RU2473908C2 - Method for quantitative analysis of specific immunoglobulins g to conjugate of formaldehyde-serum human albumil in blood serum - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: when implementing the method for quantitative analysis of specific immunoglobulins G to a conjugate of formaldehyde - human serum albumin in blood serum, test and reference nitro-cellulose substrate are prepared by placing them in various microplate wells with said test substrate prepared by sequential sorption of human serum albumin and formaldehyde thereon at room temperature for 8 hours, while the reference substrate is prepared by sorption of human serum albumin thereon. It is followed by incubation of the test and reference substrates with the analysed serum sample for 30 minutes. After the substrates are washed in a phosphate buffer, the substrates are added with monoclonal antibodies with Fc-fragments of human immunoglobulin G conjugated with horseradish peroxidase; the substrates are washed in the phosphate buffer once more, and an immunochemical complex of specific immunoglobulin G antibodies are coloured by adding chromogene and developed by adding a stop reagent, The worked out substrates are removed from the microplate wells, and optical density is recorded in the test and reference samples; they are matched with optical density of the reference samples of the known concentration of immunoglobulin G, and a difference of the content of immunoglobulin G in the test and reference samples shows a number of patient's blood serum immunoglobulin G specific to the test substance provided optical density in the test sample exceeding that of the reference sample not less than 1.5 times.

EFFECT: method enables higher accuracy and reliability of determining specific immunoglobulins G in blood serum.

2 tbl, 1 ex

Description

The invention relates to techniques for laboratory research, in particular to methods for conducting immunological analysis, and can be used in clinical immunology to determine the concentration of specific immunoglobulins G in blood serum, due to which the presence of autoimmune reactions caused by formaldehyde is diagnosed.

A known method for the diagnosis of sensitization to water-soluble industrial allergens (RF Patent No. 2323441), according to which determine the levels of allergen-specific antibodies - immunoglobulins of subclasses IgG1 and IgG4 in blood serum using a specific allergen. The specific allergization coefficient (COSALL) is calculated using a specific formula. With COSALL less than 2.5, they conclude that there is an IgG dependent sensitization to a specific industrial allergen. The known method allows to increase the accuracy of diagnosis of the presence of sensitization to the allergen and to reduce the study time.

However, the known method is intended to diagnose only sensitization (allergies) to industrial haptens, while the proposed method provides information on the formation of IgG antibodies (total indicator) in response to the ingestion of formaldehyde, and not just "allergic" (reagins), to which refers only to the IgG4 subclass.

Also known from the prior art is a method for the quantitative determination of antibodies, including immunoglobulins G, to benz (a) pyrene (article: Glushkov AN et al. [A new immunoassay of human antibodies to chemical carcinogens]. [Article in Russian]. Klin Lab Diagn. 2008 Mar; (3): 42-44) and an enzyme-linked immunosorbent assay for antibodies to chemical carcinogens of the polycyclic aromatic hydrocarbon group (PAH) (RF Patent No. 2124731). The indicated methods are almost identical. In particular, according to the method of the patent, antigen is adsorbed on the walls of the wells of a plastic tablet. Then the test biological fluid with the adsorbed antigen is incubated. Antibodies bound to the antigen are shown by anti-labeled enzymes. In this case, a conjugate of fluoromethylbenzantryl acetic acid with human serum albumin is used as antigen at a mass ratio of 1: 5.4, respectively, at a concentration of 2 μg / ml.

The disadvantages of these known methods are the following:

- when implementing the known method, the totality of antibodies is determined, including IgG (the totality of serum antibodies includes IgG + IgA + IgM, etc.), i.e. this known method lacks selectivity for immunoglobulin G;

- in the known method, it is possible to determine only arbitrary units of the content of immunoglobulins, i.e. relative value, the use of which in comparison with the absolute value increases the error of the result, because to establish a relative value, at least two indicators are used, each of which can introduce its own error;

- when implementing the known method requires dilution of blood serum with a solution of ovalbumin, which introduces additional uncertainty in the accuracy of determination of minor protein fractions and complicates the method.

The proposed method eliminates all these disadvantages of the known method, namely:

- provides selectivity due to the ability to determine the amount of immunoglobulin G to the formaldehyde conjugate - human serum albumin;

- provides the ability to establish the quantitative content of specific immunoglobulin G in absolute units - ng / ml;

- provides accurate determination by eliminating the dilution of blood serum during the implementation of the method;

- ensures the accuracy of the determination by introducing an additional criterion, namely the excess of the optical density in the experimental sample over the optical density of the control sample is not less than 1.5 times.

The technical result achieved by the present invention is to provide a method for determining the quantitative content in absolute units of immunoglobulin G in blood serum to the formaldehyde-serum human albumin conjugate.

The specified technical result is achieved by the proposed method for the quantitative determination of specific immunoglobulins G to the conjugate formaldehyde - human serum albumin in the blood serum, characterized in that they prepare the experimental and control nitrocellulose substrates, for this they are placed in different wells of the microplate, and the preparation of the experimental substrate is carried out by sequential sorption on it of serum human albumin and formaldehyde at room temperature in t 8 hours, and the preparation of the control substrate is carried out by sorption of human serum albumin on it, then the test sample of the serum is incubated for 30 minutes with the test serum sample, then after washing the substrates with phosphate buffer, monoclonal antibodies conjugated to horseradish peroxidase are added to them Fc fragment of human immunoglobulin G, again wash the substrates with phosphate buffer and the resulting immunochemical complex of specific immunoglobulin G a ntel is stained by adding chromogen and developing by stop-reagent addition, then the used substrates are removed from the microplate wells and the absorbance is recorded in the experimental and control samples, they are compared with the optical density of standard samples with a known concentration of immunoglobulin G and the difference in the content of immunoglobulin G in the experimental and control samples judge the amount of immunoglobulins G specific for the test substance in the patient’s blood serum when the simultaneous condition above optical density in the experimental sample over the optical density of the control sample is not less than 1.5 times.

The specified technical result is achieved due to the following.

It should be noted that the purpose of the proposed method indicates the determination of specific IgG to the conjugate "formaldehyde - serum human albumin" (NFA), due to the fact that formaldehyde, entering into a chemical reaction with albumin, forms the above conjugate (Patterson R. et.al. Human antibodies against Formaldehyde - Human serum albumin conjugates or Human serum albumin in individuals exposed to Formaldehyde. Int Arch Allergy Appl Immunol. 1986; 79 (1): 53-59). But this is a formal indication of purpose. However, it is formaldehyde that acts as an immunogen, which, entering the human body, forms a conjugated (bound) form of persistence in the body. And in fact, it is on the indicated immunogen that the body develops an allergic (autoimmune) reaction with the formation of specific antibodies. If not formaldehyde will act as an immunogen, then there will be no specific IgG on the formaldehyde-albumin complex. Therefore, the practically claimed method acts as a measure of the effect of formaldehyde on the formation of antibodies to the conjugate "formaldehyde - serum human albumin." And the formaldehyde-NFA conjugate is only a derivative of formaldehyde and NFA, where its specificity is manifested precisely due to formaldehyde.

Thus, there is no doubt that the antibodies to formaldehyde and antibodies to the formaldehyde + albumin complex are identical, since the latter represents the hapten (immunogenic) form of formaldehyde persistence in the body.

Due to the fact that during the implementation of the proposed method, two nitrocellulose substrates are taken: experimental (containing the test substance: formaldehyde conjugate - human serum albumin) and control (not containing the test substance), the exact concentration of specific IgG antibodies is established by cutting off non-specific antibodies that joined the control the substrate.

The application of serum human albumin (hereinafter referred to as AFA) to them provides the diagnosticum with a substrate for the conjugation of formaldehyde.

Moreover, the sequence of adding to the well with the experimental substrate, first NAV, and then the test substance is an important feature of the proposed method, because only such a sequence will provide the necessary spatial arrangement of antigenic epitopes for conjugation of specific IgG on them and will allow to achieve high accuracy and reliability.

The use of exposure for 8 hours as a mode of sorption ensures the completeness of conjugation.

Conducting a 30-minute incubation of the experimental and control substrates of one test sample of serum (and not two, as in the known methods) allows to ensure equal conditions for both substrates, which ensures measurement accuracy.

In addition, the use of the proposed method undiluted serum avoids additional error in the determination.

Adding to each well after the above incubation, horseradish peroxidase-conjugated monoclonal antibodies to the Fc fragment of human immunoglobulin G allows the identification of IgG antibodies to formaldehyde that bind to the diagnosticum, which ensures the specificity of this method.

Due to the fact that a nitrocellulose substrate, such as a paper disk, is used as the material containing the test substance, the simplicity of the method and the originality of the approach for determining specific IgG (by binding the protein and forming a protein-polysaccharide sandwich) are ensured, which ensures the accuracy of determination.

When conducting an enzyme immunoassay, reliable indicators of the amount of IgG in blood serum, and in the form of absolute values in units of ng / ml, were obtained only if the simultaneous condition of exceeding the optical density in the experimental sample over the optical density of the control sample was not less than 1.5 times. That is, the indicator of the quantitative content of IgG corresponded to reality only in the presence of this attribute as well. The choice of this numerical value "not less than 1.5 times" is due to the presence of the phenomenon of non-specific binding of ballast protein, therefore, the index of excess of the optical density of the solution of the experimental sample over the control should be significant, exceeding the "normal" randomness of this process.

The proposed method has been tested in a clinical setting.

An example of a specific implementation of the proposed method.

During the tests, blood was examined in 6 patients. After blood sampling from a vein by standard centrifugation method, serum was obtained, which was subsequently studied.

To do this, in the wells of a standard microplate for enzyme immunoassay, the strips of which were made of polystyrene, experimental and control nitrocellulose substrates were introduced, which are a filter paper disk (5.5 mm in diameter from the anesthetized white tape filter TU 6-09-1678- 86). Then, the experimental and control substrates were prepared, for which 50 μl of a 5% solution of serum human albumin (AFA) and 50 μl of a 0.025% formaldehyde solution were successively added to the well with the experimental substrate, and 50 μl 5 to the well with the control substrate % solution of NAV.

The specified preparation for sorption was carried out for 8 hours at room temperature. The results of the experimental selection of the incubation time for the formation of the sorbent for the determination of IgG to the formaldehyde conjugate - NFA allowed us to establish the optimal exposure time equal to 8 hours (experimental data are shown in table 1).

Table 1 The content of specific IgG to the formaldehyde conjugate with NFA depending on the incubation time for the formation of the sorbent (ng / ml) Incubation time 2 hours 4 hours 8 ocloc'k 10 hours 12 hours 24 hours IgG antibodies to formaldehyde conjugate with NFA 0.68 1.31 2.21 1.95 1.89 1,63

The data shown in table 1 show that incubation for 2 and 4 hours is not enough to fully bind the components of the sorbent complex, which results in a reduced yield of specific antibodies. At the same time, incubation exceeding 8-hour exposure leads to a decrease in the stability of the sorbent complex and the loss of the resulting amount of specific antibodies. Thus, the specified implementation mode of the proposed method is unexpected and non-obvious.

Then the wells were washed three times with phosphate buffer (0.02 M solution of KH ₂ PO ₄ and Na ₂ HPO ₄ with a pH of 7.6) to remove unbound formaldehyde from its surface. After sorption of antigens, a formaldehyde-serum human albumin conjugate-human serum albumin conjugate (with experimental and control substrates) was introduced with a sample of the studied serum sample in an amount of 50 μl. During incubation, Fab fragments of specific human antibodies were bound to antigenic complexes sorbed on a nitrocellulose substrate. Upon completion of the 30-minute exposure and washing of the wells with phosphate buffer in the second stage of an enzyme-linked immunosorbent assay to form a classic sandwich, 50 μl of horseradish peroxidase conjugated monoclonal antibodies to the human IgG Fc fragment were simultaneously added to the experimental and control samples. During the hourly incubation of the test sample at room temperature, competitive binding of specific antibodies to the formaldehyde conjugate with NFA occurred with specific regions of the variable domains of Fab fragments of monoclonal antibodies conjugated to horseradish peroxidase. After washing the wells with phosphate buffer and staining by adding a chromogenic substrate, for example, tetramethylbenzidine, and a stop reagent, for example 1 N sulfuric acid solution, spent substrates were removed and optical density was measured and recorded in the experimental and control samples. Then they were compared with the optical density of standard samples with a known concentration of immunoglobulin G to chicken egg protein. To obtain such standard samples, calibration disks were produced in parallel onto which samples with a known content of human IgG antibodies to chicken egg were conjugated. Standard IgG samples, conjugate, chromogenic substrate and stop reagent from the reagent kit for determining total IgG were used (see "Instructions for the use of the general IgG reagent kit - IFA-BEST" A 8662, Vector-Best CJSC, Novosibirsk, Russia) . The data obtained during the tests are shown in the table.

Based on the difference in the content of immunoglobulin G in the experimental and control samples, the number of patient-specific immunoglobulins G in the blood serum of a patient is judged (if the patient’s blood serum sample contains formaldehyde-specific conjugate - IgA, the optical density in the test sample will exceed the same value in the control sample, because the “sandwich” contains more conjugated antibodies that have found specific receptors). Moreover, this value is taken as reliable if the conditions for exceeding the optical density in the experimental sample over the optical density of the control sample are not less than 1.5 times.

The data shown in table 2 show that the results of the determination of IgG to formaldehyde conjugate - NFA by the proposed method correlate with the positive results of a chemical blood test for formaldehyde content, which is proved by the data given in the last column of the table. This column indicates the frequency of excess of formaldehyde in the patient’s blood over the reference, which is logically correlated in these patients with a high level of specific IgG antibodies to formaldehyde conjugate - NFA determined by the claimed method.

The introduction of criterion 1.5 in assessing the excess of optical density of the control sample over the experimental one allows not only to determine the quantitative value of specific IgG, but also to qualitatively characterize the patient's condition as the presence of increased antibody formation to a specific industrial chemical compound - formaldehyde.

The examples shown in table 2 indicate the presence of specific antibody formation to formaldehyde in patients under numbers 2, 5 and 6 of the experimental test (criterion 1.5) of the excess optical density (ODexperiment) of the experimental sample (immunosorbent conjugated with formaldehyde) over the corresponding optical control density (ODcont.) (Unconjugated immunosorbent). The obtained result is confirmed by the simultaneous detection of formaldehyde in a patient’s blood sample at numbers 2, 5 and 6 in a concentration exceeding the reference (permissible).

The proposed method allows, through the use of an immunosorbent diagnosticum conjugated with formaldehyde to be studied, to identify specific antibodies to the protein-antigen complex in vitro (ELISA), and the introduction of criterion 1.5 in assessing the excess optical density of the experimental sample over the control allows not only determine the quantitative value of specific IgG, but also qualitatively characterize the patient’s condition as a state of increased sensitivity of the patient’s immune system to formaldehyde guide.

To prove that the proposed method is accurate and reliable, an in-depth immunological examination of the patients under the numbers 2, 5 and 6 was carried out. An immunogram was performed for these patients, as a result of which an immunodeficiency state was detected. The presence of a diagnosis of "immunodeficiency" in patients is correlated with known information about the predominant negative health effects of formaldehyde. Thus, the health status of patients 2, 5, and 6 is characterized by symptoms typical of increased expression of specific IgG antibodies to formaldehyde.

The proposed method is intended for the diagnosis of not only sensitization (allergies) to industrial antigens, but also allows to identify the formation of the main serum immunoglobulins - IgG antibodies in response to the ingestion of formaldehyde, and not only "allergic" (reagins), which significantly expands the diagnostic capabilities of the method .

Thus, providing accuracy and reliability, the proposed method can be widely used in laboratory practice.

table 2 Estimation of the content of specific IgG to formaldehyde conjugate - NFA in children with different levels of formaldehyde in the blood Contingent, the diagnosis IgG to the formaldehyde conjugate - NFA (inventive method), ng / ml The ratio of the optical density of the test sample to the optical density of the control sample in relation to formaldehyde ODex./ ODcont. * The frequency of exceeding the reference level of formaldehyde in the patient’s blood 1. Patient, 12 years old Immunodeficiency 2.22 1.8 1.4 2. Patient, 5 years old
Atop. dermatitis 2.47 2.1 1.8 3. Patient, 8 years old
Biliary dyskinesia 1.18 0.8 0.7 4. Patient, 5 years old
Vegetative dystonia 2.21 1.75 1.2 5. Patient, 7 years old
Rep. allergosis 2.31 2.0 1.5 6. Patient, 0 years old
Rep. allergosis 2.55 2.3 2.0 Note. * Diagnostic criterion for the ratio of the optical density in the experimental sample to the optical density in the control sample

Claims (1)

A method for the quantitative determination of specific immunoglobulins G to a formaldehyde conjugate - human serum albumin in blood serum, characterized in that the experimental and control nitrocellulose substrates are prepared, for this purpose they are placed in different wells of a microplate, and the experimental substrate is prepared by sequential sorption of human serum on it albumin and formaldehyde at room temperature for 8 hours, and the preparation of the control substrate is carried out by after sorption of serum human albumin on it, then the test and control substrates are incubated for 30 minutes with the test serum sample, then after washing the substrates with phosphate buffer, monoclonal antibodies to the Fc fragment of human immunoglobulin G conjugated to horseradish are added to the well, they are produced again washing the substrates with phosphate buffer and the resulting immunochemical complex of specific immunoglobulin G antibodies are stained with chromogen and manifested by stop -reagent, then the spent substrates are removed from the microplate wells and the optical density is recorded in the experimental and control samples, they are compared with the optical density of standard samples with a known concentration of immunoglobulin G, and the difference in the content of immunoglobulin G in the experimental and control samples is used to determine the amount of specific substance for the test substance immunoglobulins G in the patient’s blood serum when the simultaneous condition of exceeding the optical density in the experimental sample over the optical density th control sample not less than 1.5 times.