RU2743426C1 - Method for determining biological macromolecules based on nmr relaxometry - Google Patents

Abstract

FIELD: molecular immunobiotechnology.

SUBSTANCE: invention relates to the field of molecular immunobiotechnology. A method for the determination of biological macromolecules is disclosed, including the sorption of molecules of a compound capable of specifically binding an analyte to be determined (analyte) on the surface of the wells of an immunological plate, sequential incubations with a sample containing an analyte and a conjugate of iron-carbon nanoparticles functionalized with recognition molecules specific to the analyte to be determined, with washes wells of PBS between each operation. In this case, the detection of the analysis results is carried out by measuring the relaxation time of protons (T2), which is inversely proportional to the number of nanoparticles, using an NMR relaxometer; the measurement process involves nanoparticles formed by the release of conjugate nanoparticles from the bound state in the structure of a specific complex and their transition into solution when NaOH solution is added to the plate wells.

EFFECT: invention improves reproducibility and reliability of analysis results.

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Description

The invention relates to the field of molecular immunobiotechnology and can be used in the production of highly sensitive test systems for the quantitative determination of any compounds capable of stereospecific interactions (antigens, antibodies, nucleic acids, biologically active macromolecules).

By now, systems of homogeneous NMR analysis are well known, based on the ability of magnetic nanomaterials to change the time of longitudinal (T1) or transverse (T2) relaxation of the surrounding water protons [1]. A key feature of these methods is the clustering (or declustering) of magnetic nano- or microparticles in the presence of the analyte (analyte) in the test sample. Individual particles and clusters have different relaxivity (the ability to change T1 or T2). Thus, the concentration of the analyte can be determined by measuring the relaxation time. The advantages of such analyzes include reduced analysis time and ease of arrangement. Despite this, such analytical methods have, as a rule, relatively low sensitivity and specificity due to the absence of washing steps that would remove impurities present in the sample, primarily protein molecules capable of non-specific binding, which, in addition, reduces reliability and reproducibility of analysis results.

From these positions, methods of heterogeneous analysis based on the sorption of analyte-binding compounds (anti-analytes) onto the solid phase are of attractive importance. These methods have a higher sensitivity and specificity, since the procedure for their formulation includes the stages of washing at each stage, as well as the stage of blocking nonspecific binding sites, which in itself significantly reduces the level of the background signal. The relevance of the development of analytical methods with high sensitivity is determined by the need to detect compounds that are in biological media in trace amounts and / or low concentrations and are markers of dangerous diseases (oncological diseases, infections of various etiologies); or substances, even small quantities of which can be life-threatening. The development of such methods of analysis can contribute not only to an increase in the efficiency of diagnostics, primarily early ones, but also to the emergence of the possibility of timely intervention in the process of ongoing therapy. In this case, the reproducibility and reliability of the analysis methods used are of particular importance.

The closest to the claimed method in technical essence and the achieved result is the enzyme-linked immunosorbent assay, in which enzyme conjugates of horseradish peroxidase with recognition molecules are most often used as detecting reagents. The detection and quantification of the analyte in this method is carried out spectrophotometrically. Today, enzyme immunoassay is the most widely used and reliable method of laboratory diagnostics.

From the standpoint of criticism, it should be noted that in the method of enzyme-linked immunosorbent assay, an enzyme, usually horseradish peroxidase, is used as a label, which has a number of disadvantages: the high cost of synthesis and exactingness to the conditions of its reduction, which also applies to the storage and transportation conditions of the finished product ( test systems) [2]. For example, it is known that the activity of horseradish peroxidase is significantly reduced when reagents are used in the analysis, which include such a widely used preservative as sodium azide. The low stability of the reagents used in the analysis procedure, as well as their sensitivity to the environment, leads to a decrease in both reliability and reproducibility of results. The unattractive aspects of ELISA also include the toxicity of the substrates required for enzymatic detection, the instability of the reaction products, and the procedural complexity of the analysis.

The inventive method allows you to solve the problem of increasing the reproducibility and reliability of the analysis results, through the use of highly stable (Fig. 1 and Fig. 2) conjugates of iron-carbon nanoparticles with recognition molecules in tandem with a highly sensitive detection method based on NMR-relaxometry in the arrangement of solid-phase heterogeneous analysis. This is essential, especially in situations where the analyte (marker) being determined is an indicator of such serious and potentially life-threatening pathological processes as, for example, malignant neoplasms. In such cases, the reliability and reproducibility of the results obtained by the proposed method plays a significant role in making decisions regarding the timely initiation of treatment and correction during the therapy.

It is known that the reliability of the reagents used plays an important role in the design of reliable analysis systems. In the implementation of the proposed method used conjugates, synthesized by the author's method [3]. To study the stability of diagnostic reagents, conjugates of magnetic iron-carbon nanoparticles with the protein G of streptococci, which have the ability to bind affinity to Fc-fragments of IgG of humans and most animals [4] and magnetic iron-carbon nanoparticles with streptavidin, which can actively bind to any biotinylated compounds, were used [4]. Conventionally, such conjugates are designated as Fe @ C-NH ₂ / BSA4 / bar G and Fe @ C-NH ₂ / BSA / streptavidin. FIG. 1.presents the results of a study of the structural stability of conjugates Fe @ C-NH ₂ / BSA / protein G in various elution solutions after 0, 1 and 2 hours of exposure. FIG. 2 illustrates the results of a study of the structural stability of the conjugates Fe @ C-NH ₂ / BSA / streptavidin during storage for 30 days at + 4 ° C in PBS.

The essence of the invention. The walls of the wells of a standard immunological plate are sensitized with a compound (anti-analyte) capable of specifically binding the compound to be determined. The described procedure is a preliminary step leading to the formation of a solid-phase reagent (immunosorbent) suitable for constructing an appropriate test system. During the implementation of the analytical procedure, a test sample containing the compound to be determined (analyte) is introduced into the wells of the plate. After appropriate incubation and washing, a detection reagent is introduced into the wells of the plate, which is a conjugate of iron-carbon nanoparticles with a compound that specifically interacts with the compound to be determined. After incubation, the wells are washed with buffer solution. As a result of the procedures carried out, a complex is formed on the walls of the wells of the plate, consisting of a binding compound, an analyte to be determined and a conjugate. Subsequently, a NaOH solution is added to the wells, which leads to the release of the conjugate nanoparticles from the bound state and their transition into solution. The wells are separated from the plate and placed in the measuring cell of an NMR relaxometer to measure the relaxation time of protons in a given sample. According to this indicator, the device quantitatively allows to estimate the content of the analyte in the sample in accordance with the previously performed calibration.

Signs of the method that meet the criterion of novelty are that the measurement of the value of the relaxation time of protons (T2), due to the amount of magnetic nanoparticles extracted from a specific complex, is carried out in a suspension devoid of impurities. In this case, the level of the measured parameter is in direct proportion to the amount of the analyte being determined. Carrying out a specific reaction in relation to the analyte to be determined in the solid-phase analysis format allows you to get rid of the impurity components contained in the analyzed sample and can significantly and irreproducibly influence the measurement result. The transfer of nanoparticles, the amount of which is proportional to the amount of the analyte to be determined, into a suspension makes it possible to measure with high accuracy the relaxation time of protons, which is directly dependent on the amount of nanoparticles, using an NMR relaxometer.

The proposed method allows you to solve any analytical (diagnostic) problem associated with the quantitative determination of any marker (analyte), which during the analytical procedure can be included in a specifically formed complex, for example, antibodies (antianalyte) -antigen (determined analyte) -antibodies / magnetic nanoparticles (conjugate). Further measurement of the relaxation time of protons using extracted from the complex of nanoparticles and NMR registration objectively does not depend on the nature of the determined marker, which ensures the versatility and multiplexity of the proposed method.

The proposed technical solution is illustrated by the following examples.

The following examples are provided only for the purpose of demonstrating the possibilities of the present invention, and in no way limit the range of compounds that can be determined using the proposed method of analysis.

The specific quantitative parameters given in the examples, even in the area of the indicated ranges, are not fixed by the claims, because they are not fundamental conditions for the implementation of the proposed method.

Example 1. Quantification of prostate-specific antigen in the format of solid-phase analysis using a conjugate of iron-carbon nanoparticles with monoclonal antibodies (clone 1A6) specific to human PSA (Fe @ C-1A6).

Mouse monoclonal antibodies (MCA) from clone 3A6 (manufacturer - Bialexa, Russia), specific to PSA, diluted in 0.2 M carbonate buffer with pH 9.6 to a concentration of 100 μl are sorbed onto the walls of the wells of a collapsible polystyrene plate for immunological reactions. 0.05 mg / ml. Sorption is carried out within 2 hours at a temperature of 37 ° C. The wells of the plate are washed three times with 250 μl of 0.15 M NaCl solution buffered with 0.015 M Na-phosphates and containing 0.1% sodium azide and 0.1% tween-20, pH 7.25 (PBS), using a device for washing microplates, then add 200 μl of blocking buffer (PBS + 2% casein + 1% BSA), the plates are incubated for 1 hour. PSA samples are added to the washed wells after blocking in a volume of 100 μl, diluted in multiples of 10 in blocking buffer, starting at a concentration of 0.01 mg / ml and incubated for 60 minutes, after which the wells washed as described above are added suspension of Fe @ C-1A6 (100 μl, 0.05 mg / ml) in PBS + 2% casein. Incubation time 60 minutes. In washed wells add 100 μl of elution solution (0.1 M NaOH) and after 60 min. the relaxation time of protons in each well is measured, for which each well is separated from the plate and placed in the measuring cell of the relaxometer. The measurement results for the given example are presented in Table 1. All stages of the analysis, with the exception of the stages of washing and measurement, are performed at + 37 ° C. The total analysis time is 180-200 minutes, excluding the preparation time for the solid phase, which can be done in advance.

Example 2. Quantitative indirect determination of antibodies (Ab) to tetanus toxoid on the solid phase using a conjugate of iron-carbon nanoparticles with protein G (Fe @ C-protein G).

Tetanus toxoid diluted in 0.2 M carbonate buffer with pH 9.6 to a concentration of 0.05 mg / ml is sorbed onto the walls of the wells of a collapsible polystyrene plate for immunological reactions from a volume of 100 μl. Sorption is carried out within 2 hours at a temperature of 37 ° C. The wells of the plate are washed three times with 250 μl of 0.15 M NaCl solution buffered with 0.015 M Na-phosphates and containing 0.1% sodium azide and 0.1% tween-20, pH 7.25 (PBS), using a device for washing microplates, then add 200 μl of blocking buffer (PBS + 2% casein + 1% BSA), the plates are incubated for 1 hour. Samples of antibodies to tetanus toxoid are added to the wells washed after blocking in a volume of 100 μl, diluted in multiples of 4 in negative rabbit blood serum (previously diluted 100 times in blocking buffer), starting at a concentration of 100 mIU / ml and incubated for 60 min., after which a suspension of Fe @ C - protein G (100 μl, 0.05 mg / ml) in PBS + 2% casein is added to the wells washed as described above. Incubation time 60 minutes. 100 μl of elution solution (0.1 M NaOH) is added to the washed wells, and after 60 minutes of incubation, the relaxation time of protons is measured, for which each well is separated from the plate and placed in the measuring cell of the relaxometer. The measurement results for the given example are presented in Table 2. All stages of the analysis, with the exception of the stages of washing and measurement, are performed at + 37 ° C. The total analysis time is 180-200 minutes, excluding the preparation time of the solid phase, which can be performed in advance.

One of the main tasks solved by the developed method was to increase the reproducibility and accuracy of the analysis results, since these analytical characteristics are important for obtaining reliable reliable data in diagnostic procedures. In order to confirm the high reproducibility and accuracy of the proposed method, appropriate studies were carried out.

To assess the reproducibility of the assay, a known amount of antibodies to tetanus toxoid was added to the negative rabbit serum, previously diluted 100 times with blocking buffer. A total of 8 samples were analyzed with a concentration of 0.2 to 80 mIU / ml in 10 replicates. In all samples, the coefficient of variation did not exceed 7.5% (table 3), which corresponds to the permissible limits [5].

To confirm the accuracy of the developed analysis, the recovery rate was assessed by the spike-recovery test. We used samples of rabbit blood serum with known antibody levels (5, 3, and 1.3 mIU / ml) against tetanus toxoid. The proposed method was used to determine the concentration of Ab. Further, the degree of extraction was calculated (table 4). The recovery, expressed as a percentage, was within acceptable limits [5].

Thus, the technological result from the use of the proposed invention is that the developed method of analysis is objectively independent of the use of expensive reagents and allows obtaining reliable results with high reproducibility and accuracy due to the use of effective reagents: conjugates of magnetic nanoparticles that are stable and do not lose their structural and functional properties during a long storage period (Fig. 1, 2), as well as due to the developed method, which provides for the ability to get rid of impurities during the analysis that affect its result.

The application of the developed analysis method can find its implementation not only in the creation of effective test systems designed for the diagnosis of various diseases and analytical studies, but also in solving such urgent problems as the development of methods for assessing environmental and food pollution by various agents.

Bibliographic list

1. Zhang Y. et al. Recent Advances on Magnetic Relaxation Switching Assay-Based Nanosensors // Bioconjugate Chemistry. 2017. Vol. 28. No. 4. P. 869-879.

2. Huang Y., Ren J., Qu X. Nanozymes: Classification, Catalytic Mechanisms, Activity Regulation, and Applications // Chemical Reviews. 2019. Vol. 119. No. 6. P. 4357-4412.

3. A method of producing a conjugate based on magnetic metal-carbon nanoparticles, suitable for diagnostic and analytical purposes, using NMR relaxometry as a detection method // RF Patent No. 2684325. 2019. Bul. No. 10. / Raev M.B., Khramtsov P.V., Bochkova M.S. and etc.

4. Khramtsov P. et al. Conjugation of carbon coated-iron nanoparticles with biomolecules for NMR-based assay // Colloids and Surfaces B: Biointerfaces, 2019. Vol. 176. P. 256-264

5. Bioanalytical Method Validation Guidance for Industry / U.S. Department of Health and Human Services, Food and Drug Administration / [Electronic resource]. - Access mode: URL: https://www.fda.gov/media/70858/download (date of access: 20.02.2020).

Claims (1)

A method for the determination of biological macromolecules, including the sorption of molecules of a compound capable of specifically binding the compound to be determined (analyte) on the surface of the wells of an immunological plate, successive incubations with a sample containing the analyte and a conjugate of iron-carbon nanoparticles functionalized with recognition molecules specific to the analyte to be determined, with washes PPRT between each operation, characterized in that the detection of the analysis results is carried out by measuring the relaxation time of protons (T2), which is inversely proportional to the number of nanoparticles, using an NMR relaxometer, while the measurement process involves nanoparticles formed due to the release of conjugate nanoparticles from the bound states in the structure of a specific complex and their transition into solution when adding NaOH solution to the wells of the plate.

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