C., suffering for a month from coughing and febrile temperature. When conducting studies for the presence of anti-TB secretory IgA, the result was 0.359 ± 0.013 units. opt. density. Comparing the obtained value with a control sample, the average optical density of which was 0.947, we determined that this sample of saliva contains the amount of secretory anti-tuberculosis IgA antibodies significantly lower than the control sample. Additional studies: Mantoux test, microscopy and PCR, also did not reveal the presence of the causative agent of tuberculosis in patient C. Fluoroscopy revealed the presence of blackouts in the middle lobe of the lung. The patient was diagnosed with pneumonia. After an enhanced course of antibiotic therapy, patient S. disappeared from all symptoms of malaise.

Table 2. The level of anti-tuberculosis secretory IgA antibodies of saliva in the group of healthy donors. №№ OD arr. / OD Counter, arr. Result one 0,07 Negative 2 0.80 Negative 3 0.63 Negative four 0.20 Negative 5 0.92 Negative 6 0.37 Negative 7 0.24 Negative 8 0.15 Negative 9 0.37 Negative 10 0.85 Negative eleven 0.49 Negative 12 0.63 Negative 13 0.20 Negative fourteen 0,03 Negative fifteen 0.44 Negative 16 0.29 Negative 17 0.10 Negative eighteen 0.60 Negative 19 0.23 Negative twenty 0.21 Negative 21 0.16 Negative 22 0,03 Negative 23 0.08 Negative 24 0.27 Negative 25 0.29 Negative 26 0,07 Negative 27 0.23 Negative 28 0.09 Negative 29th 0.02 Negative thirty 0.12 Negative 31 0.37 Negative 32 0.01 Negative 33 0.22 Negative 34 0.39 Negative 35 0.16 Negative 36 0.24 Negative 37 0.47 Negative 38 0,07 Negative 39 0.40 Negative 40 0.14 Negative 41 0.44 Negative 42 0.51 Negative 43 0.84 Negative 44 0.19 Negative 45 0.27 Negative 46 0.30 Negative 47 0.28 Negative 48 0.91 Negative 49 0.73 Negative fifty 0.64 Negative 51 0.12 Negative 52 0.56 Negative 53 0.80 Negative 54 0.54 Negative 55 0.43 Negative 56 0.20 Negative

Claims (1)

A method for the diagnosis of tuberculosis, including the study of biological fluid by conducting an enzyme-linked immunosorbent assay, determining the concentration of specific antibodies specific for mycobacterial antigens, expressed in units of optical density (OD), characterized in that as a biological fluid, saliva is examined, which is 3-4 ml in volume canned by adding sodium azide at a concentration of 0.002%, and mycobacterial antigen that specifically binds to secretory immunoglobulin is used as antigen A man m, using as conjugate specific monoclonal antibodies directed against human secretory immunoglobulin A, and a value of OD at or above the OVSF (0.946 + 0.2) diagnose the presence of tuberculosis infection.