RU2702900C1 - Method for quantitative determination of antibodies to benzo[a]pyrene in human biological fluids - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to immunology, biotechnology and clinical laboratory diagnostics and can be used to determine individual risks associated with exposure to chemical environmental carcinogens. Disclosed is a method for quantitative determination of antibodies to benzo[a]pyrene in human biological fluids, involving collection and preparation of donor blood; immobilization of human recombinant anti-idiotypic antibodies to benzo[a]pyrene on magnetic microspheres; wherein the diluted human blood serum diluted with phosphate buffer solution PBS in ratio of 1:100 is fed into the reaction mixture; incubation with biotinylated idiotypic antibodies to benzo[a]pyrene in a final concentration of 20 ng/mcl as a competitor with human blood serum for binding with magnetic microspheres; incubation of the microspheres is performed for 1 hour at room temperature; detection of binding of antibodies with microspheres is determined with the help of conjugate streptavidin-phycoerythrin and analysis of amount of bound antibodies with microspheres – by calibration curve.

EFFECT: invention provides higher accuracy, sensitivity and reproducibility of the method as compared to enzyme immunoassay, as well as reduced carcinogenic health hazard for the researcher and manufacturer of test systems and reduced non-specific binding of the antibodies of the analyzed human biological fluid.

1 cl, 2 dwg

Description

The invention relates to the field of immunology, biotechnology and clinical laboratory diagnostics and can be used to determine individual risks of diseases associated with exposure to environmental chemical carcinogens: malignant tumors, congenital malformations, autoimmune and others.

A lot of similar solutions are known, but all of them, in our opinion, have certain disadvantages. All analogues we found are dealt with below. The literature and databases do not describe the quantitative multiplex method for the determination of antibodies to polycyclic aromatic carbohydrates in biological fluids using recombinant idiotypic and anti-idiotypic human antibodies, proposed by us.

One of the well-known approaches to solving the definition of individual cancer risks is the analysis of antibodies specific for chemical carcinogens in human biological fluids. Previously, associations of antibodies to chemical carcinogens with environmental carcinogens and oncological diseases were discovered (Galati R. et al., 2001; Pauk N. et al, 2013; Glushkov A. et al, 2016).

However, these well-known studies were performed using qualitative or semi-quantitative methods of enzyme-linked immunosorbent assay of antibodies using conjugates of chemical carcinogens and their metabolites with DNA or protein. Qualitative methods do not accurately assess the specific immune response of an organism to chemical carcinogens, which is necessary for the further development of a promising cancer immunoprophylaxis strategy in humans with anticarcinogenic vaccines (Silbart L. et al, 1999; De Buck S. and Mullerc C., 2005; Schellenberger M. et al, 2011). In addition, the presence of carcinogens or their metabolites in analytical test systems poses a health hazard to users and researchers.

Analogues of the claimed invention are methods for the quantitative determination of antibodies in human biological fluids (for example, RF patents: G01N33 / 531, G01N33 / 68, G01N33 / 577, G01N33 / 53, CN108196061 (A), CN108226460 (A), CN107677810 (A); others patents: US8383350 (B1), WO2005108989 (A2), US5219730 (A), US4910131 (A), WO9901477 (A1), WO9006515 (A1), CN105785030 (A), CN105203750 (A), UA78419 (U)) based using enzyme-linked immunosorbent assay, which is an analogue of the invention. Conventional enzyme-linked immunosorbent assay involves binding of the antigen to the surface of the microplate, followed by placing the biological fluid containing antibodies on the microplate, incubation, color reaction and spectrophotometric evaluation of the color reaction. To determine the concentration of antibodies in biological fluids, a calibration curve is used - a standard preparation of antibodies of appropriate specificity with a known concentration. Another example, the use of a solid-phase carrier is granular aerosil (RF patent SU1513403, publication date 10/07/1989, IPC class G01N33 / 53), which is treated with sheep erythrocytes, incubated with test serum, and then protein is desorbed from the aerosil surface. Then, the protein concentration is determined, and the number of specific antibodies is determined by the difference between the amount of protein in the antigen-antibody complex and the amount of protein adsorbed on sheep erythrocytes.

However, the analogue (enzyme-linked immunosorbent assay) has its drawbacks that prevent its use as a quantitative method for determining antibodies in biological fluids:

1) the reproducibility of the method is about 30%;

2) low sensitivity of the method;

3) the complexity of the analysis;

4) the impossibility of conducting multiplex analysis of antibodies in one sample of the studied biological fluid.

Using enzyme immunoassay in the patents: US8383350 (B1), WO2005108989 (A2), US5219730 (A), US4910131 (A), WO9901477 (A1), WO9006515 (A1), it was proposed to use anti-idiotypic antibodies for the detection of idiotypic antibodies in human biological fluids.

Signs of a known technical solution that coincides with the essential features of the claimed invention are: 1) determination of antibodies in human biological fluids (in human serum); 2) the use of an adsorbed anti-idiotypic antibody in the wells of an immunological tablet or in the patent presented — immobilized onto the surface of magnetic microspheres; 3) incubation of the diluted test human biological fluid in the wells of the tablet or, in the present patent, with magnetic microspheres; 4) the determination of bound antibodies from human biological fluid by the intensity of the color enzymatic reaction or in the present patent - by the intensity of the fluorescence of the biotin-streptavidin complex.

Using these previously proposed methods: 1) determine the qualitative, but not quantitative levels of antibodies in human biological fluids; 2) it was proposed to use monoclonal antibodies. In addition, due to the complexity of their preparation, these patented methods are very laborious; 3) monoclonal antibodies are produced by mouse cells, i.e. they are murine, not human antibodies; 4) it was not proposed to use anti-idiotypic antibodies against polycyclic aromatic hydrocarbons.

Another analogue of the claimed invention is a method previously patented using liposomes as an immunosorbent of anti-idiotypic antibodies to determine idiotypic antibodies in human biological fluids (WO8806293 (A2)).

Another analogue of the claimed invention is a method previously patented (patent RU 2124731 G01N33 / 573, date of publication of 01.10.1999). According to this method, it is proposed to use enzyme-linked immunosorbent assay, where the conjugate of fluoromethylbenzantryl acetic acid with human serum albumin is adsorbed on the walls of the wells of the immunological tablet. Then the test biological fluid with the adsorbed antigen is incubated. Antibodies bound to the antigen are shown using anti-enzyme labeled antibodies.

The disadvantages of this method are: 1) qualitative, but not quantitative determination of antibodies against polycyclic aromatic hydrocarbons in human biological fluids; 2) low sensitivity of the method; 3) low reproducibility; 4) the use of a conjugate of modified polycyclic aromatic hydrocarbons to determine idiotypic antibodies against polycyclic aromatic hydrocarbons, which may lead to inaccurate determination of idiotypic antibodies in human biological fluid; 5) the use of conjugate of fluoromethylbenzantryl acetic acid with human serum albumin as an antigen can be accompanied in some cases by high nonspecific binding of antibodies of the studied human biological fluid not to the antigen, but to a carrier protein, which significantly limits the possibility of the method; 6) the impossibility of conducting a comprehensive (multiplex) analysis of antibodies in one sample of human biological fluid.

A well-known analogue of the claimed invention is a method previously patented using microarrays (patents: KR20180006092 (A), CN107064493 (A)), when antibodies are immobilized on a microchip to quantify antigens in human biological fluids.

The disadvantages of the patented method are: 1) it was proposed to use monoclonal antibodies. Due to the complexity and cost of obtaining them, these patented methods are very laborious and unprofitable; 2) monoclonal antibodies are produced by mouse cells, i.e. they are murine, not human antibodies; 3) determination of antigens, and not antibodies, in human biological fluid samples.

It was also proposed to use biological microchips (another analogue of the present invention) containing hydrogel elements with immobilized antibodies to protein tumor markers, hydrogel elements with immobilized glycans, hydrogel elements with immobilized antibodies against human immunoglobulins of classes A, G and M, where the concentration of the analyzed markers compared with a control sample obtained from a healthy person, indicates colorectal cancer (patent RU 2625018). Thus, the method proposed in the present invention allows the diagnosis of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins A, G and M in human blood.

The disadvantages of this method are:

1) it was proposed to use monoclonal antibodies. Due to the complexity and cost of obtaining them, these patented methods are very laborious and unprofitable;

2) monoclonal antibodies are produced by mouse cells, i.e. they are murine, not human antibodies;

3) determination of antigens, and not antibodies, in samples of human biological fluid;

4) the impossibility of multiplex analysis in one sample of the studied human biological fluid, i.e. For multiplexing, the entire hydrogel chip is used through which one sample of biological fluid is passed, in contrast to the proposed method using magnetic microspheres, where a whole set of magnetic microspheres is added to one sample of biological fluid with simultaneous and separate detection of each type of microspheres.

Known previously patented method (analogue of the present invention), using strips with antibodies immobilized on them for the detection of antigens in human biological fluids (CN108020666 (A)).

The disadvantages of this method are:

1) it was proposed to use monoclonal antibodies. Due to the complexity and cost of obtaining them, these patented methods are very laborious and unprofitable;

2) monoclonal antibodies are produced by mouse cells, i.e. they are murine, not human antibodies;

3) determination of antigens, and not antibodies, in samples of human biological fluid;

4) the impossibility of multiplex analysis in one sample of the studied human biological fluid, in contrast to the proposed method using magnetic microspheres, where a whole set of magnetic microspheres is added to one sample of biological fluid with simultaneous and separate detection of each type of microspheres;

5) qualitative, but not quantitative determination of analytes in biological fluid;

6) low sensitivity of the method.

An analogue of the claimed invention is also a method, a previously patented quantitative method for determining antibodies and antigens in human biological fluids using magnetic microspheres onto which idiotypic antibodies are immobilized to determine antigens in human biological fluids: KR101807871 (B1), CN105259344 (A), US2014170767 (A1 ), CN106290867 (A), CN106366197 (A), CN103278521 (A); immobilized anti-IgE for determination of IgE in human serum: CN101696973 (A); immobilized antigen for determination of IgE in human serum: CN105785030 (A); a combination of strips and magnetic microspheres with a combination of immobilized antibodies and antigens for the semiquantitative determination of pneumonia: CN107748252 (A) and prostatitis CN107271666 (A).

The invention adopted CN107271666 as the prototype (publication date 10/20/2017, IPC class G01N33 / 558).

Signs of a prototype that coincides with the essential features of the claimed invention are: 1) determination of analytes in human biological fluids (in human serum); 2) the use of immobilized antigen or antibody on the surface of magnetic microspheres; 3) incubation of the diluted test biological fluid with magnetic microspheres; 4) determination of bound analytes from human biological fluid by the intensity of the fluorescence of the biotin-streptavidin complex. 4) quantitative analysis of analytes in human biological fluids (in human serum); 5) high sensitivity, accuracy and reproducibility of methods.

The disadvantages of the prototype are:

1) it was proposed to use monoclonal antibodies. Due to the complexity and cost of obtaining them, these patented methods are very laborious and unprofitable;

2) monoclonal antibodies are produced by mouse cells, i.e. they are murine, not human antibodies;

3) it was not proposed to use idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons.

The technical result of the invention is to increase the accuracy, sensitivity and reproducibility of the method compared to enzyme-linked immunosorbent assay by conducting multiplex analysis (multiple determination) of antibodies in one sample of human biological fluid, while reducing the carcinogenic hazard to the health of the researcher and manufacturer of test systems, reducing non-specific binding of antibodies of the studied human biological fluid.

A method is proposed for the quantitative determination of antibodies to chemical carcinogens of the polycyclic aromatic hydrocarbon group, including the collection and preparation of donor blood, immobilization of human recombinant anti-idiotypic antibodies to benzo [a] pyrene on magnetic microspheres.

The difference is that diluted human blood serum is diluted with PBS phosphate buffer solution in a ratio of 1: 100, incubation is carried out with biotinylated idiotypic antibodies (in the amount of 20 ng / μl) as a competitor with human blood serum for binding to magnetic microspheres, incubation microspheres 1 hour is carried out at room temperature, the detection of binding of antibodies to microspheres and the analysis of the number of bound antibodies to microspheres is determined by the calibration curve.

The essence of the invention is shown in figure 1 and figure 2, where figure 1 shows the calibration curve of competitive binding of BT72 (10 ng / μl) and T72 (from 1 ng / μl to 32 ng / μl) with MV5, and in Fig. .2 - concentrations of antibodies against benzo [a] pyrene in human serum.

The proposed method is as follows:

1. To implement the method used blood serum of 10 donors. All studies were conducted with the consent of blood donors.

2. The antibodies of the invention are known and described in detail in Ustinov et al, 2015 and Arnst et al, 2017. The following were used: human recombinant idiotypic anti-benzo [a] pyrene antibody T72, and human recombinant anti-idiotypic anti-benzo [a] pyrene antibody B5 . All antibodies were expressed and isolated from E. coli.

3. B5 immobilization on magnetic microspheres was carried out by amino groups using a Bio-Plex immobilization kit from Bio-Rad (USA).

4. Biotinylation of T72 was performed using Sileks kit (Russia).

5. For the analysis of quantitative anti-benzo [a] pyrene antibodies in human blood serum, 100 μl of PBS phosphate buffer solution with 0.5% BSA were added to the wells of an opaque immunological plate to reduce non-specific binding. Then, at first 6,000 magnetic microspheres with B5 (MB5) immobilized on them were added to each well, then 50 μl of biotinylated T72 (BT72) with concentrations from 34.827 pg / μl to 16 pg / μl. Streptovidin-phycoerythrin company Bio-Rad (USA) was used to detect the MV5-BT72 complex. As control of background binding, 6000 magnetic microspheres were used without adding BT72 to them. Background binding values used were subtracted from the values of all other binding points. The half concentration of BT72 from the maximum binding of BT72 to MB5 was 10 ng / μl. This concentration was used later.

6. The calibration curve was constructed on the basis of competition between BT72 and T72 for binding to MB5 as follows: BT72 at a concentration of 10 ng / μl was added to all wells of an opaque immunological plate. Then T72 was added to the same wells with a double dilution step from a concentration of 1 to 32 ng / μl. The maximum fluorescence was 15410. The sensitivity of the calibration curve (minimum binding and, as a consequence, the sensitivity of the determination of antibodies in human serum) was in the range of less than 1 ng / μl. The calibration curve is presented in figure 1.

7. For the quantification of antibodies against benzo [a] pyrene in human serum, serum samples were diluted with phosphate buffered PBS in a ratio of 1: 100. Then, diluted human serum was added to the binding reaction (see paragraph 5 of this section) instead of T72 as a competitor to BT72 (20 ng / μl) for binding to MB5. The final volume of the reaction mixture was 50 μl in each well of an immunological opaque plate (half and half of the volume of diluted human serum and BT72). Then the immunological plate was incubated for 1 hour at room temperature. An incubation mixture of MB5 with human serum, but without BT72, was used as a negative control for nonspecific binding. This control went through all the stages of this technique. Nonspecific binding was 120 fluorescence units, which was subtracted as the background from all other fluorescence values. The concentration of antibodies against benzo [a] pyrene in human serum ranged from 8620 pg / μl to 17768 pg / μl (average value 12946.5 (9671.0: 15469.0) pg / μl) (figure 2). Moreover, the lower limit of concentration was in the center of the calibration curve (figure 1).

The invention is proposed to be used for the quantitative method of immunoassay of antibodies against polycyclic aromatic hydrocarbons in human biological fluids by the competitive method of immunoassay, in which human recombinant anti-idiotypic antibodies to polycyclic aromatic hydrocarbons immobilized on magnets microspheres are used as antigen, and human recombinant and standard recombinant anti-polycyclic aromatic antibodies levodorodov.

Biotinylated idiotypic antibodies in constant concentration and non-biotinylated idiotypic antibodies in increasing concentrations, which compete with biotinylated idiotypic antibodies for binding to immobilized anti-idiotypic ones, are added to the prepared microspheres. Streptovidin-phycoerythrin conjugate is added to the bound biotinylated idiotypic antibodies. The degree of competition is assessed by the degree of fluorescence of phycoerythrin in the streptovidin-biotin complex on a Bio-Plex 200 instrument or other analytical instruments. In separate samples, a sample of human biological fluid is added to the microspheres along with biotinylated idiotypic antibodies and the antibodies against polycyclic aromatic hydrocarbons contained in it compete with biotinylated idiotypic antibodies for binding to immobilized anti-idiotypic antibodies, after which a streptovidin-phycoerythrin complex is added to the sample. The degree of suppression of fluorescence under the action of the desired antibodies in the human biological fluid is compared with that under the action of non-biotinylated idiotypic antibodies and the amount of the desired antibodies in the human biological fluid is equal to the number of non-biotinylated idiotypic antibodies with the same fluorescence level.

At the same time, we obtained recombinant human antibodies specific for the most common environmental carcinogens - polycyclic aromatic hydrocarbons (in particular, benzo [a] pyrene), which can be used as a standard, as well as the corresponding anti-idiotypic recombinant human antibodies (in in particular, to benzo [a] pyrene), which can be used as an antigen in the development of a health-friendly quantitative method for the immunoassay of antibodies against chemical carcinogens in bi biological human fluids.

Thus, the significance and causal relationship with the claimed technical result of the development of the proposed method for the quantitative determination of antibodies to polycyclic aromatic hydrocarbons in human biological fluids is due to the following:

- the proven carcinogenic effect of polycyclic aromatic hydrocarbons in the environment;

- the ability to identify previously unknown immunological mechanisms of human adaptation to polycyclic aromatic hydrocarbons and the features of their violation in cancer;

- The practical importance of creating an informative and safe method for assessing individual cancer risks for subsequent cancer prevention.

A causal relationship with the claimed technical result is achieved due to:

1. The use as an antigen is not conjugates of native polycyclic aromatic hydrocarbons with protein, but anti-idiotypic antibodies that carry an immunological cast of antigen.

2. Use a competitive analysis method in which competition between biotinylated and non-biotinylated idiotypic antibodies is used for the calibration curve, and competition between biotinylated idiotypic antibodies and the biological fluid itself is used to determine the concentration of idiotypic antibodies in a human biological fluid.

3. To increase the accuracy, sensitivity and reproducibility of the method, multiple determination of antibodies in one sample of human biological fluid, reducing the number of samples of human biological fluid, the ability to process a large number of samples of human biological fluid at once and a more convenient quantitative method for determining antibodies is used multiplex system using fluorescent magnetic microspheres.

The proposed method is relevant, feasible and allows to achieve the claimed technical result, differs from known analogues and has clear advantages over them.

Claims (1)

A method for the quantitative determination of antibodies to benzo [a] pyrene in human biological fluids, including the collection and preparation of donated blood; immobilization of human recombinant anti-idiotypic antibodies to benzo [a] pyrene on magnetic microspheres; characterized in that for the quantitative analysis of antibodies against benzo [a] pyrene, diluted human blood serum diluted with PBS phosphate buffer solution in a ratio of 1: 100 is fed into the reaction mixture; incubation with biotinylated idiotypic antibodies to benzo [a] pyrene, at a final concentration of 20 ng / μl, as a competitor to human serum for binding to magnetic microspheres; incubation of the microspheres for 1 hour is carried out at room temperature; detection of binding of antibodies to microspheres is determined using streptavidin-phycoerythrin conjugate and analysis of the number of bound antibodies to microspheres is determined by a calibration curve.

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