RU2381505C1 - Syphilis diagnostic technique with using method of direct proteomic blood serum profiling - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: syphilis diagnostic technique provides the stage of serum protein fractionation with magnetic particles, mass spectrometre analysis of an eluate sample, and analysis of the derived mass spectrum for the presence or absence of a peak set corresponding to syphilis biomarkers. The presence of the characteristic peak set in the mass spectrum shows syphilis, while the absence of the characteristic peak set in the mass spectrum shows the absence of syphilis in the patient.

EFFECT: developed effective syphilis diagnostic technique in the patient by the method of direct proteomic blood serum profiling by detecting the syphilis biomarkers in the serum test sample.

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Description

The technical field of the present invention

The present invention relates to medicine, more specifically to the diagnosis of sexually transmitted infections, namely syphilis.

BACKGROUND OF THE INVENTION

Syphilitic infection refers to socially significant diseases, i.e. diseases that cause pronounced public resonance. In addition to economic damage to society associated with disability of the patient, certain moral and social conflicts, these diseases are fraught with the so-called delayed danger, which can manifest itself after many years in the form of impaired reproductive health, the birth of physically and mentally defective offspring, severe damage to the nervous system. systems and various internal organs. The result may be a decrease in the population (increase in mortality, a decrease in the birth rate), an increase in the number of mentally and mentally disabled citizens, people with disabilities, etc., which poses a real threat to the country's economic and social potential [44].

To prevent the long-term effects of diseases, the leading role belongs to their early detection.

Currently, for the diagnosis of syphilitic infection, direct methods are used, based on the direct detection of the pathogen from the lesion, and indirect, serological methods to detect antibodies to the causative agent of the disease.

The most convincing evidence of syphilis infection is direct detection of the causative agent of the infection, Treponema pallidum pale treponema. Currently, the most widely used microscopic examination of material in a dark field. The result of dark-field microscopy will not always give an unambiguous answer about the presence of infection. The absence of treponema in typical lesions may be due to both a decrease in their number in the lesion due to the duration of its existence and preliminary treatment of the patient (the use of antibacterial and antiseptic drugs), and poor-quality sampling or material research techniques [13, 17]. A negative result is not a reason for excluding the diagnosis of syphilis and requires further confirmation by serological diagnostic methods. At the same time, false-positive results are possible due to the difficulty of differentiating pale treponema from other saprophytic treponemas [22]. To solve the above problems, new diagnostic methods are created, and the most developed polymerase chain reaction (PCR), which currently has a research status [15, 22, 35, 123, 160].

However, a direct determination of the pathogenic Treponema pallidum can reliably establish a diagnosis only with manifest forms of the disease, i.e. when there are clinical manifestations of infection [17, 20, 113, 160]. According to official statistical reporting, latent forms of syphilitic infection have prevailed recently [45], the diagnosis of which is based on the detection of antibodies to Treponema pallidum.

Thus, at present, the main diagnostic methods are serological tests, which are divided into two groups: nontreponemal, or nonspecific (NTT), and treponemal, or specific (TT).

In NTT, an antigen of non-treponemal origin is used (usually cardiolipin - lecithin - cholesterol antigen), immunoglobulins M and G (IgM and IgG) antibodies to cell wall lipids Treponema pallidum are detected in the test serum.

Historically, the first non-treponemal serological test for syphilis was the complement binding reaction (CSC) adapted by A. Wasserman et al. (1906), in which the liver extract of newborns with congenital syphilis was used as antigen. After the work of Pangborn (1941) [149], which isolated and purified cardiolipin, which subsequently made it possible to standardize cardiolipin – lecithin – cholesterol antigenic complex, commercial preparations of cardiolipin antigen were used in CSCs. In addition to CSCs with cardiolipin antigen (CSCC), the microprecipitation reaction (RMP) and the fast plasma reactin test (RPR) are also referred to as NTTs.

NTTs are used as screening tests to screen a large number of people and evaluate the effectiveness of therapy.

In some cases, all tests in this group can give false negative results (when a negative reaction result is determined in the presence of a disease) due to the prozone phenomenon, when the antigen-antibody reaction is blocked with an excess of antibodies in the test serum, which is most often observed in the secondary period of syphilitic infection ( 1-2%) [17, 114,], as well as when examining patients with severe immune deficiency, HIV-infected [37, 80].

In addition, a significant amount (up to 2.5%) of false-positive results (DM) is recorded in NTT (when a positive reaction result is determined in the absence of disease) [29]. DM can be caused by both exogenous, depending on some external factors in relation to the human body, and endogenous, due to the processes occurring in it, causes.

Among the exogenous causes can be attributed technical errors and errors during the study: violation of the conditions and methods of the reaction [30, 49], unstable characteristics of the antigen isolated from treponem cell components [36], difference in ingredients, quality and working dose of complement, timing serum storage [31].

The endogenous causes causing LPR include the genetic characteristics of individual individuals, accompanied by the ability to nonspecific reactions [27, 70, 83, 178], physicochemical changes in blood serum [39], and autoimmune disorders [26, 49, 54, 72, 119 ], as well as shifts in the state of protein and lipid metabolism [29, 30, 46, 48, 51, 170]. The accumulation of lipids involved in reagin tests can occur in tissues as a result of the destruction of cell mitochondria and the release of cardiolipin, which is part of mitochondrial membranes, which creates the basis for the development of non-specific positivity of lipid tests [29].

False positive results of NTT are described in case of leprosy [31], tuberculosis [159], malaria [98, 161], respiratory diseases [191], diffuse connective tissue diseases [71, 101, 110], dermatoses [95, 111], diabetes mellitus [ 17], diseases of the gastrointestinal tract and liver [10, 28, 39], malignant neoplasms [29], cardiovascular and hematological pathologies [54]. Among the diseases caused by viruses, DM are registered with influenza [1], chickenpox [79], hepatitis [169, 173], and HIV infection [33, 61, 85]. DM can accompany drug addiction and chronic alcoholism [23, 67, 77, 99, 115, 154], poisoning and intoxication [29], noted in physiological conditions such as pregnancy, menstruation and advanced age [4, 29, 54, 70] , with vaccination, multiple blood transfusion [4, 54]. Decision-making is often observed in so-called practically healthy people and can be preclinical manifestations of serious diseases [150], such as systemic lupus erythematosus, rheumatism, oncopathology, inflammatory gynecological diseases, deep venous thrombosis of the extremities and necrotic purpura [55, 112, 175].

The titers of serological reactions in individuals with false positive results can be both low, fluctuating [34, 147, 157], and high [4, 49], which undoubtedly complicates the interpretation of the reaction results and complicates the diagnosis of syphilitic infection.

Attempts to differentiate the truly positive results of serological reactions to syphilis from DM have long been associated with the improvement of known serological diagnostic methods [12, 32,49, 156, 177] and the creation of fundamentally new ones. The results of the improvement of the tests did not receive wide recognition and did not solve the existing problem.

A revolution in the differentiation of the true and false-positive results of syphilis seroreactions occurred as a result of the development of treponemal tests (TT), in which the treponemal antigen is used. They are used to confirm the diagnosis, recognition of false-positive results of NTT.

The first TT proposed in 1949 by R. Nelson and M. Meyer [144] was RIBT, or RIT. The essence of this reaction is the loss of mobility of pale treponema in the presence of immobilizing anti-treponemal antibodies from the blood serum of patients with syphilis and complement under conditions of anaerobiosis. Over time, it became known that RIT, like any other biological reaction, does not have absolute specificity [2, 3, 7, 29, 31, 40, 43, 58, 116, 140]. So, Rezaykina A.V. (1979) received 0.4% of DM in RIT in the study of 2157 somatic patient sera [43], Pshenichnaya L.A. (1971) revealed similar results in malignant neoplasms (0.27%), systemic lupus erythematosus (3%), cardiovascular disease (0.19%), pneumonia (0.66%), in pregnant women (0.34%) ) [40]. Non-specific loss of positive RIT results was observed in patients with tuberculosis, generalized atherosclerosis, hyperthyroidism, systemic lupus erythematosus, rheumatic heart disease, gastric ulcer and practically healthy individuals [58], in patients with myeloma, sarcoidosis, diabetes mellitus, cirrhosis of the liver and 116 liver disease , 140]. A significant percentage of positive (2.8%) and weakly positive (2.2%) RIT results were observed in patients with leprosy [116]. According to some authors, RIT is completely unsuitable for differentiating syphilis from treponematoses (bezel, pint, etc.), in which almost the same percentage of positive results are revealed as with syphilis [29, 31]. However, RIT in some cases does not detect antisyphilitic AT in patients with known syphilis, especially in the early stages of the disease due to the small number of immobilizins [2, 93, 187].

In 1953, a modification of RSK-RSK was developed with treponemal antigen prepared from non-pathogenic culture Treponema phagedenis (Reuters strain) [81], which have common cross-reacting antigens with Treponema pallidum. This test yielded a significant amount of false-positive results when examining individuals with systemic diseases of the connective tissue, hematopoietic organs, diabetes, oncological and inflammatory diseases [29]. Until 2006, in the territory of the Russian Federation, the test was used as a qualifying test as part of a complex of serological reactions (DAC) along with DSC with cardiolipin antigen and RMP. Currently, in connection with the development of more sensitive, specific, and less labor-intensive reactions, the use of CSCs with cardiolipin and treponemal antigens is limited and replaced by modern ELISA and RPGA in combination with RMP (38).

The next reaction, which is still widely used for the diagnosis of syphilis, was the RIF, combining the properties of an immunological study that provides specificity, and luminescent microscopy, which determines the high sensitivity of the test [31]. First used by L. Borel and P. Durel in syphilidology in 1959, having high sensitivity and specificity in all forms of the disease, RIF is the “gold standard” for diagnosing syphilitic infection [17, 68]. However, it is not absolutely specific. According to Larsen S.A. (1995) in specific serum fluorescence tests, approximately 1% of people give DMD for syphilis [124]. Non-specific RIF results were noted in systemic lupus erythematosus, rheumatoid arthritis, scleroderma, diabetes mellitus, cirrhosis, genital herpes, leprosy, treponematosis, pregnancy, after vaccination, in old age, with hypergammaglobulinemia, etc. [29, 31, 52, 53, 66, 124, 132].

RIBT and RIF are complex and time-consuming tests, require the presence of vivarium, as live pathogenic pale treponemas of the Nihols strain obtained from 7-10 day old rabbit orchitis are used for their formulation.

For modern treponemal tests, ELISA and RPHA use industrial test systems. They were widely used both screening and confirmatory tests for syphilis [16, 18, 199], and, in accordance with the Order of the Ministry of Health of the Russian Federation No. 87 dated 03/26/2001 “On improving serological diagnosis of syphilis”, since 2006 in the territory Of the Russian Federation are called upon to replace the DAC [38].

The principle of ELISA consists in combining the complex "antigen-antibody" with a conjugate containing an enzyme label detected using a substrate mixture [18, 24, 31, 81, 182]. There are test systems for detecting IgM antibodies, IgG antibodies and IgM + IgG antibodies, antibodies to various components of Treponema pallidum immunoblotting. Due to the high sensitivity and specificity close to the RIF, the method has received universal recognition [6, 16, 18, 20, 22, 42, 54, 63, 143]. However, he, like the previously described treponemal reactions, was not absolutely specific. False positive results are described in 1.5-3.2% of cases [9, 125, 134, 158]. According to V.F. Lyakhov (1991) in ELISA, LPR can be obtained due to the rheumatoid factor, which is an IgM against human IgG, as well as cross-reacting antibodies with a treponemal antigen that are formed during various systemic or drug or drug-induced disorders exchange [26]. Mezzardra G. (1983) [138] points to the rheumatoid factor as a cause of DM in newborns. In addition, in the fetus or child, IgM antibodies can form against the mother’s IgG antibodies that enter the body via the transplacental route [8, 18, 26, 41, 47, 124, 138, 155]. A necessary condition for the reliability of the study is impeccable accuracy in the separation of sera into classes of immunoglobulins (IgM, IgG), since the presence of even small amounts of IgG in the serum can give a false positive reaction by the mechanism of competitive inhibition [103, 130]. The occurrence of nonspecific reactions in ELISA can be due to technical reasons and develop as a result of the interaction of whey proteins or solid phase conjugates and protein – protein interactions associated with cross-reactions between antibodies and antigens, or strong hydrophobic bonds of large complexes with proteins on the carrier [29] .

One of the simplest and quickest treponemal tests in the formulation is the reaction with sensitized red blood cells, or RPHA, first used for syphilis G. Blumental and W. Bachman in 1932 and studied in detail by T. Rathlev (1967) and T. Tomizawa et al. (1966) [31, 154, 174]. The priority for the application of this reaction in Russia belongs to V.N. Bednova and G.F. Timchenko (1987) [5]. According to many authors [18, 19, 174], RPHA is quite specific; it can be used to differentiate the positive results of CSC with cardiolipin hypertension and Reiter protein from those caused by syphilis and nonspecific. However, as it turned out, this reaction is also capable of producing LPR in a number of diseases: leprosy, diffuse connective tissue diseases, infectious mononucleosis, heterophilic reactions with unsensitized red blood cells [18, 52, 121, 167].

Thus, none of the treponemal tests currently used has become an arbitration reaction, since in some cases all of them themselves may turn out to be false positive or false negative.

In addition, it should be noted that in most patients after successful treatment of syphilis, treponemal tests remain positive for a very long time, and sometimes throughout their lives [20, 25, 38, 133]. Nontreponemal tests, by changing the positivity of which evaluate the effectiveness of antisyphilitic therapy, in some cases, as was indicated earlier, give false-positive results and make it difficult to determine further tactics of patient management.

Thus, at present, modern syphilology requires fundamentally new approaches to the diagnosis of syphilitic infection. A method is needed with which it is possible to detect the disease at any stage and in any form of the course of infection, regardless of the state of the human immune system, biological factors, burdened somatic status, technical features, and which could serve as a criterion for evaluating the effectiveness of specific treatment.

In recent years, one of the promising directions in creating diagnostic methods for various diseases is the use of proteomic technologies, the scope of which is closely related to the search for diagnostic markers of the disease [50, 60]. The main idea of such a targeted “marker” diagnosis is the identification of certain molecules or their complexes that are present only in the affected tissues or cells (but are absent in normal ones) and / or are released into the external environment or internal environment of the body. Any biological process is accompanied by cascades of enzymatic reactions, and a specific set of their protein fragment products can be found in the blood [152].

Proteomic technologies make it possible to obtain information immediately about large aggregates of proteins and to compare their sets in patients and healthy people. In the application of proteomic technologies to the identification of biomarkers, two different approaches can be distinguished [11]. The first approach uses new technical capabilities to search for biomarkers and their further identification, after which the found markers can be used in diagnostics similar to those currently used (for example, by immune methods). Another approach is based on comparing the proteomic profile as a whole for sick and healthy people using statistical methods to identify significant differences, without implying the mandatory identification of a biomarker. In this case, the composition of the proteomic profile, i.e. research and direct diagnosis can be carried out by the same method.

Proteomics is a field of molecular biology engaged in the comparative study of cellular proteomes, i.e. all proteins that can be expressed by the genome of a given cell at a certain point in time [185]. Its main task is to predict the functional role of individual proteins by experimentally comparing their qualitative and quantitative compositions in different cells, as well as establishing the relationship between the structure of the protein and its functions [108].

The total number of different proteins in the human body is in the millions, which can only be identified by direct analysis. Proteins have various properties: electric charge, size, hydrophobicity, which is used for their separation and subsequent identification.

Objects of research can be biological fluids (plasma or blood serum, cerebrospinal, synovial, amniotic, bronchoalveolar fluids, urine), cells and biopsy samples of human tissues, as well as proteomes of microorganisms - pathogens [11, 21, 62, 126].

The methodological base of proteomics includes a set of high-performance separation and analysis methods aimed at obtaining comprehensive information on the biochemical properties of proteins in living systems (expression level, post-translational modifications, their relationships with each other) [14].

Currently, in the proteomic analysis, several stages can be arbitrarily distinguished: sample preparation, protein fractionation, protein identification.

Sample preparation. The experience of proteomic studies shows that the problem of sample preparation must be given special attention, since there are objective factors that can drastically worsen protein separation and, therefore, make it impossible to interpret the results objectively [14]. So, most biopsy and autopsy samples are cells of different tissue affiliation (connective tissue, organ-specific cells, blood cells, etc.), which makes the interpretation of the results difficult. Recently, proteomic studies have begun to apply the technology of laser microdissection (LCM) (Laser Capture Microdissection) to isolate a homogeneous cell population directly from the tissues of biopsy materials [14].

Sample preparation for proteomic analysis is facilitated when body fluids (plasma and blood serum, synovial and cerebrospinal fluid, urine) or cell lysates are used, since the proteins are already dissolved in these preparations. The easiest way to disintegrate cells is to use standard lyse solutions. In some cases, the procedure is “freezing - thawing”, as well as sonication.

Fractionation of proteins. Currently, two main approaches are widely used for protein separation in proteomic studies: two-dimensional (2D) electrophoresis and multidimensional chromatography.

2D electrophoresis is based on fractionation of proteins [59, 128, 136]. When implementing the method, proteins are separated according to two different physicochemical properties: in the beginning, separation occurs in the first direction according to charge according to their isoelectric point by isoelectric focusing (IEF), and then in the second direction according to their molecular weight by electrophoresis. Both procedures are carried out in polyacrylamide gel (PAG). As a result of 2D electrophoresis, a two-dimensional “protein map” is obtained.

Up to 2,000 individual polypeptide chains can be separated at a time by two-dimensional gel electrophoresis. The resolution of this method is so great that it allows you to separate two almost identical proteins that differ in one charged amino acid.

After separation of proteins by 2D electrophoresis, they are visualized using staining: colloidal solution of Coomassie [146], silver (silver nitrate with sodium thiosulfate) [145], and special fluorescent dyes SYPRO Ruby [195].

To identify biomarkers, “proteomic maps” of samples are compared with a pathological condition and normal, revealing proteins whose expression level changes with the development of pathology [11]. Further, these proteins are excised from the gel and hydrolyzed by any endopeptidase, for example, trypsin (trypsinolysis) to obtain short (on average 10-15 amino acid residues) peptides that are identified in the next step of the analysis.

However, this method is complicated in execution, amenable to automation and therefore has low productivity [14]. In addition, one of the disadvantages regarding the search for biomarkers is the inconvenience of using two-dimensional electrophoresis for the separation of low molecular weight proteins and peptides.

Multivariate chromatography is a group of chromatographic methods using a variety of sorbents, but, as a rule, one eluent system for chromatographic separation of protein fragments after their hydrolysis (trypsinolysis) [14]. In most cases, the carrier consists of two sorbents — a strong cation exchanger and a reversed phase [186, 196].

Proteomic methods based on multidimensional chromatography are very diverse. These include high-performance liquid chromatography (HPLC), which has high productivity, which can significantly increase the resolution of chromatographic separation of peptides and increase the sensitivity of mass spectrometric determination of proteins [94], two-dimensional (2D) chromatography, which allows to significantly expand the set of proteins used for solubilization in preparation of samples of organic solvents, detergents, and surfactants [129], as well as the SELDI method (Surface-Enhanced Laser Desorbtion lonization), based on the use of protein microarrays and having high sensitivity and easy adaptation to the diagnostic format [109, 180].

Identification of proteins. Identify proteins and peptides found in biological material using mass spectrometry methods. They make it possible to obtain spectra of the distribution of proteins in the mixture by mass: each protein has a specific peak, the position of which is determined by the ratio of the mass and charge of the protein, and the intensity implicitly reflects the amount of protein.

When comparing the mass spectra of samples of biological material from sick and healthy, it is possible to identify proteins and peptides that are specific for a given disease.

In the process of mass spectrometric analysis, the studied proteins are preliminarily fragmented using proteolysis to peptides (usually trypsin), which are then subjected to ionization.

Currently, two methods are mainly used for the ionization of organic compounds, including proteins: matrix desorption laser ionization (MALDI), which results in the formation of mostly singly charged protein ions, although doubly and triple charged ions can be observed, and the method ionization in electrospray (ESI), the result of which is the formation of a pool of multiply charged ions [14].

After receiving the ions, the second stage of the mass spectrometric analysis begins - the sorting of ions by mass (more precisely, by the ratio of mass to charge, or m / z), which occurs in that part of the mass spectrometer, which is called the "mass analyzer".

There are several types of mass analyzers. In modern proteomic research, time-of-flight (TOF) mass spectrometers are widely used, which combine well with MALDI.

MALDI-TOF mass spectrometers make it possible to conduct direct mass spectrometric analysis of blood serum proteins (direct protein profiling) and obtain unique mass spectra with high accuracy and resolution that characterize the object under investigation as “fingerprints” [65]. They allow you to quickly analyze a large number of samples, require a relatively small amount of biological material, have high sensitivity and good resolution for low molecular weight proteins and peptides, which makes them especially promising for the search for biomarkers. The next step is to determine the amino acid sequence of the peptide fragments. There are databases containing amino acid sequences of proteins, and by querying a database of a known peptide sequence, a protein is identified [14].

To detect biomarkers of diseases, SELDI-TOF technology has also been developed, combining mass spectrometric detection of proteins using protein chips. As biological material for research using the SELDI-TOF method, both protein extracts from tissues and biological fluids (plasma or blood serum, urine) can be used. However, blood serum is a complex mixture in which major proteins (e.g. albumin) are present in high concentrations. And we can assume that in the conditions of competition for the site of binding to the surface of the chip, major proteins will have an advantage, while potential biomarkers present in trace amounts simply will not bind to the chip. For the same reason, the amount of bound protein, and, therefore, the peak intensity can depend not only on its amount, but also on the concentration of other proteins in serum [82]. Therefore, the real limits of the sensitivity of the method when working not with isolated proteins, but with a complex mixture, such as blood serum, have not yet been determined. At this stage, SELDI-TOF technology is widely used to develop diagnostics of various types of cancer, while remaining largely an empirical method [11].

The resolution of mass spectrometry methods can be improved by using tandem mass spectrometry (MS / MS), in which two mass spectrometers are used [14]. The principle of the method is that ionized peptides, selected by mass in the first mass spectrometer, undergo further fragmentation upon collision with gas in the second, and their amino acid sequence is established by fragments of the peptide.

Currently, one of the promising systems for solving clinical proteomics is the CLINPROT ^ТМ system manufactured by Bruker Daltonics, which includes tools for fractionation, measurement and visualization of peptide and protein profiles, and software for processing the obtained data. The fractionation method is based on various types of volume chromatography (ion exchange, metal affinity, hydrophobic interaction chromatography). Using the ClinProtRobot robot provides automation of sample cleaning, fractionation and preparation of the corresponding target for mass spectrometric analysis. The profile spectra of the separated biomolecules are recorded with a MALDI-TOF microflex mass spectrometer, which allows to distribute a large number of proteins and peptides according to the mass / charge ratio in a biological sample, or MALDI-TOF ultraflex, which allows to process each peptide and determine the sequence of amino acids that form the target peptide. The sequence of amino acids makes it possible to search in the databases for a protein that may be specific for a given disease, in fact, being its marker. Data processing is carried out using ClinProTools ^TM software, which allows you to compare large amounts of data, as well as recognize complex combinations of biomarkers. Thus, in the mass spectra, peaks are determined by which the most reliable differences between patients and healthy in relation to this disease are revealed, which, in fact, is a marker of the disease.

The resulting peaks can be used not only as an object of study, but also as a diagnostic feature [11]. The researchers' task is to select the conditions for obtaining the most informative and reproducible spectra and develop a rule according to which a particular spectrum could be classified as “health” or “disease”.

The possibilities of proteomic technologies immediately attracted the attention of researchers in the field of medicine.

The largest number of studies is devoted to the search for cancer biomarkers [56, 57, 64, 76, 84, 90, 91, 96, 97, 102, 104, 105, 122, 127, 137, 141, 162, 163, 164, 171, 172 , 184, 190, 192, 194, 197, 198]. It was shown that by comparing the “protein maps” of two-dimensional electrophoresis of proteins isolated from different tumors, it is possible to isolate proteins whose expression naturally increases or decreases in the cells of certain neoplasms. MALDI-TOF allows you to quickly identify and study all differentially expressed proteins.

To date, a number of works on the development of a method for the diagnosis of ovarian cancer using proteomic technologies have been published. Most researchers set the task of identifying significant differences in the spectra of patients and healthy people without clarifying the nature of these differences, and developing diagnostic rules based on them [120, 152, 183, 202], while others identified different peaks [142, 200].

It should be noted that the sets of potential biomarkers obtained by different researchers are completely different. Only 2 proteins are found in more than one work: a marker with a mass of approximately 11.7 kDa [142, 183], identified as serum amyloid A1 [142], and a marker with a mass of approximately 28 kDa [120, 200], identified as apolipoprotein A1 [200]. Serum amyloid A1 is a known protein of inflammation, the level of which rises with many infectious diseases, injuries and inflammatory processes [181]. There is evidence of an increase in its concentration in the blood in lung cancer [106, 117] and kidney [118]. Apolipoprotein A1 is produced by the liver and its concentration is directly related to the presence or absence of the inflammatory process [74]. The identified proteins are not products of the cancerous tissue itself, but the liver and, consequently, an increase in their concentration in the blood is a consequence of the systemic response of the body to the presence of a malignant formation. Apparently, a change in the concentration of biomarkers found can be caused not only by cancer, but also by other inflammatory processes [11].

Petricoin et al. [152] based on the results of comparing the proteomic profile of the plasma of patients with ovarian cancer and various benign formations, correctly identified patients with stage I ovarian cancer and those who have stage II-IV. Of 66 patients with benign ovarian neoplasms, 3 were diagnosed with ovarian cancer incorrectly. It can be assumed that inflammatory diseases and benign tumors have their own characteristic profiles of protein expression.

It is important to note that in almost all cases, not one or two, but a whole series of proteins, diverse in structure and functional properties, were identified as biomarkers. The use of several markers increases the sensitivity and specificity of the diagnostic method [11].

Using proteomic technologies, biomarkers are searched for in patients with nasopharyngeal cancer [78]. When comparing the proteomic profiles of the blood serum of patients with nasopharyngeal cancer and healthy people, two proteins with molecular weights of 11.6 and 11.8 kDa were found, which are two isoforms of serum amyloid A (SAA), the content of which is significantly increased in patients with malignant nasopharyngeal tumors. Long-term monitoring of patients showed that the level of SAA in serum increases significantly with relapse.

In a study by 2D electrophoresis in combination with mass spectrometry of biopsy samples of lung cancer epithelial tissue, proteins of diagnostic significance were detected. Identified proteins include: 15-hydroxy prostaglandin dehydrogenase, 3-hydroxymethylglutaryl coenzyme A synthetase, apolipoprotein J, beta-glucoronidase, flavin reductase, CuZn superoxide dismutase, cathepsin D, heat shock protein 27, etc.

During proteomic profiling of the urine of patients with kidney carcinoma, a number of metabolism-controlling enzymes were found, the expression level of which was significantly increased compared to the norm [151]. The detected proteins are involved in such important metabolic processes as glycolysis, pyruvate metabolism, and the urea conversion cycle.

In the study of the protein profile of biological samples of patients with prostate cancer, annexation proteins I, II, and VII were found, the expression level of which differs for patients and healthy people [168].

The main proteomic studies in dermatology are focused on the search for tumor growth biomarkers in diseases such as squamous cell carcinoma [148, 201] and melanoma [139, 189]. In these studies, the proteomic profile is studied both in samples of tumor tissue and in biological fluids of patients: blood and saliva [86]. Using 2-D electrophoresis in combination with mass spectrometry, 2 proteins were detected in the saliva of patients with squamous cell carcinoma: α-1-B-glycoprotein and complement factor B, which are absent in the saliva of healthy people [148]. In patients with melanoma, several types of proteins with a molecular weight of about 30 kDa, which are absent in the blood serum of healthy people, are detected. The number of peptides with molecular weights of 2.5–3.5 kDa in patients with melanoma depends on the clinical stage of the disease [86].

Proteomic studies are widely used in the study of psoriasis [73]. An analysis of the protein profile of psoriatic skin by 2D electrophoresis followed by microsequencing of keratinocyte proteins revealed 6 proteins, the expression of which during psoriasis was increased 5 or more times compared to the norm, with apparent molecular weights of 40.3 kDa, 12.4 kDa, 11 , 9 kDa, 11.6 kDa, 11.6 kDa and 10.1 kDa. When comparing the proteomic profile of different forms of psoriasis (plaque and teardrop) and healthy tissue, it was found that, regardless of the form of the disease, the expression of SCCA-2 and RhpoGDI proteins and HSP27 and 14-3-3σ proteins with plaque form is increased compared to healthy skin [69] .

In addition, studies of the proteomic profile are carried out for various autoimmune diseases [107], such as lupus erythematosus [176], scleroderma [92], vitiligo [131]. The studies used 2D electrophoresis and mass spectrometry, SELDI; blood serum, synovial fluid, tissue biopsy samples were used as a substrate. The data obtained indicate that in the pathogenesis of autoimmune diseases, a special role is played by cytokines, chemokines, as well as proteins associated with apoptosis processes.

The possibility of using proteomic technologies for the diagnosis of diseases is being actively studied in rheumatological practice. Studies of differences in the protein profile of blood serum in patients with osteoarthritis and rheumatoid arthritis have been performed [193]. Almost 90 protein subunits that were absent in patients with osteoarthritis were identified in the blood serum of patients with rheumatoid arthritis. When proteomic analysis of blood plasma proteins and synovial fluid in patients with various forms of arthritis revealed: 1) degradation products of the fibrinogen β-chain in synovial fluid in all patients; 2) proteins of calgranulin B (MRP14) and calgranulin C exclusively in the synovial fluid of patients with rheumatoid arthritis; 3) fractions of amyloid A protein in the blood plasma and synovial fluid of patients with rheumatoid arthritis [166]. In the coming years, many new biomarker molecules of peptides associated with disease-associated conditions in rheumatology are expected to be detected [153].

There are separate studies on the use of proteomic profiling of blood plasma of patients with pathology of the cardiovascular system and cerebrovascular accident [188]. It has been shown that at least 177 blood plasma proteins can be used as diagnostic markers.

There are few studies on the use of proteomic profiling of blood plasma of patients with infectious diseases. As a result of proteomic analysis of the blood plasma of patients with SARS, 38 proteins were identified, most of which are characteristic of the acute phase of the disease and appear in the blood of SARS patients due to a number of processes caused by the action of SARS-coronavirus [75]. Among them are haptoglobin, which is widely used as a marker of inflammation in the diagnosis of infectious and inflammatory lesions in veterinary medicine [87], a mannose-binding protein belonging to the family of C-type pectins and closely associated with infectious diseases [88], an immune response when infected with influenza virus A type [100] and HIV [179].

In the scientific literature there is no data on the results of using proteomic profiling of blood serum to study syphilitic infection. However, taking into account the available information on the methodology of proteomic technologies, the possibility of using blood serum as a material for the study, which does not require special equipment and does not cause inconvenience for the patient, the proteomic profiling method can be considered promising for the development of a new technology for the specific diagnosis of syphilis.

Using knowledge of the proteomic composition of serum or blood plasma in combination with a high-performance analytical tool of proteomics - mass spectrometric analysis of proteins - provides an opportunity for a fundamentally new approach to the diagnosis of syphilitic infection, which can overcome the limitations of classical methods.

Disclosure of the present invention

The essence of the present invention is a method by which it is possible to detect syphilis at any stage and in any form of flow, regardless of the state of the human immune system, biological factors, burdened somatic status, technical features and which can serve as a criterion for evaluating the effectiveness of specific treatment.

Thus, the present invention relates to a method for the diagnosis of syphilis in a patient by the method of direct proteomic profiling of blood serum based on the detection of syphilis biomarkers in an experimental serum sample, which comprises the following steps:

a) fractionation of whey proteins using magnetic particles;

b) mass spectrometric analysis of a sample of the eluate obtained in stage a);

c) analysis of the obtained mass spectrum for the presence or absence of a set of peaks corresponding to biomarkers of syphilis, moreover, the presence of a characteristic set of peaks in the mass spectrum indicates the presence of syphilis, and the absence of a characteristic set of peaks in the mass spectrum indicates the absence of syphilis in the patient.

According to one embodiment of the present invention, the magnetic particles are MB-WAX (magnetic microparticles with positively charged functional groups on the surface).

In accordance with one of the preferred embodiments of the present invention, mass spectrometric analysis of a sample of the eluate is carried out according to the MALDI-TOF method.

However, it should be understood that other types of microparticles, as well as alternative methods of mass spectrometry, can be used to implement the present invention.

In accordance with one of the embodiments of the present invention, syphilis is an early latent syphilis, a characteristic set of biomarkers of which is characterized by the ratio of molecular weight to charge (m / z) 2453.67; 4,296.44; 2729.63; 10,672.86; 2623.83; 7,199.98; 6078.82; 7,934.44; 2011.07; 2644.48.

In accordance with another embodiment of the present invention, syphilis is characterized by a manifest course and is primary or secondary syphilis, whose characteristic set of biomarkers is characterized by a ratio of molecular weight to charge (m / z) of 2452.18; 2,600.42; 2642.96; 2727.81.

An embodiment of the present invention

Study conditions determining the significance of its results

To conduct research on the search for biomarkers specific for syphilitic infection, we used 60 blood serum samples obtained from 60 patients with early forms of syphilitic infection, including 30 patients with manifest (primary and secondary) and 30 with early latent syphilis. The diagnosis was confirmed by the positive results of standard serological reactions (RPR, RPGA, ELISA), regulated in the Russian Federation for the diagnosis of the disease, the detection of the causative agent of syphilitic infection (Treponema pallidum) in the discharge of erosive / ulcer specific elements, clinical data.

The control was 30 samples of blood serum obtained from healthy individuals. The main criterion for inclusion in the control group was a negative result of standard serological reactions (RPR, RPGA, ELISA), regulated in the Russian Federation for the diagnosis of syphilis.

The study groups were comparable by gender and age. The study did not include blood serum samples obtained from patients with severe concomitant diseases of the internal organs, indicating drug dependence or constant alcohol consumption, in pregnant women.

Research Methodology:

1) sample preparation (fractionation of blood serum proteins)

2) mass spectrometry

3) analysis of the data

1. Fractionation of serum proteins

MB-WAX magnetic microparticles (magnetic microparticles with positively charged functional groups on the surface, which allows anion-exchange protein chromatography) were used for fractionation. The basis of the MB-WAX magnetic particle method is weak anion-exchange chromatography, used to fractionate proteins by ion charge.

The procedure is carried out in accordance with the manufacturer's protocol:

Preparation of magnetic particles for fractionation

1. A mixture of magnetic particles (MB-WAX) was thoroughly mixed for 1 minute until a homogeneous suspension on the vortex.

2. To 10 μl of magnetic particles, carefully pipetting was added 20 μl of activation buffer (AB).

3. The tube was placed with a mixture of magnetic particles and buffer in a magnetic separator for 20 seconds to activate.

4. The supernatant was carefully removed with a pipette without touching the precipitate of magnetic particles.

5. To balance the magnetic particles, 100 μl of binding buffer (BB) was added.

6. The tube was placed with a mixture of magnetic particles and buffer in a magnetic separator for 20 seconds to bind.

7. The supernatant was carefully removed with a pipette without touching the precipitate of magnetic particles.

8. Steps 5-7 were repeated.

9. The pellet of magnetic particles was resuspended in 20 μl of binding buffer (BB).

Protein fractionation procedure

1. 5 μl of the sample was added to the solution of resuspended magnetic particles and pipetted 5 times to mix the magnetic particles with the sample.

2. Magnetic particles were incubated with the sample for 5 minutes at room temperature.

3. After incubation, the tubes were placed in a magnetic separator for 20 seconds to separate unbound proteins from magnetic particles.

4. After 20 seconds, the tubes were removed from the magnetic separator and the supernatant was carefully removed with a pipette, without touching the resulting complex in the form of a precipitate.

5. To the pellet of magnetic particles was added 100 μl of binding buffer (BB), carefully pipetting the mixture.

6. Incubate the tube with magnetic particles and wash buffer (BB) for 20 seconds to form a magnetic deposit on the tube wall in the magnetic separator.

7. After 20 seconds, the tube was removed from the magnetic separator and the supernatant was carefully removed with a pipette, without touching the precipitate of magnetic particles.

8. Steps 5-7 were repeated two times.

9. To elute the bound proteins, 10 μl of elution buffer (EB) was added to the pellet of magnetic particles and mixed thoroughly.

10. Incubated at room temperature for 5 minutes.

11. After incubation, the tube was placed in a magnetic separator to separate the eluate from the magnetic particles for 30 seconds.

12. The tube was removed from the magnetic separator, the eluate was carefully separated from the magnetic particles, and transferred to another tube.

The isolated protein fraction was intended for mass spectrometric analysis (MALDI-TOF).

2. Mass spectrometry

Mass spectrometric analysis was performed using a MALDI-TOF (time-of-flight) Bruker Microflex mass spectrometer (Bruker Daltonics, Germany) equipped with a nitrogen laser (λ = 337 nm), an ion extraction delay system, and a microchannel detector. During operation, a laser pulse frequency of 20 Hz was used. All measurements were performed in a linear mode, detecting positively charged ions using an accelerating voltage of 20.0 kV. The parameters of the mass spectrometer were optimized for the range of the m / z ratio from 2000 to 20000, the voltage at the storage electrode was 18.6 kV and the extraction delay time was 150 ns. To obtain each mass spectrum, a sample was applied to the target of the mass spectrometer. The target was installed in the ion source of the device and mass spectra were recorded at 3000 laser pulses with a radiation power set at the minimum value sufficient for desorption-ionization of the sample. Spectra were taken in manual mode (AutoExecute ^TM , Bruker Daltonics, Germany).

To control the mass spectrometer, including setting the operating modes and recording the mass spectra, we used the flexControl 2.4 software package (Bruker Daltonics, Germany).

3. Analysis of the data

The marking of the mass spectra, the isolation of signals, the statistical analysis of the experimental data obtained in the form of mass spectra, and the construction of classifier models were carried out using the ClinProTools version 2.1 clinical mass spectrometric analysis software package (Bruker Daltonics, Germany) corresponding to all the main requirements for the search for biomarkers in complex biological mixtures and their validation. The ClinProTools bioinformatics system (version 2.1) implements two methods for constructing classification models (classifiers) for analyzing the distribution of biomarkers between groups: the Support Vector Machine-SVM method and the genetic algorithm (GA), which are generally recognized and effective methods of mathematical analysis mass spectrometric data for solving problems associated with the construction of classifiers. The determination of biomarkers detected in models using these algorithms as mass spectrometric signals is carried out by selecting the optimal parameters of the algorithms:

the number of specified mass spectrometric signals (peaks) and the number of “neighbors” (for both algorithms), the number of iterations (for the genetic algorithm) to achieve the highest Cross-Validation and Recognition Capability.

As a result of mass spectrometric analysis of serum proteins / peptides of patients with early latent syphilis using the SVM classifier, 20 significant mass spectrometric signals (peaks) were determined using the GA classifier 10. It was found that in both models constructed using the above classifiers, 10 peaks coincide with the average value of the ratio of molecular weight to charge (m / z): 2453.67; 4,296.44; 2729.63; 10,672.86; 2623.83; 7,199.98; 6078.82; 7,934.44; 2011.07; 2644.48. The peaks obtained in both models are shown in Tables 1 and 2. It should be noted that all ten peaks obtained using the GA classifier are among the twenty peaks obtained using the SVM classifier, and therefore correspond to the most reliable markers of early hidden syphilis, a characteristic set of biomarkers of early latent syphilis.

Table 1
Significant mass spectrometric signals (peaks) determined in the blood serum of patients with early latent syphilis using the SVM classifier
Cross Validation 97.22%
Recognition Ability 100% Index Weight Initial mass Final mass The weight one 2011.07 2006.73 2020.85 1.35 2 2026.94 2020.85 2040.09 1,56 5 2453.67 2442.43 2464.72 1.82 9 2601.86 2585.76 2617.74 1.3 10 2623.83 2617.74 2633.98 1.23 eleven 2644,48 2633.98 2659,02 1.3 12 2729.63 2717.01 2735.59 1,52 17 3374.78 3353.33 3392.82 0.63 27 4296.44 4280.05 4320.27 1.47 29th 4632.38 4604.17 4644.8 0.77 thirty 4650.96 4644.8 4687,02 1.44 35 5388.68 5371.04 5414.91 0.79 36 6078.82 6051.76 6107.85 1.29 38 6312,23 6,296.65 6,352.95 1.01 43 7199.98 7139.37 7255.42 0.99 45 7779.19 7700.1 7838.11 1.09 46 7934.44 7872.75 7997.07 1,54 48 8,229.41 8208.18 8257.63 1,02 51 8950.31 8882.53 9018.95 0.89 56 10672.86 10582,2 10765.91 1.89

table 2
Significant mass spectrometric signals (peaks) determined in the blood serum of patients with early latent syphilis using the GA classifier
Cross Validation 98.65%
Recognition Ability 100% Index Weight Initial mass Final mass The weight 5 2453.67 2442.43 2464.72 1.79 27 4296.44 4280.05 4320.27 1,08 12 2729.63 2717.01 2735.59 1.33 56 10672.86 10582,2 10765.91 1,63 10 2623.83 2617.74 2633.98 1.36 43 7199.98 7139.37 7255.42 1,1 36 6078.82 6051.76 6107.85 0.54 46 7934.44 7872.75 7997.07 0.74 one 2011.07 2006.73 2020.85 2.17 eleven 2644,48 2633.98 2659,02 1.84

As a result of mass spectrometric analysis of serum proteins / peptides in patients with the manifest (primary and secondary) course of syphilis using both classifiers (SVM and GA), 10 significant mass spectrometric signals (peaks) were determined. It was found that in both models built using the above classifiers, 4 peaks coincide with the average value of the ratio of molecular weight to charge (m / z): 2452.18; 2,600.42; 2642.96; 2727.81, which correspond to the most reliable markers of manifest syphilis, a characteristic set of biomarkers of manifest syphilis. Peaks obtained in both models on the blood serum of patients with the manifest course of syphilis are shown in tables 3 and 4.

Table 3
Significant mass spectrometric signals (peaks) determined in the blood serum of patients with manifest syphilis using the SVM classifier
Cross Validation 98.96%
Recognition Ability 100% Index Weight Initial mass Final mass The weight eleven 2727.81 2715.41 2734.94 1.48 9 2600,42 2588.57 2616.09 1.51 49 10665.83 10576.45 10753.13 1.3 25 4293.37 4277.3 4317.81 0.94 37 7194.95 7136.09 7257.55 1.01 6 2452.18 2440.55 2463.17 1.73 3 2282.63 2275.82 2289.09 0.51 28 4648.65 4641.81 4694.86 0.6 10 2642.96 2632,43 2657.46 1.78 17 3446.07 3432.64 3463.24 0.26

Table 4
Significant mass spectrometric signals (peaks) determined in the serum of patients with manifest syphilis using the GA classifier
Cross Validation 98.96%
Recognition Ability 100% Index Weight Initial mass Final mass The weight one 2009,99 2005.89 2019.69 1,11 2 2026.04 2019.69 2038.92 1.14 6 2452.18 2440.55 2463.17 2.06 9 2600,42 2588.57 2616.09 2.15 10 2642.96 2632,43 2657.46 1.92 eleven 2727.81 2715.41 2734.94 2,37 13 2755.22 2744.56 2770.11 0.91 39 7776.04 7697.31 7834.03 0.92 44 8946.38 8889.09 9015.46 0.9 46 9307.83 9,248.55 9360,99 1,02

Claims (3)

1. A method for the diagnosis of syphilis in a patient by direct proteomic profiling of blood serum based on the detection of syphilis biomarkers in the serum sample, which includes the following stages:
a) fractionation of whey proteins using magnetic particles;
b) mass spectrometric analysis of a sample of the eluate obtained in stage a);
c) analysis of the obtained mass spectrum for the presence or absence of a set of peaks corresponding to biomarkers of syphilis, moreover
the presence in the mass spectrum of a set of peaks characterized by the values of the ratio of molecular weight to charge (m / z): 2453.67; 4,296.44; 2729.63; 10,672.86; 2623.83; 7,199.98; 6078.82; 7,934.44; 2011.07; 2644.48, indicates the presence of early latent syphilis in the patient,
the presence in the mass spectrum of a set of peaks, characterized by the values of the ratio of molecular weight to charge (m / z): 2452.18; 2,600.42; 2642.96; 2727.81, indicates the presence of primary or secondary syphilis in the patient, and
the absence of these sets of peaks in the mass spectrum indicates the absence of syphilis in the patient. 2. The method according to claim 1, wherein the magnetic particles are MB-WAX. 3. The method according to claim 1, whereby mass spectrometric analysis of a sample of the eluate is carried out according to the MALDI-TOF method.