RU2296335C2 - Set of reagents for quantitative assay of secretory immunoglobulin a in serum and human body secrets by single-step solid-phase immune enzyme analysis - Google Patents

Abstract

FIELD: medicine, analytical immunology.

SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.

EFFECT: improved assay method.

3 tbl, 5 ex

Description

The invention relates to immunobiotechnology, in particular to the creation of a diagnostic kit that is intended for immunoanalytical methods for the quantitative determination of secretory immunoglobulin A in the serum and secrets of the human body when establishing the status of local immunity, identifying secondary immunodeficiency states, detecting dysbiotic intestinal disorders, monitoring bacteriotherapy and antibiotic therapy.

It is known that the mucous membranes of open biotopes (respiratory, intestinal, urogenital tracts, oral cavity, conjunctiva of the eye, etc.) are the entrance gate to the causative agents of infection. Therefore, the development of infectious diseases largely depends on the level of nonspecific resistance and the level of local immunity of the mucous membranes. It has been shown that IgA deficiency underlies many chronic inflammatory diseases of the mucous membranes and various types of allergies (1).

Determination of the level of secretory immunoglobulin A in serum and secrets (saliva) is carried out using the Mancini method. The method is based on the ability of antibodies and antigen to form an insoluble immune complex in an area of equivalent quantitative ratios. This method underlies the semiquantitative determination of IgA by radial immunodiffusion (RID) in a gel (1). The duration of the RID reaction is 18-48 hours. There are no standardized and officially registered diagnostic reagent kits for RID in Russia or abroad, therefore, the different levels of IgA levels in serum and saliva are ambiguous (1-5). In the literature there are no data on the indicators of local immunity of human mucous secrets normal. However, the presence of significant changes in the system of local immunity in pathological processes has been noted by many researchers.

Laboratory assessment of dysbiotic intestinal disorders is based on microbiological analysis of feces (6, 7). The collection of material in an amount of from 0.1 to 1 g for research is carried out in sterile disposable backpacks and delivered to the laboratory for research within 1-2 hours. Sample storage is not permitted. Microbiological sowing of faecal material is carried out from a series of standard ten-fold dilutions in physiological saline by the drop method and by the method of distribution over the surface of the nutrient medium.

Microbiological crops are taken into account without isolation of pure cultures according to the results of a quantitative assessment and a description of the morphology of the grown colonies after 24, 48, 72 and 120 hours of temperature control according to the growth characteristics of each particular type of microorganism. To evaluate the results of microscopy of lactobacilli and bifidobacteria, staining is carried out by simple staining using carbol fuchsin, methylene blue, as well as Gram differential staining (8).

Microbiological criteria are the state of bifidobacteria and lactoflora, a decrease in the number of Escherichia, the appearance of E. coli strains with altered properties, an increase in the number of cocci, the detection of conditionally pathogenic gram-negative rods, as well as fungi. There is no single point of view in assessing the degree of dysbiosis, since different clinical and laboratory criteria are often used (9-12). When assessing the results of microbiological analysis of feces, they proceed from indicators of the conditional norm (11).

The bacteriological method for the diagnosis of dysbiosis has a number of significant drawbacks, which include not only its complexity and the duration of the necessary bacteriological tests, the relative subjectivity of the results, but also the underestimation of the role of uncultivated representatives of intestinal microbiocenosis, as well as the possibility of obtaining inconsistent results in a second study, such as a consequence of the different growth of bacteria in the host organism and on artificial nutrient media . Studies to identify anaerobic microorganisms are time-consuming and require special nutrient media that are not produced by the domestic industry.

The above methods can serve as analogues of the claimed invention.

The prototype of the claimed invention can serve as reagent kits for quantitative enzyme-linked immunosorbent assay of sIgA in the secrets of the body, described in 13 and 14.

The kits include tablets with immobilized monoclonal antibodies to sIgA. After incubation with serum or other body fluids, the presence of sIgA is detected by binding of a goat polyclonal antibody conjugate to sIgA α-chain with horseradish peroxidase. The concentration of sIgA (μg / ml) in different biological fluids using this reagent kit (13):

Serum (normal) (n = 40) - 0.94-2.73

Women's milk (n = 6) - 1374-15100

Bile (n = 7) - 25.4-89.1

Saliva (n = 8) - 81.5-130

Flushing (100 ml of saline) of the jejunum (n = 4) - 38.5-95.5

Urine (n = 5) - 0-58.4

Tears (n = 4) - 0.51-1.18

Bronchoalveolar flush (150 ml of saline) (n = 6) - 0.19-48.3

Vaginal flush (10 ml of saline) (n = 7) - 0.186-22.3

The reagent kit designed for the quantitative determination of sIgA in various biological fluids contains plates with immobilized monoclonal antibodies to the secretory component, which binds sIgA of the analyzed sample, and peroxidase-labeled monoclonal antibodies against the sIgA α chain reveal sIgA bound to the solid phase. The concentration of sIgA in μg / ml of biological fluids of healthy individuals using this reagent kit (14):

Serum - 2.86 ± 1.25

Saliva - 207.5 ± 92.2

Urine - 14.6 ± 9.5

Lacrimal fluid - 76.04 ± 17.58

Bronchoalveolar lavage - 7.3 ± 6.0

Vaginal secretion - 85.03 ± 27.16

The purpose of the invention is to develop a method for the quantitative determination of sIgA in serum, human secrets, suitable for assessing the status of local immunity of mucous membranes of open biotopes, in feces, suitable for assessing functional disorders of the large intestine ecosystem, choosing adequate complex therapy, evaluating its effectiveness, and also for dynamic monitoring of the adaptation of the body to various kinds of influences.

The goal is achieved:

1) our comprehensive study, including the study of microbiocenosis and sIgA level in feces. We showed that the process of adaptation of the cosmonaut's organism to pre-flight preparation, space flight and landing is realized through a series of changes: a decrease in the protective groups of microorganisms, the emergence of opportunistic microflora, the formation of dysbiosis against the background of an increase in sIgA level in feces. A similar picture was obtained in model experiments on primates. This means that the level of sIgA can practically serve as a convenient marker of stressful changes in microflora and an indicator of the physiological state of the body under stress, determining the degree of deviation of the body's natural resistance from the individual physiological norm (15);

2) using a pair of highly specific murine monoclonal antibodies to sIgA produced by strains of hybrid cultured animal cells of Mus.musculus L No. RKKK (P) 676D and 677D.

Our kit is intended for the quantitative determination of sIgA in serum, the secrets of open biotopes of the body. The results of determination of sIgA content can be used to establish the status of local immunity, identify immunodeficiency states, assess functional impairments of the large intestine ecosystem, and control antibiotic therapy and bacteriotherapy. The invention relates to biology and medicine and can be used to assess the functioning of the local serum immunity system and open biotopes (intestines, urogenital tract, upper respiratory tract, etc.).

The kit contains a tablet with immobilized monoclonal antibodies, five calibration samples (containing 0; 1.0; 5.0; 10.0; 20.0 μg / ml sIgA), a conjugate of mouse monoclonal antibodies with horseradish peroxidase, and reagents for the enzymatic reaction. Murine monoclonal antibodies produced by the strain of hybrid cultured animal cells of Mus.musculus L No. RKKK (P) 676D were immobilized onto the carrier. The conjugate contains murine monoclonal antibodies produced by the strain of hybrid cultured animal cells of Mus.musculus L No. RKKK (P) 677D. Human colostrum sIgA with 1% serum albumin is used as calibration samples.

Analysis

Release tablet from packaging. In duplicate, add 20 μl of each calibration sample and 25 μl of the control sample in duplicate. In the remaining wells, add in duplicates 20 μl of the studied samples. Add 100 μl of a solution of conjugate of antibodies to sIgA with peroxidase to all wells and incubate at 37 ° C for 1 h. After incubation, remove the contents of the wells and wash 4 times with a working solution for washing the plates, 200 μl per well. Add 100 μl of the developing solution to all wells, incubate at 37 ° С for 15 minutes. Stop the reaction by adding 100 μl of 4.8% sulfuric acid to all wells. Measure the optical density in the wells on a vertical scanning photometer at a wavelength of 450 nm. The main characteristics of the kit are shown in table 1.

It should be noted that a diagnostic kit for quantitative enzyme-linked immunosorbent assay of sIgA in secrets (including feces) was not previously known, the technical solution we found was not described in the literature and worked out as a result of a large series of experiments, i.e. the present invention meets the criteria of originality, novelty and inventive step.

Examples of practical applications of the invention (tables 2 and 3).

Preparation of samples for analysis.

Samples for analysis were collected from the first portion of freshly excreted feces. For microbiological studies, the material was delivered no later than 2 hours after collection. The state of intestinal microbiocenosis was evaluated by 6.

To determine the sIgA level, coprofiltrates were prepared on saline with a concentration of 30-50 mg feces / ml saline, stirred on a vortex mixer for 1 h, centrifuged at 3000 rpm. The level of sIgA was determined in supernatants using this kit.

Saliva was collected in test tubes, frozen at -20 ° C and stored at this temperature until analysis. Before analysis, they were thawed and centrifuged at 3000 rpm. SIgA was determined in the supernatant.

Bronchoalveolar lavage was obtained by administering 100 ml of physiological saline followed by active aspiration of the flush and subsequent centrifugation at 3000 rpm. SIgA was determined in the supernatant.

Example 1. In feces 15 children aged 1.5 months. up to 5 years without deviations in the intestinal microbiocenosis (eubiosis), the level of sIgA was 82.63 ± 56.37 μg / g feces.

Example 2. In the feces of 67 frequently ill children aged 1 month to 8 years (frequent acute respiratory diseases, signs of atopic dermatitis, various deviations in the microflora of the large intestine: dysbiosis from 1 to 3 severity), the level of sIgA was 184.23 ± 84 , 02 mcg / g feces.

Example 3. In the feces of 33 frequently ill children with abnormalities in the intestinal microflora, after a three-week course of bacteriotherapy with probiotics, the sIgA level was 99.2 ± 54.62 μg / g feces.

Example 4. In the feces of 51 patients of the medical unit of the airport with various diseases (frequent acute respiratory infections, gastritis, peptic ulcer, colitis, neurodermatitis, pancreatitis, cholecestitis), complicated by dysbiosis grade 1-4, sIgA level was 875 ± 119 μg / g feces .

Example 5. In the feces of 26 patients of the airport medical unit with various diseases (frequent acute respiratory infections, gastritis, peptic ulcer, colitis, neurodermatitis, pancreatitis, cholecestitis), complicated by dysbiosis 1-4 degrees of severity, after a two-week course of bacteriotherapy with bifidumbacterin and lactobacterin s level 288 ± 101 μg / g feces.

Table 1. Key features of the sIgA quantification diagnostic kit. Measured parameter Characteristic Principle of analysis Solid-phase enzyme-linked immunosorbent single-stage Solid phase Polystyrene microplates measurement range 1-20 mcg / ml Analysis time 1 h + 15 min Analysis sample volume 25 μl Sensitivity 0.2 mcg / ml KB 4,5

Table 2. SIgA levels in feces of different patient groups No. Groups of subjects n sIgA (M ± m) μg / g one. Children aged 1.5 months to 5 years without deviations in the microflora (eubiosis) fifteen 82.63 ± 56.37 2. Often sick children aged 1 month to 8 years with various deviations in the microflora of the large intestine (dysbiosis from 1 to 3 severity) 67 184.23 ± 84.02 3. Patients (airport ground workers) of the airport medical unit with various diseases complicated by dysbiosis 51 875 ± 119 four. Patients (airport ground personnel) of the airport medical unit with various diseases complicated by dysbiosis after a two-week course of bacteriotherapy 26 288 ± 101 5. Often sick children with deviations in the intestinal microflora after a three-week course of bacteriotherapy 33 99.20 ± 54.62

Table 3. SIgA levels in secrets of healthy people Secret sIgA (M ± m) μg / g Saliva (n = 24) 130.1 ± 45.5 μg / ml Serum (n = 28) 0.602 ± 0.17 μg / ml Bronchoalveolar flush (n = 7) 46.3 ± 2.2 μg / ml Feces (children from 1.5 months to 5 years) (n = 15) 82.63 ± 56.37 mcg / g

Literature

1. Khaitov P.M., Pinegin B.V., Istamov H.I. Ecological immunology. (1995), "VNIRO", M., 218 p.

2. Kiryukhin A.V., Parfenova N.A., Maksimova T.A., Shenogina N.A., Lvov A.V., Chumakova M.M., Andronova T.M. Optimization of treatment for frequently and long-term ill children: immunocorrection with lycopid. Immunology (2003), No. 1, pp. 47-50.

3. Masternak Yu.A., Luss L.V. The effect of polyoxidonium on the indicators of the immune status of the elderly. Immunology (2002) No. 6, p. 343-346.

4. Clinical Guide to Laboratory tests. (Ed. Tietz NW) 3 ^d issue. WBSaunders Company (USA), 1995, p. 354.

5. Pershin B. B., Kuzmin S. N., Kochkurkin V. N. and others. Local immunity reactions in swimmers of the national team. J. mic., Epid. and immunobiol. 1996, No. 1, p. 53-57.

6. Methodological recommendations of the Medical Center of the Office of the President of the Russian Federation, "Comprehensive diagnosis, treatment and prevention of intestinal dysbiosis in the clinic of internal diseases", M., 1997.

7. A manual for doctors and students of MMA named after Sechenov "Dysbacteriosis in children." Edited by A.A. Vorobyov and S.G. Pak, M., 1998.

8. Methods of General Bacteriology, Ed. Gerhard, M., 1986, 1 vol., Pp. 31-33, 67-68.

9. Kuvaeva IB, Ladodo KS "Microbiological classification of colonic dysbiosis";

10. Goncharova G.I., Gracheva N.V. "Classification of dysbiosis according to severity", M., 1986.

11. Bondarenko V.M. "The composition of the intestinal microflora is normal", M., 1998.

12. Kopanev Yu.A., Sokolov A.L. Intestinal dysbiosis: microbiological, immunological and clinical aspects of microecological disorders in children. M., 2002, 146 p.

13. Vincent C., Revillard J.P. Sandwich-type ELISA for free and bound secretory component in human biological fluids. J. Immun.Meth. 1988, v. 106, p. 153-160.

14. Galkina O.V., Gryazeva I.V., Klimovich V.B. et al. Quantitative determination of secretory immunoglobulin A in biological fluids using monoclonal antibodies. Medical immunology. 2000, vol. 2, No. 2, p. 155.

15. Viha G.V. Clinical and experimental rationale for the development of test systems for identifying stress-dependent markers for studying the mechanisms of adaptation of an organism in experimental situations. Abstract. dis ... doctor biol. Sciences, M., 2002.

Claims (1)

A set of reagents for the quantitative one-step enzyme-linked immunosorbent assay for secretory immunoglobulin A (sIgA) in blood serum and secrets, containing a carrier with the first mouse monoclonal antibodies to human sIgA immobilized, a conjugate with peroxidase of second mouse monoclonal antibodies to sIgA, calibration samples containing s in that, as the first antibody, it contains murine monoclonal antibodies produced by a strain of hybrid cultured animal cells of Mus.musculus LN PK K (R) 676D, and as the second antibody used murine monoclonal antibodies produced by hybrid strain cultured animal cells RKKK Mus.musculus L N (R) 677D.