RU2744521C1 - Method and a kit for an immunoenzymometric assay of the quantitative content of antitoxic anti-diphtheria igg antibodies in human serum - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: invention relates to immunology. To detect antitoxic IgG antibodies in quantitative terms (mcg/ml), a standard calibration sample containing 80 mcg/ml of anti-diphtheria antitoxic IgGs is put into the wells of a microplate with an occluded antigen (10 mcg/ml). The test samples of human blood serum are incubated. Then a conjugate of the horseradish peroxidase enzyme and rabbit antibodies against human IgGs are put into the wells at a dilution of 1: 16000. Incubation is carried out at a temperature of 37 ° C for 30 minutes in a thermal shaker with a frequency of 700 rpm. The contents of the wells are poured and washed four times with a phosphate buffered saline with Tween-20. After that a tetramethylbenzidine solution is added and incubated. A stop reagent is added to all wells of the plate and the formed immune complexes are detected at a wavelength of 450 nm. Calculation of the concentration of the determined antitoxic IgGs is carried out using a standard calibration sample with a known concentration of antitoxic IgGs taking into account the dilution of the samples. The kit for the determination of antitoxic anti-diphtheria antibodies of a person contains: a flat-bottomed microplate with the occluded antigen (10 mcg/ml), a standard calibration sample containing anti-diphtheria antitoxic IgGs (80 mcg/ml), a conjugate of the horseradish peroxidase enzyme with rabbit antibodies against human IgGs, a concentrate of the phosphate buffered saline with the detergent Tween-20, a tetramethylbenzidine solution, a stop reagent, and instructions.

EFFECT: invention increases specificity, sensitivity and reproducibility of results. It also reduces analysis time.

2 cl, 2 tbl, 3 ex, 2 dwg

Description

The invention relates to medicine, namely to immunology, and can be used for the diagnosis of antitoxic anti-diphtheria antibodies in the blood serum of people (children and adults): healthy, sick, vaccinated with DPT vaccine, AD-M toxoid, bacterial carriers of the causative agent of diphtheria.

A known method for determining antitoxic antidiphtheria antibodies by the method of passive hemagglutination reaction (RPHA), which is used in the Russian Federation. For this, "Diagnosticum erythrocyte diphtheria liquid" is used. RPHA is based on the formation of immune agglutinates as a result of the interaction of specific antibodies with an antigen (toxoid) fixed on the surface of sheep erythrocytes. The method provides for the production of carriers loaded with antigen: treatment of sheep erythrocytes, sensitization with diphtheria toxoid. The next stage is the introduction of erythrocytes sensitized with diphtheria toxoid into the test serum. If it contains anti-diphtheria antitoxic antibodies, agglutination occurs. The agglutination reaction is taken into account visually. BIOMED them. I.I. Mechnikov. 2008, 4 p.

The disadvantages of the RPHA method include its low sensitivity, as well as the detection of total antibodies of different isotypes.

The known method allows you to determine the quantitative content of anti-diphtheria antitoxic antibodies in human sera using a commercial enzyme-linked immunosorbent assay (ELISA) used in world practice. The advantage of this test system in comparison with RPHA is the use of an antigen (toxoid) pre-sorbed on the wells of the plate, which is stored for a long time under normal conditions, as well as the presence of a ready-made calibration sample with a known IgG concentration, converted to IU / ml, which makes it possible to determine the content of specific IgG in quantitative relative units IU / ml. VaccZyme Diphtheria Toxoid IgG Enzyme Immunoassay Kit. Instructions for use. Version 16. The Binding Site Group Ltd. 2009, 11 p., Diphtheria IgG ELISA. Enzyme immunoassay for the quantitative determination of IgG antibodies against Diphtheria in human serum and plasma. Instructions for use. IBL International GMBH. 2011, 6 p.

The disadvantage of the described methods is the expression of the results obtained in relative qualitative (RPHA) and relative quantitative (ELISA) indicators of the content of antitoxic antibodies in sera.

The closest to the proposed invention in terms of the technical essence and the achieved results are a method and a kit for enzyme immunoassay, which allows to determine the quantitative content of antitoxic IgG in absolute terms (μg / ml). Specific antitoxic anti-diphtheria IgG antibodies are isolated from the γ-globulin fraction of human blood serum using the method of affinity chromatography. This method is based on the ability of biologically active substances to specifically and reversibly bind other substances. Affinity chromatography includes several stages. The chromatographic column is filled with a carrier (activated with cyanogen bromide sepharose) covalently bound to a ligand (purified diphtheria toxoid). Then the analyzed mixture of substances (γ-globulin fraction of human blood serum containing antidiphtheria antitoxic IgG) is passed through the column. As a result, due to biospecific interaction, a complex of the immobilized ligand with specific antibodies is formed. The rest of the components pass through the column without lingering. A specifically adsorbed substance (anti-diphtheria antitoxic IgG) is released from the complex after washing with an acidic pH buffer. Firsova T.N. Antidiphtheria, antitoxic and antibacterial antibodies in human serum. Abstract of the thesis. dis. for a job. uch. Art. Ph.D. M., 2012, 36 p.

The disadvantages include those that the stages of preparing the kit and conducting ELISA are not optimal: in terms of the time and conditions of incubation during ELISA, the stability and safety of the reagents used.

The technical objective of the claimed invention is to develop a method and kit for the enzyme immunoassay for determining the amount of antitoxic antidiphtheria antibodies (IgG) in human blood serum in μg / ml, which has good reproducibility of results, specificity, sensitivity, and also a shorter analysis time.

The technical result achieved from the implementation of the claimed invention is to increase the specificity, sensitivity and reproducibility of the results, as well as reduce the analysis time.

The technical result is achieved by the fact that in the method of enzyme immunoassay for the quantitative content of antitoxic anti-diphtheria IgG antibodies in human blood sera, according to the invention, a standard calibration sample containing antitoxic anti-diphtheria IgG antibodies at a concentration of 80 μg is introduced into the wells of a plate with a sorbed antigen at a concentration of 10 μg / ml / ml, obtained as a result of the isolation of specific antitoxic anti-diphtheria IgG antibodies from the γ-globulin fraction of the pool of blood serum using the method of affinity chromatography, and the studied samples of human blood serum are incubated at 37 ° C for 30 min in a thermal shaker with a frequency of 700 rpm min, pour out and wash the contents of the wells four times with phosphate-buffered saline with Tween-20, then add horseradish peroxidase enzyme conjugate with rabbit antibodies against human IgG at a dilution of 1: 16000, incubate at 37 ° C for 30 min in a thermal shaker withfrequency of 700 rpm, pour out and wash the contents of the wells four times with phosphate-buffered saline with Tween-20 detergent, add a solution of tetramethylbenzidine as a substrate buffer solution, incubate at room temperature in the dark for 15 minutes, add as a stop reagent to all wells of the plate 2 M sulfuric acid solution, the formed immune complexes are detected at a wavelength of 450 nm and the quantitative content of antitoxic anti-diphtheria IgG antibodies in the test sera is calculated according to a calibration graph constructed using data on the optical density and concentration of successive two-fold dilutions of the standard calibration sample and taking into account the dilution of the test samples.

Thus, a more reliable determination of the quantitative content of antitoxic anti-diphtheria IgG antibodies in human blood serum in μg / ml is achieved by the proposed immune enzymatic method.

The technical result is also achieved by the fact that the kit for implementing the method for determining antitoxic anti-diphtheria IgG antibodies in human blood serum contains:

- polystyrene flat-bottomed plate with sorbed antigen at a concentration of 10 μg / ml - 1 pc .;

- standard calibration sample with the content of antitoxic anti-diphtheria IgG antibodies at a concentration of 80 μg / ml, obtained as a result of the isolation of specific antitoxic anti-diphtheria IgG antibodies from the γ-globulin fraction of the blood serum pool using the method of affinity chromatography

- 1 bottle;

- conjugate of horseradish peroxidase enzyme with rabbit antibodies against human IgG - 1 vial;

- concentrate of phosphate-buffered saline solution with detergent Tween 20 - 1 vial;

- tetramethylbenzidine solution - 1 vial;

- stop reagent - 1 fl.;

- instruction - 1 pc.

The proposed method is based on a standard calibration sample with a known concentration of anti-diphtheria anti-diphtheria IgG antibodies (μg / ml), which is obtained as a result of the isolation of specific anti-diphtheria anti-diphtheria IgG antibodies from the γ-globulin fraction of the blood serum pool using the method of affinity chromatography. The concentration of antitoxic anti-diphtheria IgG antibodies in a standard sample is 80 μg / ml.

Specific antitoxic anti-diphtheria IgG antibodies are isolated from the γ-globulin fraction of human blood serum using the affinity chromatography method described in the prototype. FIG. 1 shows a scheme for the isolation of antitoxic antidiphtheria IgG antibodies using affinity chromatography. Sepharose activated by cyanogen bromide is used as a matrix. The ligand is sorbed on it - purified diphtheria toxoid. The γ-globulin fraction is applied to the column, then antibodies not bound to the antigen are washed off. After that, the elution of specific antibodies bound to the antigen ligands, on which the fraction of specific immunoglobulins was sorbed, is carried out. The collection of fractions of anti-diphtheria anti-diphtheria IgG antibodies is stopped when the optical density curve reaches a constant minimum value.

The quantitative content of anti-toxic anti-diphtheria IgG antibodies isolated from the γ-fraction (in the fractions obtained after affinity chromatography) is carried out by enzyme immunoassay using a commercial kit "Total IgG - ELISA - BEST" (Vektor-Best) under the conditions described in the prototype. For this, first, calibration samples with a known concentration of IgG antibodies and analyzed samples of fractions are incubated in the wells of a striped plate with immobilized monoclonal antibodies (MCAT) to IgG. Then, the IgG bound in the wells is treated with a conjugate of MKAT to IgG with horseradish peroxidase. The formed immune complexes are identified by color reaction with a tetramethylbenzidine solution, the optical density of the samples is measured. Using the calibration graph, calculate the quantitative content of antitoxic IgG in the analyzed fractions in mg / ml. According to the results of the analysis, for subsequent work, the fractions with the highest concentration of antitoxic IgG are selected, which serve as a standard calibration sample for the proposed method and kit. Get a standard sample with a concentration of antitoxic anti-diphtheria IgG antibodies of 80 μg / ml.

As a result of the implementation of the claimed method and the kit for its implementation, a high correlation of the indicators of the detected antitoxic anti-diphtheria IgG antibodies (in μg / ml) and the imported ELISA test system (in IU / ml) is achieved in comparison with the previously known prototype, when the data obtained were compared. using two test systems and determined the correlation between them using the Pearson correlation coefficient. Correlation determines the degree to which the values of two variables are proportional to each other. The correlation is high if the dependence on the graph can be represented as a straight line (with a positive or negative slope). If the value of the correlation coefficient is in the range of 0.9-1, then this indicates a strong correlation dependence.

The invention is also illustrated by examples of its implementation

Example 1. Comparison of the quantitative content of anti-diphtheria antitoxic antibodies detected using the developed kit and a commercial ELISA test system (Binding Site, Italy).

The study of antitoxic antibodies was carried out in the blood sera of 40 healthy adults aged 18-50 years. To compare the indicators of the level of antitoxic antidiphtheria antibodies, determined using two sets of ELISA, we used a correlation analysis, including the determination of the Pearson correlation coefficient and the construction of a scatter diagram. Zaitsev T.M. Methodology for biometric calculations. M: Nauka, 1973 .-- P. 89.

The method of correlation analysis confirmed the legitimacy of using both test systems to detect the content of antitoxic antibodies in absolute (μg / ml) or relative (IU / ml) values. Optimization of the conditions for each stage of the creation of the proposed set and the production of ELISA led to an increase in the reaction sensitivity. FIG. 2 shows a scatter diagram of the indicators of antitoxic antidiphtheria antibodies detected in the blood serum of healthy adults using a commercial test system and a developed kit. The Pearson correlation coefficient was 0.967, while the previously developed test system provided a lower degree of correlation with the indicators detected using the imported set (the Pearson correlation coefficient was 0.873).

Thus, the indicators of the revealed antitoxic anti-diphtheria IgG antibodies in the blood serum of healthy adults according to the proposed method correlate more reliably with the indicators of the commercial test system than the indicators of the antitoxic anti-diphtheria IgG determined by the known method.

Example 2. Determination of the quantitative content of antitoxic anti-diphtheria IgG antibodies in the blood serum of healthy adults aged 20 years and older (living in the Russian Federation) using the developed kit.

The study of antitoxic antidiphtheria IgG antibodies was carried out in 67 blood sera.

Determination of anti-diphtheria IgG antibodies in the blood serum of healthy adults includes the following stages:

1) in a flat-bottomed polystyrene plate with sorbed antigen (diphtheria toxoid) at a concentration of 10 μg / ml, add a standard calibration sample with a concentration of antitoxic anti-diphtheria IgG antibodies of 80 μg / ml and test samples. To wash the plate and to dilute the standard calibration sample, the test samples and the conjugate, use a phosphate-buffered saline solution with Tween-20 pH 7.5 (manufactured by Pierce ThermoFisher Scientific, USA), for the preparation of which to 50 ml of a phosphate buffered saline concentrate with Tween-20 add 950 ml of distilled water. In the first two strips of the plate, successive two-fold dilutions of the standard calibration sample are added: in wells A1 and A2 - 200 μl of the sample with a dilution of 1: 4, in wells B1 -G1 and B2-G2 - 100 μl of phosphate-buffered saline with Tween detergent -20 (FSB + Twin-20); Transfer from wells A1 and A2, 100 μl of solution to B1 and B2, respectively, from B1 and B2 to C1 and C2, etc. while mixing the contents thoroughly. Wells H1 and H2 contain pure PBS + Tween-20 - these wells are negative controls; the studied serum samples are introduced in a dilution of 1:20 in two replicates;

2) the plate is closed with a lid and incubated at 37 ° C for 30 min in a thermal shaker with a frequency of 700 rpm;

3) the contents of the wells are removed, the plate is washed four times with PBS + Tween-20, dried; 100 μl of a solution containing a conjugate of horseradish peroxidase enzyme with rabbit antibodies against human IgG in a dilution of 1: 16000 (manufactured by Sorbent Ltd., it is permissible to use conjugates from other manufacturers with similar characteristics) are added to all wells. For dilution, use phosphate-buffered saline with detergent Tween-20 (FSB + Tween-20);

4) the plate is closed with a lid and incubated at 37 ° C for 30 min in a thermal shaker with a frequency of 700 rpm;

5) the contents of the wells are removed, the plate is washed four times with PBS + Tween-20, dried. In all wells add 100 μl of tetramethylbenzidine solution (ready-made commercial substrate mixture containing a stabilized solution of 3,3 ', 5,5'-tetramethylbenzidine, ThermoFisher Scientific, USA), cat. No. 34028); incubate the plate for 15 min at room temperature in the dark;

6) stop the enzymatic reaction by adding 100 μl of 2 M sulfuric acid solution to all wells as a stop reagent;

7) after 2 min, measure the optical density at a wavelength of 450 nm. The quantitative content of antitoxic IgG in μg / ml in the test samples is calculated taking into account their dilution based on the calibration graph constructed using the data on the optical density and concentration of successive two-fold dilutions of a standard calibration sample containing 80 μg / ml of antitoxic IgG.

Table 1 shows the results of determining the quantitative content of antitoxic IgG in the studied sera. With increasing age, the concentration of antitoxic antidiphtheria IgG in the blood of healthy adults decreases. A high concentration of antitoxic antibodies is observed in the blood of adults 20-30 years old (89.0 ± 8.0 μg / ml).

Example 3. Determination of the quantitative content of antitoxic IgG in the blood serum of healthy adults aged 20 years and older (living in Italy) using the developed kit. The study of antitoxic antibodies was carried out in 41 blood serum. The stages of setting are similar to the stages from example 2.

Table 2 shows the results of determining the quantitative content of antitoxic antibodies in the studied sera. With age, the concentration of antitoxic IgG in the blood of healthy adults decreases. An increased concentration of antitoxic antibodies, as in the Russian Federation, is noted in the blood of adults 20-30 years old (51.5 ± 16.0 μg / ml).

It follows from the above examples that the developed method and kit can be used to detect protective titers of antitoxic IgG in human sera by bacteriological laboratories of infectious diseases hospitals and diagnostic centers, as well as to monitor population anti-diphtheria immunity among certain groups of people (children, adolescents, adults) stipulated by the sanitary and epidemiological rules of the Federal Service for Supervision of Consumer Rights Protection and Human Wellbeing of the Russian Federation.

Claims (9)

1. A method for determining the quantitative content of antitoxic anti-diphtheria IgG antibodies in human blood sera, characterized in that a standard sample with a content of antitoxic anti-diphtheria IgG antibodies at a concentration of 80 μg / ml is introduced into the wells of a plate with a sorbed antigen at a concentration of 10 μg / ml. As a result of the isolation of specific antitoxic anti-diphtheria IgG from the γ-globulin fraction of the pool of blood serum using the method of affinity chromatography, the test samples of human blood serum are incubated at 37 ° C for 30 min in a thermal shaker with a frequency of 700 rpm, poured out and washed four times wells with phosphate buffered saline with Tween-20, then add horseradish peroxidase enzyme conjugate with rabbit antibodies against human IgG at a dilution of 1: 16000, incubate at 37 ° C for 30 min in a thermal shaker with a frequency of 700 rpm, pour out and wash the contents of the wells four times with phosphate saline buffer solution with Tween-20, add a solution of tetramethylbenzidine as a substrate buffer solution, incubate at room temperature in the dark for 15 min, add 2 M sulfuric acid solution as a stop reagent to all wells of the plate, identify the formed immune complexes at a wavelength of 450 nm and the quantitative content of anti-diphtheria anti-diphtheria IgG antibodies is calculated according to a calibration graph constructed using data on the optical density and concentration of a standard calibration sample with an anti-toxic anti-diphtheria IgG content of 80 μg / ml, taking into account the dilution of the samples. 2. A kit for determining the quantitative content of antitoxic anti-diphtheria IgG antibodies in human sera according to claim 1, characterized in that it contains: - polystyrene flat-bottomed plate with sorbed antigen - 10 μg / ml - 1 pc .; - standard calibration sample with a known content of antitoxic anti-diphtheria IgG antibodies at a concentration of 80 μg / ml - 1 vial; - conjugate of horseradish peroxidase enzyme with rabbit antibodies against human IgG - 1 vial; - concentrate of phosphate-saline buffer solution with Tween-20 detergent - 1 vial; - tetramethylbenzidine solution - 1 vial; - stop reagent - 1 vial; - instruction - 1 pc.

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