RU2599890C2 - Multi-parameter diagnostic test system intended for detecting and monitoring therapy of breast cancer and ovarian cancer, and analysis procedure using said method - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, biotechnology and medicine, in particular, to analytical biochemistry, immunochemical analysis and diagnostic agents for oncology, and concerns a multi-parameter diagnostic test system that is intended for detecting and monitoring therapy of breast cancer and ovarian cancer. System is a kit of reagents, including a biological microchip (biochip) for simultaneous determination of concentration of several tumor-associated antigens: alpha fetoprotein, HCG, oncodevelopmental antigen CA125, CA15-3 and CA19-9 in a human blood serum sample set of calibration samples in a lyophilized form, made on the basis of stabilized pooled donor blood serum and consisting of several calibration samples that simultaneously contain all analyzed tumor markers, and a zero calibration sample that does not contain any of the above markers; it is a stabilized pooled blood serum of healthy donors that is absorbed on affine sorbents. System also includes a displaying system, set of buffer solutions and an instruction for carrying-out analysis.

EFFECT: offered test system can be used to detecting the most common forms of oncological diseases of female reproductive system, in particular breast cancer and ovarian cancer, including early stages of tumor formation; for screening of population as well as for monitoring the effectiveness of therapy.

7 cl, 4 tbl, 7 ex, 5 dwg

Description

FIELD OF THE INVENTION

The invention relates to the field of biochemistry, biotechnology and medicine, in particular to analytical biochemistry, immunochemical analysis and diagnostic tools for oncology, and relates to a multi-parameter diagnostic immunoanalytic test system (set of reagents) for the simultaneous quantitative determination of several tumor-associated antigens (tumor markers) in blood serum human, as well as the method of multi-parameter analysis of the sample carried out with its help. The diagnostic test system is designed to detect and monitor the treatment of breast cancer and ovarian cancer.

The proposed diagnostic test system can be used in the field of medical diagnostics and analytical biochemistry and is designed to identify the most common forms of cancer of the female reproductive system, namely breast cancer and ovarian cancer, including in the early stages of tumor formation; for screening the population, as well as for monitoring the effectiveness of therapy.

State of the art

Timely differential diagnosis and the ability to monitor the treatment of breast cancer and ovarian cancer significantly reduce mortality from these cancers. The modern literature describes methods for diagnosing tumors of the female reproductive system, including breast cancer and ovarian cancer: hardware, in vivo and in vitro examinations, including biopsy, as well as serological ones.

Describes the use for in vivo diagnosis of tumors of various localization (preferably gliomas), as well as breast and ovarian cancer, nanoparticles smaller than 0.1-0.3 microns, consisting of an oxide coated magnetic metal with a metal chelating polymer (gelatin) with covalent bound molecules capable of binding a tumor-specific cell marker and a contrast agent for X-ray imaging or magnetic resonance imaging (MRI) or a fluorescent dye. Nanoparticles can also be used for X-ray imaging or magnetic resonance imaging (MRI) and for the transport of drugs (Polymer nanoparticles coated with magnetic metal oxide and their use. Application for invention RU 2010116167/15).

For the diagnosis of breast and ovarian cancer, it is proposed that the patient's biopsy biological sample of cancer cells be analyzed in vitro by flow cytometry using a panel of agents that bind to extracellular surface markers (Multiplex detection of tumor cells using a panel of agents that bind to extracellular markers. Application for invention RU 2009135780/15). Monoclonal antibodies, polyclonal antibodies, Fab fragments, (Fab ′) ₂ fragments, Fv fragments, peptides, proteins, DNA molecules, polysaccharides or any combination thereof may be agents specific for various extracellular markers of cancer. CA15-3, CA125, SBA, Ep-CAM, NY-ESO-1, PRL-R, RCAS1, STN and TA-90 and B2M are offered as markers for breast cancer cells. Application of the database allows identification of cancer cells and identification of metastases.

Currently, immunological methods based on the quantitative or semi-quantitative determination of a number of antigens called tumor markers, the level of which in the blood correlates with the localization of the tumor and with the stage of the pathological process, are widely used in serological diagnosis of tumors. The diagnostic significance of a tumor marker depends on its sensitivity and specificity. It is known that an increased concentration of one tumor marker cannot serve as absolute evidence of the presence of a tumor (Maruvada P, Wang W, Wagner PD, Srivastava S. / Biomarkers in molecular medicine: cancer detection and diagnosis. // Biotechniques. 2005. Suppl: 9-15) , therefore, the most informative is the integrated determination of several tumor markers in the sample {Belokhvostoe AS, Rumyantsev AG Oncomarkers. Molecular genetic, immunochemical, biochemical analyzes. Manual for doctors. // 2003. M.: Max Press; Alekseeva M.L., Gusarova E.V., Mullabaeva S.M., Ponkratova T.S. / Oncomarkers, their characteristics and some aspects of clinical diagnostic use. // 2005. Problems of reproduction. Number 3. from. 65-79; Karpishchenko A.I., Antonov V.G., Butenko A.B., Belokhvostoe A.S., Shelepina E.P. Oncomarkers and their diagnostic value. // 1999, St. Petersburg, 47 p .; Kushlinsky N.E. / Opportunities, failures and prospects for the study of tumor markers in a modern cancer clinic. Part 1 // 1999. Clinical laboratory diagnostics. No. 3, p. 25-32; Kushlinsky N.E. / Opportunities, failures and prospects for the study of tumor markers in a modern cancer clinic. Part 2. // 1999. Clinical laboratory diagnostics. No. 4, p. 25-32).

The simultaneous determination of several tumor markers increases the effectiveness of the analysis and the possibility of differential diagnosis of cancer (Clinical Guide to Laboratory Tests. / Ed. By N.U. Tits // Unimed-Press, Moscow, 2003, 942 pp .; Kishkun A.A. / Immunological and serological studies in clinical practice. // Moscow, MIA LLC, 2006, 536 p., pp. 239-271).

Several types of immunoassay systems for the detection of cancer markers are described, including automatic analyzers, and many simpler test systems developed on the basis of the principles of enzyme-linked immunosorbent assay (ELISA), affinity microcolumns, analytical strips, microspheres and biological microarrays. Immunoanalytic test systems differ in the method of detection: using enzymes (chromogenic and luminescent substrates), radioactive and fluorescently labeled compounds, and various types of mass spectrometry (SELDI, ICP-MS, MALDI-TOF MS). The term “test system” in most sources means the device itself for analysis (microspheres, biochip, analytical strips, affinity columns, ELISA plate, etc.), although in some cases we mean a set of reagents that combines all components and the protocol immunoassay.

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 Vector-Best company offers a set of reagents "ONKO-SKRIN-IFA-BEST T-8462" for the simultaneous detection of four tumor markers by the method of a single-stage sandwich-ELISA, designed for the initial selection (primary screening) of cancer patients ("A set of reagents for the simultaneous detection of tumor markers CA125, CEA, prostate-specific antigen (PSA) and AFP in serum. ”. Http://www.vector-best.mhost.ru/prod/index.php?page=instr). However, this test system is not multiplexed, but a mechanical combination of four analyzes.

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 which are an open system for any methods and reagents. For example, a ChemWell analyzer uses standard microplates for all types of reactions and is able to conduct 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, UBC (bladder cancer) 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. Immunohemilyuminestsentny stripovoj analyzer Lumi Stat - CLIA used to determine the 11-and tumor markers CEA, PSA _commonly, PSA _free, CA125, AFP, prostatic acid phosphatase, CA19-9, CA15-3, NMP22 (nuclear matrix protein), beta-2- microglobulin, cytokeratin 18. Biomerieux mini VIDAS, France, is a multi-parameter automatic immunoassay based on enzyme-linked immunofluorescence assay (ELFA) technology and is 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 analytes (including cancer 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 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 a specific device), as well as their high cost (from several tens to hundreds of thousands of US dollars), the bulkiness of the equipment, high requirements for the qualifications of personnel, which does not allow them to be implemented in wide clinical practice.

Patent application WO / 2005/016126 (Multifactorial assay for cancer detection. University of Pittsburgh of the commonwealth system of higher education, USA) proposes a method for diagnosing ovarian cancer using multiplex immunoassay on the microspheres of one or more tumor associated antigens (EGF, G-CSF, IL-6, IL-8, CA125; VEGF, MCP-1, antibodies against interleukins IL6, IL8, antibodies against CA125, c-myc, p53, CEA, antibodies against CA15-3, MUC-1, Survivin, β-hCG , osteoponin, PDGF, Her2 / neu, Aktl, cytokeratin 19, as well as antigens cytokeratin 19, EGFR, CEA, kallikrein-8, M-CSF, FasL, ErB2 and Her2 / neu) in a patient's blood serum with fluorimetric th detection method or quantum dots using a flow hemocytometer. The method allows to diagnose ovarian cancer before its clinical manifestation, as well as to discriminate various forms and localization of the malignant process. The disadvantage of the test system is the need to use expensive equipment, highly qualified personnel, as well as the bulkiness of the equipment.

Recently, the most intensively developing method of analysis associated with the use of biological microarrays. Microchips are arrays of individual trace elements deposited on a solid substrate and containing various probes - DNA, RNA, oligonucleotides, sugars, polypeptides, proteins, cells and tissues, etc. Microchips allow simultaneous parallel analysis of the sample for many analytes, which significantly increases the efficiency of the analysis, allows miniaturization of research and significantly reduces the required amount of the studied material (Mirzabekov A.D. / Biochips in biology and medicine of the XXI century. // Herald of the Russian Academy of Sciences, 2003, t. 73, No. 5, p. 412-422). There are a number of manufacturing companies involved in the development of biochips, while there is still no clearly dominant manufacturing technology. The largest manufacturers are, for example, Affymetrix, Inc., USA - only DNA chips, RANDOX Laboratories, Ireland, - DNA and protein chips for the simultaneous determination of a number of analytes (Kricka LJ, Master SR, Joos TO, Fortina P. / / Current perspectives in protein array technology. // 2006. Ann Clin Biochem. 43 (Pt 6): 457-67; Information from the website: http://www.randox.com/tumour%20markets.php) and others.

Tripath Imaging, Inc. (USA) a screening method for identifying patients at risk with an increased susceptibility to ovarian cancer based on the detection of increased expression of a number of biomarkers (e.g., HE4, CA125, glycodelin, MMP7, Muc1, PAII, CTHRC1, inhibin A, PLAUR, prolactin, KLKIO , KLK6, SLPI ₅ and α1-antitrypsin). In this case, the principle of specific binding of antigen-antibody (the method of immunoassay on microplates) is used to detect the proteins themselves in the blood serum, or the DNA / RNA hybridization technique (Affymetrix microchips) to identify the nucleotide sequences encoding the expressed proteins. A two-stage scheme is proposed: first, patients with obviously positive analyte content are detected (cut-off by cut-off), and then quantitative analysis and statistical processing of data is carried out (Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefore. / 2007/090076).

Zhang X. et al. showed that in the microchip format simultaneous immunoanalytical determination of several tumor markers (AFP, R-hCG and CEA) and total human IgG in plasma with ICP-MS mass spectrometric detection using laser technology using antibodies labeled with rare-earth metal salts is possible (Sm ³⁺ , Eu ³⁺ ) and colloidal gold. Analytical characteristics are determined - the detection limit, the measurement range and the variation coefficients within the series and between series (Zhang S, Zhang C, Xing Z, Zhang X. / Simultaneous determination of alpha-fetoprotein and free beta-human chorionic gonadotropin by element-tagged immunoassay with detection by inductively coupled plasma mass spectrometry. // Clin Chem. 2004 Jul; 50 (7): 1214-21; Hu S, Zhang S, Hu Z, Xing Z, Zhang X. / Detection of multiple proteins on one spot by laser ablation inductively coupled plasma mass spectrometry and application to immuno-microarray with element-tagged antibodies. // Anal Chem. 2007 Feb 1; 79 (3): 923-9). The detection ranges for AFP and β-hCG were 4.6-500 and 5.0-170 ng / ml, respectively, the analytical sensitivity was 1.2 and 1.7 ng / ml, (defined as the analyte concentration corresponding to the value of the signal from zero sample plus 3 standard deviations). A comparison of the method with IRMAs for AFP and β-hCG showed correlation coefficients (R ² ) of 0.97 and 0.95, respectively.

Song S.-P. et al. (Song S.-P., Li B., Hu J., Li M.-Q. I Simultaneous multianalysis for tumor markers by antibody fragments microarray system. // Analytica Chimica Acta. 2004.510: 147-152) developed a microchip for the simultaneous determination of six markers of cancer. Six Fab fragments of antibodies against AFP, CEA, R-hCG, CA125, CA19-9 and CA15-3 markers were applied on silylated slides containing aldehyde groups. A sandwich immunoassay using an avidin-biotin system was used to amplify the fluorescent signal. Despite the low sensitivity (less than 12 ng / ml for AFP, CEA and β-hCG, and less than 17 IU / ml for CA125, CA19-9 and CA15-3) compared with traditional immunoassay systems - ELISA and RIA, the authors received a good correlation with radioimmunoassay data.

In the work of Wilson M.S. et al. (Wilson MS, Nie W. / Multiplex measurement of seven tumor markers using an electrochemical protein chip. // Anal. Chem. 2006, 78, 6476-6483) describe a sensor chip consisting of electrode elements with immobilized antigens, with with the help of which the simultaneous determination of the concentrations of seven tumor markers is carried out: AFP, ferritin, CEA, β-hCG, CA15-3, CA125 and CA19-9 by the method of competitive enzyme immunoassay. The chip showed good accuracy and reliability of the results in comparison with the independent quantitative determination of the same tumor markers in ELISA tests: the coefficient of variation was 1.9-8.1% within the series; the sensitivity was not more than 2 ng / ml (or 2 IU / ml) for most analytes.

Patent application WO / 2005/083440 describes a method for diagnosing cancer and discrimination between healthy individuals and cancer patients based on the simultaneous identification of many biomarkers, the production of which in the body increases dramatically in cancer. To predict and monitor ovarian cancer, breast cancer and colorectal cancer, a number of biomarkers of leptins, prolactin, chymotrypsin enzymes, kallikreins, tumor markers CA125, CA15-3, CA19-9, MUC1, OVX1, CEA, M-CSF, OPN were proposed and IGF-II, prostatin, CA54-61, CA72, HMFG2, interleukins IL-6, IL-10, LSA, M-CSF, NB70K, PLAP, TAG72, factors TNF, TPA, UGTF, VEGF, CLDN3, NOTCH3, E2F and others that are identified using a unique new screening technology. A pre-established negative standard is the level of expression of these biomarkers in healthy individuals and the level of expression of these biomarkers in healthy tissues of cancer patients. The expression of selected biomarkers was determined by ELISA, RCA immunoassay, chemiluminescence, an optical biosensor in thin films, proton resonance, etc. A chip was created to identify 169 biomarkers and 46 serum samples were tested on it (18 patients with ovarian cancer and 28 healthy donors) by X-ray immunoassay, and of the 169 biomarkers, 35 markers were selected as the most significant. In the next series of experiments (100 cancer patients and 106 healthy donors), the 4 most important parameters for the diagnosis and monitoring of ovarian cancer and breast cancer (leptin, prolactin, OPN, IGF-11) were selected, to which CA 125 (Identification of cancer protein biomarkers using proteomic techniques, patent application WO / 2005/083440).

In (Balachandra K, Laisupasin P, Dhepakson P, Warachit J, Jantraraksri U, Issaragrisil S, Yang XL, Hus GX. I Preliminary clinical evaluation of a protein chip for tumor marker serodiagnosis of various cancers // Asian Ras J Allergy Immunol. 2003 Sep; 21 (3): 171-178) describes the use of a protein microchip for serodiagnostic determination of several tumor markers. The sensitivity and specificity of the created microchip were investigated and the principal possibility of its clinical use among the Thai population for 10 types of cancer was shown. Chip diagnostics is applicable for cancer of the lungs, neck, breast, ovaries, prostate, pancreas, liver, esophagus, stomach, colorectal cancer. 165 cancer patients and 50 healthy people were examined. The sensitivity and specificity of the serodiagnostic protein chip were 82.4% and 94.0%, respectively. The percentage of successful attempts to diagnose cancer ranged from 57% to 100% for all 10 types of cancer. The sensitivity of the determination on the chip of each of the tumor markers in the mixture was not lower than the sensitivity of its individual determination. Thus, the protein chip is very promising both for screening and for examining cancer patients up to the stage of metastases.

Sun et al. (Sun Z, Fu X, Zhang L, Yang X, Liu F, Hu G. IA protein chip system for parallel analysis of multi-tumor markers and its application in cancer detection. // Anticancer Res. 2004 Mar- Apr; 24 (2C): 1159-65) developed a microchip for the simultaneous immunological determination of 12 tumor markers (C12 biochip). The chip is intended for screening examination of cancer patients and population groups with a high risk of malignant diseases. Twelve monoclonal antibodies were immobilized on the nitrocellulose membrane against the CA125, CA15-3, CA19-9, CA242, CEA, AFP, PSA _total , PSA _free , hCG, β-hCG, NSE and ferritin markers. 1147 blood serum samples of cancer patients with a clinically confirmed diagnosis were tested. The microchip-based test system turned out to be highly sensitive to liver cancer, pancreatic cancer, and ovarian cancer, and low-specific to gastrointestinal cancer. Further, with the help of this microchip, the authors conducted a screening examination of 15867 people, and in 436 examined patients an elevated level of one or another tumor marker was revealed. Of these, only 138 patients underwent further examination, in 16 patients cancer of various localization was detected, in 41 patients - diseases of a non-cancerous nature. 81 patients did not have any diseases.

This C12 biochip was used to diagnose and monitor various types of cancer diseases (Yu DY. / Application of multi-tumor markers protein chip assistance diagnostic system for detection of various tumors // China Trop Med 2005; 5: 414-16; Liang Z, Wang HF, Wu AZ, Fu SM. I Clinical value of 12 tumor markers protein biochip detection of digestive system neoplasm. // China Trop Med 2005; 5: 407-09; Jiang ZJ, Zhou WX, Lin W, Huang QXI Application of multi-tumor markers protein chip diagnose system in the diagnosing of intestinal neoplasms // J Mod Lab Med 2005; 20: 17-19; Mou JH, Li ZP, Wang D, Ma Y, Xiao HL, Zhang QH. I Combined detection of multiple tumor marker and its diagnostic value in colorectal cancer. // J Digest Surg 2005; 4: 268-70; Zhong ZY, Wang D, Li ZP, Li MX, Dai N, Cao XJ, et al. / Clinical significance of C -12 multiple tumor marker protein chip detective system in diagnosing colorectal c arcinoma // Chongqing Med 2007; 36: 2406-08). We studied the level of 12 tumor markers in the blood serum of 170 patients with colorectal cancer and showed that the C12 biochip can detect 41.8% of cancer cases, while the percentage of detection in the early stages (0-I) was lower - 12.9%. Among all analyzed tumor markers, the sensitivity to this type of cancer was highest in CEA - 36.5%. The use of various combinations of tumor markers did not lead to a significant improvement in test sensitivity; the levels of tumor markers most informative for colon cancer (CEA, CA19-9, CA242) correlated well with each other. It was also shown that the levels of CA242 and β-hCG increased in patients with metastases to the lymph nodes. The authors concluded that the C12 biochip is suitable for the diagnosis and monitoring of late stages of colorectal cancer, but is not effective for its early diagnosis (Chen C, Chen LQ, Yang GL, Li Y. / The application of C12 biochip in the diagnosis and monitoring of colorectal cancer: Systematic evaluation and suggestion for improvement. // J Postgrad Med. 2008.54 (3): 186-190).

The same authors used the C12 biochip for the diagnosis and monitoring of gastric cancer. The level of tumor markers was analyzed in 100 patients with various stages of gastric cancer. The diagnostic sensitivity of the system for the detection of gastric cancer based on the C12 biochip was 37%. For patients with stage I cancer, the sensitivity was only 7.8%, for stage II, III and IV - 29.4%, 35.5% and 50%, respectively. Among all 12 tumor markers, CA19-9 had the best sensitivity (23%). The C12 microchip was less sensitive to the early stages of gastric cancer and is suitable for the diagnosis and monitoring of the late stages of this disease (Chen C, Chen LQ, Chen LD, Yang GL, Li Y. / Evaluation of tumor markers biochip C12 system in the diagnosis of gastric cancer and the strategies for improvement: analysis of 100 cases. // Hepatogastroenterology. 2008, 55 (84): 991-997).

Relatively new for the serological diagnosis of breast cancer is the determination by the microchip with the applied glycans of the presence of antibodies in the patient’s blood to a specific and sensitive combination of tumor-associated glycans against the individual characteristics of a particular patient (Method for the diagnosis of breast cancer. Patent RU 2504785 C1). Diagnosis is by the presence of an antibody level at the same time to all glycans above the threshold, which can improve the sensitivity and specificity of the diagnosis of breast cancer.

All of the above test systems use "two-dimensional" chips, where antibodies are immobilized on the surface of a solid-phase carrier with some loss of biological activity due to aggregation, denaturation, steric hindrance, restricting analyte access to the active centers of the immobilized protein (Zhang S. / Hydrogels: Wet or let die. // Nat Mater. 2004 Jan; 3 (1): 7-8; Rubina A.Yu., Kolchinsky A., Makarov AA, Zasedatelev AS I Why 3-D? Gel-based microarrays in proteomics. // Proteomics 2008, Feb; 8 (4): 817-831, Jonkheijm P., Weinrich D., Schroder K, Niemeyer C. M., Waldmann H. I Chemical Strategies for Generating Protein Biochips // Angew. Chem. Int. Ed. 2008, 47, 9618-9647).

A number of works described the creation of “three-dimensional” gel microchips, which have several advantages compared to “two-dimensional” chips (Rubina A., Dementieva E., Stomakhin A., Darii E., Pan′kov S., Bar sky V., Ivanov S., Konovalova E., Mirzabekov A. / Hydrogel-based protein microchips: manufacturing, properties, and applications. // BioTechniques, 2003, 34 (5), 1008-1022; Rubina A.Yu., Kolchinsky AM, Makarov AA , Zasedatelev AS / Why 3-D? Gel-based microarrays in proteomics // Proteomics, 2008, v. 8, p. 817-831). Thus, the use of tumor-associated markers for identification and analysis (von Willebrandt factor, IgM, α1-antichymotrypsin, IgG) for the diagnosis of prostate cancer, 3D microarrays containing antibodies immobilized in a polyacrylamide gel showed a 6-fold increase in the discrimination rate (ratio the magnitude of the fluorescent signal of the sample to the magnitude of the fluorescent signal of noise) compared to 2D chips in which antibodies are immobilized on the surface of glass treated with poly-lysine (Miller J. C, Zhou K, Kwekel J., Cavallo R., Burk e J., Butler E. B., Teh B. S., Haab B. B. I Antibody microarray profiling of human prostate cancer sera, // Proteomics, 2003, 3, 56-63).

The use of 3D microarrays for the simultaneous quantitative determination of several tumor-associated markers is described in the works of the group Rubina A.Yu. et al. (Dementieva E.I., Rubina A.Yu., Darii E.L. et al. / Use of protein microarrays for the quantitative determination of tumor-associated markers // Dokl. Akad. Nauk, 2004, v. 395, p. 542 -547, Konovalova E.V., Savvateeva E.N., Dementieva E.I., Filippova M.A., Turygin A.Yu., Osipova T.V., Ryabykh T.P., Rubina A.Yu. , Zasedatelev AS / Development of a biochip for the quantitative determination of two forms of prostate-specific antigen using an internal calibration curve // Molecular Biology, vol. 41, No. 4, July-August 2007, pp. 734-738; Savvateeva E. N., Dementieva E.P., Tsibul skaya M.V., Osipova T.V., Ryabykh T.P., Turygin A.Yu., Yurasov R.A., Zasedatelev A.S., Rubina A.Yu. / Biological microchip for simultaneous quantitative immunological analysis of markers oncological diseases in human serum // Bull. Exp. Biol. Med., 2009, t. 147, No. 6, S. 679-683).

In the patent of the Russian Federation No. 2363955 (biological microchip for multiple parallel immunological analysis of compounds and methods of immunoassay in which it is used, IMB RAS named after V.A. Engelhardt, RF) the creation and use of a hydrogel microchip for various types of multiple parallel immunoassay (direct, competitive sandwich analysis).

In addition to the above sources regarding the description of microchips for the simultaneous quantification of several tumor markers, there are several patents that describe reagent kits, including a microchip, a developing system, a calibration sample panel for quantitative analysis, positive and negative controls, and wash buffers. For example, Bayer Healthcare Lie (USA) and Mayo Foundation For Medical Education And Research (USA) describe a combined chip (DNA protein) and a set of reagents for determining the expression level of polypeptides or nucleotide sequences encoding tumor-specific markers that can be used for diagnosis and monitoring of malignant diseases, including cancer of various types (adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, leukemia) and various localization (breast cancer, ovarian cancer, colon cancer, and many others). The kit also contains positive and negative controls, various combinations of showing fluorescently labeled specific sequences and / or antibodies and instructions for analyzing and interpreting the results (Use of differentially expressed nucleic acid sequences as biomarkers for cancer, patent application WO / 2005/044990).

Shanghai Health Digit Co., Ltd (China) has described a detection test system consisting of a protein chip showing mixtures of various proteins labeled with peroxidase, washing buffer (0.9% NaCl, 1.21%) Tris, 0.1% Tween20, pH 7.5), a buffer for diluting samples of biological fluids (0.9% NaCl, 1.21%) Tris, 0.1%) Tween20, 0.1% tyrosine, pH 7.5), a chemical composition that provides the chemiluminescence reaction , detergent and a series of calibration samples containing detectable markers, sandwich immunoassay protocol. The protein chip contains monoclonal antibodies immobilized on the surface of the nitrocellulose membrane (Protein chips, method producing it and detection system of the protein chips, and operating method of the detection system. Patent CN 01105023.3).

The continuation of this patent is patent application WO / 2004/038413 (Protein chips detecting system which can simultaneously detect multi target) of the Chinese company Shanghai Health Digit Co. Ltd (China), which describes a diagnostic test system for the simultaneous parallel analysis of multiple indicators based on protein microarrays, including a protein microchip, a developing system (a ready-made mixture of proteins containing luminescent labels), a series of standard samples (mixed proteins in known increasing concentrations ) and a solution for washing protein chips. As an example, the test system for the simultaneous determination of the parallel six tumor markers of AFP, CEA, PSA _{commonly, svob} PSA, CA125, CA15-3, wherein the probes are antibodies to six oncomarkers deposited on the substrate. In the described test system, the authors use "two-dimensional" chips, describe in detail the methods for their manufacture and use, but there is no data on the immunoassay of patient sera, and the technical and analytical characteristics of the diagnostic test system have not been determined.

In patent application WO 03/104808, Chengdu Kuachang Science And Technology Co., Ltd (China), Human Plasma Product Services Ltd (Hong Kong) and Chengdu Kuachang Medical Industrial Co., Ltd (China) describe a multiplex test system consisting of a biological a chip exhibiting systems (fluorescent, chemiluminescent, electroluminescent, radioactive or magnetic tags), mediators and signal amplification systems (colloidal gold with the addition of silver salts, avidin-biotin system, protein A, protein G, psoralen, digoxin), negative and positive controls reference material, puff Cleaners, etc. As an example, a biochip has been proposed for the simultaneous analysis of six tumor markers (hCG, CEA, CA50, CA242, CA15-3, CA125) and one antibody (against EBV virus) associated with tumors in one reactor. The negative control was serum, selected according to the test results using 7 ELISA test kits. Positive control is not described (Biochip kit containing biochip based on antigen-antibody reactions, and its usage, patent application WO 03/104808, USA 20050153382, patent of the Russian Federation 2316000).

In addition to test systems (a set of reagents) for the simultaneous quantitative determination of several tumor markers in one sample, the calibration samples necessary for constructing calibration curves are also described. Calibration samples should have sufficient storage stability, stability and reproducibility for all analytes. A patent search for methods of manufacturing a panel of calibration samples used to build calibration curves for the simultaneous quantitative determination of several analytes revealed the following patent documents relating to the creation of calibration samples for various test systems:

In the patent of the Russian Federation 2242006 RONTS them. N.N. Blokhin described a kit for the quantitative determination of PSA _total in human serum using a one-step enzyme-linked immunosorbent assay and a method for creating a panel of calibration samples necessary for constructing calibration curves for the quantitative interpretation of immunoassay results containing known PSA concentrations (0; 2.5; 5; 10 and 30 ng / ml PSA). Calibration samples were made in Tris buffer with 1% bovine serum albumin. The disadvantages of this method include the fact that it is designed for only one tumor marker, as well as the presence in the matrix medium of a foreign protein for humans - bovine albumin.

RF patent application 2007106670 LLC Scientific and Production Association "Diagnostic Systems" (RF) provides for the manufacture of a lyophilized industry standard sample (CCA) of hepatitis B virus surface antigen (HBsAg) to control the quality of test systems and quantify the content of antigen in serum (plasma) and human blood preparations by ELISA. TOC includes a 1% aqueous BSA solution containing 0.75% glycine, 0.01% thimerosal, and a plasma or recombinant HBsAg hepatitis B virus surface antigen to a concentration of 20 IU / ml. The technical result of the claimed invention is to increase the duration of storage and ensure the possibility of repeated use of the drug. The advantage is the presence of a solution for the dilution of CCA - negative human serum. The disadvantage of this method is that the described invention relates to only one analyte - HBsAg hepatitis B virus surface antigen. In addition, the kit contains only one bottle containing CCA and 9 bottles with a solution for diluting CCA, that is, there is no whole panel of calibration samples, but there is only a stock bottle from which it is suggested to the user to prepare calibration samples independently, which will inevitably lead to manual errors.

Patent Application WO / 2004/038413 of the Chinese company Shanghai Health Digit Co. Ltd (China), as part of a protein-chip-based test system, describes a method for creating a panel of calibration samples for the quantitative parallel analysis of several tumor markers — standard antigen solutions containing a mixture of two or more proteins in precisely known increasing concentrations. Standard solutions of antigens are made in two stages. First, a stock solution of a mixture of antigens with a high concentration is prepared in a buffer solution containing 40% -60% fetal calf serum and 0.002-0.01% NaN ₃ , or in K, Na-phosphate buffer: 0.05 M KH ₂ PO ₄ - Na ₂ HPO ₄ , pH 7.0-7.8, containing 2-30% BSA, 1.5-2.5% sucrose, 0.002-0.01% NaN ₃ . As an example, the manufacture and stability test for lyophilization and storage of standard antigen solutions for the simultaneous parallel determination of three tumor markers (AFP, CEA and NSE) are described. Antigen calibration solutions were prepared by adding 60 μl of a stock solution of a mixture of antigens to a large volume of buffer in two buffer systems: in a buffer solution based on 60% fetal calf serum and 0.005% NaN ₃ or in K, Na-phosphate buffer: 0, 05M KH ₂ PO ₄ -Na ₂ HPO ₄ , pH 7.0-7.8, containing 15% BSA, 2.5% sucrose, 0.005% NaN ₃ . Serially diluted standard antigen solutions were lyophilized and stored for 3 months. at + 4 ° C. After storage, lyophilized standard antigen solutions were analyzed on microarrays. After immunoassay, the level of signals recorded using an automatic chemiluminescence analyzer did not decrease using both buffer systems.

Known diagnostic test systems (reagent kits) and calibration sample panels for the simultaneous quantitative determination of several tumor markers in human biological fluids have several disadvantages.

disadvantages

Most of the non-invasive methods for detecting and monitoring the treatment of breast cancer and ovarian cancer described in world practice, carried out using multi-parameter diagnostic test systems (reagent kits), are not sufficiently developed and reliable. Most immunochemical test systems can detect the content of only one marker in one clinical sample. The simultaneous determination of several tumor markers increases the effectiveness of screening analysis and expands the possibilities of differential diagnosis of cancer.

The disadvantage of the existing "closed" diagnostic systems - analyzers for conducting multi-parameter analysis (USA, Japan, China) is their high cost, the bulkiness of the equipment and the requirement to use reagents and components only from the manufacturer, which does not allow them to be introduced into wide clinical practice.

The disadvantages of the currently described diagnostic multi-parameter test systems with proteins immobilized on the carrier surface (RF, USA, China), for example, based on microspheres, are their high cost and the fact that there are no commercially available test systems among them. The disadvantage of diagnostic test systems based on planar microchips is their low reproducibility and insufficient storage stability.

In most inventions, only the microchip itself is patented, a method for its manufacture and use is described, but the developing system, calibration samples used to construct calibration curves, and solutions for diluting samples are not described. For example, in the patent of the Russian Federation No. 2363955 IMB RAS named after V.A. Engelhardt (Biological microchip for multiple parallel immunological analysis of compounds and methods of immunoassay in which it is used) to interpret the results, it is proposed to construct an individual calibration curve for each analyte, which greatly complicates the procedure of multi-parameter analysis.

The creation of diagnostic test systems should be accompanied by an assessment of such technical and analytical characteristics - sensitivity, specificity, linearity, accuracy and reproducibility of the analysis (Tigranyan R.A., Kalita N.F., Roganov A.S. / Methodological guide for quality control of sets reagents for enzyme immunoassay. // Moscow, 1994). The lack of an assessment of these most important characteristics of immunoanalytic test systems, as well as data on their use for diagnostic purposes, on the correlation analysis of patient sera, is a significant drawback of most of the described inventions.

The disadvantages of the prior art description of calibration samples (RF patent 2242006 and patent application WO / 2004/038413 of the Chinese company Shanghai Health Digit Co. Ltd.) include the fact that in the first patent the proposed reagents allow the determination of only one tumor marker - PSA, and in the second, the composition of the calibration samples is designed to determine three tumor markers of AFP, CEA and NSE.

Thus, there is an urgent need to develop a reliable diagnostic multi-parameter test system for the simultaneous quantitative determination of a number of tumor markers in human serum. Such a test system should be fully characterized in accordance with the standard requirements for immunoanalytic test systems, and be favorably distinguished from the prior art solutions by the simplicity of analysis and low cost for the patient.

The problem is solved by the invention.

Disclosure of invention

The invention is a multi-parameter diagnostic test system (kit of reagents), designed to detect and monitor the treatment of breast cancer and ovarian cancer. The test system allows the simultaneous quantitative determination of several tumor-associated antigens (tumor markers) in human serum, which are diagnostically significant for the detection and monitoring of treatment of breast cancer and ovarian cancer. The invention also includes a method for conducting multi-parameter analysis of a human serum sample using this test system.

The first aspect of the invention is a diagnostic test system, which is a biological microchip (biochip) for the simultaneous determination of the concentrations of several tumor-associated antigens in a human serum sample and a combination of components: a developing system, a set of calibration samples, a set of buffer solutions and instructions for analysis. The biochip-based immunoanalytical test system is based on the principle of sandwich immunoassay with fluorescence signal recording. When conducting an immunoassay on a biochip inside gel cells, the formation of triple complexes of an immobilized antibody - antigen - manifesting antibody occurs. The value of the fluorescent signal recorded from each gel cell containing a specific immobilized antibody is proportional to the content of the corresponding tumor marker in the solution.

Diagnostic test system (set of reagents) includes:

a) a biological microchip (biochip) as the main component of the test system for the simultaneous quantitative determination of several tumor markers — an ordered array of three-dimensional hydrogel elements of a given volume formed on a solid-phase substrate according to the technology developed at the Institute of Molecular Biology named after V.A. Engelhardt RAS, the method of photo-induced copolymerization (Mirzabekov A.D., Rubina A.Yu., Pankov S.V., / Method for the polymerization immobilization of biological macromolecules and composition for its implementation // RF Patent No. 2216547 (B.I.P. M No. 32, 11/20/2003). In each individual cell of the biochip, only one individual compound is immobilized (for example, an antibody specific for one tumor marker);

b) a developing system, which is a mixture of antibodies against all analyzed tumor markers labeled with a fluorescent dye. The main requirements for the developing system are the ability to obtain high significant fluorescence signals as a result of the analysis with a low background of non-specific binding, as well as stability over the entire shelf life of the kit;

c) a set of calibration samples: each calibration sample contains simultaneously all the analyzed tumor markers in precisely known concentrations, and a zero calibration sample that does not contain the analyzed compounds. The main requirements for a set of calibration samples is that the fluorescence signals obtained after analysis should maintain linearity in the range of determination of each of the analyzed tumor markers, as well as the stability of calibration samples both throughout the shelf life of the kit and under conditions of immunoassay;

g) a set of buffer solutions:

- a solution for diluting serum, which is a buffered isotonic solution containing stabilizing additives. The main requirement for the serum dilution buffer is the absence of a background of non-specific binding, the absence of a matrix effect, as well as its stability over the entire shelf life of the kit and stability under the conditions of immunoassay;

- a solution for diluting developing antibodies, which is a buffered isotonic solution containing stabilizing additives. The main requirements for a buffer solution for dilution of developing antibodies are the absence of a matrix effect, stability throughout the shelf life of the kit and stability under conditions of immunoassay;

- a solution for washing microchips from unbound proteins, which is a buffered isotonic solution containing surface-active and stabilizing additives. The main requirements for the solution for washing microchips are the ability to destroy all forms of non-specific protein interactions, with maximum preservation of the formed immune complexes, as well as stability throughout the shelf life of the kit;

e) instructions for the analysis.

The set of calibration samples included in the test system allows the construction of several calibration curves simultaneously for analysis to quantify the concentration of several tumor markers in the sample. Each calibration sample simultaneously contains all of the analyzed tumor markers in the form of a mixture in exactly known concentrations (for example, see table. 1). In order to adequately determine the concentration of each tumor marker in blood serum, it is important to avoid the so-called "matrix effect", i.e. the influence of the analyte microenvironment on the result of the analysis. That is, it is necessary that the analyte in the calibration samples interact with antibodies immobilized on the biochip under the same conditions as in the sample. Therefore, calibration samples are made on the basis of pulsed serum from healthy donors diluted with a stabilizing solution.

To prepare a zero calibration sample, use is made of stabilized, pulsed serum from healthy donors, from which all analyzed tumor markers are reliably removed. Calibration samples may be in lyophilized form.

The object of the invention is also a test system in which the analyzed tumor markers are AFP, hCG, CEA, CA125, CA15-3 and CA19-9, including:

a) a biological microchip (biochip) obtained by photoinduced copolymerization (Mirzabekov A.D., Rubina A.Yu., Pankov S.V., / Method for polymerization immobilization of biological macromolecules and composition for its implementation, // RF Patent No. 2216547 ( B.I.P.M.M. 32, 11/20/2003), in the cells of which antibodies against AFP, hCG, CEA, CA125, CA15-3 and CA19-9 are immobilized;

b) a developing system, which is a mixture of fluorescently labeled antibodies against all analyzed tumor markers: AFP, hCG, CEA, CA125, CA15-3 and CA19-9;

c) a set of calibration samples, consisting of several calibration samples containing simultaneously all analyzed tumor markers: AFP, hCG, CEA, CA125, CA15-3 and CA19-9 - in precisely known concentrations, and a zero calibration sample that does not contain analyzed tumor markers;

g) a set of buffer solutions:

- a solution for diluting serums, which is a buffered isotonic solution containing stabilizing additives;

- a solution for diluting developing antibodies, which is a buffered isotonic solution containing stabilizing additives;

- a solution for washing microchips, which is a buffered isotonic solution containing surface-active and stabilizing additives;

e) instructions for the analysis.

Another aspect of the invention is a method for simultaneously quantifying several tumor-associated antigens - markers of breast cancer and ovarian cancer - in a human serum sample using a multi-parameter diagnostic test system. The method includes carrying out several stages:

- incubation of a biological microchip with a calibration sample or with an analyzed sample;

- washing biochips from an excess of unreacted substances;

- incubation of a biological microchip with a developing system;

- washing biochips from an excess of unreacted substances;

- registration and interpretation of analysis results.

It is allowed to add developing antibodies to the analyzed sample (or to the calibration sample), which reduces the number of stages of analysis.

The values of fluorescent signals from all gel elements of all biochips are determined and calibration graphs are constructed for all analyzed tumor markers - the curves of the dependence of the value of the fluorescent signal on the corresponding cells with immobilized antibodies to each antigen on the concentration of the same antigen in calibration samples. Then, from these graphs, the unknown concentration of each analyte in the sample is determined.

The principal novelty of the multi-parameter diagnostic test system and the method of simultaneous quantitative analysis of several tumor markers carried out with its help is the possibility of a multi-parameter analysis of several tumor markers with high sensitivity and specificity. This result is achieved thanks to correctly selected antibody pairs, concentrations of immobilized and developing antibodies, calibration samples and instructions for analysis, which, in turn, provides the possibility of detecting breast and ovarian cancer in the early stages and monitoring the effectiveness of the therapy.

The advantage of the invention is the ease of analysis, the involvement of highly qualified personnel is not required. The minimum amount of a human serum sample required for simultaneous quantitative immunoassay of several tumor markers does not exceed 130 μl in volume. The inventive method of immunoassay on biochips allows simultaneous quantitative analysis of several tumor markers in the concentration range required for clinical analysis. Simultaneous quantitative analysis of each of the tumor markers using biochips has a sensitivity that is not inferior to the sensitivity of its individual determination using commercial diagnostic ELISA kits of well-known manufacturers (for example, Fujirebio Diagnostics / CanAg (Sweden); NovaTec (Germany); " Alpha Diagnostic Intl. "(USA);" Abazyme "(USA);" Immunotech "," Amerkard "," Hema-Medica "," Alkor-Bio "," Vector-Best (Russia)).

Brief Description of the Figures

Figure 1 shows an example of a biochip structure for simultaneous quantitative immunoassay of six tumor markers (AFP, hCG, CEA, CA125, CA15-3 and CA19-9). Photo of a biochip in transmitted light. The biochip contains gel elements with a diameter of 100 μm and a volume of 0.1 nl (four identical elements in a row) with immobilized monoclonal antibodies against six tumor markers and control cells that do not contain biomaterial (empty gels). Marker points containing the immobilized dye Su5 are located at the edges of the biochip.

Figure 2 shows the result of a simultaneous quantitative immunoassay of six tumor markers (AFP, CEA, hCG, CA125, CA15-3 and CA19-9). The fluorescence image of the biochip after analysis:

A - healthy donor serum;

B - calibration test "KP ₅ ";

B - a patient’s serum containing an increased concentration of CEA and high concentrations of three tumor markers: CA 125, CA 15-3 and CA 19-9;

G - serum of an oncological patient, containing high concentrations of three tumor markers: AFP, hCG and CEA.

Figure 3 shows an example of constructing calibration curves during a two-stage simultaneous quantitative sandwich immunoassay of six tumor markers (AFP, CEA, hCG, CA125, CA15-3 and CA19-9) on a biochip. Each point of the calibration curve is the average value from measurements on ten biochips. The vertical segments show the standard deviations.

Figure 4 illustrates the determination of analytical sensitivity for one of the analytes (by the example of CEA) according to a calibration curve obtained in a simultaneous sandwich immunoassay of six tumor markers on a biological microchip. A - calibration curve, B - calibration curve in the region of low concentrations.

Figure 5 shows the result of a correlation analysis of the concentrations of six tumor markers (AFP, hCG, CEA, CA125, CA15-3 and CA19-9) obtained after simultaneous quantitative sandwich immunoassay on a biochip, and their concentrations obtained by an independent ELISA in an individual analysis with using commercial ELISA systems.

The implementation of the invention

The present invention is a multi-parameter diagnostic test system (kit of reagents) designed to detect and monitor the treatment of breast cancer and ovarian cancer, characterized in that the detection and monitoring are carried out by simultaneous quantification of several tumor-associated antigens (tumor markers) in human serum, and including a biological microchip (biochip) for the simultaneous determination of the concentrations of several tumors associated antigens in a human blood serum sample, a developing system, a set of calibration samples, a set of buffer solutions and instructions for analysis, as well as a method for multi-parameter analysis of a sample using this test system, which is a sandwich immunoassay of a serum sample on a biological microchip human blood with fluorescent signal recording.

The biochip is a microarray located on the surface of the substrate, consisting of hemispherical gel cells with covalently immobilized antibodies, each of which is specific to one of the analyzed tumor markers, and only one individual compound is immobilized in each individual cell. The antibodies used are mono- and polyclonal antibodies, mini-antibodies, antibody fragments and similar compounds that form specific complexes with the analyzed tumor markers.

The developing system is a concentrated mixture of antibodies against all analyzed tumor markers labeled with a fluorescent dye. The antibodies used are mono- and polyclonal antibodies, mini-antibodies, antibody fragments and similar compounds that form specific complexes with the analyzed tumor markers and capable of forming triple complexes “immobilized on a biochip antibody” - “tumor marker” - “developing antibody”.

As fluorescent dyes, it is possible to use labels such as Texas Red, dyes of the cyanine series and others well known to one skilled in the art. Other methods for visualizing the results of the analysis are possible, for example, the introduction of a luminescent or radioactive label, peroxidase with a chemiluminescent substrate, etc.

The set of calibration samples used to simultaneously construct several calibration curves includes several calibration samples, each of which simultaneously contains all analyzed tumor markers in certain precisely known concentrations, and a zero calibration sample that does not contain the analyzed compounds. To avoid the manifestation of the matrix effect, calibration samples are made on the basis of stabilized, pulled human donor blood serum. To prepare a zero calibration sample, pulsed serum from healthy donors is used, from which all analyzed tumor markers diluted with a stabilizing buffer solution are reliably removed. Calibration samples may be in lyophilized form.

The set of buffer solutions includes a solution for diluting the sample, a solution for diluting developing antibodies and a solution for washing microarrays.

The object of the invention is also a test system for the simultaneous quantitative analysis of six tumor markers, which are markers of breast and ovarian cancer: AFP, hCG, CEA, CA125, CA15-3 and CA19-9. The test system includes:

a) biological microchip (biochip) - a microarray located on the surface of the substrate, consisting of hemispherical gel cells with covalently immobilized antibodies, each of which is specific to one of the 6 analyzed tumor markers (AFP, hCG, CEA, CA125, CA15-3 and CA19- 9);

b) a developing system, which is a mixture of fluorescently labeled antibodies against all analyzed tumor markers: AFP, hCG, CEA, CA125, CA15-3 and CA19-9;

c) a set of calibration samples, consisting of several calibration samples containing simultaneously all analyzed tumor markers: AFP, hCG, CEA, CA125, CA15-3 and CA19-9 - in precisely known concentrations, and a zero calibration sample that does not contain analyzed tumor markers;

g) a set of buffer solutions:

- a solution for diluting serums, which is a buffered isotonic solution containing stabilizing additives;

- a solution for diluting developing antibodies, which is a buffered isotonic solution containing stabilizing additives;

- a solution for washing microchips, which is a buffered isotonic solution containing surface-active and stabilizing additives;

e) instructions for the analysis.

A method for the simultaneous quantitative immunoassay of several tumor markers is a sandwich immunoassay with fluorescence registration of signals and includes the sequential conduct of several stages, namely:

a) incubation of a biological microchip with a sample of human blood serum for the formation of binary immune complexes: antibody immobilized on a microchip — an analyzed tumor marker;

b) washing biochips from an excess of unreacted substances;

c) incubation of a biological microchip with a developing system for the formation of triple immunocomplexes: an immobilized antibody - an antigen - a developing antibody;

d) washing biochips from an excess of unreacted substances;

e) recording the results - detecting the level of fluorescence of the complexes formed in the corresponding cells of the biochip, and determining the values of the fluorescence signal using a special program;

f) interpretation of the results and quantitative determination of the concentration of each analyte.

To carry out the quantitative determination of analytes (tumor markers), stages a) to e) are carried out using calibration samples included in the test system, and calibration curves are constructed for the dependence of the intensity of the fluorescent signal of the corresponding biochip cell - the concentration of the tumor marker in the calibration sample. The fluorescence signals of the biochip cells are recorded after analysis of the sample, and the concentration of the tumor marker in the blood serum sample is calculated from the obtained calibration curves.

Stage a) -c) can be combined into one stage, i.e. a developing solution may be added to the sample to be analyzed.

The following examples demonstrate the effectiveness of the developed test system for the simultaneous quantitative determination of several tumor-associated antigens (tumor markers) in human serum and its technical characteristics: sensitivity, specificity, range of determined concentrations and reproducibility of data are given.

Examples

Example 1. The manufacture of biochip

The basis of the claimed multi-parameter diagnostic test system is a biological microchip (biochip) for the simultaneous determination of the concentrations of several analytes. To create a biochip for detecting and monitoring the treatment of breast and ovarian cancer, the tumor-associated antigens were selected as the analyzed compounds: α-fetoprotein (AFP), cancer embryonic antigen (CEA), human chorionic gonadotropin (hCG), cancer antigen 125 (CA125), cancer antigen 19-9 (CA19-9), cancer antigen 15-3 (CA15-3), diagnostically significant for these diseases.

Hydrogel biochips were manufactured using the patented polymerization immobilization technology developed under the guidance of Academician A.D. Mirzabekov at the Institute of Molecular Biology named after V.A. Engelhardt RAS (Mirzabekov A.D., Rubina A.Yu., Pankov S.V., / Method for the polymerization immobilization of biological macromolecules and composition for its implementation. // RF Patent N 2216547 (B.I.P.M. N 32 , 11/20/2003).

The biochip for the simultaneous quantitative determination of several tumor markers contained gel cells with immobilized monoclonal antibodies to all analyzed tumor markers: AFP, CEA, hCG, CA125, CA15-3 and CA19-9, and in each individual cell only one individual antibody was immobilized in a certain concentration. To enable reliable quantitative interpretation of the data using the median method of counting, four identical gel cells with an immobilized monoclonal antibody for each analyte were used in the microarray array. Marker points containing a fluorescent dye were applied along the edges of the biochip to orient the biochip during measurements.

A polymerization mixture containing gelling monomers and proteins to be immobilized was applied to the pre-treated surface of the glass substrate in the form of microdrops with a diameter of 100 μm and a volume of about 0.1 nl using a robot with a pin application technology (e.g., QArray, Genetix, UK). The gel was polymerized by irradiation with UV light with a maximum radiation of 350 nm in a stream of nitrogen. Microchips were washed from unpolymerized monomers and proteins in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl and 0.2% Tween-20. To reduce nonspecific interactions, the prepared microchips were treated with a blocking solution: 1% polyvinyl alcohol in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl for 1 h, rinsed with water and dried in a stream of air. Thus, the biochip is fully prepared for immunoassay. The biochips thus prepared were stored at + 4 ° C for at least 6 months without loss of activity.

The biochip structure for the simultaneous determination of six tumor markers is shown in FIG. one.

Example 2. The manufacture of calibration samples

The set of calibration samples, which is part of the test system for the simultaneous quantitative analysis of six tumor markers, consists of several calibration samples, each of which simultaneously contains all the analyzed tumor markers: AFP, CEA, hCG, CA125, CA15-3 and CA19-9 - exactly known concentrations (see table. 1), and a zero calibration sample that does not contain analyzed tumor markers.

To avoid the manifestation of the matrix effect, calibration samples were prepared on the basis of healthy donated serum diluted with a stabilizing buffer solution containing 0.14 M sodium chloride, 0.01 M sodium hydrogen phosphate, pH 7.2, 0.15% polyvinyl alcohol, 0.15 % polyvinylpyrrolidone and 0.01% methylisothiazolone hydrochloride.

To prepare the zero calibration test “KP ₀ ”, specially treated pulled serum from healthy donors was used. Normally, the serum of each person contains all of the analyzed tumor markers in low concentrations (see table. 1). In order to achieve zero concentrations of the analyzed tumor markers, the procedure for absorbing the pulled serum of healthy donors on affinity sorbents was carried out. The reliability of the removal of the analyzed tumor markers from the serum intended for the manufacture of the KP ₀ zero calibration sample was proved by testing it using commercially available test kits: to determine the content of AFP, CEA, CA125, CA15-3 and CA19-9, kits of the company “ Fujerebio Diagnostic "/" CanAg "(Sweden), for determination of hCG content - a set of the company" DSL "(USA).

For the manufacture of calibration samples “KP ₁ ” - “KP ₆ ”, commercially available antigens AFP, CEA, hCG, CA15-3 (Hema-Medica, Russia), CA125 and CA19-9 (Fujerebio Diagnostic / CanAg ", Sweden. The concentration of tumor markers AFP, CEA, CA125, CA15-3 and CA19-9 in the obtained samples was determined using independent methods.

Example 3. Verification of calibration samples

Each batch of manufactured calibration samples must undergo a verification procedure, that is, a reliable determination of the concentrations of all analyzed tumor markers in each calibration sample.

The batch of calibration samples, intended to be a component of the diagnostic test system, consisted of six calibration samples - “KP ₁ ” - “KP ₆ ” and a zero sample “KP ₀ ”. All calibration tests “KP ₀ ” - “KP ₆ ” were using commercially available immunoassay immunoassay kits from Fujerebio Diagnostic / CanAg (Sweden) for AFP, CEA, CA125, CA15-3, CA19-9, and DSL "(USA) - for hCG, according to the manufacturer's instructions. An example of the concentration composition of a set of calibration samples leading to a diagnostic test system is given in table. one.

Calibration samples can be lyophilized without significant changes in the initial activity of all analyzed tumor markers. The manufactured calibration samples (see Example 2) were lyophilized and tested in two versions: using the developed test system (see Examples 4-7) and an independent method using commercially available immunoassay ELISA kits from Fujerebio Diagnostic / " CanAg "(Sweden) and" DSL "(USA) according to the manufacturer's instructions. It was shown that after lyophilization, the magnitude of the fluorescent signal for each of the six antigens remains practically unchanged (Table 2). Lyophilized calibration samples are stable during transportation and storage at a temperature of + 4 ° C for at least 6 months (Table 3).

Example 4. Multiparameter diagnostic test system for the simultaneous quantitative determination of six tumor-associated antigens (tumor markers) in human serum

For the detection and monitoring of therapy for breast cancer and ovarian cancer, the tumor-associated antigens AFP, hCG, CEA, CA125, CA19-9, CA15-3 are the diagnostically significant tumor markers. Therefore, the biochip (see Example 1) is a microarray of hemispherical gel cells with covalently immobilized monoclonal antibodies against AFP, hCG, CEA, CA125, CA19-9, CA15-3, and in each individual cell only one individual antibody is immobilized in a certain concentration .

The developing system is a 20-fold concentrate of a mixture of six antibodies against six tumor markers AFP, hCG, CEA, CA125, CA19-9, CA15-3, labeled with Su5 fluorescent dye. Each fluorescently labeled antibody is taken in a specific concentration.

The set of calibration samples includes at least 3 sets of lyophilized calibration samples, each of which contains 7 calibration samples “KP ₀ ” - “KP ₆ ” (see Example 3, table 1) for the possibility of simultaneously constructing six calibration curves for six tumor markers in three different experiments.

The set of buffer solutions includes a solution for diluting the sample, a solution for diluting developing antibodies and a solution for washing microarrays. The serum dilution solution is a stabilized buffer solution containing 0.14 M sodium chloride, 0.01 M sodium hydrogen phosphate, pH 7.2, 0.15% polyvinyl alcohol, 0.15% polyvinylpyrrolidone and 0.01% methylisothiazolone hydrochloride. A solution for diluting developing antibodies is a stabilized buffer solution containing 0.14 M sodium chloride, 0.01 M sodium hydrogen phosphate, pH 7.2, 0.15% polyvinyl alcohol, 0.15% polyvinyl pyrrolidone and 0.01% methylisothiazolone hydrochloride. The solution for washing microchips from unbound proteins is a 5-fold concentrate of buffered saline containing 1% Tween-20. Before use, the buffer solution for washing microchips is diluted to the required number of times with distilled water. All kit components are stable at + 4 ° C for at least 6 months.

Example 5. Obtaining calibration curves during simultaneous quantitative immunoassay of six tumor markers (AFP, hCG, CEA, CA125, CA15-3 and CA19-9) using a multi-parameter diagnostic test system for the detection and monitoring of treatment of breast cancer and ovarian cancer.

A diagnostic test system (see Example 4) was tested in an immunoassay. As the analyzed sample, the calibration samples “KP ₀ ” - “KP ₆ ” included in the kit were used (see Example 3). 130 μl of the sample was applied to the biochip located under the chamber. The biochip chambers were sealed with sealing tape. Biochips were incubated for 20 h at 37 ° C. After incubation, the reaction chambers were removed. The biochip was washed for 20 min with a buffer for washing biochips, then it was rinsed with distilled water and dried in a stream of air. After that, 50 μl of a mixture of developing antibodies against all six tumor markers diluted 20 times with a buffer for diluting developing antibodies was applied to the biochip. Biochips were incubated for 2 hours at 37 ° C. After incubation, the biochips were washed for 30 min with the biochip washing buffer, then they were rinsed with distilled water and dried in a stream of air, then fluorescent signals were recorded.

Fluorescence measurements were carried out using a portable fluorescence biochip analyzer (IMB, Moscow) with laser excitation using 650/670 nm filters (excitation / registration) (Barsky V., Perov A., Tokalov S., Chudinov A., Kreindlin E ., Sharonov A., Kotova E., Mirzabekov A. I Fluorescence data analysis on gel-based biochips. // J. Biomol. Screening 7 (2002) 247-257). The fluorescence signal intensity from each cell of the biochip was calculated using special software (ImaGelResearch, IMB, Moscow). When analyzing the data obtained, the fluorescence intensity was calculated as the median signal from four identical gel cells.

In one experiment, six calibration curves of the dependence of the fluorescent signal from gel cells containing the corresponding immobilized antibodies on the concentration of antigen in solution are simultaneously obtained for each tumor marker.

In one experiment on 70 biochips, calibration samples were analyzed and 10 calibration curves were constructed for each of the six analytes. In the graphs shown in FIG. 3, each point represents the arithmetic mean of 10 measurements carried out under the same conditions. As can be seen, for each of the six analytes in the selected concentration range, the value of the fluorescent signal from cells with immobilized antibodies increases in proportion to the concentration of the corresponding antigen in the sample.

Example 6. Determination of the analytical characteristics of a multi-parameter diagnostic test system for the detection and monitoring of breast and ovarian cancer therapy based on a biochip.

The main characteristics of any diagnostic test system are its sensitivity, specificity and reproducibility of the analysis results. The high specificity of the simultaneous determination of the concentration of each of the six analytes: AFP, hCG, CEA, CA125, CA15-3 and CA19-9 - is achieved by rigorous selection of antibodies used for immobilization and developing antibodies for each analyte.

To assess the reproducibility and analytical sensitivity of the analysis of the developed diagnostic test system (see Example 4), a series of experiments was carried out on seventy biochips and ten calibration curves were constructed for each of the six tumor markers. The average calibration curves obtained on biochips simultaneously for all six tumor markers are shown in FIG. 3.

(см. фиг. 4). Полученные значения приведены в таблице 4. Как видно из таблицы, тест-система позволяет проводить анализ онкомаркеров в диапазоне, необходимом для клинического анализа, с чувствительностью, не уступающей коммерческим ИФА - системам.Analytical sensitivity i.e. the smallest detectable concentration of each tumor marker was calculated as the concentration corresponding to a fluorescence value exceeding by two standard deviations the value of the fluorescence signal of a 10-fold zero-measured calibration sample: $${\overline{F}}_0+2SD$$

(see Fig. 4). The obtained values are shown in table 4. As can be seen from the table, the test system allows the analysis of tumor markers in the range required for clinical analysis, with a sensitivity not inferior to commercial ELISA systems.

To assess the reproducibility of the results of the determination of six tumor markers (AFP, hCG, CEA, CA125, CA15-3 and CA19-9) in one experiment, several blood serum samples of cancer patients were analyzed 10 times and three calibration curves were also constructed for each of the six tumor markers. From the average calibration curves, the concentration of each of the six analytes in the samples was determined 10-fold (see Example 7). Then, statistical data processing was carried out. The value of the coefficient of variation for various concentrations of the determined tumor markers AFP, hCG, CEA, CA125, CA15-3 and CA19-9 did not exceed 15%.

Example 7. Determination of the content of six tumor markers in blood serum

The developed diagnostic test system (see Example 4) was used for the simultaneous quantitative determination of six tumor markers (AFP, hCG, CEA, CA125, CA15-3 and CA19-9) in the blood serum of cancer patients and healthy donors. For this, in one experiment, this sample (or several samples) and 3 parallels from 7 calibration samples “KP ₀ ” - “KP ₆ ” were analyzed (see Examples 2, 3). The calculation of the fluorescence signal intensity from each cell of the biochip and determination of the concentrations of all tumor markers were performed using special software (ImageAssay, IMB, Moscow). When analyzing the data obtained, the fluorescence intensity was calculated as the median signal from four identical gel elements.

Based on the measurement results for six tumor markers, 6 average calibration curves were constructed at the same time, where each point represents the arithmetic average of three measurements of the fluorescence intensity of each of the three calibration samples obtained in one experiment.

To quantify the concentration of each analyte, the fluorescent signal observed in the cell with an immobilized antibody to this analyte after analysis of the sample was compared with the fluorescent signal on the calibration curve for this analyte obtained in the same experiment. The quantitative determination of the content of each of the tumor markers was carried out by piecewise linear interpolation.

In parallel, the content of each marker was determined in an individual analysis of the same sera using standard ELISA test systems: Fujirebio Diagnostics (CanAg), Sweden for AFP, CEA, CA125, CA15-3 and CA19-9, and DSL, USA - for hCG.

The results of the simultaneous determination of six tumor markers in human serum on a biochip (see Fig. 5) correlate well with the results obtained by individually determining the concentration of each of the tumor markers using standard ELISA test systems. Correlation coefficients were: 0.993 (n = 33, p <0.01) for AFP, 0.998 (n = 28, p <0.01) for CEA, 0.992 (n = 21, p <0.01) for hCG, 0.967 (n = 18, p <0.01) for CA 19-9, 0.986 (n = 17, p <0.01) for CA 15-3 and 0.975 (n = 26, p <0.01) for CA 125.

Claims (7)

1. A multi-parameter diagnostic test system designed to detect and monitor the treatment of breast and ovarian cancer, characterized in that it is a set of reagents, including a biological microchip (biochip) for the simultaneous determination of the concentrations of several tumor-associated antigens: AFP, hCG, CEA, CA125, CA15-3 and CA19-9 in a sample of human blood serum, a set of calibration samples in lyophilized form, made on the basis of stabilized, pulled donor serum human blood count, consisting of several calibration samples containing simultaneously all analyzed tumor markers, and a zero calibration sample that does not contain the indicated tumor markers and is a stabilized, pulsed serum of healthy donors absorbed on affinity sorbents, showing a system, a set of buffer solutions and instructions for analysis . 2. The test system according to claim 1, characterized in that the biochip is a microarray located on the surface of the substrate, consisting of hemispherical gel cells with covalently immobilized antibodies, each of which is specific to one of the analyzed tumor markers, and is immobilized in each individual cell only one individual substance. 3. The test system according to claim 1, characterized in that the set of calibration samples used to simultaneously construct several calibration curves includes several calibration samples, each of which simultaneously contains all the analyzed tumor markers AFP, hCG, CEA, CA125, CA15 -3 and CA19-9, and a zero calibration sample, reliably not containing the indicated tumor markers. 4. The test system according to claim 1, characterized in that the developing system is a mixture of several fluorescently labeled monoclonal antibodies against tumor markers AFP, hCG, CEA, CA125, CA15-3 and CA19-9. 5. The test system according to claim 1, characterized in that the set of buffer solutions includes a solution for diluting the sample, a solution for diluting developing antibodies and a solution for washing microarrays. 6. A method for conducting multi-parameter analysis of a sample using the test system according to claim 1, intended for the detection and monitoring of therapy for breast and ovarian cancer, which is a biological microchip sandwich immunoassay of a human serum sample with fluorescence detection of signals. 7. The method according to p. 6, characterized in that the analysis includes several stages: incubation of the biochip with a sample or with a calibration sample; washing the biochip from an excess of unreacted substances; incubation of a biochip with a developing system; washing the biochip from excess unreacted substances, recording and interpreting the results.

Images