WO2009154510A1 - Système de test permettant de diagnostiquer le cancer de la prostate et méthode de diagnostic du cancer de la prostate - Google Patents

Système de test permettant de diagnostiquer le cancer de la prostate et méthode de diagnostic du cancer de la prostate Download PDF

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WO2009154510A1
WO2009154510A1 PCT/RU2009/000288 RU2009000288W WO2009154510A1 WO 2009154510 A1 WO2009154510 A1 WO 2009154510A1 RU 2009000288 W RU2009000288 W RU 2009000288W WO 2009154510 A1 WO2009154510 A1 WO 2009154510A1
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hepsin
reaction
substrate
prostate cancer
buffer
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PCT/RU2009/000288
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English (en)
Russian (ru)
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Евгений Сергеевич СЕВЕРИН
Сергей Евгеньевич СЕВЕРИН
Екатерина Михайловна КУЗНЕЦОВА
Мария Владимировна САВВАТЕЕВА
Евгений Сергеевич БУДОРАГИН
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Автономная Некоммерческая Организация "Иhctиtуt Молекулярной Диагностики"
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Publication of WO2009154510A1 publication Critical patent/WO2009154510A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59—Transmissivity

Definitions

  • the invention relates to medicine, in particular, oncology and molecular biology, and can be used for early diagnosis of prostate pathologies, including prostate cancer.
  • prostate cancer is one of the most common tumor diseases in men, with a frequency of occurrence in 4th place after cancer of the lungs, stomach and colon.
  • PCa prostate cancer
  • the incidence of prostate cancer in Russia amounted to 12.8 million people, mortality - 8.2 million, while the ratio of the number of deaths from prostate cancer to the number of cases is 64%.
  • the incidence of prostate cancer increases, and from the age of 45 it grows exponentially.
  • the age of 70 the disease, according to histological data, occurs in more than 60% of men, after 70 years - in 83%.
  • the clinical picture of prostate cancer does not have pronounced symptoms and is often asymptomatic.
  • the main symptoms of the disease - erectile dysfunction, a change in the nature of urination - are similar such for a benign tumor of the prostate gland, prostatitis, cystitis and other diseases of the genitourinary system.
  • the prior art knows several basic diagnostic methods for prostate cancer, which can be divided into invasive and non-invasive and invasive.
  • the first include a biopsy of the prostate gland, the second - digital rectal examination (PRI), transrectal ultrasound (TRUS) and measurement of the level of prostate-specific antigen (PSA) in blood serum.
  • PRI second - digital rectal examination
  • TRUS transrectal ultrasound
  • PSA prostate-specific antigen
  • the second group is preferable because of the less traumatic nature.
  • analysis by TRUS and PRI methods places high demands on the level of qualification and experience of diagnosticians, while the probability of recognition of prostate cancer at an early stage is close to zero.
  • Closest to the claimed invention is a method for diagnosing prostate cancer based on the measurement of PSA by the enzyme-linked immunosorbent assay as environmentally safer (compared to radioimmune) and less expensive (compared to chemiluminescent).
  • An enzyme-linked immunosorbent assay requires a set of equipment, the so-called ELISA, which includes a shaking device - an incubator, a washing device and a microplate or cuvette photometer.
  • PSA is a glycoprotein with a molecular weight of about 32,000 Da, consisting of one polypeptide chain.
  • PSA is a serine protease produced exclusively by the epithelium of the human prostate. Normally, PSA is secreted into the seminal fluid in high concentrations, where it is enzymatically active, and is directly involved in liquefaction. seed clot. PSA is present in the bloodstream in low concentrations. An increase in the concentration of PSA in serum indicates pathologies of the prostate, such as benign hyperplasia and malignant degeneration of prostate tissue. The definition of PSA is widely used to detect and monitor patients with prostate cancer.
  • the level of total PSA in patients with prostatitis is extremely rare (only 4.5% of cases) exceeds the value of 4.0 ng / ml, and these values do not go beyond the "gray zone" (4.0-19.9 ng / ml) .
  • the average level of total PSA is 4.1 ng / ml and the frequency of “falling” PSA values in the “gray zone” increases to 33%.
  • the average content of total PSA is 100 times higher than that of patients with adenoma and amounts to 419.7 ng / ml, and the maximum values of total PSA in individual patients can exceed 7000.0 ng / ml.
  • the level of total PSA is within normal limits, in 1/3 of patients the level of PSA is in the “gray zone”, in most patients (53.3% of cases) the PSA values are higher 30.0 ng / ml.
  • the OncoIFA-General PSA reagent kit is intended for the quantitative determination of total prostate-specific antigen (PSA) in blood serum by enzyme-linked immunosorbent assay.
  • the OncoIFA-General PSA kit is designed for analysis in duplicates of 40 unknown, 6 calibration samples, one control serum sample and one sample to determine the optical density of the TMB solution (tetramethylbenzidine) using all strips simultaneously.
  • the “Ono IFA-General PSA” kit uses a “sandwich” -variant of enzyme-linked immunosorbent assay. To implement this option, two monoclonal antibodies with different epitope specificity for PSA were used. One of them is immobilized on the solid phase (the inner surface of the holes), the second is conjugated to horseradish peroxidase.
  • the solution stains in the wells.
  • the degree of color is directly proportional to the amount of PSA in the test sample.
  • the concentration of PSA in the determined samples is calculated based on the calibration graph.
  • the prototype test system includes a set of twelve eight-well strips in a frame with anti-PSA antibodies immobilized on the inner surface of the wells, calibration samples based on blood serum containing known amounts of PSA (indicated on the label of the vials), conjugate anti-PSA peroxidase, concentrated buffer solution (buffer P) for washing the wells, tetramethylbenzidine solution (TMB solution), a stop reagent (IH hydrochloric acid solution), • a control serum with a known content of PSA, • spectrophotometer vertical ck nirovaniya capable of measuring absorbance of the solution in the mixer strips at a wavelength of 450 nm; single-channel pipettes with variable volumetric fluid withdrawal, eight-channel pipette, frame shaker with strips (thermostatic shaker), which allows shaking at a speed of 500-800 rpm at a temperature of +37 0 C 5 measuring cylinders - glass glasses, distilled water, filter paper, medical protective gloves.
  • the range of PSA concentrations up to 4 ng / ml was determined as normal, the judgment of the presence of non-malignant urological disease is made when the PSA concentration is from 4 ng / ml to 10 ng / ml, with PSA values above 10 ng / ml, the presence of malignant prostate tumors is judged .
  • PSA concentrations corresponding to normal it is recommended to clarify the values of PSA concentrations corresponding to normal.
  • All reagents are mixed before analysis and brought to room temperature, the wells are labeled according to their purpose - to measure the optical density of the TMB solution, for calibration samples and for control serum.
  • an anti-PSA peroxidase conjugate solution is added in all wells, except for those intended for measuring the optical density of the TMB solution.
  • Calibration samples and control serum are added to the corresponding wells, and the test blood serum to the remaining ones. strips are incubated with shaking in a thermostatically controlled shaker at a temperature of + 37 ° C at a speed of 500-800 rpm. After decanting the contents of the wells and washing them, a solution of tetramethylbenzidine is introduced into all wells.
  • the strips are incubated in the dark for a period of time, the duration of which depends on the degree of color development.
  • a stop reagent is added to the wells to stop the enzymatic reaction.
  • the vertical scanning photometer measures the optical density in the wells at 450 nm. From the values of optical densities of the remaining wells b the values obtained for the wells designed to measure the optical density of the TMB solution are subtracted, a plot of the optical density versus PSA concentration in the calibration samples is constructed for calibration samples and, on its basis, a judgment is made about the presence of non-malignant urological diseases, prostate tumors
  • the disadvantage of the method for diagnosing prostate cancer based on measuring the PSA level is its lack of effectiveness, since PSA is a predictive, but not predictive marker due to its insufficient specificity for the diagnosis of prostate cancer, especially in the diagnosis at the early stages of the disease. So, for example, in the range below 10 ng / ml it can be false negative in 20% and false positive in 70% of patients.
  • An increase in PSA levels in addition to prostate cancer may be due to benign hyperplasia, the presence of inflammation, infection and ischemia in the prostate gland.
  • the test system also has its drawbacks - the need to use both an anti-PSA peroxidase conjugate, a solution of tetramethylbenzidine and a stop reagent.
  • the analysis takes a long time - about 4.5 hours, in addition, the development of the detected pathology can be predicted only when repeated analyzes are carried out for a long time (about 18 months).
  • the technical result to which the invention is directed is to increase the reliability of the analysis results while simplifying and reducing the cost of the base used, reducing the time required for diagnostics.
  • Hepsin can be used as a marker for diagnosing prostate cancer.
  • Hepsin is a protein from the group of transmembrane serine proteases of type II, which is absent on the surface of prostate cells in normal, but found in varying amounts with benign hyperplasia and a malignant tumor and plays a role in the process of oncogenesis of a prostate tumor. Normally, this protein is found mainly in hepatocytes. This property allows the use of hepsin for the diagnosis of prostate cancer with a high degree of reliability.
  • this marker has enzymatic activity and is localized on the cell surface. It was experimentally found that when using monoclonal antibodies to bind hepsin on the cell surface, the tumor's ability to metastasize was significantly reduced. It is believed that this phenomenon is associated with the activation of extracellular matrix components by hepsin, which, when in active form, contribute to the enhancement of invasive tumor growth.
  • a test based on the determination of the enzymatic function of hepsin most fully characterizes the stage of the disease and the development of the tumor process, in contrast to standard polymerase chain reaction methods using the RNA reverse transcription method (RT-PCR), which determines the total amount of hepsin mRNA , and ELISA (enzyme-linked immunosorbent assay), which determines the total amount of hepsin protein on the cell surface in active and inactive form.
  • RT-PCR RNA reverse transcription method
  • ELISA enzyme-linked immunosorbent assay
  • the essence of the invention is to use as a substance to test the quality of the reaction in the test system of the recombinant protein hepsin produced from the producer strain pHPNTM "A N ° B-10098, using when testing and setting the reaction as reaction buffer O 5 IM Tris- imidazole, and as a substrate - the tetrapeptide L-lysine P 2 Pz L-arginine, where P 2 -L-asparagine, L-leucine or L-threonine, P 3 L-lysine or L-glutamine conjugated to a chromogen, and judgment on the presence of prostate cancer and its stage by hepsin Ao activity in nmol / l * h, determined by the average value of the rate of change in minute optical density of the mixture of cell lysate with added substrate and reaction buffer ⁇ Eo / min, in accordance with the following dependence:
  • V is the total volume of the reaction mixture, i.e. the volume of the treated urine sample with the substrate and buffer added to it, ml;
  • L is the optical path length, the optical path length of the tablet, cm,
  • V is the total volume of the reaction mixture, i.e. mixtures of control hepsin with substrate and reaction buffer added to it, ml;
  • L is the optical path length of the tablet, cm,
  • ⁇ Ek / min optical units / min, is the average rate of change in optical density in 1 min;
  • the activity of Ak of the control hepsin in the test system intended for diagnosis is compared with the reference activity of Ae of the control hepsin, when the Ak values differ from Ae by more than 10 percent in the direction of increase or decrease, the values of Ao are excluded from the number of analyzes on the basis of which the diagnosis is carried out, in other cases when the value of Ak differs from Ae by no more than 10 percent in the direction of increase or decrease, the judgment on the presence of cancer gland according to the obtained reliable value of Ao is made in accordance with the above dependence for Ao.
  • the value of Ao is less than or equal to 0.17 nmol / l * min, a judgment is made about the absence of pathology in the prostate gland.
  • urine samples are taken after rectal massage of the prostate gland, carried out with interruptions of 1-2 minutes after each session, with a total massage duration of 3 to 15 minutes.
  • the substrate was created taking into account the fact that the serine protease hepsin has a narrow substrate specificity and hydrolyzes protein bonds with arginine in the position L-lysine P 2 P 3 L-arginine j, where P 2 and P 3 are amino acids with polar, uncharged radical groups.
  • the substrate is a tetrapeptide L-lysine P 2 P 3 L-arginine (where P 2 -L-asparagine, L-leucine or L-threonine, Pz - L-lysine or L-glutamine) conjugated to a chromogen (this can be a particular case be paranitroanilide or aminomethylcoumarin), the optical density of which lies within the operating ranges of existing spectrometers and colorimeters and fluorimeters. So, for Microrlate Reader model 680 manufactured by Bio-Rad, He ⁇ ios Alrha manufactured by ThermoSpectronic, paranitroanilide with an absorption range of 405-415 nm can be used as a chromogen.
  • Hepsin in the active form has the ability to hydrolyze the bond between the peptide part of the substrate and the chromogenic, resulting in the release of free chromogen into the solution and staining of the solution, with an intensity proportional to the activity of the enzyme, which allows you to determine the reaction rate and on its basis to judge the activity of hepsin.
  • Hepsin activity values determining the limits of differences between normal and pathology were obtained based on the data shown in Table 1. In the statistical processing of the results, extreme values were excluded in all the analyzed groups. The results of the studies confirm that the use of hepsin for the diagnosis of prostate pathologies makes it possible to distinguish prostate cancer from prostate adenoma and chronic prostatitis and cases of absence of pathology with a high degree of certainty.
  • Isolation of the cell fraction from urine samples includes the following stages: deposition of cells, which should be carried out in a centrifuge with a bucket rotor, and twice washing the precipitate with phosphate-buffered saline with physiological pH. At the same time, you should try to get rid of the mucus, which may interfere with the analysis, for this purpose, you should clearly visually separate the dense cellular sediment from the gelatinous mucosa and carefully separate the latter without a pipette. Urine sediment containing red blood cells is not allowed for analysis.
  • the determination of hepsin activity in the obtained sample includes, in turn, the following stages: treatment of the cell fraction with a lysing solution, centrifugation, buffering of the supernatant with a pH buffer from 7.4 to 8.1, adding a substrate, thermostating, measuring the optical density of the mixture, calculating the activity.
  • a buffer taking into account the ability of hepsin in active form to hydrolyze the bond between the peptide and chromogenic parts of the substrate, 0.1 M tris-imidazole is used.
  • the recombinant hepsin protein is obtained from pHPNTM strain A deposited in the Russian collection of microorganisms under number B-10098 under the following conditions:
  • hepsin - IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the culture medium upon receipt of a sufficient number of cells, the culture medium is subjected to centrifugation.
  • the supernatant containing the target protein is incubated with an ion exchange chromatographic carrier;
  • control hepsin is mixed with a substrate and reaction buffer, and the absorbance EK of the resulting mixture is measured with an interval of at least 10 minutes over the entire measurement interval (it was experimentally determined that the measurement interval is sufficient from the point of view of reliability of the results Stu in 60 minutes).
  • V is the total volume of the reaction mixture, i.e. mixtures of control hepsin with substrate and reaction buffer added to it, ml;
  • V I the volume of control hepsin, ml
  • L is the optical path length of the tablet, cm,
  • ⁇ Ek / min optical units / min, is the average rate of change in optical density in 1 min.
  • the Ak activity value obtained in the test system is compared with a reference Ae deposited on a vial with a control sample containing control hepsin obtained from the producer strain pHPNTM "A JVbB-10098 and intended to judge the quality of the reaction.
  • test system contains a set of reagents "Photo-Hepsin” and is intended for the quantitative determination of the enzymatic activity of a marker protein of prostate cancer of the serine protease hepsin in biological material obtained non-invasively (in urine obtained after rectal massage of the prostate gland).
  • the “Photo-Xepsin” kit is designed for analysis in duplicates of 45 unknowns, and 6 control samples and one sample to determine the optical density of the buffer lysis solution using all strips simultaneously.
  • the “Photo-hepsin” kit used the colorimetric method for determining the kinetic activity of the enzyme. To implement this option, the following were used: lysis buffer to prepare urine samples for examination, buffering solution, hepsin substrate. In the wells, when the test sample, the buffer solution and the substrate solution are mixed during incubation, the bond between the L-arginine of the substrate and its chromogenic part is cleaved, and as a result, the solution is stained, the intensity of which is proportional to the activity of the hepsin enzyme. After measuring the optical density of the solution in the wells, the activity of the enzyme (A) in the determined samples is calculated according to the above formula.
  • the test system includes a set of twelve eight-well strips in a frame, a control sample based on recombinant hepsin protein obtained from producer strains B-10098 containing an enzyme with known activity (Ae, is indicated on the label of the vial), lyophilized hepsin substrate, lysis buffer for processing urine sediment, Tris-imidazole buffer for buffering test samples, weighed phosphate-saline buffer for processing urine sediment, weighed protease inhibitors for the treatment of urine sediment, a vertical scanning spectrophotometer that allows you to measure the optical density of the solution in strips at a wavelength of 405 nm, single-channel pipettes with a variable volume of fluid sampling, an eight-channel pipette, a frame shaking device with strips (a thermostatically controlled shaker) that allows shaking with the oscillation amplitude of 0.5 mm and a frequency of 9-18 Hz at room temperature + (18-25) ° C; temperature-maintaining thermostat + (37 ⁇ 1) 0 C; household
  • the specificity of the analysis is 100%, which is due to the specificity of hepsin cleavage of the peptide-chromogen bond in the used artificial substrate.
  • Sensitivity of the kit the minimum activity of hepsin in the urine lysate of a person reliably determined by the kit does not exceed 0.05 nmol / l * min. Accuracy: this analytical parameter is checked by the test for "discovery”. The opening percentage is 90-110%.
  • Linearity the dependence of hepsin activity in urine lysates, taking into account the urine dilution factor, is linear in the range of activities 0.11 - 5.5 nmol / l * min and is 90-110%.
  • hepsin activity 0 - OD 7 nmol / l * min was determined as normal, the judgment on the presence of a process leading to the early development of prostate pathology is made at hepsin activity 0.175-0.25 nmol / l * min; a judgment on the presence of an average degree of development of prostate pathology is made when the value of hepsin activity is 0.25-0.9 nmol / l * min; with values of hepsin activity of 0.9 - 5.0 nmol / l * min and higher, a judgment is made about the presence of malignant prostate tumors.
  • All reagents before analysis are prepared according to the instructions and brought to room temperature, the wells are labeled in accordance with their purpose - to measure the optical density of the buffer solution, for control samples, for samples.
  • Buffer solution is added to all wells.
  • Test wells and control samples are introduced into the wells according to the marking.
  • a substrate solution is introduced in all wells, except for those designed to measure the optical density of the buffer solution.
  • the samples are mixed on a shaker for tablets and the optical density at 405 nm (E 0 ) is determined on a vertical spectrophotometer using a blank as a comparison cell. If for any urine lysate E 0 exceeds the optical density of 1, 0, the sample should be diluted 10 times.
  • Urine samples are obtained from men with suspected prostate cancer.
  • Urine samples are centrifuged at 450-500g for 15-20 minutes at room temperature; remove the supernatant; wash the precipitate with phosphate-saline buffer of physiological pH (FSB from Pan-Eco was used); re-centrifuge the samples and repeat the washing procedure; completely remove the supernatant.
  • FAB phosphate-saline buffer of physiological pH
  • a lysis solution (used RIPA buffer company ⁇ Verizon, Inc.
  • the prepared urine lysate is frozen at -2O 0 C.
  • urine lysates are thawed at room temperature and centrifuged. The supernatant is used to determine the activity of the hepsin enzyme.
  • the activity determination procedure consists of the following steps:
  • V is the total volume of the reaction mixture, i.e. a mixture of urine cell lysate with a substrate and buffer added to it, ml;
  • V I the volume of the cell lysate of urine, ml
  • L is the optical path length of the tablet
  • the values of the measured hepsin activity in the lysate are divided by the urine concentration factor (200) and the final hepsin activity in the test urine sample is obtained.

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Abstract

La présente invention relève du domaine de la médecine, plus spécifiquement de l'oncologie et de la biologie moléculaire, et peut servir au diagnostic précoce de pathologies de la prostate, y compris du cancer de la prostate, au moyen d'une méthode non invasive. Cette invention se caractérise en ce qu'on utilise une protéine hepsine recombinante produite à partir d'une souche productrice pHPNTM-A N°B-100978 comme substance permettant de vérifier la qualité de la réaction dans le système de test, du 0,1M tris-imidazole comme tampon de réaction pour vérifier et effectuer la réaction, ainsi qu'un tétrapeptide L-lysine P2 Р3 L-arginine conjugué à un chromogène comme substrat, P2 représentant L-asparagine, L-leucine ou L-thréonine, P3 représentant L-lysine ou L-glutamine; en ce que la présence d'un cancer de la prostate ainsi que son stade sont déterminés en fonction de l'activité de l'hepsine Ao exprimée en nmol/l⋅h, laquelle est définie en fonction de la valeur moyenne ΔЕо/min de la vitesse de variation par minute de la densité optique d'un échantillon d'urine traité auquel le substrat et le tampon de réaction ont été ajoutés. Les résultats obtenus lorsque l'activité de l'hepsine contrôlée Ak, vérifiée à l'aide du système de test, diffère des valeurs de référence Ae de plus de 10 % sont exclus des résultats d'analyse vérifiés à l'aide de ce même système de test.
PCT/RU2009/000288 2008-06-19 2009-06-05 Système de test permettant de diagnostiquer le cancer de la prostate et méthode de diagnostic du cancer de la prostate WO2009154510A1 (fr)

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EA200801822A EA011694B1 (ru) 2008-06-19 2008-06-19 Тест-система для диагностики рака предстательной железы и способ диагностики рака предстательной железы

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EP3431490A1 (fr) * 2017-07-17 2019-01-23 Uniwersytet Gdanski Composés chimiques utilisés comme marqueurs diagnostiques d'inflammations et de tumeurs, procédé de synthèse de composés chimiques, kit de diagnostic à utiliser dans le diagnostic de processus inflammatoires et de néoplasies épithéliales et procédé de diagnostic in vitro de processus inflammatoires et des néoplasies épithéliales
WO2020017984A1 (fr) * 2018-07-14 2020-01-23 Uniwersytet Gdanski Composés chimiques destinés à être utilisés en tant que marqueurs de diagnostic d'une inflammation et de néoplasmes

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