RU2625018C2 - Method for colorectal cancer diagnosis/screening based on simultaneous quantification of protein nature tumour markers, glycans antibodies, g, a and m immunoglobulins in human blood on biological microchip - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves the simultaneous quantification of protein nature tumour markers, glycan antibodies, G, A and M immunoglobulins in human blood on a biological microchip. For this purpose, the biochip contains hydrogel elements with immobilized antibodies to protein oncomarkers, hydrogel elements with immobilized glycans, hydrogel elements with immobilized antibodies against human immunoglobulins of G, A, M classes. The change in the level of the analyzed markers in comparison with the level in the serum of healthy donors indicates colorectal cancer. The group of inventions also refers to a biological microchip, which is an array of three-dimensional hydrogel elements.

EFFECT: application of this invention allows colorectal cancer diagnosis, screening, simultaneous quantification of protein markers, antibodies to glycans, G, A and M immunoglobulins in human blood on a biological microchip that allows successive reactions of various types.

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Description

FIELD OF THE INVENTION

(Technical Field)

The invention relates to biochemistry and medicine, in particular, to analytical biochemistry and immunochemical analysis, and relates to a method for the diagnosis of colorectal cancer based on the simultaneous quantitative determination of the concentration of tumor markers of protein nature, levels of antibodies to cancer-associated glycans, levels of immunoglobulins G, A, M in human serum on a biological microchip. A biological microchip based on hydrogels contains immobilized antibodies to protein oncomarkers, immobilized glycans (including oncological associated ones), immobilized antibodies against human immunoglobulins G, A, M. The present invention can find application in analytical biochemistry, immunology and medicine, in particular in the diagnosis of colorectal cancer.

State of the art

(Background art)

Colorectal cancer (CRC) ranks third in terms of prevalence among the population of industrial countries (Claudia Allemani, Hannah K Weir, Helena Carreira, Rhea Harewood, Devon Spika, Xiao-Si Wang, Finían Bannon, Jane V Ahn, Christopher J Johnson, Audrey Bonaventure , Rafael Marcos-Gragera, Charles Stiller, Guiñar Azevedo e Silva, Wan-Qing Chen, Olufemi J Ogun MPC, Group * and the CW. Global surveillance of cancer survival 1995-2009: analysis of individual data for 25 676 887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet 2014; (November 26). doi: http: //dx.doi.org/10.1016/S0140-6736 (14) 62038-9). Statistics from developed countries of the world indicate a steady growth of newly diagnosed cases of colon and rectal cancer compared with malignant tumors of any other localization except lung cancer. Mortality from CRC is also very high, for example, in the United States 10% of all cancer deaths occur in CRC, for Japan this figure is 13%.

Russia is one of the countries with a high incidence of mortality and cancer mortality. In 2010, more than 58 thousand new cases of CRC were registered in our country; in the structure of oncological morbidity in 2008, colon cancer was in 4th place (6.5% of cases), colorectal cancer was in 5th place (4.9%). Accordingly, mortality is increasing. Thus, in 2008, the share of colon cancer in the structure of total mortality was 7.0% (3rd place), and the share of colon cancer - 5.8% (4th place) (Chissov V.I. et al. Malignant neoplasms in Russia in 2010: morbidity and mortality // Federal State Budgetary Scientific Research Institute named after P. Gretsen. - 2012; 1: 17-136). The main risk factors for the development of CRC are genetic predisposition, inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, diverticulitis. In the early stages, CRC develops with minimal clinical symptoms. At the same time, it is believed that mortality from CRC can be reduced through early diagnosis.

Adenomas are considered the most important precursor to sporadic CRC. However, it has been proven that 50% of people will develop colorectal adenoma over time, but only 6% will transform into CRC (Siegel R, Naishadham D, Jemal A. Cancer Statistics, 2012. CA Cancer J Clin 2012; 62: 10 -29). Jagged polyps (i.e., jagged adenomas and large hyperplastic polyps) are increasingly recognized as the source of probable precancerous lesions. An estimated 20% - 30% CRC arises from dentate polyps and not from adenomas (Noffsinger AE. Serrated polyps and colorectal cancer: new pathway to malignancy. Annu. Rev. Pathol. 2009; 4: 343-364).

Jagged polyps are usually located on the right side, more common among older people, and less endoscopic than adenomas (Groff RJ, Nash R, Ahnen DJ. Significance of serrated polyps of the colon. Curr. Gastroenterol. Rep. 2008; 10 (5): 490-498.). They grow faster than adenomas and can progress faster in CRC (Noffsinger, A.E. Serrated polyps and colorectal cancer: new pathway to malignancy. Annu. Rev. Pathol. 2009; 4: 343-364). The development of dentate polyps is associated with BRAF mutations, which are rarely present in adenomas. Until now, there is no single criterion that should be used to identify a high risk of developing CRC from a dentate polyp, except, perhaps, for one criterion - the size of the polyp is more than 1 cm.

Today, the following invasive methods are used to detect colorectal cancer - colonoscopy, flexible sigmoidoscopy, double-contrast irrigoscopy, computed tomographic colonography ("virtual colonoscopy").

Flexible sigmoidoscopy allows you to examine the inner surface of the colon at a distance of 60 cm from the anus. Using this method, colorectal polyps and tumors can be detected. However, the obvious drawback of this method is the possibility of examining only the left part of the colon, and its right part remains unexplored. While the specificity of flexible sigmoidoscopy is very high (98-100%), sensitivity for detecting CRC is low from 35% to 70% due to the presence of a large number of right-sided adenomas.

Colonoscopy allows you to identify and remove polyps, to conduct a biopsy of a tumor located in the colon. The specificity and sensitivity of detecting polyps and CRC are high. There are no promising randomized trials evaluating the impact of colonoscopy on morbidity and mortality. However, according to mathematical modeling, the long-term results of a polyectomy (United States National Polyp Study) show an almost 90% reduction in the incidence of deaths and deaths from it (Regula J., Rupinski M., Kraszewska Ε., Polkowski Μ. And other. Colonoscopy in colorectal cancer screening for detection of advanced neoplasia. N. Engl. J. Med. 2006; 355: 1863-1872).

Colonoscopy is the "gold standard" for detecting CRC, all patients with a positive result of other screening studies should be subsequently referred for colonoscopy.

Double-contrast irrigoscopy (DCI) allows you to examine the entire colon, its sensitivity and specificity below the diagnostic parameters obtained during colonoscopy. Even in the presence of large polyps and tumors, IDK has a significantly lower sensitivity (48%) than colonoscopy. In addition, IDK gives more than coloscopy false positive results (artifacts defined as polyps) (Winawer SJ, Stewart E.T., Zauber AG, Bond JH, and other. A comparison of colonoscopy and double-contrast barium enema for surveillance after polypectomy. National Polyp Study Work Group. N. Engl. J. Med. 2000; 342: 1766-1772). Patients who have been diagnosed with IDK have a pathology followed by a colonoscopy. Despite these shortcomings, IDK is widespread and the fact that with its help it is possible to identify up to 50% of large polyps, determines its use in the absence of the possibility of more accurate studies.

Computed tomographic colonography (CPC). A layered spiral computed tomographic scan of the abdominal cavity and pelvis, followed by digital processing and image analysis, can create both two- and three-dimensional reconstruction of the lumen of the colon (“virtual colonoscopy”). This study requires insufflation of air to inflate the intestine to the extent that the patient can tolerate. Meta - analysis of studies in which CPC was used to detect colorectal polyps and cancer showed high sensitivity (93%) and high specificity (97%) in the presence of polyps 10 mm or more in size. However, with a combination of polyps of large and medium sizes (over 6 mm), the sensitivity of the method decreased to 86% and specificity also to 86%. In the study of polyps of different sizes, the scatter of sensitivity indicators (from 45% to 97%) and specificity (from 26% to 97%) became too large. A major obstacle to using CPC for early detection of CRC is that polyps of 6 mm-9 mm and flat lesions in the intestine can be skipped (Kim DH, Pickhardt PJ, Taylor AJ, Leung WK and other. CT colonography versus colonoscopy for the detection of advanced neoplasia. The New England journal of medicine. 2007; 357: 1403-1412). The big disadvantage of CPC is that for its repeated conducting the patient must be re-exposed to ionizing radiation.

The invasive methods described above are not suitable for the early diagnosis of CRC, as None of the above studies can capture the full picture of the development and spread of CRC.

For molecular diagnosis of CRC, it is necessary to control the release of tumor markers at various stages of tumor growth and in various biological media (Ahlquist DA, Harrington JJ, Burgart LJ, Roche PC. Morphometry analysis of the "mucocellular layer" overlying colorectal cancer and normal mucosa: relevance to exfoliation and stool screening. Hum. Pathol. 2000; 31 (1): 51-57).

The widespread non-invasive methods for determining CRC used in the clinic include stool analysis for occult blood, immunochemical determination of hemoglobin in the patient’s stool, determination of DNA markers in the patient’s stool, determination of protein tumor markers in the patient’s blood serum.

Fecal occult blood analysis is based on the determination of hemoglobin in the patient’s stool (Allison JE, Sakoda LC, Levin TR et al. Screening for colorectal neoplasms with new fecal occult blood tests: update on performance characteristics. J Natl Cancer Inst 2007; 99: 1462- 1470). The most common tests are Hemoccult II (Smith Kline Diagnostics) and Hemoccult II Sensa (Smith Kline Diagnostics). If there is blood in the stool, the guaiac test shows a blue color. The color change is related to the peroxidase-like activity of hemoglobin in the stool. The guaiac test is characterized by a large proportion of false-positive reactions, low specificity for detecting CRC. False positive signals can occur due to a diet violation in preparation for analysis, for example, eating red meat, plant peroxidases (some fruits and vegetables), eating high doses of vitamin C. The presence of hemorrhoids and angiodysplasias can also cause false positive results. Current recommendations for Hemoccult testing include nutritional restrictions (Ransohoff D.F., Lang S.A., Part I: Suggested technique for fecal occult blood testing and interpretation in colorectal cancer screening. Ann Intern Med 1997 126: 808).

Due to its low diagnostic sensitivity, this test did not detect the early stages of cancer (Heresbach D, Manfredi S, D'Halluin Ρ Ν, Bretagne JF, Branger B. Review in depth and meta-analysis of controlled trials on colorectal cancer screening by fecal occult blood test. Eur J Gastroenterol Hepatol 2006; 18 (4): 427-433).

The immunochemical detection of hemoglobin in the patient’s stool is based on the interaction of hemoglobin with specific antibodies, followed by signal registration by the immunochemical method (ELISA). These tests do not react with non-human hemoglobin or plant peroxidases, which allows dietary restrictions not to be observed (Cole SR, Young GP. Effect of dietary restriction on participation in fecal occult blood test screening for colorectal cancer. Med J Aust 2001; 175 (4): 195-198). Examples of such tests are HemeSelect (SmithKline Diagnostics, USA) and InSure (Enterix Inc., USA).

The quantitative determination of hemoglobin in the patient’s stool is the basis of the FOB Gold test (Sentinel Diagnostics, Italy). The FOB Gold test allows you to accurately determine the quantitative content of hemoglobin in the stool without first observing a strict diet by patients. The method is based on the antigen-antibody agglutination reaction between human hemoglobin and anti-hemoglobin antibody on latex particles. Agglutination is measured by absorption at 570 nm, the increase in absorption units is proportional to the amount of human hemoglobin in the sample. This test is fully automatic. A special sample collection tube is designed as a container for collecting stools and fits directly into a biochemical analyzer. The disadvantages of the method include the low sensitivity to detect CRC.

A method for detecting DNA markers in a patient’s stool has been rapidly developing since the beginning of 2000 (Ahlquist DA. Colorectal cancer screening by detection of altered human DNA in stool: Feasibility of a multitarget assay panel. Gastroenterology 2000; 119 (5): 1219-1227). The feasibility of testing stool for DNA to detect CRC was first demonstrated when, in 1992, the relationship between CRC and the only marker, the mutant gene KRAS (Sidransky D, Tokino Τ, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 1992; 256 (5053): 102-105). The DNA test allows up to 52% of invasive CRC tumors to be detected, compared with 13% detected using a conventional guaiac test (P = 0.003), with a comparable level of specificity. In another study (Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann. Intern. Med. 2008; 149 (7): 441-450), the simultaneous use of three DNA markers revealed 46% of cases of adenomas, while the guaiac test found only 10% (P <0.001), and the specificity of the DNA test was 17% higher.

However, the DNA test has a lower level of sensitivity compared to occult blood feces (Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann. Intern. Med. . 2008; 149 (7): 441-450.).

Despite the wide variety of molecular approaches to colorectal cancer screening described above, there is still a need for a method with high diagnostic potential to increase the efficiency of screening / diagnosis of CRC and allowing staff to work with the patient's blood serum.

In clinical practice, for the diagnosis of CRC, it is customary to determine the concentration of two protein tumor markers: carcinoembryonic antigen (CEA) and carbohydrate antigen CA 19-9 in the patient's blood serum by ELISA (Gold Ρ, Freedman SO. Specific carcinoembryonic antigens of the human digestive system. J. Exp .Med. 1965; 122 (3): 467-481. Locker GY, Hamilton S, Harris J, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J. Clin. Oncol. 2006; 24 (33): 5313-5327), however, the sensitivity of the method is not high. Traditionally, the concentration of tumor markers in the blood serum of patients is determined by the method of solid-phase immunoassay of ELISA. The most common commercial test systems based on solid-phase ELISA (Fujirebio Diagnostics - CanAg, Sweden, NovaTec, Germany, Abazyme, Needham, USA, Biosourse Diagnostics, Belgium, Alpha Diagnostic Intl., Texas, USA; Vector-Best, Immunotech, Amercard and Hema-Medica, Moscow, Russia, Alkor-Bio, St. Petersburg, Russia), allow to determine the content of only one marker in the sample.

The main directions of further development and improvement of in vitro methods for the diagnosis of CRC are their miniaturization, increased sensitivity and the possibility of simultaneous multi-parameter analysis. Many companies produce automatic analyzers that can simultaneously detect several protein tumor markers in the patient's blood serum. Concentrations of tumor markers are automatically quantitatively calculated by calibration curves obtained during immunoassay of the studied solutions and control samples. Multiparameter testing of a sample in this system is achieved by simultaneously conducting a large number of individual analyzes for each tumor marker.

For example, to determine the content of several tumor markers, automatic analyzers are used, for example, Awareness Technology Inc., USA, operating on the basis of the ELISA principle and representing an open system for any methods and reagents. The ChemWell analyzer uses standard microplates for all types of reactions and is capable of conducting several parallel studies, including enzyme-linked immunosorbent assay of tumor markers: AFP, CEA, PSA, CA125, CA15-3, CA19-9, CA242, ferritin, hCG, NSE, tissue polypeptide antigen, beta 2-microglobulin in 4 wells. The fully automated microplate ELISA analyzer Stat Fax 3200 provides the construction of calibration curves, the procedure for calculating the results, subtracting the background, etc., allows up to 12 parallel tests simultaneously. The Lumi Stat immunochemiluminescent strip analyzer - CLIA is used to determine 11 tumor markers of CEA, PSAobs, PSAsobvod, CAI25, AFP, prostatic acid phosphatase, CAI 9-9, CAI 5-3, NMP22 (nuclear matrix proteins), beta-2- microglobulin, cytokeratin 18. Biomerieux mini VIDAS, France, is a multi-parameter automatic immunoassay based on enzyme-linked immunofluorescence assay technology and designed to perform “single” tests. The determination of analytes occurs with high sensitivity (exceeding the sensitivity of ELISA by several orders of magnitude), which can significantly reduce the analysis time (up to 30 minutes). Instruments of this class represent a mechanical combination of several analyzes, but not a simultaneous multi-parameter analysis of several analytes in one sample.

The simultaneous determination of several protein tumor markers using fully automated Elecsys systems from Roche Diagnostics, USA / Switzerland, Luminex, USA / Netherlands, and AxSYM from Abbott, USA, is described. The Elecsys system uses the principle of heterogeneous immunochemical analysis based on electrochemiluminescent diagnostics - analytical strips or microcolumns for affinity chromatography. The Luminex analyzer (USA) is based on the principle of flow fluorimetry of microspheres with ligands immobilized on the surface (using various types of interaction: complementary interaction of nucleic acids and bases, ligand-receptor interaction, formation of immunocomplexes, enzymatic reactions), which allows simultaneous analysis of up to 100 different analytes. AxSYM automated systems are based on enzyme-linked immunosorbent assay on microparticles and / or fluorescence polarization enzyme-linked immunosorbent assay. They provide quick time to obtain the result (8-30 minutes) and high performance (from 80 to 120 tests per hour). The disadvantage of Roche Diagnostics, Luminex and Abbott analyzers is that they are "closed" systems consisting of an analyzer and a set of reagents for it (tubes, microcolumns, strips and other reagents of only this company are used, designed for this system or this specific device), as well as their high cost (from several tens to hundreds of thousands of US dollars), high requirements for staff qualifications, which does not allow them to be introduced into wide clinical practice. The disadvantages of all analyzers include a large amount of blood serum taken for analysis (over 100 μl per tumor marker).

Diagnosis of oncological diseases by the presence of antibodies to cancer-associated glycans in human blood serum is a promising area of cancer diagnostics, has been actively developing since 2010. Antibodies to cancer-associated glycans (AGA) have proven to be promising markers of a number of diseases, for example, ovarian cancer (Jacob, Francis et al. 2012. " Serum Antiglycan Antibody Detection of Nonmucinous Ovarian Cancers by Using a Printed Glycan Array. "International Journal of Cancer 130 (1): 138-46).

Given the fact that most protein markers are not specific for the location and type of tumor, it is necessary to combine them into groups in specific combinations - the so-called diagnostic and prognostic signatures (combinations of protein tumor markers and antibodies to glycans) that can solve the problem of specificity, early and more accurate diagnosis of CRC.

Search and selection of specific antibodies to glycan structures in CRC is of high clinical importance and opens up new possibilities for the diagnostic support of the oncotherapy process, determines the treatment strategy and tactics, the course and possible outcome of the disease. This is because antibodies to glycans are found in the blood serum in the early stages of cancer (Chapman C, Murray A, Chakrabarti J, et al. Autoantibodies in breast cancer: Their use as an aid to early diagnosis. Ann. Oncol. 2007; 18 (5): 868-873. Chapman CJ, Murray A, McElveen JE, et al. Autoantibodies in lung cancer: possibilities for early detection and subsequent cure. Thorax. 2008; 63 (3): 228-233).

Currently, solid-state plate immunoassays (Shilova NV, Galanina OE, Pochechueva TV, et al. High molecular weight neoglycoconjugates for solid phase assays. Glycoconj. J. 2005; 22:43 -51).

As in the case of the analysis of protein tumor markers, for the determination of AGA antibodies there is a need for a quick and automatic analysis of the patient's blood serum. For example, SCIEX (USA) has released an automatic analyzer for the determination of AGA antibodies using magnetic particles. The disadvantages of this method include the possibility of determining only AGA for N-glycans.

Currently, the task of conducting multi-parameter analysis with a minimum volume of the test sample is solved using biological microarrays - arrays of elements containing immobilized biological probes (DNA, proteins, oligosaccharides, oligonucleotides, etc.). The creation of biochips allowed the transfer of the macro variant of immunochemical analysis into the microchip format and the simultaneous quantitative analysis of a biological sample to determine the concentrations of several analytes, which replaces several individual immunochemical tests. The use of microarray technology for laboratory research allows us to miniaturize and simplify the analysis procedure, in particular, for the analysis of protein tumor markers and antibodies to onco-associated glycans in blood serum, in comparison with other in vitro tests. The development of microarrays for the diagnosis / screening of CRC is closely related to the development of biochip technology that allows for the multiplex study of the analyte for the presence of protein tumor markers and for the presence of antibodies to glycans.

The immunochemical principle underlies the multiplex analysis of anti-glycan antibodies on biochips, the cells of which contain immobilized glycans. Activity in this area has increased dramatically thanks to the development of a methodology that allows several hundred different glycans to be applied to a microchip (25x75 mm in size) (Blixt, Ola et al. 2004. "Printed Covalent Glycan Array for Ligand Profiling of Diverse Glycan Binding Proteins." Proceedings of the National Academy of Sciences of the United States of America 101 (49): 17033-38. Jacob, Francis et al. 2012. "Serum Antiglycan Antibody Detection of Nonmucinous Ovarian Cancers by Using a Printed Glycan Array." International Journal of Cancer 130 (1): 138-46. Pochechueva, Tatiana, Francis Jacob, Andre Fedier, and Viola Heinzelmann-Schwarz. 2012. "Tumor-Associated Glycans and Their Role in Gynecological Cancers: Accelerating Translational Research by Novel High-Throughput Approaches." Metabolites 2 (4): 913-39. Galanina, O.E., M. Mecklenburg, NE Nifantiev, G . V Pazynina, and Ν. V Bovin. 2003. "GlycoChip: Multiarray for the Study of Carbohydrate-Binding Proteins." Lab on a chip 3 (4): 260-65).

In the majority of works devoted to the analysis of antiglycan antibodies, the immunochemical determination of antibodies is carried out in the sera of healthy donors, microchips are two-dimensional, i.e. immobilized glycans are on the surface of the carrier (glass, plastic); wherein the glycans can be either adsorbed or covalently bonded to the substrate. Two basic methods are used to immobilize glycans: chemical immobilization on a solid surface and immobilization of glycan through a spacer (linker). Currently, there are several platforms that are used for printing glycan microchips.

The Functional Glycomics Cosortium (www. Functionalglycomic.org), for example, uses NHS-activated glass slides to print glycans containing an amino group. Other research groups use epoxy-activated slides (Pohl NL. Fluorous tags catching on microarrays. Angew Chem Int Ed Engl. 2008; 47: 3868-3870.), Fluoride slides (Ko KS, Jaipuri FA, Pohl NL. Fluorous-based carbohydrate microarrays. J Am Chem Soc. 2005; 127: 13162-13163; Feizi T, Chai W. Oligosaccharide microarrays to decipher the glyco code. Nat Rev Mol Cell Biol. 2004; 5: 582-588.), Slides coated with nitrocellulose (Feizi Τ , Fazio F, Chai W, et al. Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics. Curr Opin Struct Biol. 2003; 13: 637-645; Fukui S, Feizi T, Galustian C, et al. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat Biotechnol. 2002; 20: 1011-1017; Padler-Karavani V, Song X, Yu H, et al. Cross-comparison of protein recognition of sialic acid diversity on two nov el sialoglycan microarrays. J Biol Chem. 2012; 287: 22593-22608.). Moreover, for different conditions for determining AGA, different platforms for immobilizing glycans are required. Biochips printed on different platforms from the same glycan libraries often show different signals for glycan binding proteins, different specificity and sensitivity of the analysis (Liu Y, Childs RA, Palma AS, et al. Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses. Methods Mol Biol. 2012; 808: 117-136.), direct comparison of signals from two different platforms is often misleading. For printing glycans on nitrocellulose (Edwards HD, Nagappayya SK, Pohl NLB. Probing the limitations of the fluorous content for tag-mediated microarray formation. Chem Commun. 2012; 48: 510-512.) And fluoride platforms (Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, ME, Alvarez, R., Bryan, MC, Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, DJ, Skehel, JJ, van Die, I., Burton, DR, Wilson, IA, Cummings, R., Bovin, N., Wong, CH, and Paulson, JC Printed covalent glycan array for ligand profiling of diverse glycan binding proteins . Proc. Natl. Acad. Sci. USA. 2004; 101: 17033-17038.) A lipid component is required so that glycan can bind to GBPs. The advantages of glycochips on a nitrocellulose membrane include the three-dimensional arrangement of immobilized glycan, but this statement is debatable, because it is necessary to monitor the humidity of the chamber where the glycochip is stored. Drying of the microchip leads to false results of subsequent immunoassay of GBPs.

International patent application Vuskovic M., M. Huflejt (US 2014/0087957 A1) describes a method for diagnosing and monitoring dozens of oncological diseases by changing the level of anti-glycan antibodies nAbs to more than 500 glycan structures in various human biological fluids, and glycans with any spacer groups can be immobilized on a die, biochips or magnetic particles. Determination of the level of antibodies to glycans in blood serum is carried out by the method of solid-phase immunoassay. Cases of detecting CRC are not described in this patent application; the disadvantages also include the lack of a simultaneous quantitative determination of the content of protein tumor markers, levels of immunoglobulins G, A, M and antibodies to glycans in the patient's blood serum.

International patent Blixt O., Head S. (US 2007/0059769 A1) describes a biological microchip with immobilized glycans for the simultaneous analysis of nAbs antibodies to more than 1000 glycan structures and immunoassay methods in which the biochip is used. The biochip described in the patent cannot be used for the quantitative analysis of antibodies to glycans, while quantitative analysis of protein tumor markers, levels of immunoglobulins G, A, M, i.e. for the diagnosis of CRC.

The disadvantages of two-dimensional chips include denaturation of biological samples on the surface of the carrier and at the interface, leading to a loss in the functional activity of biological samples, insufficient concentration of active glycans in the cells of the microchip, and, as a result, low analysis sensitivity. Moreover, the disadvantages of the technology of two-dimensional glycochips include the impossibility of simultaneously immobilizing glycans and proteins on the same substrate.

Commercial biochip test systems for determining antiglycan antibodies are not on the market.

An alternative to two-dimensional chips for determining protein tumor markers and antibodies to glycans are hydrogel-based microchips.

The technology is being developed at the Institute of Molecular Biology named after V.A. Engelhardt RAS (IMB RAS) and is based on the simultaneous formation of hydrogel cells with the immobilization of molecular probes. In this case, the molecular probes are uniformly distributed throughout the cell volume. Due to the developed surface of the hydrogel, molecular probes are immobilized with a sufficient immobilization density, however, at a considerable distance from each other to level steric effects. The gel structure prevents contact of the compounds with the hydrophobic substrate, and provides a native aqueous environment of biological molecules. Obviously, this method of immobilization is great for working with molecular probes of protein and glycan nature and in its characteristics compares favorably with the classical methods of planar immobilization (Rubina, a Yu et al. Hydrogel-Based Protein Microchips: Manufacturing, Properties, and Applications. BioTechniques . 2003; 34 (5): 1008-1014; Dyukova VI, Dementieva EI, Zubtsov DA, Galanina OE, Bovin NV, Rubina AY Hydrogel glycan microarrays. Anal. Biochem. 2005; 347 (1): 94-105).

After completion of the polymerization procedure, the molecular probes with sufficient efficiency are covalently fixed in the gel network and distributed evenly on it (Zubtsova Zh.I., Tsybulskaya MV, Savvateeva EN, Butvilovskaya VI, etc. Analysis of nine serological tumor markers on a hydrogel biochip. Bioorganic Chemistry. 2013; 39 (6): 693-704).

The disadvantages of the methods for diagnosing colorectal cancer, as well as the disadvantages of microarrays for the analysis of protein oncomarkers and antiglycan antibodies, known from the prior art, are overcome by the present invention, which provides a method for the diagnosis of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins G, A and M in human blood on a biological microchip.

Disclosure of invention

(Disclosure of Invention)

The first aspect of the invention is a method for the diagnosis of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins G, A and M in human blood on a biological microchip containing hydrogel elements with immobilized antibodies to protein oncomarkers, hydrogel elements with immobilized glycans hydrogel elements with immobilized antibodies against human immunoglobulins of classes G, A, M, where the change in the level of analyzed m rkerov compared to the level of the markers in the serum of healthy donors suggests colorectal cancer.

In one of the embodiments, a method for the diagnosis of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins G, A and M in human blood on a biological microchip, is carried out in the format of a sandwich immunoassay and includes several stages:

a) incubation of the biological microchip in the reaction medium, including a sample containing compounds to be analyzed, for the formation of immune complexes with ligands immobilized on the microchip;

b) detection of the resulting complex;

c) quantification of the analyte.

In the case of sandwich immunoassay of protein tumor markers, the biological microchip contains immobilized antibodies, each of which specifically interacts with one or another analyte being determined, the reaction medium in stage a) contains the analyte, and at this stage a specific complex of antigen - immobilized antibody is formed. If necessary, this stage is carried out under stirring conditions, which greatly accelerates the immunoassay.

After complex formation in step b), the microchip is developed with labeled antibodies specific for another epitope of the analyte. The higher the concentration of the analyte in the sample, the higher the signal recorded from the corresponding gel cells of the microchip.

In the case of a sandwich immunoassay of antibodies to glycans, the biological microchip contains immobilized glycans against the analyte, the reaction medium in step a) contains the analyte, and a specific antibody-immobilized glycan complex forms. After complex formation, the microchip is developed with labeled antispecies antibodies specific for another epitope of the analyzed antibody. The higher the concentration of the analyte in the sample, the higher the signal recorded from the corresponding gel cells of the microchip.

For the quantitative determination of the analyzed compounds in stage c), stages a) -b) are carried out with standard (reference) samples containing known concentrations of the analyzed compound and the calibration dependence of the recorded signal on the concentration of the analyzed compound is constructed (calibration curve), which determines the content of the analyzed compound in sample.

The detection of immunoassay results is carried out fluorimetrically. Antibodies and other compounds used for detection may contain fluorescent labels, biotin, or conjugates to proteins or other compounds capable of participating in reactions leading to light emission (chemiluminescence or bioluminescence).

The sensitivity of the proposed multiple parallel immunoassay of compounds on a biological microchip is not inferior to the sensitivity of the standard variant of immunoassay, for example, in the format of a microtiter tablet (Table 1)

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 G, A and M in human blood on a biological microchip 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 G, A, M, g de change in the concentration of the analyzed markers in comparison with the control sample obtained from a healthy person indicates colorectal cancer.

A second aspect of the invention is a biological microchip for the diagnosis of colorectal cancer. The biological microchip is an array of three-dimensional hydrogel elements located on a substrate of glass, metal plastic. Three-dimensional hydrogel elements of the microchip are covalently attached to the surface of the substrate and are separated from each other by the hydrophobic surface of the substrate, and an individual compound is immobilized in each individual cell. Each gel element contains an immobilized ligand of a protein or non-protein nature, which, when the microchip is incubated in a reaction medium including a sample containing compounds to be analyzed, forms a specific antigen-antibody immune complex. At this stage, the analyzed compounds are separated from the mixture by their ability to specifically bind to immobilized ligands, which allows simultaneous, parallel analysis of several objects on the same microchip.

Three-dimensional gel elements have a significantly higher capacity compared to elements of two-dimensional microarrays, which increases the sensitivity of the analysis. The hydrophilic environment of the ligands immobilized in the hydrogel structure stabilizes their structure, which allows them to fully maintain their biological activity.

Ligands immobilized in the hydrogel cells of a biological microchip are selected from the group consisting of antibodies of any type and antigens of various nature.

The following are preferably proposed as test subjects for the quantitative immunological analysis of colorectal cancer according to the invention:

- tumor associated antigens (CEA, CA 19-9, CA 125, SCCA1 (SPB 3), SCCA2 (SPB 4), PSA total., PSA free., hCG, AFP, CA 242), which are markers of cancer;

- human immunoglobulins of classes G, A, M;

- antibodies to various glycans (SiaT _n , TF, T _n , Le ^C , Le ^Y , SiaLe ^A , Manβ1-4GlcNAc, Galal-4Galβ1-4GlcNAcβ).

For immunoassay of tumor-associated antigens - markers of colorectal cancer, antibodies to the indicated tumor-associated antigens and / or tumor-associated antigens themselves are used as immobilized ligands. For immunological determination of human immunoglobulins of classes G, A, M, for example, when determining the immune status of an organism, antibodies against immunoglobulins G, A, M, human and / or immunoglobulins G, A, M, human are used as immobilized ligands of a biological microchip. For immunoassay of antibodies to various glycans, glycans are immobilized ligands of a biological microchip.

A biological microchip allows simultaneous and parallel immunochemical reactions, such as the simultaneous interaction of an immobilized antibody with a protein tumor marker present in blood serum, the interaction of immobilized glycan with an antibody present in the same blood serum, the simultaneous interaction of an immobilized antibody to a human immunoglobulin, for example, class G with an immunoglobulin of this class located in the same serum sample.

In another embodiment, sequential immunochemical reactions are provided, for example, an immobilized antibody with a protein oncomarker present in the blood serum on one biochip cluster and the interaction of the immobilized glycan with an antibody present in the blood serum, simultaneously with the interaction of the immobilized antibody to human immunoglobulin, for example, class G with immunoglobulin in the same serum sample.

Further, the invention will be disclosed in more detail with reference to the figures and examples, which are given solely for the purpose of illustrating and explaining the essence of the claimed invention, but which are not intended to limit the scope of claims.

The implementation of the invention

The present invention provides a method for the diagnosis of colorectal cancer based on the simultaneous quantitative determination of protein markers of oncology, antibodies to glycans, class G, A and M immunoglobulins in human blood on a biological microchip 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 G, A, M, where the level change is analyzed markers compared with the level of markers in the blood serum of healthy donors indicates colorectal cancer.

A method for the diagnosis / screening of colorectal cancer, based on the simultaneous quantitative determination of the concentration of tumor markers of protein nature, levels of antibodies to glycans, levels of immunoglobulins G, A, M in human blood serum on a biological microchip, consists in conducting immunoassay of human blood serum on a biological microchip, and provides :

a) the interaction of a sample of human blood serum with biochip cells under conditions providing for the formation of specific immune complexes — compounds immobilized in hydrogel cells of the microchip with compounds contained in the analyzed sample;

b) detection of the formed immune complexes;

c) determination of the levels of protein tumor markers, antibodies to glycans and human immunoglobulins of class G, A, M.

d) the diagnosis of colorectal cancer by comparing the concentration of protein tumor markers, antibodies to glycans, immunoglobulins G, A, M in the analyzed sample with the corresponding levels of markers in the blood serum of healthy donors.

As the reaction medium providing the formation of specific immune complexes, choose solutions that are usually used in various types of immunoassays, for example, phosphate buffer, Tris-HCl, etc. To reduce nonspecific interactions, polyvinyl alcohol, sucrose, and bovine serum albumin are added to the reaction medium , milk powder proteins and other compounds well known to those skilled in the art.

The general procedure for immunoassay using biological microarrays is described in example 1.

The detection of the formed immune complexes is preferably carried out by the fluorimetric method. Antibody conjugates of antibodies against protein oncomarkers, conjugates of antispecies antibodies and conjugates of antibodies against human immunoglobulins of classes G, A, M with fluorescent dyes (Example 2) or conjugates of antibodies against protein oncomarkers, conjugates of antivirus antibodies and conjugates of antibodies against human immunoglobulins are used as developing antibodies. G, A, M with biotin and fluorescently-labeled avidin (streptavidin) (Examples 3, 5). As fluorescent dyes use, for example, cyanine 3 (Cy3), cyanine 5 (Cy5), fluorescein, Texas Red, but not limited to. Fluorescence images of microarrays after analysis are shown, for example, in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2B. The registration of fluorescence signals from the cells of the microchip is carried out using fluorescence microscopes (Example 1, Example 4) or scanning microscopes of various types (Example 2), allowing to record the signal from the used fluorescent label.

As samples for analysis, diluted or undiluted human blood serum, diluted or undiluted human blood plasma are used.

When determining the levels of various immunoglobulins by the method of the present invention, steps a) and b) are carried out with known concentrations of the analyzed compounds and the calibration dependencies “signal from the corresponding elements of the microchip — concentration of the analyzed compound” are constructed, which determine the levels of the analyzed compounds (Fig. 3A, Fig. 3B )

When determining the concentration of protein tumor markers by the method of the present invention, steps a) and b) are carried out with known concentrations of the analyzed compounds and the calibration dependencies “signal from the corresponding elements of the microchip — concentration of the analyzed compound” are constructed, which determine the levels of the analyzed compounds (Fig. 4A, Fig. 4B )

The following describes a method for the diagnosis of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins G, A and M using biological microarrays, which are preferred embodiments of the present invention.

A biological microchip containing elements with immobilized antibodies against tumor markers of protein nature, glycans, antibodies against human immunoglobulins of classes G, A and M.

Stage a) When the microchip interacts with a sample of human blood serum in elements with immobilized glycans, the formation of immune complexes with immunoglobulins G, A, M specific to these glycans occurs simultaneously. At the same time, when a microchip interacts with a sample of human blood serum in elements with immobilized antibodies against protein tumor markers, the formation of immune complexes with tumor markers simultaneously occurs from a human blood sample specific to these antibodies. At the same time, when a microchip interacts with a sample of human blood serum in elements with immobilized antibodies against human immunoglobulins of classes G, A, M, the formation of immune complexes with immunoglobulins G, A, M from a blood serum sample specific to these antibodies occurs.

Stage b) When processing (developing) conjugates of antibodies with a fluorescent dye against each of the tumor markers, signals from microchip elements containing immobilized antibodies against the determined tumor marker are recorded and these fluorescence signals are used further in step c) to determine the concentration of the tumor marker.

When processing (developing) conjugates of labeled antibodies against immunoglobulins G, A, M, the signal from this microchip element (with immobilized antibodies to immunoglobulins G, A, M) determines the level of immunoglobulins G, A, M.

When processing (developing) conjugates of labeled antibodies against immunoglobulins G, A, M by the signal from this element of the microchip (with immobilized glycans), the level of antibodies to glycans is determined.

To simultaneously determine the levels of tumor markers, antibodies to glycans and immunoglobulins G, A, M, the developing solution may include a mixture of antibodies against tumor markers, immunoglobulins G, A, M, which are conjugates with fluorescent dyes that emit light in different spectral regions, for example, conjugates with Su3 and Su5. The simultaneous determination of tumor markers, antibodies to glycans, immunoglobulins G, A, M is also possible by sequentially processing the microchip with solutions containing first labeled antibodies and then other labeled antibodies, and in this embodiment, the developing antibodies may contain different or the same fluorescent labels .

Stage c) To build the calibration dependences, the biological microchip is incubated with solutions containing known concentrations of tumor markers, and the dependences “signal from the corresponding elements of the microchip — concentration of the analyte” are built, which determine the levels of the analyzed tumor markers using fluorescence signals obtained in Stage b).

To build the calibration dependences, the biological microchip is incubated with solutions containing known concentrations of immunoglobulins G, A, M and the dependences “signal from the corresponding elements of the microchip — concentration of the analyte” are constructed, according to which the concentration is determined using fluorescence signals obtained in Stage b) analyzed immunoglobulins G, A, M and levels of antibodies to glycans.

Stage g) Diagnosis of colorectal cancer (Example 3) is carried out by ROC analysis, by comparing the concentration of protein tumor markers, antibodies to glycans, immunoglobulins G, A, M (Fig. 6A, 6B) in the analyzed sample with the corresponding levels of markers in healthy blood serum donors (Fig. 5A, 5B).

The results of determining the diagnostic effectiveness of a method for detecting colorectal cancer based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins G, A and M in human blood on a biological microchip containing hydrogel elements with immobilized antibodies to protein oncomarkers, hydrogel elements with immobilized glycans , hydrogel elements with immobilized antibodies against human immunoglobulins of classes G, A, M are presented in Table e 2.

The present invention provides a biological microchip for the diagnosis of colorectal cancer. A microchip is an array of three-dimensional hydrogel elements in the form of microdroplets of a given volume located on a substrate of glass, plastic or metal. The number and volume of array elements is determined by the number of analyzed protein tumor markers, glycans, immunoglobulins and the equipment used to obtain microchips (for example, the size of a robot pin, applying droplets of a solution to a substrate) and the method of recording signals (fluorescence microscope, biochip analyzer, scanner, etc. ) Biological microchips for immunological analysis of compounds are made by polymerization immobilization using a patented technology (A.D. Mirzabekov, A.Yu. Rubin, S.V. Pankov, Method for polymerization immobilization of biological macromolecules and composition for its implementation, RF Patent No. 2216547 (B. I.P.M. No. 32, 11/20/2003). International Application PCT / RU 01/004204, WO 03/033539), which allows to obtain microchips containing immobilized proteins, glycans and other compounds. The binding of the ligand to the hydrogel matrix is possible both directly during its immobilization in the process of gel formation, and indirectly. As examples of indirect binding of a ligand to a hydrogel matrix, immobilization through the formation of specific complexes of the avidin type (streptavidin) - biotin can serve as, for example, the formation of a bond between avidin immobilized on a hydrogel matrix and a biotinylated antibody, or immobilization through the formation of specific metal-chelator complexes, for example , the formation of a bond between nitrilotriacetic acid immobilized on a hydrogel matrix with coordination bonds a nickel volume and recombinant protein containing a polyhistidine fragment, etc.

Ligands immobilized in gel cells may include compounds of protein and non-protein nature, such as monoclonal antibodies, polyclonal antibodies, recombinant antibodies, various types of mini antibodies, including Fab fragments and single chain antibodies (scFv and others), immunoglobulins of any type (IgG, IgM, IgA, IgD, IgE), antigens of various nature (proteins, glycoproteins, polysaccharides, etc.), low molecular weight substances. The type of ligands used in the analysis is determined by a number of factors, for example, the affinity of antibodies to a given antigen, stability, the possibility of immobilization of antibodies and the introduction of a label (fluorescent label, enzyme label by obtaining conjugates, etc.), as well as the method of analysis (direct , competitive, sandwich analysis, etc.).

A biological microchip allows sequential reactions of various types. For example, the first reaction can be the interaction of an antigen with an antibody to form a double antigen-antibody complex or an antibody-antigen-antibody triple complex for protein antigens (immunological reaction). Then, if the antibody or antigen contains an enzyme label, an enzymatic reaction is carried out, for example, the oxidation of luminol with hydrogen peroxide in the presence of amplifiers, catalyzed by horseradish peroxidase, which results in the emission of light (chemiluminescence). If the antibody or antigen is a conjugate with biotin, after the immunological reaction, it is possible to bind to the conjugate of streptavidin with a fluorescent dye directly on the microchip.

As the analyzed objects for a method for the diagnosis of colorectal cancer based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, immunoglobulins of classes G, A and M in human blood on a biological microchip according to the invention, it is preferably proposed:

tumor-associated antigens that are markers of cancer, including colorectal cancer, for example:

- alpha-fetoprotein (AFP) - a marker of liver cancer; prostate-specific antigen (PSA), general and free, is a marker of prostate cancer; cancer embryonic antigen (CEA) - a marker of gastrointestinal cancer; cancer antigen 125 (CA 125) - a marker of cancer of the ovary, uterine body; cancer antigen 19-9 (CA 19-9) - a marker for pancreatic cancer; SCCA 1 squamous cell carcinoma antigen (SPB 3) - a marker of squamous cervical cancer, head and neck cancer; SCCA 2 squamous cell carcinoma antigen (SPB 4) - a marker for squamous cell carcinoma of the cervix uteri, head and neck cancer; human chorionic gonadotropin (hCG) is a marker of cancer; CA 242 cancer antigen, a marker of bowel cancer;

- human immunoglobulins of classes G, A, M;

- antibodies against glycans (SiaT _n , TF, T _n , Le ^C , Le ^Y , SiaLe ^A , Manβ1-4GlcNAc);

The biochip for the diagnosis of colorectal cancer can contain either one cluster containing immobilized antibodies to protein tumor markers, glycans, antibodies to human immunoglobulins of classes G, A, M for simultaneous immunological reactions (Example 3), and two. The presence of two clusters allows for independent parallel immunological reactions on a single biochip (Example 4, Example 5).

Immunoassay of tumor-associated protein tumor markers is carried out using biological microarrays (Fig. 1A, 1B) containing immobilized antibodies against these tumor-associated antigens. In the immunological determination of human immunoglobulins of classes G, A, M, antibodies against human immunoglobulins G, A, M are used as immobilized ligands of the biological microchip (Fig. 1A, 1B). For immunoassay of antibodies against various glycans, the immobilized ligands of the biological microchip are glycans (Fig. 1A, 1B).

The general procedure for the immunoassay using biological microarrays is described in Example 1. For the simultaneous immunoassay of tumor-associated protein tumor markers, antibodies to these tumor-associated protein tumor markers were used as immobilized ligands of the biological microchip (examples 4).

Example 5 describes a biological microchip for simultaneous immunoassay of levels of antibodies to glycans and human immunoglobulins of classes G, A, M in a blood serum sample using glycans and antibodies against immunoglobulins G, A, M as immobilized ligands. The results of testing immunoglobulins G, A, M in the studied groups of patients are shown in FIG. 6A and FIG. 6B. The results of determining the diagnostic efficacy of individual antibodies to glycans are shown in FIG. 7 and Table 3.

The following examples are not intended to limit claims, but solely to illustrate individual embodiments of the present invention.

Example 1. Immunoassay of molecular markers of colorectal cancer using hydrogel microarrays

For immunoassay, microarrays were used, the structure of which is shown in Fig. 1A, Fig. 1B, containing elements with immobilized antibodies against protein tumor markers, antibodies against human immunoglobulins of classes G, A, M, glycans. To reduce nonspecific interactions in all analysis variants, microarrays were preincubated in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1% polyvinyl alcohol (PVA-50), or in the same buffer containing 3% bovine serum albumin (BSA) and 4% sucrose (“blocking buffer”), 40 min. at room temperature. Before analysis, the antigen (sample) solutions were, if necessary, diluted with 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.15% PVA-50 and 0.15% polyvinylpyrrolidone 360.

For a sandwich immunoassay, 100 μl of antigen solution was applied to biochips. In the case of analysis of protein tumor markers, undiluted blood serum was used, incubation was carried out at 37 ° C for 20 hours. To determine antibodies to glycans, blood serum was diluted with 1/5 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl , 0.15% PVA-50 and 0.15% polyvinylpyrrolidone 360, incubation was carried out for 2 hours at 37 ° C. After a short (20 min.) Washing in 0.01 M phosphate buffer, pH 7.2, 0.15 M NaCl, 0.1% Tween-20, microarrays were shown by conjugates of developing antibodies with biotin (or fluorescently-labeled antibodies) specific for another epitope antigen (60 minutes at 37 ° C). After washing (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 20 min, room temperature), they were incubated with a streptavidin solution with a fluorescent dye (15 min., 37 ° C, 0.05 mg / ml), the intensity of the fluorescent signals was measured. After washing (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 15-30 min, room temperature), registration of fluorescent signals was started. Antibodies, streptavidin, and protein-derived antigens labeled with fluorescent labels (e.g., Su 5) were prepared according to known methods (e.g., described in G.T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996).

Fluorescence signals were measured using a fluorescence microscope developed in the biochip laboratory of the IMB RAS (V. Barsky, A. Perov, S. Tokalov, A. Chudinov, Ε. Kreindlin, A. Sharonov, Ε. Kotova, A. Mirzabekov, Fluorescence data analysis on gel-based biochips, J. Biomol. Screening, 2002, 7, 247-257). A microscope equipped with a CCD camera and computer programs for image visualization and processing of obtained data (IMB RAS). The microscope allows you to simultaneously analyze data from all elements of the microchip and to obtain two-dimensional and three-dimensional images of the fluorescent signal from the gel elements. It is also possible to use fluorescent scanners (for example, a Genepix 4200 A scanner (Molecular Devices, USA)). The values of fluorescence signals were calculated using the software Genepix Pro 6.0 (Molecular Devices, USA). To interpret the results, the F635 median fluorescence intensity obtained in the program Genepix Pro 6.0 was used.

For all analysis options, a plot was made of the dependence of the fluorescence intensity on the concentration of antigen in solution. The smallest detectable antigen concentration was calculated as the concentration corresponding to the signal intensity, 3 times the spread of the background signal.

Example 2. Quantitative immunoassay of antibodies to glycans using hydrogel microarrays

To simultaneously determine the levels of antibodies to glycans and total class G, A, M immunoglobulins in a human serum sample, microchips were used, the structure of which is shown in Fig. 1A, Fig. 1B. Before analysis, to reduce nonspecific interactions, the microarrays were incubated in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1% polyvinyl alcohol (“blocking solution”), 40 min. at room temperature.

Microchips were incubated with the test sample (blood serum) or with calibration samples containing known concentrations of human immunoglobulins of classes G, A, M, for 2 hours at 37 ° C. After washing in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1% Tween 20 (20 min, room temperature), the microchips were treated with a solution of mouse antibodies against human immunoglobulins G, A, M with fluorescent dye Cy5 (Pierce, USA) (1 h, 37 ° C) .

After incubation and washing were completed, fluorescent signals were recorded in (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 15-30 min, room temperature) from non-specifically bound components. A Genepix 4200 A microarray scanner (Molecular Devices, USA) with software was used to measure fluorescent signals. The fluorescence image of the microchip after interaction with the blood serum of a patient with CRC is presented in Figure 2 B.

To increase the reliability of the data obtained, all immobilized glycans and antibodies in the microchip are presented in four identical gel elements, and the fluorescence intensity was calculated as the median signal from four identical gel elements.

A calibration dependence was built: “the intensity of the fluorescent signal of the elements of the microchip containing antibodies against immunoglobulins G, A, M is the concentration of immunoglobulin G in the calibration sample (Fig. 3 B), which determined the levels of glycan-specific and total immunoglobulins G, A, M in the studied sample.

The smallest detectable antigen concentration was calculated as the concentration corresponding to the signal intensity, 3 times the spread of the background signal.

Example 3. Diagnosis of colorectal cancer based on the simultaneous determination of the concentration of protein oncomarkers, antibodies to glycans, immunoglobulins of classes G, A, M in a blood serum sample on a biological microchip.

3.1. Statement of analysis

To simultaneously determine the concentration of protein tumor markers, antibodies to glycans, immunoglobulins of classes G, A, M in a human serum sample, microarrays were used, the structure of which is shown in Fig. 1A. Before analysis, to reduce nonspecific interactions, the microarrays were incubated in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1% polyvinyl alcohol (“blocking solution”), 40 min. at room temperature.

Microchips were incubated with the test sample (blood serum) or with calibration samples containing known concentrations of protein tumor markers, human G class immunoglobulins for 20 hours at 37 ° C. After washing in 0.01 M phosphate buffer, pH 7.2, containing 0 , 15 M NaCl, 1% Tween 20 (20 min, room temperature) the microchips were treated with a mixture of conjugates of antibodies against all tumor markers with biotin, a conjugate of antibodies against human immunoglobulins of classes G, A, M with biotin (1 h, 37 ° C) . After washing in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1% Tween 20 (20 min at room temperature), the microchips were treated with a solution of streptavidin with fluorescent dye Cy5 (Pierce, USA) (15 min. , 37 ° C). After incubation and washing were completed, fluorescent signals were recorded in (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 15-30 min, room temperature) from non-specifically bound components.

3.2. Data processing

Fluorescence signals were measured using a fluorescence microscope equipped with a CCD camera and software for image visualization and data processing, developed at the IMB RAS (V. Barsky, A. Perov, S. Tokalov, A. Chudinov, Ε. Kreindlin, Α. Sharonov, Kotova, A. Mirzabekov, Fluorescence data analysis on gel-based biochips, J. Biomol. Screening, 2002, 7, 247-257) or a GenePix 4200A microchip scanner (USA). The fluorescence image of the microchip after analysis of the blood serum of a patient with CRC is shown in FIG. 2A, healthy donor - in FIG. 2C.

To increase the reliability of the data obtained, all immobilized probes in the microchip are presented in four identical gel elements, and the fluorescence intensity was calculated as the median signal from four identical gel elements.

Cells with immobilized immunoglobulin G act as a positive control of the health of the diagnostic tool.

The calibration dependence “intensity of the fluorescent signal of microarray elements containing antibodies against the protein tumor marker — the concentration of the protein tumor marker in the calibration sample (Fig. 4) was constructed, which was used to determine the concentration of the protein tumor marker in the test sample. The determination results are presented in Table 4.

3.3. Analysis of the diagnostic effectiveness of signatures for the diagnosis of colorectal cancer using statistical data processing and ROC analysis.

Statistical processing of analysis data (concentration of tumor markers, fluorescence signals from immobilized glycans and antibodies to human immunoglobulins of classes G, A, M, obtained after immunoassay on chips of the design of Fig. 1A, 1B was carried out using MedCalc Version 15.4 programs. A comparative analysis of the diagnostic effectiveness of different factors for multiplex analysis on chips was performed using multi-parameter log-regression analysis (Hosmer DW, Lemeshow S. Applied logistic regression. Wiley Series in probability and statistics, 3rd ed. New York: Wiley-Interscienc e, 2013.528 p) and ROC curves (Receive Operating Characteristic curve analysis) (Bradley AP. The use of the area under the ROC curve in the evaluation of machine learning algorithms. Pattern Recognition 1997; 30: 1145-59; Vuskovic MI, Xu H, Bovin NV, et al. Processing and analysis of serum antibody binding signals from Printed Glycan Arrays for diagnostic and prognostic applications. Int J Bioinform Res Appl 2011; 7: 402-26). The larger the area under the ROC curve, the higher the diagnostic effectiveness of this factor. The method for determining the diagnosis is considered excellent if the area under the ROC curve AUC = 0.9-1.0; very good at AUC = 0.8-0.9; good at AUC = 0.7-0.8; satisfactory at AUC = 0.6-0.7; and unsatisfactory at AUC = 0.5-0.6. Random prediction corresponds to AUC = 0.5. The number of correctly predicted positive diagnoses (TP,%) and the share of correctly classified diagnoses (accuracy,%) were also used to assess the diagnostic effectiveness of the developed method for determining colorectal cancer (CRC).

The following models were considered: the Oncology model (CRC = 1, IBD = 0, HD = 0); "Disease" (CRC = 1, IBD = 1, HD = 0); and "CRC / IBD" (CRC = 1, IBD = 0). The Oncology Model allows you to highlight cases of CRC; "Disease" allows you to distinguish sick patients from healthy ones; the CRC / IBD model distinguishes cases of CRC from inflammatory bowel disease.

The fractions of truly positive and false positive examples were calculated using formulas (1) and (2).

Percentage of truly positive examples of TPR (True Positives Rate):

False positive examples FPR (False Positives Rate):

where TP (True Positives) - correctly classified positive diagnoses;

TN (True Negatives) - correctly classified negative diagnoses;

FN (False Negatives) - positive examples falsely classified as

negative (the disease is not erroneously detected);

FP (False Positives) - negative examples that are falsely classified as positive (false detection of the disease).

The sensitivity and specificity of the binary ROC analysis model was calculated using formulas (3) and (4).

The sensitivity of the model (Sensitivity) is the proportion of truly positive cases:

Specificity of the model (Specificity) - the proportion of truly negative cases that were correctly identified by the model:

The sensitivity and specificity of the binary model for determining the disease tends to 100% (the area under the ROC curve tends to 1) if the number of false-negative or false-positive diagnoses of the disease detected by the model tends to zero.

In the MedCalc Version 11.4.2.0 program, AUC was calculated for the signatures (Fig. 7, Fig. 6B., Fig. 5).

The results of the diagnostic efficacy of signatures for the detection of colorectal cancer in the blood serum of patients from three cohorts are shown in Table 2.

Example 4. Simultaneous analysis of several protein tumor markers on one microchip

Microchips with immobilized antibodies were made to all studied markers CEA, CA 19-9, CA 125, SPB 3, SPB 4, PSA total., PSA St., hCG, AFP, CA 242, (Fig. 1A, 1B). For analysis of protein tumor markers, undiluted blood serum was used, incubation was performed at 37 ° C for 20 hours. After short-term (20 min.) Washing in 0.01 M phosphate buffer, pH 7.2, 0.15 M NaCl, 0.1% Tween -20, the microarrays showed conjugates of developing antibodies with biotin (or fluorescently-labeled antibodies) specific for another epitope of the antigen (60 min, at 37 ° C). After washing (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 20 min, room temperature), they were incubated with a streptavidin solution with a fluorescent dye (15 min., 37 ° C, 0.05 mg / ml). After washing (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 15-30 min, room temperature), registration of fluorescent signals was started.

As can be seen from FIG. 2B, in the case of analysis of a sample of a patient with colorectal cancer, bright signals were observed in gel cells containing specific antibodies (CEA, CA 19-9, CA242). The detection limits of tumor markers in the case of their parallel analysis were the same as in the determination of each marker separately (Table 5).

Example 5. Simultaneous determination of levels of antibodies to glycans and human immunoglobulins of classes G, A, M in a blood serum sample

Used microchips, the structure of which is shown in FIG. 1B. Before analysis, the microchips were treated with a blocking solution, as described in Example 1.

Microchips were incubated with a test serum sample or with calibration samples containing known concentrations of immunoglobulin G for 2 hours at 37 ° C; after washing (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 20 min, room temperature), the microarrays were incubated with a conjugate of biotinylated polyclonal antibodies against immunoglobulins G, A, M (1 h, 37 ° C). Incubated with a streptavidin solution with a fluorescent dye (15 min., 37 ° C, 0.05 mg / ml). After washing (0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1% Tween-20, 15-30 min, room temperature), registration of fluorescent signals was started, as described in Example 1. The fluorescence image of the microchip after analysis of the blood serum of a healthy donor is shown in FIG. 2D.

A calibration dependence was constructed, the fluorescence signal intensity of the microchip elements containing antibodies against immunoglobulins G, A, M - the concentration of immunoglobulin G in the calibration sample (Fig. 3B), which determined the levels of specific and total immunoglobulins G in the test blood serum.

Brief Description of the Figures

Brief Description of Drawings)

FIG. 1A and FIG. 1B show the appearance of a hydrogel biological microchip for the simultaneous quantitative determination of protein markers of oncology, antibodies to glycans, immunoglobulins G, A and M in human blood on a biological microchip. Each immobilized compound is represented in four identical elements.

FIG. 1A shows a view of a single cluster hydrogel biological microchip.

FIG. 1B shows a view of a two-cluster hydrogel biological microchip.

Microchip scheme for the diagnosis of colorectal cancer. The array of hydrogel elements contains elements with immobilized glycans, antibodies to protein tumor markers and elements with immobilized antibodies against human immunoglobulins of classes G, A, M.

Molecular Probes:

Antibodies to protein tumor markers: 1) Elements with immobilized antibodies against AFP, 2) Elements with immobilized antibodies against hCG, 3) Elements with immobilized antibodies against CEA, 4) Elements with immobilized antibodies against CA 19-9, 5) Elements with immobilized antibodies against CA 125, 6) elements with immobilized antibodies against PSA, 7) elements with immobilized antibodies against SPB3, 8) elements with immobilized antibodies against SPB4, 9) elements with immobilized antibodies against CA 242;

Glycans: 10) T _n ; 11) SiaT _n ; 12) SiaLe ^A ; 13) Le ^C ; 14) Le ^Y ; 15) TF; 16) Manβ1-4GlcNAc;

17) elements with immobilized antibodies against immunoglobulins G, A, M,

18) elements with immobilized human immunoglobulin class G,

19) elements that do not contain biomaterial.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D represent fluorescence images of biological microarrays after analysis of the blood sera of patients diagnosed with CRC and a healthy donor. Serum levels of protein tumor markers, antibodies to glycans, immunoglobulins G, A, M were determined. Microarrays were incubated with a blood serum sample and developed with a mixture of biotinylated antibodies against protein tumor markers, biotinylated antibodies against immunoglobulins G, A, M followed by development of streptavidin labeled with fluorescent dye Su5.

FIG. 2A. An image of a single cluster microchip whose structure is shown in FIG. 1 A, after analysis of the serum of a patient diagnosed with colorectal cancer.

FIG. 2B. An image of a single cluster microchip whose structure is shown in FIG. 1 B, after analysis of the serum of a patient diagnosed with colorectal cancer.

FIG. 2C. An image of a two-cluster microchip, the structure of which is shown in FIG. 1A, after analysis of the serum of a healthy patient.

FIG. 2D. An image of a two-cluster microchip, the structure of which is shown in FIG. 1 V, after serum analysis of a healthy patient.

FIG. 3A, FIG. 3B are graphs of the dependence of the intensity of the fluorescent signal obtained from microarray elements containing immobilized antibodies against immunoglobulins of classes G, A, M on the concentration of immunoglobulins G, A, M in solution. Each point of the calibration dependence is the average value of measurements on ten microchips. The vertical segments show the standard deviations. Gauge dependencies were constructed using piecewise linear interpolation.

FIG. 3A is a graph of the dependence of the fluorescence signal intensity of microarray elements containing immobilized antibodies against class G, A, M immunoglobulins on the concentration of immunoglobulins G, A, M in dilutions of the patient's blood serum with known immunoglobulin G.

FIG. 3B is a graph of the intensity of the fluorescent signal of microarray elements containing immobilized antibodies against immunoglobulins of classes G, A, M on the concentration of immunoglobulin G in solution.

FIG. 4A, FIG. 4B are graphs of the dependence of the intensity of the fluorescent signal of the elements of the microchip containing immobilized antibodies against protein tumor markers CEA and CA 19-9 on the concentration of CEA and CA 19-9 in solution. Each point of the calibration dependence is the average value of measurements on ten microchips. The vertical segments show the standard deviations. Gauge dependencies were constructed using piecewise linear interpolation.

FIG. 4A is a graph of the intensity of the fluorescent signal of microarray elements containing immobilized antibodies against the CEA protein tumor marker on the concentration of CEA in solution.

FIG. 4B is a graph of the intensity of the fluorescent signal of microarray elements containing immobilized antibodies against the protein oncomarker CA 19-9 on the concentration of CA 19-9 in solution.

FIG. 5A and FIG. 5B. demonstrate the results of a statistical analysis (ROC analysis) of the diagnostic efficiency of detecting CRC while determining molecular signatures in blood serum. Signature consisting of 4 antibodies to glycans: Tn, TF, SiaLeA and Manβ1-4GlcNAc; The signature, consisting of 6 tumor markers: CEA, CA 19-9, AFP, hCG, CA 125, CA 15-3.

FIG. 5A. demonstrate the results of a statistical analysis (ROC analysis) of the diagnostic efficacy of detecting CRC with the simultaneous determination of six tumor markers in blood serum compared with the diagnostic effectiveness of detecting CRC with the simultaneous determination of CEA and C19-9 concentrations in the blood serum of patients diagnosed with CRC (33 pcs.) , inflammatory diseases of the intestine (27 pcs.) and healthy donors (69 pcs.) (the Oncology model was used to detect CRC (CRC = 1, IBD = 0, HD = 0)). Construction of ROC-curves, statistical processing of analysis data was carried out using the MedCalc Version 11.4.2.0 software.

FIG. 5B. Demonstrates the dependence of the diagnostic efficiency of detecting CRC (the area under the ROC-curve (AUC)) on the composition of the diagnostic signatures. Signature consisting of 4 antibodies to glycans: Tn, TF, SiaLeA and Manβ1-4GlcNAc; The signature, consisting of 6 tumor markers: CEA, CA 19-9, AFP, hCG, CA 125, CA 15-3.

FIG. 6A and FIG. 6B show the results of measuring the content of immunoglobulins G, A, M in the studied groups of patients diagnosed with colorectal cancer (CRC), healthy donors (HD), with a diagnosis of inflammatory bowel disease (IBD).

FIG. 6A presents a Box-and-wiskers chart (“mustache box”) of the results of the determination of the levels of immunoglobulins G, A, M in the blood serum of patients diagnosed with CRC, inflammatory bowel disease (IBD) and healthy donors (HD). The distribution of fluorescent signals obtained on microarrays from cells containing immobilized antibodies to human immunoglobulins (IgG, IgA, IgM) for three groups of patients is shown. The boundaries of the box are the 25th and 75th percentiles, respectively, the line in the middle of the box is the median (50th percentile). The ends of the whiskers are the edges of a statistically significant sample.

FIG. 6B show the results of a ROC analysis to identify the level of total immunoglobulins G, A, M in the blood serum of patients diagnosed with CRC, inflammatory bowel disease, and healthy donors in the following models:

A - “Oncology” model (CRC = 1, IBD = 0, HD = 0), the model allows only colorectal cancer to be detected;

B - “Disease” model (CRC = 1, IBD = 1, HD = 0), the model allows only healthy donors to be identified.

The study was carried out in three cohorts of patients: patients diagnosed with CRC (33 pcs.), Inflammatory bowel diseases (27 pcs.), Healthy donors (69 pcs.) (Construction of ROC-curves, statistical analysis of the analysis data was carried out using MedCalc Version 15.4 programs )

FIG. 7 shows the results of a statistical analysis (ROC analysis) of the diagnostic efficacy of antibodies to glycans for detecting CRC. The study was carried out in the blood serum of patients diagnosed with CRC (33 sera), inflammatory bowel disease (27 sera) and healthy donors (69 sera) (the Oncology model was used to detect CRC (CRC = 1, IBD = 0, HD = 0) ) Construction of ROC-curves, statistical processing of analysis data was carried out using the MedCalc Version 15.4 software.

Claims (15)

1. A method for the diagnosis of colorectal cancer, based on the simultaneous quantitative determination of tumor markers of protein nature, antibodies to glycans, class G, A and M immunoglobulins in human blood on a biological microchip containing hydrogel elements with immobilized antibodies to protein oncomarkers, hydrogel elements with immobilized glycans, hydrogel elements with immobilized antibodies against human immunoglobulins of classes G, A, M, where the change in the level of the analyzed markers compared to the level eat markers in the blood serum of healthy donors, indicates colorectal cancer. 2. The method according to p. 1, including a) the interaction of a sample of human blood serum with biochip cells under conditions providing for the formation of specific immune complexes — compounds immobilized in hydrogel cells of the microchip with compounds contained in the analyzed sample; b) detection of the formed immune complexes; c) determination of the concentration of protein tumor markers, antibodies to glycans, immunoglobulins G, A and M; d) the diagnosis of colorectal cancer by comparing the levels of protein tumor markers, antibodies to glycans and immunoglobulins of classes G, A, M in the analyzed sample with the corresponding levels of markers in the blood serum of healthy donors. 3. The method according to p. 2, in which stage a) is carried out with known concentrations of protein tumor markers, immunoglobulins G, A, M, and a calibration dependence of the signals on the cells of the biological microchip is built on the concentration of compounds that is used to determine the concentration of protein tumor markers, immunoglobulins G, A, M, the concentration of antibodies to glycans in the analyzed sample. 4. The method according to p. 2, in which the detection of the formed immune complexes in stage (b) is carried out fluorimetrically. 5. The method according to p. 1, in which CEA, CA 19-9, CA 125, SCCA1 (SPB 3), SCCA2 (SPB 4), PSA total., PSA free, hCG, AFP, CA are used as protein tumor markers 242. 6. The method according to claim 1, wherein antibodies to SiaTn, TF, Tn, LeC, LeY, SiaLeA, Manβ1-4GlcNAc are used as antibodies to glycans. 7. A biological microchip for the diagnosis of colorectal cancer, which is an array of three-dimensional hydrogel elements, characterized in that it includes: - elements containing immobilized antibodies to tumor markers of protein nature, - elements containing immobilized glycans capable of inducing immune reactions in humans with the participation of immunoglobulins of classes G, A and M during the development of colorectal cancer - elements containing immobilized antibodies against human immunoglobulins of classes G, A and M. 8. The biological microchip of claim 7, wherein each element of the array with immobilized glycans contains antibodies to spacer glycans and / or a neoglycoconjugate and / or glycan isolated from natural sources.

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