RU62709U1 - TEST SYSTEM FOR SEMI-QUANTITATIVE IMMUNO-ENZYME DETERMINATION OF ALLERGEN-SPECIFIC IgE ANTIBODIES IN HUMAN BLOOD SERUM (IgE-AT-ELISA) - Google Patents

Abstract

The utility model relates to medicine, namely to laboratory diagnostics. The task is carried out through the proposed test system IgE-AT-ELISA. The utility model is a kit, which includes a set of reagents, including a solid-phase carrier with immobilized domestic broad-spectrum allergens, calibrators ready for use and with a known content of IgE antibodies, washing solution, conjugate, substrate-chromogenic mixture, ready to use, stop reagent and instructions for use, packed in one box.

Description

The utility model relates to medicine, namely to laboratory diagnostics.

According to the WHO data, allergic diseases also belong to diseases caused by insufficiency of the immune system, the number of which is increasing every year. The main reasons for the significant growth of such pathologies around the world are industrialization, chemicalization, and environmental pollution. The prevalence of such diseases has higher values in regions (districts) with an ecologically unfavorable environment caused by anthropogenic environmental impacts, and depends both on the nature of the alergenic environment and climatogeographic features (Food allergy in children. Modern aspects. - Revyakina V. A., Borovik T.E., Russian Allergological Journal. N2, 2004.)

Therefore, the problem of allergic diseases is an important medical and social problem of our time, the importance of which will increase in the coming years. Ways to solve these problems are reflected in the Order of the Ministry of Health and Social Development of Russia No. 342 of May 16, 2005, where in Appendix No. 1 “PROVISION ON ORGANIZING THE ACTIVITIES OF AN ALLERGOLOGIST-IMMUNOLOGIST” in the Diagnostics section, the determination of specific IgE antibodies to various types was included in the mandatory paragraph allergens.

Thus, the determination of the level of specific antibodies of the IgE class in the diagnosis of allergic diseases seems to be an important criterion in the complex of diagnostic measures. This means that the solution of practical medical issues of this problem largely depends on the quality of specific in vitro allergic diagnostics.

Among laboratory methods in the specific diagnosis of allergies, the most informative are the immunological ones: the radio allergy sorbent test (PACT) and the enzyme-linked immunosorbent assay (ELISA), which identify with great certainty specific IgE and IgG antibodies to various food allergens. The diagnostic value of these methods is very high and amounts to 87-85%, while the informational content of skin testing with food allergens is only 49%. (Allergic diseases. Diagnosis and treatment. Edited by R. Patterson. M., Geotar Med., 2000, p. 733.). According to the researchers, a semi-quantitative assessment is more acceptable for clinical use (Komarova M.V. Sinelnikova L.V. Fufygina E.G. On the question of the study of food intolerance by immediate and delayed mechanisms. - Collection of scientific papers: "Ekaterinburg Consultative and Diagnostic center: the results of 15 years of activity in practical healthcare ", - Ekaterinburg, 2004. - P.229-230).

In the Russian market, the determination of the level of specific IgE antibodies is widely represented by test systems of foreign companies. From domestic manufacturers, test systems for the determination of specific IgE antibodies to various types of allergens are released at Allergen Federal State Unitary Enterprise. city of Stavropol.

Hitachi Chemical Diagnostics Inc. USA) offers a chemiluminescent method for the determination of specific IgE and IgG - a multiple allergy-absorbent test using chemiluminescent analysis (MAST-CLA). The essence of the MAST-CLA method is to conduct an allergen-specific IgE (IgG) reaction followed by labeling the resulting complex with a photoreagent and determining its luminosity, the intensity of which is proportional to the concentration of specific IgE (IgG) in the patient's serum.

Dade Behring (USA) offers the measurement of specific blood immunoglobulins based on the nephelometric method. The method is based on the precipitation reaction of the “protein - highly specific serum” complex. To do this, measurements of the lateral scattering of laser radiation at a wavelength of 480 im. using an automatic analyzer.

The Dutch company Medland Systems offers the determination of specific IgE and IgG in blood serum using the CLA-1 electro-diagnostic chemiluminescent device.

The allergens proposed for specific diagnostics are immobilized on a solid-phase carrier, the role of which is a nitrocellotose disc (allergodisc) of the diameter of a microplate well (when determining specific IgE antibodies) or the surface of plastic tubes or microplate wells (when determining specific IgG antibodies).

Milenia-Biotec, Germany, offers test systems in which the diagnosed allergens are presented in the form of test cassettes.

Closest to the technical solution is a test system for determining specific IgE antibodies to various allergens of FSUE Allergen, Stavropol.

Principle of the method: allergens are immobilized on a solid-phase carrier (polystyrene 96-well plates type-setting or solid). Calibrators are pre-diluted by titration. Blood serum and diluted calibrators of 100 μl are added to the wells of the tablet. Allergens bind to specific antibodies present in the patient's blood serum. Excess antibodies are removed at the washing stage. At the next stage, enzyme-labeled antibodies bind to the allergen-antibody complex. After washing and removing unbound components, a substrate solution is added, consisting of orthophenylenediamine and a citrate buffer solution, interacting with the enzyme, resulting in the formation of a color complex. The optical density of the resulting solution is directly proportional to the concentration of specific IgE antibodies in the serum sample.

However, this test system for the determination of specific immunoglobulins of class E has a number of significant disadvantages:

- additional time and reagent costs for preliminary dilution and titration of calibrators;

- the use of large (100 μl) patient serum per 1 allergen, which makes it difficult to diagnose a wide range of allergens in one patient;

- the use of orthophenylenediamine, a toxic substance, as a chromogen;

- high costs of the substrate mixture.

The above complicates the widespread use of the test system in practical health care.

The task is to develop a test system for the semi-quantitative immunofermeitis determination of specific IgE antibodies to a wide range of domestic allergens in the blood serum of patients (IgE-AT-ELISA).

The task is carried out through the proposed test system IgE-AT-ELISA, which includes the following reagents, packed in one box:

Reference reagent 1 (P1) - 96-well stacked stripped plates of size 125 × 85, made of a transparent polymeric material (Cat. No. 95029100 Thermo Labsystems, Cat. No. 70507 Greiner) with horizontal and vertical rows of holes (8 × 12). used as a solid-phase carrier on which monoclonal anti-IgE antibodies are immobilized. The proposed stacked tablets have high sorption quality, the optical density of each well of the tablet is standardized and does not exceed 0.04 units of optical density (OD). In the wells of the vertical row No. 1, there are immobilized monoclonal antibodies (MKAT) anti-IgE, and in the holes of the vertical rows No. 2 to No. 12 there are immobilized allergens purified, standardized by the nitrogen content of the protein (OJSC I. Biomed named after I.I. Mechnikov )

Reference reagent 2 (P2) - calibration samples: KPA, KPV, KPS, KPO, A KPO - containing a known amount of IgE and corresponding to OP - 4, 3, 2, 1, 0 classes (calibration according to WHO 67/87 standard) , liquid, ready to use.

Reagent 3 (P3) - polystyrene typesetting strips from Thermo Lab-systems (Finland) or Greiner (Germany) coated with various allergens with a concentration of 40 μg / ml by physical sorption - 22 stryls (rows) of 11 strips (rows) ) of different specificity in each tablet. The use of typesetting strips of these firms allows the use of single wells, which makes it possible to avoid the period of accumulation of patient sera necessary for whole or strip 96-well plates and to reduce the time for diagnosis.

Reagent 4 (P4) - conjugate (monoclonal diagnostic antibodies against light and heavy polypeptide chains of human immunoglobulins E with

horseradish peroxidase enzyme. Working dilution of the conjugate 1: 100 (manufactured in St. Petersburg FSP No. 42-0190-0593-00 from 01.16.2002). The use of this conjugate allows you to capture only specific IgE antibodies.

Reagent 5 (P5) - washing solution, concentrated, pH 7.4. The components of the trio buffer are selected taking into account the absence of cross-reactions with other reagents of the test system, which excludes the registration of non-specific reactions

Reagent 6 (P6) - substrate-chromogenic mixture [hydrogen peroxide (GOST 177-88), 3 mM, in 31 mM citrate (citric acid TU 6-09-584-75) buffer: tetramethylbenzidine - TMB (CJSC Diaplus) , Cat. No. 0763600)], the TMB-substrate system provides about 10 times higher sensitivity for detecting peroxidase than the OFD-CFR system (orthophenylenediamine - citrate-phosphate solution) and, in addition, is less toxic.

Reagent 7 (P7) - stop reagent (sulfuric acid (GOST 4204-79). 1N].

The sensitivity of the semi-quantitative analysis method is estimated by the minimum level of IgE antibodies (class 1), at which cuie can detect a difference in optical density between KPO and KPO that does not contain IgE antibodies in the enzyme-linked immunosorbent assay.

Accuracy is determined by the ability of the test system to give the same results when testing the same serum on the same strip. It is expressed by the coefficient of variation of various specificities in 8 replicants of each serum sample.

Before starting work, the kit is removed from the refrigerator and kept for 30 minutes. at room temperature. Reagents are thoroughly mixed before use and prepare working solutions:

The washing solution, a 10-fold concentrate, is diluted 10 times with distilled water. To one volume of P5 add 9 volumes of distilled water, pH 7.4.

A MKAT anti-IgE conjugate stock solution is prepared by diluting 100 times with a wash solution.

The working solution is the substrate of the chromogenic mixture is prepared by mixing equal parts of P6 and P7. The solution is stable for 1 hour after preparation.

90 μl of washing solution are added to all strips. In wells A-H of the first row contribute 10 μl of calibrators in duplicates. 8 remaining wells contribute 10 μl of test sera. The tablet is covered with a lid, stirred on a shaker or manually for 1 min, then placed in a thermostat for 1 hour at t ° + 37 ° C.

The contents of the wells are removed by shaking from an inverted tablet. 150 μl of the washing solution are added to all wells of the plate and removed by shaking. The procedure is repeated 3 times. 100 μl of the conjugate working solution are added to all wells. The plates are incubated for 1 hour at a temperature of + 37 ° C. The washing procedure is repeated 3 times with a washing solution and 2 times with distilled water.

100 μl of the substrate of the chromogenic mixture are added to all wells of the plate. It is kept in a dark place at room temperature for 5-30 minutes.

Add 50 µl stop reagent to each well of the plate. Accounting is carried out on a spectrophotometer vertical shuffling at a wavelength of 450 nm. An analysis is considered valid if:

- OP KPO does not exceed 0.2;

- OP KPA exceeds 1.5;

- OP KPO exceeds the performance of OP KPO.

The arithmetic mean between the OD values of the takes of each of the tested serum samples is calculated. If the OL of the test serum is higher than the OD of the KPA solution, then the level of the content of allergen-specific IgE antibodies corresponds to class 3, etc. Taking into account the results of the reaction, in order to control specific and non-specific therapy, it is proposed to determine the quantitative content of specific IgE antibodies according to a calibration graph. Optical density indicators are plotted on linear graph paper on the Y axis, and calibrator concentration values on the X axis.

A comparative analysis of patient serum studies in the proposed utility model was performed with studies of the same serum samples using a diagnostic kit for the determination of specific IgE antibodies to food allergens from Allerren Federal State Unitary Enterprise, Stavropol.

We studied the serum of 100 patients, men and women aged 18 to 60 years (mean age 35 ± 3.1). The examined patients were divided into 2 groups in accordance with the diagnoses:

- 1 group consisted of 70 people (average age 31 ± 3.1) with a diagnosis of allergic rhinitis;

- group 2 - 30 people (average age 34 ± 2.1) were almost healthy employees of the laboratory of the Center for Specialized Types of Medical Care.

The results of the determination of specific IgE antibodies between the IgE-AT-ELISA test system and the Allergen test system in the control and experimental groups are presented in Tables 1 and 2, respectively. Significant differences noted in the results of assessments of house and library dust allergens. sunflower, ryegrass, wormwood, birch, alder may be due to higher sensitivity of the substrate-chromogenic mixture. Otherwise, there were no significant differences in the indicators.

Thus, the comparison results allow us to conclude that the proposed utility model in the form of a test system for the semi-quantitative enzyme-linked immunosorbent assay of specific IgE antibodies in human serum (IgE-AT-ELISA) can compete with both foreign and domestic analogues. The minimum amount of serum consumed per 1 allergen (10 μl) allows us to recommend the use of the test system both in practical health care in general and in pediatrics in particular.

Suggested Utility Model:

1. Allows you to define specific IaE antibodies to a wide range of domestic allergens, defined as "regional";

2. Cheap, with a fairly short staging time, which allows its use in screening studies;

3. Safe in diagnosis;

4. Highly sensitive and reproducible;

5. The use of the proposed utility model is possible in any clinical laboratory having a tablet or strip spectrophotometer.

Table 1 The level of total IgE in patients with allergic rhinitis (n = 70) The level of total IgE (M ± m, ME / ml) The number of patients with food allergies 146.9 ± 14.3 34 356.3 ± 15.2 23 604.7 ± 28.8 13 table 2 The level of total IgE in the control group (n = 30) The level of total IgE (M ± m, ME / ml) t Number of subjects 42.7 ± 14.2 16 136.5 ± 9.3 13 > 200 one

Table 3 Comparison of the diagnostic effectiveness of the IgE-AT-ELISA test system and a kit for the determination of specific IgE antibodies to food allergens from the Federal State Unitary Enterprise Allergen, Stavropol Name of allergen IgG-AT-ELISA - classes (detection rate,%) FSUE Allergen classes (detection rate,%) 0 one 2 3 four 0 one 2 3 four n % n % n % n % n % n % n % n % n % n % Orange 12 17.1 21 thirty 27 38.6 9 12.9 7 1.4 10 14.3 25 35.7 28 40 7 10 0 0 Mandarin fourteen twenty 25 35.7 29th 41,4 2 2.9 0 0 16 22.8 23 32.8 thirty 42.8 3 4.3 0 0 Lemon 9 12.9 eighteen 25.7 29th 41,4 12 17.1 2 2.9 eleven 15.7 19 27.1 26 37.1 12 17,2 2 2.9 Chicken Egg Protein 10 14.3 23 32.8 21 thirty eleven 15.7 four 5.7 13 18.6 21 thirty 22 31,4 9 12.9 5 7.1 Chicken Egg Yolk 25 35.7 fourteen twenty 29th 41,4 2 2.9 0 0 24 34.3 16 22.8 27 38.6 2 2.9 one 1.4 House dust 19 27.1 twenty 28.6 23 32.9 6 8.6 2 2.9 28 40 twenty 29th twenty 29th 2 2.9 0 0 Library dust 21 thirty 21 thirty 24 34.3 3 4.3 one 1.4 23 32.8 23 32.8 22 31,4 2 2.9 0 0 Ryegrass 17 24.3 21 thirty 23 32.8 7 10 2 2.9 twenty 29th 24 34.3 twenty 29th 5 7.1 one 1.4 Sagebrush 28 40 fourteen twenty 17 24.3 10 14.3 one 1.4 36 51,4 fifteen 21,4 fourteen twenty 5 7.1 0 0 Birch 24 34.3 17 24.3 twenty 29th 8 11,4 one 1.4 21 thirty 21 thirty 24 34.3 four 5.7 0 0 Alder 24 34.3 12 17,2 33 48.1 one 1.4 one 1.4 21 thirty fourteen twenty 35 fifty 0 0 0 0 Hake 19 27.1 23 32.8 23 32.8 four 5.7 one 1.4 22 31,4 21 thirty 24 34.3 2 2.9 one 1.4 Sunflower 21 thirty 21 thirty 19 27.1 7 10 2 2.9 26 37.1 22 31,4 eighteen 25.7 four 5.7 0 0 Buckwheat 26 37.1 23 32.8 12 17.1 9 12.9 0 0 27 38.6 25 35.7 fourteen twenty four 5.7 0 0 Oat groats 27 38.6 22 31,4 fifteen 21,4 6 8.6 0 0 27 38.6 26 37.1 eleven 15.7 5 7.1 one 1.4 Wheat flour 22 31,4 eighteen 25.7 24 34.3 5 7.2 one 1.4 25 35.7 twenty 29th 21 thirty four 5.7 0 0 Rye flour 16 22.8 19 27.1 27 38.6 6 8.6 2 2.9 19 27.1 27 38.6 16 22.8 7 10 one 1.4 Rice cereal 23 32.8 12 17,2 23 32.8 9 12.9 3 4.3 21 thirty 13 18.6 24 34.3 eleven 15.7 one 1.4 Barley groats 24 34.3 23 32.8 19 27.1 3 4.3 one 1.4 25 35.7 25 35.7 17 24.3 3 4.3 0 0 Note: allergens are marked in bold, the indicators of which differ significantly from the indicators of the test system of the Federal State Unitary Enterprise Allergen

Claims (1)

Test system for the semi-quantitative enzyme-linked immunosorbent assay for allergen-specific IgE antibodies in human serum (IgE-AT-ELISA), which is a reagent kit and includes a solid-phase carrier with immobilized allergens, calibrators, washing solution, conjugate, substrate, chromogen, stop reagent, different the fact that the test system is located in a box, the solid-phase carrier is a strip tablet, the calibrators are liquid calibration samples, ready for use, and as Mauguin chromogenic substrate-use mixture.