WO2012123527A1 - Procédé et marqueurs de diagnostic de formes infracliniques et cliniques du rejet tubulo-interstitiel à médiation lymphocytaire t après une transplantation rénale - Google Patents

Procédé et marqueurs de diagnostic de formes infracliniques et cliniques du rejet tubulo-interstitiel à médiation lymphocytaire t après une transplantation rénale Download PDF

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WO2012123527A1
WO2012123527A1 PCT/EP2012/054526 EP2012054526W WO2012123527A1 WO 2012123527 A1 WO2012123527 A1 WO 2012123527A1 EP 2012054526 W EP2012054526 W EP 2012054526W WO 2012123527 A1 WO2012123527 A1 WO 2012123527A1
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markers
rejection
polypeptide
sample
amplitude
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Harald Mischak
Jochen Metzger
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Mosaiques Diagnostics and Therapeutics AG
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Mosaiques Diagnostics and Therapeutics AG
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to the diagnosis of a subclinical or clinical T-cell-mediated tubulointerstitial acute rejection in patients after kidney transplantation by means of a polypeptide pattern comprising urinary frequency and / or amount distinguishable peptide markers which both the local inflammatory response and the tubule and in the Interstitium-induced structural changes even before the infiltration of T cells.
  • Acute rejection is the most critical factor for long-term function and long-term survival of the graft (Meier-Kriesche et al., Am J. Transplant., 2004, Vol. 4, pp. 378-383). In about 15-30% of cases, patients undergoing kidney transplantation experience one or more acute rejection episodes within the first year after transplantation (Nankivell et al., Transplantation, 2004, Vol. 15, pages 434-441; Paraskevas et al., Clin. Transplant., 2006, Vol. 20, pages 667-672). T-cell-mediated tubulointerstitial rejection is the most common cause of kidney rejection. In contrast, T cell-mediated vascular and hor- moral forms of acute rejection are correspondingly less important.
  • the diagnosis of acute kidney transplant rejection requires biopsy removal of a tissue sample of the transplanted allogeneic kidney. This is usually performed as an indication biopsy for clinically indicated functional impairment or as a protocol biopsy during postoperative follow-up. Limited local access through biopsy in transplanted patients with early signs of graft impairment results in undetected early manifestations of graft injury (Racusen, Clin. J. Am. Soc. Nephral., 2006, Vol -147). In contrast, postoperative follow-up protocol biopsies can detect rejection at the earlier subclinical stage and allow the immediate initiation of drug therapy to maintain the transplant (Rush et al., Curr. Opin. Nephral. Hypertens., 1998, Vol. 7, pages 691-694). However, protocol biopsy implies that patients with stable graft function and no signs of rejection are unnecessarily exposed to this invasive procedure. In addition, renal biopsies are associated with the risk of procedural complications and high costs.
  • CE-MS Capillary electrophoresis coupled mass spectrometry
  • the peptide marker shows the same differences in regulation as in the training cohort.
  • a peptide marker shows a further increase in the regulation (frequency and / or average amplitude) in a cohort of patients with clinical kidney transplant rejection that is conspicuous in the indication biopsy and an increase in the serum creatinine level above 20%.
  • Preselected peptides were then elucidated by means of tandem mass spectrometry in their amino acid sequence and assigned to the corresponding m otorp r t e n e n u n d s c h n i tts t e l l n n-specific endoproteases.
  • the latter are responsible for the terminal cutting motifs of the identified marker peptides and, as so-called surrogate markers, may be indicative of the differentiation of specific proteases.
  • Alpha-2-HS Glycoprotein also known as fetuin-A, inhibits the metalloproteinase meprin (Hedrich et al., Biochemistry, 2010, vol. 49, pages 8599-8607) which occurs on the brush border membrane of proximal tubular tubules. The latter is involved in the breakdown of the extracellular matrix in its secretory form (Berthier and Marti, Swiss Med. Wkly., 2007, Vol. 137 Suppl 155, page 109S-114S).
  • fetuin A also forms a cell surface anchor for the matrix metalloproteinases 2 and 9 and activates them in the cell-bound form (Ochieng et al., Arch. Biochem. Biophys., 1995, Vol. 322, pages 250-255). , Ultimately, this points to the potential role of fetuin A as a modulator of extracellular matrix turnover.
  • cystatin-B depending on the degree of differentiation of the cell, a localization of this protein restricted to the nuclear and cytoplasmic compartment and a vesicular / lysosomal structure has been described (Calkins et al., J. Histochem., Cytochem., 1998, Vol. Page 745-751), which could be related to this post-translational modification.
  • Cystati inhibits a variety of cathepsins and thus prevents cell damage by these lysosomal enzymes.
  • Elevated levels of the specific cystatin B peptide indicate increased degradation of active cystatin B induced by initial cathepsin D cleavage (Lenarcic et al., Biochem. Biophys. Res. Commun., 1988, Vol. 154, page 765- 772), as well as release of specific cystatin B fragments from the cell and subsequent excretion into the urine.
  • PGP tripeptides released from collagen fragments serve as a chemotactic for attracting granulocytes to the local site of inflammation (Gaggar et al., J. Immunol., 2008, Vol. 80, p. 5662-5669).
  • This group can be further subdivided into elevated peptide levels with c-terminal Gly-Ala-Arg-Gly or Gly-Glu-Arg-Gly end sequence [6a], lowered peptide levels with c-terminal Gly-Gln-Gly end sequence [6b ] (Assignment of the terminal sequence motifs according to merops.sanger.ac.uk) and other differentially regulated fragments derived from type 1 collagen chains in the range of n- or c-terminal MMP interfaces already described in the literature [6c] (Zhen et al., Arthritis Rheum., 2008, Vol. 58, pages 2420-2431).
  • the differential excretion of the collagen type 1-derived peptides is ascribed to the activity of specific MMPs, such as MMP-8 and -9, but not MMP-2 (Ermolli et al., Transpl. Immunol., 2003, Vol 11, pages 137-145).
  • MMPs such as MMP-8 and -9
  • MMP-2 Ermolli et al., Transpl. Immunol., 2003, Vol 11, pages 137-145.
  • MMP-derived cleavage motifs on collagen type I-derived peptides in the urine is indicative of a local inflammatory reaction occurring at the tubulointerstitium of the transplanted kidney.
  • the latter is derived from observations in the animal model that correlate a MMP activity in a causal relationship to tissue-specific Inflammatory Responses (Van Lint et al., J. Immunol., 2005, Vol.
  • tubule epithelia tubulitis
  • Group 7 contains all peptide markers which were also found in the statistical analysis to be indicative of rejection of the transplanted kidney, but for which the amino acid sequence could not yet be determined. Due to the synergistic nature of the regulation with the peptide markers known from the sequence, however, a causal relationship between their differential regulation and the local inflammatory process prior to infiltration of immune cells into the transplanted kidney is also assumed for these peptides.
  • the object is achieved by a method for the diagnosis of subclinical or clinical T-cell mediated tubulointerstitial rejection in patients after renal transplantation comprising the step of determining a presence or absence or amplitude of at least three polypeptide markers in a urine sample, wherein the polypeptide marker for the diagnosis of a subclini - see or clinical T cell mediated tubulointerstitial rejection from the markers that are characterized in Table 1 by values for molecular mass and migration time.
  • Table 1 below lists the peptides in the urine of patients after renal transplantation with differential regulation in subclinical and / or clinical acute tubular graft rejection.
  • Small letters p and k are hydroxyproline and hydroxylysine, respectively.
  • the frequency of the markers in the rejection or control group is given in% and the mean amplitude in ion counts.
  • the control group is a kidney transplant without any signs of rejection in the protocol biopsy.
  • the evaluation of the measured polypeptides can be based on the presence or absence or amplitude of the markers taking into account the following limits:
  • Peptide-peptide frequency mean frequency middle group ID rejection amplitude control amplitude
  • Specificity is defined as the number of actual negative samples divided by the sum of the number of actual negatives and the number of false positives. A specificity of 100% means that a test identifies all healthy persons as healthy, ie. no healthy person is identified as ill. This does not say anything about how well the test detects sick patients.
  • Sensitivity is defined as the number of actual positive samples divided by the sum of the number of actual positives and the number of false negatives. A sensitivity of 100% means that the test detects all patients. He does not say how well the test detects healthy people.
  • the markers according to the invention make it possible to achieve a specificity of at least 70%, preferably at least 80%, more preferably 85%, for the diagnosis of acute transplant rejection.
  • the markers according to the invention make it possible to achieve a sensitivity of at least 70%, preferably at least 80%, more preferably 85%, for the diagnosis of acute transplant rejection.
  • a particularly preferred marker is the marker with the peptide ID 3796, which is distinguished in subclinical forms of rejection by a sensitivity of 95% with a specificity of 61%.
  • this marker is combined with at least two further, preferably even more markers.
  • the migration time is determined by capillary electrophoresis (CE) - such. B. in example under point 2 - determined.
  • CE capillary electrophoresis
  • the eluent used is, for example, 20% acetonitrile, 1% (v / v) formic acid in water.
  • CE migration time can vary. However, the order in which the polypeptide labels elute is typically the same for any CE system used under the conditions indicated. To the- To compensate for any differences in migration time, the system can be normalized using standards for which migration times are known. These standards may, for. Example, the polypeptides given in the examples be (see example point 3). Variation in CE time is relatively small between individual measurements, typically in the range of ⁇ 2 min, preferably in the range of ⁇ 1 min, more preferably ⁇ 0.5 min, even more preferably ⁇ 0.2 min or 0.1 minute
  • the characterization of the polypeptides shown in Tables 1 and 2 was determined by capillary electrophoresis mass spectrometry (CE-MS), a method which was described e.g. In detail by Neuhoff et al. ⁇ Rapid Communications in mass spectrometry, 2004, Vol. 20, pages 149-156).
  • CE-MS capillary electrophoresis mass spectrometry
  • the variation of molecular masses between individual measurements or between different mass spectrometers is relatively small with exact calibration, typically in the range of ⁇ 0.1%, preferably in the range of ⁇ 0.05%, more preferably ⁇ 0.03%, even more preferably ⁇ 0.01% or 0.005%.
  • polypeptide markers according to the invention are proteins or peptides or degradation products of proteins or peptides. They may be chemically modified, e.g. by post-translational modifications such as glycolization, phosphorylation, alkylation or disulfide bridging, or by other reactions, e.g. B. in the context of degradation, be changed.
  • polypeptide markers molecular mass and migration time
  • polypeptides of the invention are used to diagnose acute tubulo-interstitial rejection after kidney transplantation.
  • Diagnosis is the process of gaining knowledge by assigning symptoms or phenomena to a disease or injury.
  • the presence or absence of certain polypeptide markers is also used differential diagnosis.
  • the presence or absence of a polypeptide marker can be measured by any method known in the art. Methods that can be used are exemplified below.
  • a polypeptide marker is present when its reading is at least as high as the threshold. If its reading is below that, the polypeptide marker is absent.
  • the threshold value can either be determined by the sensitivity of the measurement method (detection limit) or defined based on experience.
  • the threshold is preferably exceeded when the sample reading for a given molecular mass is at least twice that of a blank (eg, only buffer or solvent).
  • the polypeptide marker (s) is / are used to measure its presence or absence, the presence or absence being indicative of the early diagnosis of acute tubulo-interstitial rejection of the allogeneic renal transplant.
  • polypeptide markers which are typically present in kidney-transplanted patients without acute tubulo-interstitial rejection, but which occur more rarely or not at all in transplanted patients with acute tubulo-interstitial rejection.
  • polypeptide markers that are present in kidney transplanted patients with acute tubulointerstitial rejection but are not or only rarely present in patients without acute tubulointerstitial rejection.
  • amplitude markers can also be used for diagnosis.
  • Amplitude markers are used in a manner that does not determine the presence or absence, but decides the magnitude of the signal (amplitude) in the presence of the signal in both groups.
  • the tables show the mean amplitudes of the corresponding signals (characterized by mass and migration time) over all measured samples. Two nomination procedures are possible in order to ability to reach between differently concentrated samples or different measurement methods. In the first approach, all peptide signals of a sample are normalized to a total amplitude of 1 million counts. The respective mean amplitudes of the single markers are therefore given as parts per million (ppm).
  • amplitude markers via an alternative standardization procedure: in this case, all peptide signals of a sample are scaled with a common normalization factor. For this purpose, a linear regression is formed between the peptide amplitudes of the individual samples and the reference values of all known polypeptides. The increase in the regression line just corresponds to the relative concentration and is used as a normalization factor for this sample.
  • the decision to make a diagnosis depends on how high the amplitude of the respective polypeptide markers in the patient sample is compared to the mean amplitudes in the control group or the "sick" group. If the value is close to the mean amplitude of the "sick" group, it is to be assumed that there is an acute tubulointerstitial rejection of the kidney transplant, it corresponds more to the mean amplitudes of the control group, is not to be assumed that acute tubulointerstitial rejection of the kidney transplant.
  • the distance to the mean amplitude can be interpreted as a probability of belonging to a group.
  • the distance between the measured value and the mean amplitude can be considered as a probability of belonging to a group.
  • a frequency marker is a variant of the amplitude marker, in which the amplitude is low in some samples. It is possible to convert such frequency markers into amplitude markers in which, in the calculation of the amplitude, the corresponding samples in which the marker is not found, with a very small amplitude - in the range of the detection limit - is included in the calculation.
  • the individual from whom the sample is derived, in which the presence or absence of one or more polypeptide markers is determined, may be any individual in whom acute tubuiointerstitial rejection of the kidney graft may occur.
  • the subject is a mammal, most preferably a human.
  • the sample measuring the presence or absence of the polypeptide marker (s) of the invention may be any sample recovered from the subject's body.
  • the sample is a sample having a polypeptide composition suitable for making statements about the condition of the individual.
  • it may be blood, urine, synovial fluid, tissue fluid, body secretions, sweat, cerebrospinal fluid, lymph, intestinal, gastric, pancreatic, ga, tuberculosis, a G ewe be p ro be, S pe rm a, vaginal fluid or a stool sample.
  • it is a liquid sample.
  • the sample is a urine sample.
  • Urine samples may be known as known in the art.
  • a mid-jet urine sample is used.
  • the urine sample may e.g. by means of a catheter or also with the aid of a urination apparatus, as described in WO 01/74275.
  • the presence or absence of a polypeptide marker in the sample can be measured by any method known in the art useful for measuring poly peptide markers is suitable to be determined. The person skilled in the art knows such methods. In principle, I can n the presence or absence of a polypeptide marker by direct methods, such as. As mass spectrometry, or indirect methods such. B. by ligands determined.
  • the sample of the individual e.g.
  • the urine sample may be pretreated by any suitable means before measuring the presence or absence of the polypeptide marker (s), and e.g. B. purified or separated.
  • the treatment may, for.
  • a purification, separation, dilution or concentration include.
  • the methods may be, for example, centrifugation, filtration, ultrafiltration, dialysis, precipitation or chromatographic methods such as affinity separation or separation by ion exchange chromatography, or electrophoretic separation.
  • a subsample is obtained if, according to specific properties - eg. Size, weight, affinity, charge - an enrichment occurs in at least two different fractions, which are then measured separately. Due to the division of the sample, the corresponding subsample is less complex and therefore easier to analyze.
  • An example of subsamples would be fractions obtained from chromatographic or capillary electrophoretic separations. In some cases it may be useful to split the sample into a larger number of subsamples. 10, for example, but to examine only some of the samples, for example 5, in which the relevant markers are expected.
  • the sample is separated by electrophoresis prior to its measurement, purified by ultracentrifugation and / or separated by ultrafiltration into fractions containing polypeptide labels of a specific molecular size.
  • a mass spectrometric method is used to determine the presence or absence of a polypeptide marker, which method may precede purification or separation of the sample.
  • the mass spectrometric analysis has the advantage over current methods that the concentration of many (> 100) polypeptides of a sample can be determined by a single analysis. Any type of mass spectrometer can be used. With mass pectrometry it is possible to routinely measure 10 fmoles of a polypeptide marker, ie 0.1 ng of a 10 kDa protein with a measurement accuracy of approximately ⁇ 0.01% from a complex mixture. In mass spectrometers, an ion-forming unit is coupled to a suitable analyzer.
  • electrospray ionization (ESI) interfaces are mostly used to measure ions from liquid samples, whereas matrix assisted laser desorption / ionization (MALDI) techniques are used to obtain ions from a sample crystallized with a matrix measured. For analysis of the resulting ions z.
  • ESI electrospray ionization
  • MALDI matrix assisted laser desorption / ionization
  • quadrupoles ion traps or time-of-flight (TOF) analyzers can be used.
  • electrospray ionization the molecules present in solution ia. is sprayed under the influence of high voltage (eg 1-8 kV), whereby charged droplets become smaller, which become smaller as a result of evaporation of the solvent. Close I come by so-called.
  • Coulomb explosions to form free ions, which can then be analyzed and detected.
  • TOF analyzers have a very high scanning speed and achieve a very high resolution.
  • Preferred methods for determining the presence or absence of polypeptide markers include gas phase ion spectrometry, such as laser desorption / ionization mass spectrometry, MALDI-TOF-M S, SE LDI-TOF-MS (surface enhanced laser desorption ionization), LC-MS ( Liquid chromatography mass spectrometry), 2D-PAGE-MS and capillary electrophoresis mass spectrometry (CE-MS). All of the methods mentioned are known to the person skilled in the art.
  • gas phase ion spectrometry such as laser desorption / ionization mass spectrometry, MALDI-TOF-M S, SE LDI-TOF-MS (surface enhanced laser desorption ionization), LC-MS ( Liquid chromatography mass spectrometry), 2D-PAGE-MS and capillary electrophoresis mass spectrometry (CE-MS). All of the methods mentioned are known to the person skilled in the art.
  • CE-MS in which capillary electrophoresis is coupled with mass spectrometry. This method is in detail z.
  • the CE-MS technique allows to determine the presence of several hundreds of polypeptide markers of a sample simultaneously in a short time, a small volume and high sensitivity. After a sample has been measured, a pattern of the measured polypeptide markers is prepared. This can be compared with reference patterns of ill or healthy individuals. In most cases, it is sufficient to use a limited number of polypeptide markers for the diagnosis of acute tubulointerstitial rejection of a kidney transplant. More preferred is a CE-MS method which includes CE coupled online to an ESI-TOF-MS.
  • the use of volatile solvents is preferred, and it is best to work under essentially salt-free conditions.
  • suitable solvents include acetonitrile, methanol and the like.
  • the solvents may be diluted with water and an acid (eg, 0.1% to 1% formic acid) added to protonate the analyte, preferably the polypeptides.
  • an acid eg, 0.1% to 1% formic acid
  • Capillary electrophoresis makes it possible to separate molecules according to their charge and size. Neutral particles migrate at the rate of electroosmotic flow upon application of a current, cations are accelerated to the cathode and anions are retarded.
  • capillaries in electrophoresis is the favorable ratio of surface area to volume, which enables a good removal of the Joule heat arising during the current flow. This in turn allows the application of high voltages (usually up to 30 kV) and thus a high separation efficiency and short analysis times.
  • fused silica capillaries with internal diameters of typically 50 to 75 m are normally used. The used lengths are 30-100 cm.
  • the capillaries usually consist of plastic-coated quartz glass.
  • the capillaries may be both untreated, i. on the inside show their hydrophilic groups, as well as be coated on the inside. A hydrophobic coating can be used to improve the resolution.
  • a pressure which is typically in the range of 0-1 psi may also be applied. The pressure can also be created during the separation or changed during the process.
  • the markers of the sample are separated by capillary electrophoresis, then directly ionized and transferred online to a mass spectrometer coupled thereto for detection.
  • polypeptide markers can advantageously be used for diagnostics.
  • Preferred is the use of at least 5, 6, 8, or 10 markers.
  • 20 to 50 markers are used.
  • Urine was used to detect polypeptide markers for diagnosis. Urine was collected from kidney transplant patients with no clinical and biopsy evidence of acute graft rejection (control group) and from kidney transplant patients with clinical and biopsy evidence of acute transplant rejection.
  • the proteins such as albumin and immunoglobulins, which are also present in urine of patients in higher concentrations, had to be separated by ultrafiltration.
  • 700 .mu.l of urine were removed and mixed with 700 .mu.l Filtration Buffer (2M urea, lOm M ammonia, 0.02% SDS).
  • 700 .mu.l Filtration Buffer 2M urea, lOm M ammonia, 0.02% SDS.
  • These 1.4 ml sample volumes were ultrafiltered (20 kDa, Sartorius, Gottingen, DE).
  • the UF was carried out at 3000 rpm in a centrifuge until 1.1 ml ultrafiltrate was obtained.
  • CE-MS measurements were performed with a capillary electrophoresis system from Beckman Coulter (P / ACE MDQ System, Beckman Coulter Inc, Fullerton, USA) and a Bruker ESI-TOF mass spectrometer (micro-TOF MS, Bruker Daltonik, Bremen, D).
  • the CE capillaries were purchased from Beckman Coulter, they had an ID / OD of 50/360 ⁇ and a length of 90 cm.
  • the mobile phase for the CE separation consisted of 20% acetonitrile and 1% (v / v) formic acid in water. 30% isopropanol with 0.5% formic acid was used for the sheath flow at the MS, here with a flow rate of 2 ⁇ / min.
  • the coupling of CE and MS was performed by a CE-ESI-MS sprayer kit (Agilent Technologies , Waldbronn, DE).
  • the duration of the injection was 99 seconds. With these parameters about 150 nl of the sample were injected into the capillary, this corresponds to about 10% of the Kapillarvolum ens.
  • a "stacking" technique was used, injecting an IM NH 3 solution for 7 sec (at 1 psi) before sample injection, after the sample injection Sample injection for 5 sec of a 2M formic acid solution After applying the separation voltage (30 kV), the analytes between these solutions are automatically concentrated.
  • CE separation was performed with a pressure method: 0 psi for 40 minutes, 0.1 psi for 2 min, 0.2 psi for 2 min, 0.3 psi for 2 min, 0.4 psi for 2 m , finally 32 min at 0.5 psi.
  • the total duration of a separation run was thus 80 minutes.
  • the "Nebulizer gas” was set to the lowest possible value.
  • the voltage applied to the spray needle to generate the electrospray was 3700 - 4100 V.
  • the remaining settings on the mass spectrometer were optimized according to the manufacturer's instructions for peptide detection. The spectra are recorded in a m ore 400 m / z 3000 and accumulated every 3 sec.
  • the proteins / polypeptides are each used in a concentration of 10 pmol / ⁇ in water.
  • REV, ELM, KINCON and GIVLY are synthetic peptides.
  • the molecular masses of the peptides and the m / z ratios of the individual charge states that are visible in the MS are given in the following Table 3:
  • the most probable assignment is the one in which Shift for the peptide 1 and for the peptide 2 is a substantially linear relationship.
  • An amplitude of 0 means that the marker was not found. From the frequency and amplitude data, scoring for belonging to the groups rejection and control (ie without rejection) was derived. A reading that was from the frequency / amplitude to the group Rejection, received a positive score; a reading that belonged to the frequency / amplitude control group got a negative score. The closer the value to the values in Table 2, the greater the score. Subsequently, the individual scores were combined.
  • Marker 3796 was found in subject 1 with an amplitude of 857.126. This marker is found more frequently in patients with a rejection and the amplitude is larger in these patients. The fact that this marker was found to have a high amplitude gives a positive score for rejection.
  • Marker 113765 was not found in the sample from subject 1. This marker is less common in the rejection group than in the control group. Therefore, his absence is an indicator of rejection; this gives another positive score.
  • Marker 151296 was found to have an amplitude of 8021.8, just above the mean amplitude of the rejection group. This results in another positive score.
  • marker 3796 was not found. This marker is found less frequently in the control group and with smaller amplitudes. That the marker is not found therefore gives a negative score.
  • Marker 113765 was found to have an amplitude of 229.344. Its presence is an indicator of belonging to the control group. The amplitude is also significantly above the rejection group at this altitude. This gives another negative score.
  • Marker 151296 was not found in the sample. This marker is relatively common in both the rejection and control groups. His absence is therefore a weak indicator of belonging to the control group, so that there is no small additional negative score.
  • Subject 3 shows an amplitude of 30.612 for marker 3796.
  • the presence of the marker is more likely to be part of the rejection group, but the low amplitude is a sign of belonging to the control group. This results in two different scores: one slightly positive and one slightly negative, which equalize.
  • Marker 113765 was found to have an amplitude of 54,786. The presence of the marker indicates that it belongs to the control group. The amplitude is closer to the group without rejection. This results in a negative score.
  • Marker 151296 shows an amplitude of 228.195.
  • the presence of this marker basically means that it belongs to the rejection group. However, the low score is a sign of belonging to the control group. Overall, this marker has a negative score.
  • the total score of the three markers is thus -0.269.

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Abstract

L'invention concerne un procédé de diagnostic d'un rejet tubulo-interstitiel aigü après une transplantation rénale, comprenant l'étape de mesure de la présence ou l'absence ou bien de l'amplitude d'au moins trois marqueurs polypeptidiques dans un échantillon d'urine, les marqueurs polypeptidiques sont sélectionnés parmi les marqueurs qui, dans le tableau 1, sont caractérisées par des valeurs pour la masse moléculaire et le temps de migration.
PCT/EP2012/054526 2011-03-15 2012-03-15 Procédé et marqueurs de diagnostic de formes infracliniques et cliniques du rejet tubulo-interstitiel à médiation lymphocytaire t après une transplantation rénale Ceased WO2012123527A1 (fr)

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WO2024159239A1 (fr) * 2023-01-27 2024-08-02 Bio Preventive Medicine Corp. Dosage et dispositif, kits et procédés associés

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014114787A1 (fr) * 2013-01-25 2014-07-31 Lagerstedt Jens Peptides dérivés d'apolipoprotéine a-i pour le traitement de l'hyperglycémie
WO2024159239A1 (fr) * 2023-01-27 2024-08-02 Bio Preventive Medicine Corp. Dosage et dispositif, kits et procédés associés

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