RU2465600C2 - Method for evaluation of natural antibody level in human biological fluids by immune-enzyme assay - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: level of natural autoantibodies in human biological fluids is evaluated by immune-enzyme assay with the use of a solid phase of physical sorption coated with streptavidin. The solid phase of physical sorption is pre-processed with a biotin-modified antigen and a blocking agent for closing nonspecific binding sites. That is ensured by using proteins biotin-modified by standard technique; a conjugate-containing solution is presented by monoclonal or polyclonal enzyme-marked antibodies reacting with one or all isotopes of human immunoglobulins. A test sample is pre-dissolved in a phosphate-buffered saline containing proteins used for closing nonspecific binding sites on the solid phase of physical sorption, thimerosal and Triton-X100. Then it is thermally treated. A reference solid phase of physical sorption whereon the biotin-modified antigen is not immobilised is applied for the test sample. The number of natural autoantibodies is evaluated by a calibration curve.

EFFECT: safety and effectiveness of evaluating the natural autoantibody level in human biological fluids.

4 tbl, 2 dwg, 2 ex

Description

The invention relates to medicine, in particular to laboratory diagnostics, and can be used to increase the efficiency and reliability of quantitative determination of the concentration of natural autoantibodies in human biological fluids.

The prior art method for the quantitative determination of the level of autoantibodies to endogenous proteins in human biological fluids by enzyme-linked immunosorbent assay (ELISA) (EN 2137134 C1, G01N 33/53, 1999). However, this method is not suitable for the quantitative determination of the level of natural autoantibodies in human biological fluids.

There is also a method for enzyme-linked immunosorbent assay, including adsorption of antigens on the solid phase of physical sorption, incubation of test biological samples, incubation of a conjugate-containing solution, spectrophotometric analysis of the reaction of extinction of a chromagent solution (RU 2014610 C1, G01N 33/535, 1994).

In addition, an enzyme-linked immunosorbent assay is also known to determine the level of natural autoantibodies, including adsorption of antigens on a solid phase of physical sorption, incubation of test biological samples, incubation of a conjugate-containing solution, and spectrophotometric analysis of the extinction reaction of a chromagent solution (BMS217TEN Human anti-IFN-alpha ELISA, Bender MedSystems GmbH, Austria). Despite the sufficient simplicity, sensitivity and specificity of this solution, the results obtained can be false positive, due to the non-specific and low-affinity binding of various serum or plasma immunoglobulins to the antigen adsorbed on the ELISA plate. In addition, the presence in the blood of polyreactive immunoglobulins, which are not natural antibodies, also leads to false positive results due to the non-specific binding of polyreactive immunoglobulins to the ELISA components of the tablet. Moreover, only a fraction of free natural autoantibodies is detected in ELISA, while most of the natural autoantibodies are present in the blood in the complex associated with its antigen.

The invention is aimed at creating a technologically simple, sensitive and specific method for the quantitative determination of the level of natural autoantibodies in human biological fluids by the ELISA method.

The solution of this problem is ensured by the fact that in a method for quantitatively determining the level of natural autoantibodies in human biological fluids by enzyme immunoassay, which includes treating the solid phase of physical sorption with an antigen, introducing test biological samples, treating the solid phase with a conjugate-containing solution, separating solid and liquid phases and spectrophotometric analysis of the reaction for extinction of a chromagent solution, according to the invention, I use the solid phase of physical sorption as the solid phase of physical sorption coated with streptavidin, and the processing of the solid phase of physical sorption is performed with a biotinylated antigen and a blocking agent to close the sites of nonspecific binding on the solid phase of physical sorption, using proteins biotinylated according to the standard procedure, using monoclonal or polyclonal conjugate-containing solution enzyme-labeled antibodies reacting with one or all isotypes of human immunoglobulins, when volume of the tested biological fluid is preliminarily diluted in a buffer containing proteins used to close non-specific binding sites on the solid phase of physical sorption, as well as substances that protect natural autoantibodies from degradation during heat treatment, and are subjected to heat treatment, the corresponding biological fluid is used control solid phase of physical sorption, on which the biotinylated antigen is not immobilized (which makes it possible to measure specific esky and nonspecific for natural autoantibodies spectrophotometric signal), and the amount of natural autoantibodies is determined using a calibration curve which is standardized by monoclonal or polyclonal antibodies to the antigen.

Due to the combination of the use of the solid phase of the physical sorption coated with streptavidin and biotinylated antigens as a solid phase of physical sorption, all epitopes remain free for binding of natural autoantibodies to them in an immobilized antigen, thereby increasing the sensitivity of the claimed method, while direct immobilization of the antigen leads to conformational changes and destruction of antigen epitopes, which reduces the sensitivity of the ELISA method. In addition, the use of antibodies labeled with an enzyme and reacting with one or all isotypes of human immunoglobulins, allows you to determine the level of a particular isotype or all isotypes of natural autoantibodies and, accordingly, expands the functionality of the claimed method. Preliminary dilution of the test sample in a buffer containing proteins present in the blocking buffer and used to close the sites of non-specific binding on the solid phase of physical sorption minimizes the possibility of non-specific binding of the antibodies of the test sample to the solid phase on which the proteins are immobilized, which also increases the specificity and sensitivity of the claimed way. Using heat treatment of the test sample allows you to destroy the complex of natural autoantibodies with antigens and anti-idiotypic antibodies or to ensure the availability of antibody paratopes to epitopes of antigens due to various unmasking effects, and preliminary dilution of the test sample in the buffer protects the natural autoantibodies from destruction during heat treatment, which allows to determine the overall level of natural autoantibodies (i.e., as free and associated with antigen forms of natural utoantibodies or natural autoantibodies with closed paratopes), and thereby reduces the possibility of obtaining false negative results, increases the sensitivity, efficiency and functionality of the claimed method. The use of an individual control solid phase of physical sorption for each of the studied biological fluid samples on which the biotinylated blocking protein is immobilized allows the measurement of a spectrophotometric signal specific for natural autoantibodies and a non-specific spectrophotometric signal, which also reduces the possibility of obtaining false-positive results and increases the sensitivity of the claimed method.

In addition, the claimed combination of essential features, which provides the ability to quantify the level of natural autoantibodies, expands the arsenal of technical means to solve the problem.

The claimed method for quantitative determination of the level of natural autoantibodies in human biological fluids is implemented as follows.

The solid phase of physical sorption (wells) of a standard well plate is coated with streptavidin or its analogues (for example, avidin, etc.), the solid phase of physical sorption is blocked with a blocking agent and incubated with biotinylated antigen, to which the level of natural autoantelles must be determined, or (control wells) with biotinylated blocking protein (for example, bovine serum albumin, ovalbumin, human serum albumin, rabbit serum albumin, gelatin, etc.), which is a non-control ificheskogo binding. The antigen is biotinylated with a minimum amount of biotin (antigen: biotin molar ratio is 1: 2) to minimize the destruction of epitopes.

A test biological fluid is prepared for analysis (for example, blood serum, blood plasma, etc.), while the test samples are diluted (in the range from 50 to 200,000 times) in a buffer solution containing proteins that are part of the blocking buffer (for example, bovine serum albumin, ovalbumin, human serum albumin, rabbit serum albumin, gelatin, etc.), preservatives (for example, thimerosal, etc.), surfactants (for example, Triton-X100, etc.) and carry out the heat treatment of the resulting biolo solution liquid by incubation in the temperature range from 50 to 80 ° C. Then, a heat-treated solution of the studied biological fluid is introduced into the wells in the same way as the antigen-specific antibodies used to construct the calibration curve, which are used monoclonal or polyclonal antibodies to antigens with known concentrations. For the manifestation of the formed immune complex (antigen - natural autoantibodies), a conjugate of polyclonal or monoclonal antibodies (for example, goat polyclonal antibodies, sheep polyclonal antibodies, mouse monoclonal antibodies, etc.) to the light chains of a certain or all human immunoglobulins with an enzyme (for example, alkaline phosphatase) is used , horseradish peroxidase, etc.). The amount of natural autoantibodies is determined, taking into account the reaction of extinction of a chromagent solution that changes color depending on the amount of chromagent isolated from the substrate when it is decomposed by an enzyme (for example, alkaline phosphatase, etc.) - the substrate produces a soluble product, the color characteristics of which can be measured spectrophotometrically at a specific wavelength.

Example 1

To confirm the feasibility of obtaining a technical result in the implementation of the claimed method for quantitative determination of the level of natural autoantibodies to human gamma interferon, serum samples were taken from twenty healthy donors who did not receive interferon therapy, and, therefore, there are no autoantibodies to human gamma interferon, but natural autoantibodies are present to interferon human gamma.

Greiner bio-one GmbH (Cat. No.655990) introduced recombinant human interferon gamma manufactured by eBiosciences (Cat. No. 34-8319-85) into a portion of the wells of a 96 well plate coated with streptavidin, and into another part (control wells for the subjects samples) - bovine serum albumin manufactured by Sigma Aldrich (Cat. No. A-3803), biotinylated according to the standard procedure of U-CyTech bioscience (SOP No. UCT-127) at a rate of 100 μl / well and incubated for 2 hours at 37 ° C and at 100% humidity.

Blood serum samples were diluted 20 times (100 μl of serum was diluted in 1900 μl of phosphate-buffered saline containing 1% Triton-X100 and 0.002% thimerosal) and incubated for 20 minutes at a temperature of 75 ° C.

Then, the incubated solution was further diluted 5 times (100 μl of the incubated solution was diluted in 400 μl of phosphate-buffered saline containing 1% bovine serum albumin (BSA), 1% Triton-X100 and 0.002% thimerosal) and were subsequently used for adding to the wells tablet (100 μl per well).

To build a calibration curve, standard antibody solutions were prepared (mouse monoconal antibodies to human gamma interferon, clone MD-2) in the concentration range from 16 to 0.5 U / ml. 100 pg / ml of murine monoclonal antibodies was taken as 1 U / ml.

Prepared standard antibodies, a solution used as a negative control (phosphate-buffered saline containing 1% BSA, 1% Triton-X100 and 0.002% thimerosal) and solutions of the test samples were added to the wells of the plate as indicated in table 1 and incubated with temperature of 37 ° C for 2 hours. At the end of the incubation, the liquid was removed from the plate by decantation and the plate was washed 5 times with standard washing solution (phosphate-buffered saline containing 0.05% Tween-20 and 0.01% thimerosal).

Table 1 shows the scheme of introducing samples into the wells of the tablet.

Then, 100 μl of a solution containing a mixture of goat conjugated goat anti-human IgA, IgM, IgG antibodies (Sigma Aldrich; Cat. No. A-3313) was added to all wells of the plate, which makes it possible to determine the level of all isotypes of natural autoantibodies to human gamma interferon or goat antibodies to mouse IgG (Sigma AIdrich; Cat. No. A-3562) conjugated to alkaline phosphatase to determine the level of monoclonal antibodies to human gamma interferon (MD-2) used to construct a calibration curve. Then the plate was incubated for 1 hour at a temperature of 37 ° C. At the end of the incubation, the liquid was removed from the plate wells by decantation and the plate was washed 5 times with standard washing solution (phosphate-buffered saline containing 0.05% Tween-20 and 0.01% thimerosal).

15 minutes before the end of incubation with the conjugate-containing solution, a chromagent solution was prepared by dissolving 1 tablet of substrate buffer and 1 tablet of substrate (para-nitropheniphosphate), manufactured by Sigma (Cat. No. N-2770) in 20 ml of distilled water.

100 μl of the prepared chromagent solution was added to each of the wells of the plate and the plate was incubated for 30 minutes at 37 ° C.

At the end of the incubation, a stopping solution (3N sodium hydroxide) manufactured by Merck KgaA (Cat. No. 1.06495) was added to all wells of the plate and the extinction was measured at a wavelength of 405 nm using a standard photometer.

According to the obtained measurement results, the level of natural autoantibodies to interferon gamma was calculated. For this, the true optical density (OD) value of standard antibodies and the true OD value of the studied samples were determined as follows:

1. The mean OD for the negative control was calculated (arithmetic mean OD in wells 1G, 1H, 2G, 2H).

2. The mean OD for standard antibodies was calculated (arithmetic mean OD in the wells of the corresponding standard P1-P6).

3. The true OD values of standard antibodies were calculated as the difference between the average OD values of standard antibodies and the average OD values of the negative control.

The calculation results are shown in table 2.

table 2 one 2 → Average OD values: → Average OD st. minus the average OD neg. control: P1 BUT 1,704 1,554 1,629 1,521 P2 AT 1,122 1,202 1,162 1,054 P3 FROM 0.818 0.801 0.810 0.702 P4 D 0.497 0.453 0.475 0.367 P5 R 0.285 0.266 0.276 0.168 P6 F 0.201 0.188 0.195 0,087 N G 0.115 0.108 0.108 N N 0.106 0.102

4. Based on the obtained values of the true OD of standard antibodies, a calibration curve was constructed (see Fig. 1), where the OD values are located on the ordinate (y) axis, and the concentration of antibodies to gamma interferon on the abscissa (x) axis. The constructed calibration curve can be described by the following equation: y = -0.0052x ² + 0.177x + 0.0172.

6. The true OD value for the test samples was calculated as the difference between the average OD value for the test samples measured in the wells of the plate in which biotinylated IFN-γ was immobilized and the average OD value for the test samples measured in the wells of the tablet in which biotinylated BSA was immobilized. For example, for the test sample S1, we subtract from the average OD value measured in the wells of plate A3, B3, the average OD value measured in the wells of tablet A4, B4.

7. Using the calibration curve (figure 1), the concentration of diluted natural autoantibodies to human gamma interferon in the test samples was determined. To obtain the true concentration of natural autoantibodies to human gamma interferon in the studied samples, the result was multiplied by the degree of dilution of the samples (by 100).

8. The results are presented in table 3.

Table 3 Test sample The concentration of natural autoantibodies to human interferon gamma, UE / ml S1 780.1 S2 774.6 S3 288.3 S4 977.1 S5 509.0 S6 688.1 S7 794.5 S8 572.4 S9 453.5 S10 753.1 S11 527.9 S12 298.8 S13 345.6 S14 443.5 S15 621.8 S16 575.2 S17 758.9 S18 857.5 S19 656.2 S20 753.5

Example 2

Serum samples were taken from twenty patients with infectious mononucleosis, in which the level of natural autoantibodies to human interferon gamma increases.

All stages of determining the level of natural autoantibodies to human gamma interferon were similar to those described in Example 1, except that the test samples were diluted 1000 times, human serum albumin was used as a blocking agent, and the incubation temperature was 56 ° C.

The calibration curve is presented in figure 2, the results are shown in table 4.

Table 4 Test sample The concentration of natural autoantibodies to human interferon gamma, UE / ml S1 1675.9 S2 1591.7 S3 1897.5 S4 1480.0 S5 1924.4 S6 1974.6 S7 1537.4 S8 1221.1 S9 1624.5 S10 1569.9 S11 1874.6 S12 1530.2 S13 1870.8 S14 1798.9 S15 2151.8 S16 1882.2 S17 1505.0 S18 1810.2 S19 1526.6 S20 1451.4

Thus, the above examples using blood serum in which natural autoantibodies to human gamma interferon are present confirm the effectiveness and reliability of quantitative determination of the level of natural autoantibodies to human gamma interferon when implementing the claimed combination of features.

Claims (1)

A method for quantitatively determining the level of natural autoantibodies in human biological fluids by enzyme-linked immunosorbent assay, which includes treating the solid phase of physical sorption with an antigen, introducing test biological samples, treating the solid phase with a conjugate-containing solution, separating solid and liquid phases and spectrophotometric analysis of the reaction for extinction of a chromagent solution, characterized in that as a solid phase of physical sorption, a solid phase of physical sorption coated with streptavidi is used nom, and the treatment of the solid phase of physical sorption is performed with a pre-biotinylated antigen and a blocking agent to close non-specific binding sites on the solid phase of physical sorption, which are biotinylated according to the standard procedure, monoclonal or polyclonal antibodies labeled with an enzyme are used as conjugate-containing solutions with one or all isotypes of human immunoglobulins, while the test biological fluid is preliminarily thimerosal and Triton-X100 are diluted in phosphate-buffered saline containing proteins used to close non-specific binding sites on the solid phase of physical sorption, and subjected to heat treatment, for each of the tested sample of biological fluid, a control solid phase of physical sorption is used, on which biotinylated antigen is immobilized, and the number of natural autoantibodies is determined using a calibration curve that is constructed using monoclonal or polyclonal antigen antigen.

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