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EP4010704A1 - Dosage amélioré de détection d'auto-anticorps - Google Patents

Dosage amélioré de détection d'auto-anticorps

Info

Publication number
EP4010704A1
EP4010704A1 EP20749923.7A EP20749923A EP4010704A1 EP 4010704 A1 EP4010704 A1 EP 4010704A1 EP 20749923 A EP20749923 A EP 20749923A EP 4010704 A1 EP4010704 A1 EP 4010704A1
Authority
EP
European Patent Office
Prior art keywords
receptor
phospholipase
bead
secondary antibody
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20749923.7A
Other languages
German (de)
English (en)
Inventor
Cornelia DÄHNRICH
Sarah KONITZER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Euroimmun Medizinische Labordiagnostika AG
Original Assignee
Euroimmun Medizinische Labordiagnostika AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Euroimmun Medizinische Labordiagnostika AG filed Critical Euroimmun Medizinische Labordiagnostika AG
Publication of EP4010704A1 publication Critical patent/EP4010704A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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

Definitions

  • the present invention relates to the use of a secondary antibody comprising a chemilumi nescent label for increasing the diagnostic reliability of an assay comprising the step detect ing an autoantibody against phospholipase-A2-receptor and a A kit comprising a bead comprising a secondary antibody recognizing IgG class antibodies and/or phospholipase-A2- receptor or a variant thereof, wherein the antibody and/or the phospholipase-A2-receptor comprises a chemiluminescent label, and a calibrator defining a lower chemiluminescence detection limit of at least 4, preferably a lower limit of at least 3, more preferably a lower limit of at least 2 CU/ml, as well as one or more, preferably all from the group comprising one or more calibrator solutions, a sample dilution buffer, a washing solution, a positive control, a negative control and a chemiluminescence trigger solution.
  • Membranous Nephropathy is an autoimmune disease with a prevalence of 1 to 2/100.000 persons/year and the most common cause of nephrotic syndrome in Caucasian adults.
  • the initial clinical symptoms are edema due to increased renal protein loss, defined as pathologic proteinuria, which is induced by the damage of the renal glomerular filtration barrier.
  • the clinical course of the disease is variable and ranges from spontaneous remission of proteinuria to end-stage renal disease (ESRD). Patients with spontaneous remission (about
  • a commercial ELISA for the semi-quantitative determination of phospholipase-A2-receptor autoantibody levels is available from EUROIMMUN Medizinische Labordiagnostika AG (“Anti-phospholipase-A2-receptor ELISA IgG Test instruction”, EA 1254-9601 G). According to the test manual, the results should be interpreted such that a concentration of ⁇ 14 RU/ml represents a negative result, a concentration between 14 and 20 RU/ml is borderline, and a result of 20 or more RU/ml represents a positive result, and that a titer increase, decrease or disappearance as detected by the present ELISA assay precedes a change in the clinical status.
  • concentrations of more than 1500 RU/ml may be determined.
  • the test manual teaches detecting phospholipase-A2-receptor autoantibodies in patients suffering from MN or those having undergone a kidney transplantation as part of monitoring the outcome of their therapy, but not in healthy subjects or patients without clinically active disease, for example after remission.
  • a commercial immunofluorescence assay for detecting a phospholipase-A2-receptor autoantibody is also available, for example FA 1254-1003-40 (EUROIMMUN Medizinische Labordiagnostika AG), and has a higher reported sensitivity, but immunofluorescence is neither a high-throughput method, nor can it be used to resolve small concentration increas es.
  • Well trained scientists, medical doctors or technicians are required to carry out the assay, since the cytoplasm is stained, as is the case in the presence of anti-nuclear antibod ies or antibodies against mitochondria.
  • US2013/0280738 discloses various assay formats and reagents including chemiluminescent dyes for detecting an autoantibody to phospholipase-A2-receptor. It is suggested that assay formats other than chemiluminescence such as gold labeling or ELISA based on the use of advance fluorophores lead to particularly sensitive results, though.
  • Timmermans et at. increased the sensitivity from 63% to 72% in a cohort of 109 patients, with a control cohort of only 33 patients, by lowering the cut off to 2 RU/ml (Timmermans, S. A, Dampiping, J. G., Heerings-Rewikel, P. T., Aylaon, R., Beck, L. H., Schlumberger, W., Salant, D. J., van Passen, P, Tervaert, J. W. (2014) Am. J. Clin. Pathol. 142, 29-34).
  • ELISA-based assays are suitable for high-throughput testing, such assays are less sensitive than immunofluorescence methods. Therefore, a problem underlying the present invention is to provide a more sensitive assay in a high-throughput format, ideally suitable for the prediction of active MN, both in subjects without MN symptoms and in patients in danger of developing a relapse.
  • the problem underlying the present invention is solved by the subject-matter of the attached independent and dependent claims.
  • the problem underlying the present invention is solved by a use of a secondary antibody and/or phospholipase-A2-receptor or a variant thereof comprising a chemiluminescent label for increasing the diagnostic reliability of an assay comprising the step detecting an autoantibody against phospholipase-A2-receptor.
  • the problem is solved by a use of a secondary antibody and/or phospho- lipase-A2-receptor or a variant thereof comprising a chemiluminescent label for the early diagnosis of an autoimmune disease, preferably a nephrological autoimmune disease, more preferably MN.
  • a kit comprising a bead comprising a secondary antibody recognizing IgG class antibodies and/or phospholipase-A2-receptor or a variant thereof, wherein the antibody and/or the phospholipase-A2-receptor comprises a chemilumi nescent label, and a calibrator defining a lower chemiluminescence detection limit of at least 4, preferably a lower limit of at least 3, more preferably a lower limit of at least 2 CU/ml, as well as one or more, preferably all from the group comprising one or more calibrator solu tions, a sample dilution buffer, a washing solution, a positive control, a negative control and a chemiluminescence trigger solution.
  • the problem is solved by a method for detection an autoantibody to phospho- lipase-A2-receptor, comprising the steps a) contacting a sample from a patient comprising antibodies with a polypeptide compris ing phospholipase-A2-receptor or a variant thereof under conditions allowing for the for mation of a complex comprising the polypeptide and an autoantibody in the sample, b) immobilizing the complex in step a) on a bead, c) separating the bead from the sample, d) washing the bead, e) contacting the bead with a secondary antibody binding to IgG class antibodies or with a polypeptide comprising phospholipase-A2-receptor or a variant thereof, wherein the secondary antibody or polypeptide is labeled with a chemiluminescent label, under condi- tions allowing for the binding of the secondary antibody or polypeptide to the complex such that it is also immobil
  • the secondary antibody recognizes class IgG, preferably class lgG4 antibodies. In a preferred embodiment the secondary antibody recognizes human antibodies.
  • the chemiluminescence of the chemiluminescent label is detect ed for 1 to 60 seconds, preferably for 2 to 20 seconds, more preferably 3 to 15 seconds following initiation of the chemiluminescent detection reaction. In another preferred embodi ment the chemiluminescence of the chemiluminescent label is detected for at least 0.5, 1, 1.5, 2, 2.5 or 3 seconds.
  • the ratio of sensitivity to specificity is increased, preferably compared to an ELISA assay.
  • the sensitivity is increased in samples that are negative accord ing to ELISA assay analysis.
  • the autoantibody binds to a bead, preferably a magnetic bead.
  • the chemiluminescent label emits a chemiluminescence signal which is detected in a detection range having a lower limit of at least 4, preferably a lower limit of at least 3, more preferably a lower limit of at least 2 CU/ml.
  • the increase of the diagnostic reliability is in a high throughput process.
  • the increase relates to samples that appear negative in an ELISA assay based on a cutoff of 20, preferably 14, more preferably two RU/ml.
  • a chemiluminescence signal is detected over a range of 250 to 600 nm, preferably 300 to 500 nm.
  • At least two samples taken at different time points are processed, wherein the period between the time points is preferably at least one month.
  • an increase of at least 1, 2, 3, 4 or 5 CU/ml is detected in a concentration window comprising the range 2.5 to 10 CU/ml.
  • the problem underlying the present invention is solved by a use of a second ary antibody and/or phospholipase-A2-receptor or a variant thereof comprising a chemilumi nescent label for increasing the diagnostic reliability of an assay comprising the step detecting an autoantibody against phospholipase-A2-receptor.
  • the problem is solved by a use of a secondary antibody and/or phospho- lipase-A2-receptor or a variant thereof comprising a chemiluminescent label for the early diagnosis of an autoimmune disease, preferably a nephrological autoimmune disease, more preferably MN.
  • a kit comprising a bead comprising a secondary antibody recognizing IgG class antibodies and/or phospholipase-A2-receptor or a variant thereof, wherein the antibody and/or the phospholipase-A2-receptor comprises a chemilumi nescent label, and a calibrator defining a lower chemiluminescence detection limit of at least 4, preferably a lower limit of at least 3, more preferably a lower limit of at least 2 CU/ml, as well as one or more, preferably all from the group comprising one or more calibrator solu tions, a sample dilution buffer, a washing solution, a positive control, a negative control and a chemiluminescence trigger solution.
  • the problem is solved by a method for the detection an autoantibody to phospholipase-A2-receptor for diagnosing MN, comprising the steps a) calibrating a phospholipase-A2-receptor autoantibody detection immunoassay at a concentration range with a lower detection limit of no more than 1.0 x A, preferably 0.75 x A, 0.5 x A, more preferably 0.25 x A, wherein A is the output value of the immunoassay corresponding to the average amount of phospholipase-A2-receptor autoantibody in a cohort of healthy control subjects, b) providing a sample comprising antibodies from a subject suspected of suffering from an autoimmune disease, preferably MN, and c) contacting the sample with a polypeptide comprising phospholipase-A2-receptor or a variant thereof, and d) detecting the autoantibody in the concentration range calibrated in step a) and converting the detection signal into a quantitative or
  • the immunoassay is selected from the group comprising ELISA, Western blot and chemiluminescence.
  • the autoantibody is bound to a bead coated with a polypeptide comprising phospholipase-A2-receptor or a variant thereof.
  • the bead is a magnetic bead.
  • the immunoassay is chemiluminescence and the concentration has a lower limit of no more than 4, no more than 3, no more than 2 or no more than 1 CU/ml.
  • the immunoassay is ELISA and the concentration range has a lower limit of no more than 1.5, no more than 1, no more than 0.6, no more than 0.5 or no more than 0.25 or is 0 RU/ml.
  • the samples is diluted 1:10 to 1:1000, preferably 1:10 to 1:500.
  • the value obtained in step d) is converted into an electronic signal and communicated as such to a recipient, preferably via an e-mail.
  • the problem is solved by a method for detection an autoantibody to phospho- lipase-A2-receptor, comprising the steps a) contacting a sample from a patient comprising antibodies with a polypeptide compris ing phospholipase-A2-receptor or a variant thereof under conditions allowing for the formation of a complex comprising the polypeptide and an autoantibody in the sam ple, b) immobilizing the complex in step a) on a bead, c) separating the bead from the sample, d) washing the bead, e) contacting the bead with a secondary antibody binding to IgG class antibodies or with a polypeptide comprising phospholipase-A2-receptor or a variant thereof, wherein the secondary antibody or polypeptide is labeled with a chemiluminescent label, under condi tions allowing for the binding of the secondary antibody or polypeptide to the complex such that it is also immobil
  • the secondary antibody recognizes human antibodies.
  • the chemiluminescence of the chemiluminescent label is detect ed for 1 to 60 seconds, preferably for 2 to 20 seconds, more preferably 3 to 15 seconds following initiation of the chemiluminescent detection reaction. In another preferred embodi ment the chemiluminescence of the chemiluminescent label is detected for at least 0.5, 1, 1.5, 2, 2.5 or 3 seconds.
  • the ratio of sensitivity to specificity is increased, preferably compared to an ELISA assay.
  • the sensitivity is increased in samples that are negative accord ing to ELISA assay analysis.
  • the autoantibody binds to a bead, preferably a magnetic bead.
  • the chemiluminescent label emits a chemiluminescence signal which is detected in a detection range having a lower limit of at least 4, preferably a lower limit of at least 3, more preferably a lower limit of at least 2 CU/ml.
  • the increase of the diagnostic reliability is in a high throughput process.
  • the increase relates to samples that appear negative in an ELISA assay based on a cutoff of 20, preferably 14, more preferably two RU/ml.
  • a chemiluminescence signal is detected over a range of 250 to 600 nm, preferably 300 to 500 nm.
  • at least two samples taken at different time points are processed, wherein the period between the time points is preferably at least one month.
  • an increase of at least 1, 2, 3, 4 or 5 CU/ml is detected in a concentration window comprising the range 2.5 to 10 CU/ml.
  • the present invention is based on the inventors’ surprising finding that using a bead-based assay format and the use of chemiluminescence for detection increases the sensitivity of the assay considerably, while the specificity is surprisingly maintained.
  • the present invention is based on the inventors’ surprising that several months before the onset of clinical MN, autoantibodies may be detected in the patients’ samples and indicate that clinical MN symptoms are likely to emerge.
  • the inventors have surprisingly found that an increase in the concentration of autoantibodies, even in the range of concentrations typically found in samples from healthy subjects, indicates the clinical MN symptoms are likely to emerge.
  • the present invention is based on the inventors’ surprising that several months before the onset of clinical MN, autoantibodies may be detected in the patients’ samples and indicate that clinical MN symptoms are likely to emerge, in particular at a low concentration previously believed to be diagnostically irrelevant.
  • the inventors have surprisingly found that an increase in the concentration of autoantibodies, even in the range of concentrations typically found in samples from healthy subjects, indicates the clinical MN symptoms are likely to emerge.
  • the chemiluminescent label is a chemiluminescent enzyme, preferably selected from the group comprising luciferase, peroxidase, alkaline phosphatase and D-galactosidase or a variant thereof, which may turn over a chemiluminescent substrate without being consumed itself (Kricka, L. J. (2003). Clinical applications of chemilumines- cence. Analyticazia acta, 500(1): 279-286).
  • the chemiluminescent label is a small organic compound having no enzymatic activity catalyzing a chemiluminescence reaction, which emits a chemiluminescence signal upon being de graded when contacted with a chemiluminescence trigger solution which comprises inorgan ic and/or non-enzymatic organic compounds that are required for emitting the signal.
  • the small organic compound having no enzymatic activity is selected from the group comprising acridinium esters (Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K., Woodhead, J. S. (1983) Acridinium esters as high specific activity labels in immunoassay.
  • the trigger solution comprises H 2 0 2 at a high pH.
  • a mixture of H 2 0 2 and sodium hydroxide is frequently used. The small organic compound is consumed upon emission of the chemiluminescence signal.
  • the term “diagnostic reliability” is the ratio of sensitivity and specificity.
  • sensitivity is defined as the proportion of samples from MN patients identified as positive. For example, if 100 patients suffer from MN and 80 of their samples are identified as positive, then the assay’s sensitivity is 80%.
  • specificity is defined as the proportion of negative results among samples from patients who do not suffer from MN, preferably control patients such as blood donors. For example, if 96 of 100 samples from healthy blood donors give negative results in an assay, then the assay’s specificity is 96%.
  • beads for numerous applications are commercially available, mainly based on carbohydrate, for example sepharose or agarose, or plastic. They may comprise active or activatable chemical groups such as a carboxyl or ester group, which can be utilized for the immobilization of a means for specifically capturing an antibody.
  • the beads are beads having an average diameter of from 0.1 pm to 10 pm, from 0.5 pm to 8 pm, from 0.75 pm to 7 pm or from 1 pm to 6 pm.
  • the beads can be coated with the means for specifically capturing an antibody directly or via affinity ligands, for example biotin or glutathione and streptavidin or GST, respectively.
  • the bead may be coated with biotin or glutathione and the antigen may be fused with streptavidin or glutathione-S-transferase or a variant thereof, respectively.
  • the bead is provided in the form of an aqueous suspension having a bead content of from 10 to 90%, preferably from 20 to 80%, preferably from 30 to 70%, more preferably from 40 to 60% (w/w).
  • lyophilized beads are provided or used. They may be resuspended in incubation buffer before contacting them with the sample.
  • the beads are paramagnetic beads, which can be easily concentrated on a surface with the aid of a magnet by applying a temporary magnetic field.
  • commercial paramagnetic beads usually contain a paramagnetic mineral, for example iron oxide.
  • a multiplicity of suitable paramagnetic beads is commercially available.
  • a bead may be labeled with a detectable label.
  • the primary output of chemiluminescence measurements are relative light units (RLU).
  • RLU relative light units
  • a chemiluminescence detection device Prior to running assays, a chemiluminescence detection device needs to be calibrated, and these calibrations may have to be repeated regularly.
  • a standard calibration curve and a set of at least two calibrators or a larger set of calibrators are required for calibrating the device.
  • the calibrated device is able to convert primary data in the form of RLU (relative light units) into a device-independent unit usually referred to as CU (chemiluminescence units), CU/ml or arbitrary units/ml or the like, which may be compared with reference data, preferably in the form of the calibration curve or a cut off value.
  • Example 2 In a cohort of healthy blood donors, the inventors found a background of an average of 3.8 CU/ml, as detailed in Example 2.
  • values in CU/ml may therefore be converted to units used by other systems by dividing them by 3.8, thus giving a value herein referred to as A, and multiplying this value with the CU/ml or CU or arbitrary units/ml or similar value used in that other system that corresponds to the average in such a cohort, preferably at least 150 individuals, of healthy blood donors.
  • the calibrator according to the present invention is a buffered liquid solution comprising an antibody binding to phospholipase-A2-receptor, which is recognized by a secondary antibody, preferably a secondary antibody binding to IgG class, more preferably lgG4 class antibodies.
  • the antibody may be a human antibody, for example a diluted sample from a patient.
  • the antibody may be a monoclonal and/or recombinant antibody.
  • the calibrator may comprise one or more, preferably all from the group comprising a buffering reagent, for example phosphate, a stabilizing agent, for example BSA, a preservative, for example azide, a detergent such as TweenTM and a dye.
  • the dye does essentially not interact with any other component, but may be used to confirm by simple visual inspection that the calibrator has been added to a mixture.
  • a set of calibrators comprising at least two calibrators may be used together with a standard calibration curve. In the absence of a calibration curve, more than two calibrators, for example three, four, five, six or more calibrators may be used.
  • the first of the at least two calibrators and the second calibrator comprise the antibody at different concentrations.
  • the first calibrator defines, when used for the calibration according to the present invention, a value in the range of 1 to 50 CU/ml, preferably 1.5 to 25 CU/ml, more preferably 2 to 20 CU/ml, more preferably 2.5 to 15 CU/ml, and for that purpose, comprises an amount of antibody to phospholipase-A2-receptor corresponding to such values.
  • the first calibrator comprises an amount of antibody and is processed such that the concentration of the antibody during the detection step equals a sample antibody concentration of 1 to 50 CU/ml, preferably 1.5 to 25 CU/ml, more preferably 2 to 20 CU/ml, more preferably 2.5 to 15 CU/ml.
  • the second calibrator comprises an amount of antibody and is processed such that the concentration of the antibody during the detection step equals a sample antibody concentration of 100 to 1500 CU/ml, preferably 150 to 1000 CU/ml, more preferably 250 to 800 CU/ml, more preferably 300 to 500 CU/ml.
  • the second calibrator defines, when used for the calibration according to the present invention, a value in the range of 100 to 1500 CU/ml, preferably 150 to 1000 CU/ml, more preferably 250 to 800 CU/ml, more preferably 300 to 500 CU/ml, and for that purpose, comprises an amount of antibody to phospholipase-A2-receptor corre sponding to such values.
  • such a calibration enables the person skilled in the art to run the assay according to the invention, detecting the phospholipase-A2-receptor autoantibody in a concentration window comprising the range 1 to 1500, preferably 1.5 to 1000, more prefera bly 2 to 750, more preferably 2 to 500 CU/ml, more preferably 2.5 to 400 CU/ml.
  • the linear detection range of the concentration window starts with a lower limit of no more than 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 CU/ml.
  • the concentration range of 1 to 20, 2 to 15 and 2 to 10 CU/ml, in particular, is included.
  • the person skilled in the art may obtain samples from a sufficiently large group, preferably comprising at least 150 individuals, of healthy control subjects, preferably blood donors, determine the average phospholipase-A2-receptor antibody concentration in CU/ml (A) and detect the phospholipase-A2-receptor autoantibody in a concentration window comprising 0.25 x A to 400 x A, more preferably 0.52 x A to 263 x A, more preferably 0.52 x A to 131.6 x A.
  • the concentra tion range of 0.25 to 5.26, 0.5 to 3.94 and 0.5 to 7.9 CU/ml, in particular, is included.
  • the linear detection range of the concentration window begins with a lower limit of no more than 1.3, 1.18, 0.92, 0.78, 0.66, 0.52, 0.39 or 0.25 x A.
  • the term “high throughput assay”, as used herein, refers to an assay which is carried out in an automated manner, wherein there is no need for a trained staff member to be involved in the recording and interpretation.
  • the data is recorded in the absence of a trained staff member and is, more preferably, a value that is within or outside a reference range, which indicates whether there is an increased likelihood that the patient suffers from or is likely to develop MN.
  • the secondary antibody and consequently the autoantibody binding to it is detected using chemiluminescence, based on the signal emitted by the chemiluminescent label of the secondary antibody, which is more preferably selected from the group comprising acridinium esters, acridinium sulfonamides, luminol and isoluminol as well as their chemiluminescent derivatives.
  • chemiluminescence based on the signal emitted by the chemiluminescent label of the secondary antibody, which is more preferably selected from the group comprising acridinium esters, acridinium sulfonamides, luminol and isoluminol as well as their chemiluminescent derivatives.
  • chemiluminescent label of the secondary antibody which is more preferably selected from the group comprising acridinium esters, acridinium sulfonamides, luminol and isoluminol as well as their chemiluminescent derivative
  • the chemiluminescent label is chosen such that it emits, upon triggering of its reaction, sufficient photons within 1 to 60, more preferably 2 to 20, more preferably 3 to 15 seconds, and this is how long the chemiluminescence is preferably detected.
  • the chemiluminescence is detected in a wavelength range that com prises 300 to 500 nm, preferably 250 to 600 nm.
  • a magnet bead is used and washed or incubated in buffer by applying a magnetic field to concentrate and immobilize the beads, following removal of the buffer present and addition of new buffer. The magnetic field may then be discontinued to make the suspension of the beads in the new buffer more efficient.
  • a buffer may be any solution used according to the present invention including a diluted patient sample, an incubation buffer or a buffer comprising a secondary antibody.
  • the emission of chemiluminescence is initiated by rapid mixing of a trigger solution with a solution comprising any washed complexes comprising phospholipase-A2-receptor and the secondary antibody comprising the chemiluminescent label.
  • the mixing begins less than 10, preferably 6, 5, 4, 3, 2, 1 or 0.5 seconds before the chemilumi nescence is detected.
  • the sample is a sample from a mammalian, preferably human patient and comprises antibodies, typically a mixture comprising both autoantibodies and other antibodies.
  • the patient may be a Caucasian patient.
  • the sample may be blood, plasma, serum or CSF and is preferably serum.
  • polypeptides in particular a polypeptide comprising the native sequence of phospholipase-A2-receptor referred to in this application explicitly, for example by function, name, sequence or acces sion number, or implicitly, but also using variants of such polypeptides or nucleic acids.
  • the term “autoantibody against phospholipase-A2-receptor”, as used herein also with exchangeable terms such as “autoantibody to phospholipase-A2- receptor” or “autoantibody binding to phospholipase-A2-receptor”, refers to a mammalian, preferably human autoantibody to phospholipase-A2-receptor, wherein phospholipase-A2- receptor is represented by the amino acid sequence in NP_001007268 or SEQ ID N01, preferably SEQ ID N01.
  • any data base codes cited refers to the Uniprot or other data base, more specifically the version available on the earliest priority date or filing date of this application.
  • phospholipase-A2-receptor or variants thereof may be provided in any form and at any degree of purification, from liquid samples, tissues or cells comprising said polypeptide in an endogenous form, more prefera bly cells overexpressing the polypeptide, crude or enriched lysates of such cells, to purified and/or isolated polypeptide which is optionally essentially pure.
  • a lysate comprising mammalian, preferably human kidney cells is used.
  • the polypeptide is a native polypeptide, wherein the term “native polypeptide”, as used herein, refers to a folded polypeptide, more preferably to a folded polypeptide purified from tissues or cells, more preferably from mammalian cells or tissues, optionally from non recombinant tissues or cells.
  • the polypeptide is a recom binant protein, wherein the term “recombinant”, as used herein, refers to a polypeptide produced using genetic engineering approaches at any stage of the production process, for example by fusing a nucleic acid encoding the polypeptide to a strong promoter for overex pression in cells or tissues or by engineering the sequence of the polypeptide itself.
  • a polypeptide is pure if at least 60, 70, 80, 90, 95 or 99 percent of the polypep tide in the respective sample consists of said polypeptide as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection.
  • the term “variant”, as used herein, may refer to at least one fragment of the full length sequence referred to, more specifically one or more amino acid or nucleic acid sequences which are, relative to the full-length sequence, truncated at one or both termini by one or more amino acids.
  • a fragment comprises or encodes for a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 successive amino acids of the original sequence or a variant thereof.
  • the total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 750, 1000 or more amino acids.
  • variant relates not only to at least one fragment, but also to a polypeptide or a fragment thereof comprising amino acid sequences that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity, for example the ability of an antigen to bind to an (auto)antibody, or the fold or structure of the polypeptide are deleted or substituted and/or one or more such essential amino acids are replaced in a conservative manner and/or amino acids are added such that the biological activity of the polypeptide is preserved.
  • the variant is a linear, non-folded polypeptide, which is optionally denatured.
  • polypeptide and variants thereof may, in addition, comprise chemical modifications, for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
  • chemical modifications for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
  • Any modification is designed such that it does not abolish the biological activity of the variant.
  • variants may also be generated by fusion with other known polypeptides or variants thereof and comprise active portions or domains, preferably having a sequence identity of at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % when aligned with the active portion of the reference sequence, wherein the term "active portion”, as used herein, refers to an amino acid sequence, which is less than the full length amino acid sequence or, in the case of a nucleic acid sequence, codes for less than the full length amino acid sequence, respectively, and/or is a variant of the natural sequence, but retains at least some of the biological activity.
  • the variant of the polypeptide has biological activity.
  • biological activity is the ability to bind specifically to a phospholipase-A2-receptor autoanti body, as found in a patient suffering from MN.
  • whether or not a variant of phospholipase-A2-receptor has such biological activity may be checked by determining whether or not the variant of interest binds to an autoantibody from a sample of a patient which autoantibody binds to wild type phospholipase-A2-receptor, preferably as determined by ELISA using the commercial assay.
  • the variant may be flanked C-terminally or N-terminally by amino acids or amino acid sequences derived from phospholipase-A2-receptor or from any other proteins which do not prevent sterical access to the phospholipase-A2-receptor binding epitope comprised by the variant, for example linkers and/or folded domains.
  • Behnert et al. have studied and mapped the epitopes of the receptor (Behnert, A., Fritzler, M. J., Teng, B., Zhang, M., Bollig, F., Haller, H., Skoberne, A., Mahler, M., and Schiffer, M.
  • the methods, reagents, kits and uses according to the present invention may be used for a diagnosis, aiding a diagnosis or preparing or examining a device that may be subsequently used for a diagnosis of a disease, preferably a nephrological autoimmune disease, more preferably MN.
  • the term ..diagnosis refers to any kind of procedure aiming to obtain information instrumental in the assessment whether a patient suffers or is likely or more likely than the average or a comparative subject, the latter preferably having similar symptoms, to suffer from a certain disease or disorder in the past, at the time of the diagnosis or in the future, to find out how the disease is progressing or is likely to progress in the future or to evaluate the responsiveness of a patient with regard to a certain treatment, for example the administration of immunosuppressive drugs, or to find out whether a sample is from such a patient.
  • Such information may be used for a clinical diagnosis, but may also be obtained by an experimental and/or research laboratory for the purpose of general research, for example to determine the proportion of subjects suffering from the disease in a patient cohort or in a population.
  • diagnosis comprises not only diagnosing, but also prognosticating and/or monitoring the course of a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or candidate drug, for example to determine its efficacy. While the result may be assigned to a specific patient for clinical diagnostic applications and may be communicated to a medical doctor or institution treating said patient, this is not necessarily the casefor other applications, for example in diagnostics for research purposes, where it may be sufficient to assign the results to an sample from an anonymized patient.
  • the methods and products according to the present invention may be used for interaction studies, including determining whether a drug candidate or other compound may interfere with the binding of an autoantibody to the NMDA receptor or may affect any downstream process or the strength of their binding to their target such as the NR1 subunit.
  • the mere detection in other words determining whether or not detectable levels of the antibody are present in the sample, is sufficient for the diagnosis.
  • autoantibodies are detectable by chemiluminescence but not by ELISA. If the autoanti body can be detected, this will be information instrumental for the clinician’s diagnosis and indicates an increased likelihood that the patient suffers from a disease.
  • detecting the phospholipase-A2-receptor autoantibody by chemiluminscence above the cut off value indicates an increased likelihood that the patient is likely to suffer from or develop MN.
  • an increase in the concentration of phospholipase-A2-receptor autoantibody by at least 1, preferably, 2, more preferably 3, more preferably 4, more preferably 5 CU/ml indicates an increased likelihood that the patient is likely to suffer from or develop MN.
  • an increase in the concentration of phospholipase-A2-receptor autoantibody by at least 0.26 x A, preferably 0.52 x A, more preferably 0.78 x A, more preferably 1.04 x A, more preferably 1.3 x A indicates an increased likelihood that the patient is likely to suffer from or develop MN.
  • the sample is derived from a subject showing no active clinical disease, which means, in a more preferred embodiment, that the 24 h urinary protein excretion, as determined by dipstick measurement, is less than 3.5 g/day, preferably less than 3 g/day, less than 2.5 g/day, less than 2 g/day, less than 1.75 g/day, less than 1.5 g/day, less than 1 g/day, less than 0.5 g/day, most preferably less than 3.5 g/day.
  • the sample is derived from a subject in spontaneous or therapeutical ly-induced remission, as evidenced by clinical picture, in particular a marked reduction in proteinuria and/or edema.
  • the sample is from a subject at the time of kidney biopsy.
  • the sample may be from a patient at risk of or suspected of developing MN.
  • the method according to the invention is carried out when the patient is in spontaneous or therapeutically-induced remission, which is preferably the case when he no longer shows the active disease.
  • the method is carried out using a sample from a healthy subject or to screen a healthy subject to determine whether he is at risk of developing MN.
  • a sample comprising antibodies from a subject suspected of suffering or to be likely to develop an autoimmune disease, preferably MN.
  • the sample is contacted with a polypeptide comprising phospholipase-A2-receptor or a variant thereof such that any complex comprising the polypeptide and the autoantibody is formed immobilized in a bead, followed by removal of the sample and washing of the bead.
  • the bead is then contacted with a secondary antibody comprising a detectable label, which preferably binds to the constant region of the autoantibody, or with a phospholipase-A2-receptor or a variant thereof comprising a detectable label, which may bind to a free binding site of the autoanti body.
  • the bead may then be washed to remove any excess of labeled polypeptide or secondary antibody, followed by detection of the autoantibody by way of detecting the detectable label.
  • the detection signal is converted to a value in a suitable unit such as CU/ml, CU, RU/ml, arbitrary unit/ml or the like.
  • the value is communicated, preferably via internet, for example in the form of an email, to a medical doctor examining and/or treating the patient who donated the sample.
  • the patient sample is diluted prior to contacting the sample with a reagent such as a bead or polypeptide comprising the phospholipase-A2-receptor or a variant thereof. More preferably, the sample is diluted 1:5 to 1:1000, preferably 1:8 to 800, 1:10 to 600 or 1:15 to 300 or 1:15 to 200.
  • the autoantibody against phospholipase-A2-receptor is detected using chemiluminescence.
  • the autoantibody captured by phospholipase- A2-receptor or a variant thereof on a bead is contacted with a secondary antibody labeled with a detectable chemiluminescent label.
  • the secondary antibody recognizes an IgG class antibody, preferably lgG4, from a mammal, preferably from a human.
  • the type of chemiluminescent label and the number of labels are chosen such that sufficient light is emitted within 1 to 60, preferably 2 to 20, more preferably 3 to 15 seconds.
  • the bead is coated with a secondary antibody which is a class IgG, preferably lgG4 antibody that binds all IgG antibodies including an autoantibody to be detected, followed by labeling of the complex using a polypeptide comprising phospholipase-A2-receptor or a variant thereof which carries a chemiluminescent label.
  • a secondary antibody which is a class IgG, preferably lgG4 antibody that binds all IgG antibodies including an autoantibody to be detected
  • a polypeptide comprising phospholipase-A2-receptor or a variant thereof which carries a chemiluminescent label.
  • phospholipase- A2-receptor or a variant thereof is coated on a bead and may bind to the autoantibody in the sample, followed by decoration of said autoantibody by a secondary antibody carrying the chemiluminescent label.
  • a first antibody to phospholipase- A2-receptor or a variant thereof is coated on a bead and binds to the autoantibody to be detected in the sample, followed by incubation with phospholipase-A2-receptor or a variant thereof, which carries the chemiluminescent label.
  • a chemiluminescence detection kit which may comprise one or more, preferably all from the group comprising a bead, preferably magnetic bead comprising phospholipase-A2-receptor or a variant thereof, a secondary antibody or polypeptide comprising phospholipase-A2-receptor or a variant thereof, comprising a chemiluminescent label, one or more calibrator solutions, each preferably comprising an antibody to phospholipase-A2-receptor at a different concentration, more preferably includ ing one calibrator solution eliciting a calibrator signal between 2 and 25, preferably 5 and 20 CU/ml and preferably one calibrator eliciting a signal of at least 100 CU/ml, preferably 150 to 500 CU/ml, a sample dilution buffer, a washing solution, a positive control, a negative control and a chemiluminescence trigger solution.
  • the kit may comprise instructions detailing how to carry out the inventive assay
  • the methods or products according to the present invention are used to predict the onset of active MN disease, preferably a relapse, more than three months, preferably 4, 5, 6, 8, 10, 12, 24, 36, 48 or 60 months before the onset.
  • the methods or products according to the present invention are used to predict the onset of active MN disease, preferably a relapse, 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48 or 60 months after the remission of MN.
  • the remission may be spontaneously or therapeutically induced.
  • the method or use according to the invention may be for confirming the reliability of an antibody detection assay or for calibrating a chemilumines cence detection device for such an assay or use according to the present invention and may involve detecting an antibody to phospholipase-A2-receptor in a solution, which is not a sample from a patient, but is known to comprise the autoantibody, preferably at a known relative or absolute concentration, preferably in two calibrator solutions, each corresponding to a set CU/ml value.
  • the solution may be a negative control not comprising the antibody to check the background.
  • Such method may be run in parallel with, after or before a diagnostic method.
  • any method or use according to the present invention may be intended for testing in vitro the efficiency of a medical device designed to remove a phospho- lipase-A2-receptor autoantibody from a patient’s blood, wherein the testing is performed on a liquid other than patient’s blood.
  • its capacity to remove autoantibody may be checked by running a solution comprising a phospholipase-A2-receptor antibody through the device, followed by use of the method according to the present invention to confirm that less or no antibody is in the solution that has been passed through the device, i.e. showing that the device still has the capacity to remove antibody from the solution.
  • the present invention provides an apparatus for analyzing a sample from a patient to detect an autoantibody against phospholipase-A2-receptor indicat ing an increased likelihood of MN or of developing MN, comprising: a.
  • a carrier which contains a means for capturing the autoantibody from the sample when the sample is contacted with the carrier, wherein the carrier is a bead
  • a detectable means capable of binding to the antibody captured by the carrier when the detectable means is contacted with the carrier, wherein the detectable means is a labeled secondary antibody capable of binding to the antibody captured on the car rier, wherein the secondary antibody is labeled with a chemiluminescent label
  • a detecting device for detecting the presence of the detectable means and converting the results into an electrical signal, preferably a luminometer, and e.
  • the linear range for the detection comprises the range 3 to 150 CU/ml, preferably 2.5 to 200, more preferably 2 to 250, more preferably 1.5 to 300.
  • the autoantibody is detected using a chemiluminescent label is a chemiluminescent enzyme, preferably selected from the group comprising luciferase, peroxidase, alkaline phosphatase and D-galactosidase or a variant thereof, which may turn over a chemiluminescent substrate without being consumed itself (Kricka, L. J. (2003). Clinical applications of chemiluminescence. Analyticazia acta, 500(1): 279-286).
  • a chemiluminescent enzyme preferably selected from the group comprising luciferase, peroxidase, alkaline phosphatase and D-galactosidase or a variant thereof, which may turn over a chemiluminescent substrate without being consumed itself (Kricka, L. J. (2003). Clinical applications of chemiluminescence. Analyticazia acta, 500(1): 279-286).
  • the chemiluminescent label is a small organic compound having no enzymatic activity catalyzing a chemiluminescence reaction, which emits a chemiluminescence signal upon being degraded when contacted with a chemiluminescence trigger solution which comprises inorganic and/or non-enzymatic organic compounds that are required for emitting the signal.
  • the small organic compound having no enzymatic activity is selected from the group comprising acridinium esters (Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K., Woodhead, J. S. (1983) Acridinium esters as high specific activity labels in immunoassay.
  • the trigger solution comprises H 2 0 2 at a high pH.
  • a mixture of H 2 0 2 and sodium hydrox ide is frequently used. The small organic compound is consumed upon emission of the chemiluminescence signal.
  • the term “calibrating” means, as used herein, that, using stand ard reagents with known relative or absolute concentrations, referred to as calibrators, an immunoassay detection system is configured such that concentration values in the concen tration range calibrated can be trusted to correspond to genuine concentrations of the analyte, preferably autoantibody, in that range in the sample. The main benefit is that such values can then be compared to reference data.
  • calibrators CAL1 Product number: LC 1254-10110 G
  • CAL2 Product number LC 1254- 10210 G
  • RA Analyzer 10 EUROIMMUN Medizinische Labordiagnostika AG
  • product number YG 0710-0101 Other reagents are available and preferably used as described in the example section.
  • test kit LA 1254-10010 G
  • cartridge LS 1254-10010 G
  • beads LM 1254-10010 G
  • labeled secondary antibody LK 0711-10010 G
  • dilution buffer LL 9511-10010
  • control set LR 1254-20210 G
  • positive control LC 1254-20110 G
  • negative control LC 1254-20910 G
  • concentration range for another type of assay needs to be defined to practice the invention, then a sample having the concentration of interest is confirmed as having this concentration by this ELISA, and the concentration of antibodies in this sample may then be determined by the other assay to yield a value in the relevant unit for such assay.
  • the present application comprises a range of sequences, more specifically:
  • SEQ ID N01 human phospholipase-A2-receptor
  • SEQ ID N02 HA-tagged human phospholipase-A2-receptor
  • Fig. 1 Anti-phospholipase-A2-receptor reactivity as determined in 155 pMN patients and in 154 disease controls using (A) ChLIA (chemiluminescence immunoassay), (B) ELISA and (C) RC-IFA (recombinant cell-based immunofluorescence assay). To avoid excessive overlap of data points at the distinct titer classes (negative, 1:10, 1:32, 1:100, 1:320, 1:1,000), the results of RC-IFA are indicated as absolute frequencies. Dashed lines repre sent the cut-off values for positivity.
  • ChLIA chemiluminescence immunoassay
  • B ELISA
  • C RC-IFA
  • Fig. 2 Venn diagram showing the correlation between ChLIA, ELISA and RC-IFA for the detection of anti-phospholipase-A2-receptor autoantibodies in a total of 309 sera (155 pMN, 154 disease controls). Percent values indicate the overall qualitative agreement between two adjacent assays.
  • Fig. 3 Correlation between anti-phospholipase-A2-receptor levels in 155 pMN patients measured by (A) ChLIA versus ELISA and (B) ChLIA versus RC-IFA. Axes are displayed in logarithmic scale. Dashed lines represent cut off values for positivity. Correlation coefficients and P-values were calculated using the Spearman’s rank correlation test.
  • Fig. 5 shows the results of chemiluminescence measurements of three samples from MN patients. The concentration of the original samples was detected by ELISA.
  • ROC receiver operating characteristics
  • Figs. 6 to 8 depict the prognosis of a relapse of MN using a sensitive detecting method based on serological determination of phospholipase-A2-receptor autoantibodies in three patients after a therapeutically-induced remission, more specifically Western blot, compared to conventional ELISA.
  • Example 1 Developing and characterizing a chemiluminescence-based immunoassay for the detection of an autoantibody against phospholipase-A2-receptor:
  • FSGS Focal segmental glomerular sclerosis
  • Systemic lupus erythematosus 34 31/3 42 (20-79) a Information on sex was not available for two pMN patients. b Information on age (at the time of blood sampling) was not available for eight pMN patients. c Among the 34 patients classified as LN class V, 28 had pure membranous LN, while six showed membranous and additional proliferative features. LN class V represents a subtype of sMN. Control samples were obtained from Tenon Hospital (Paris, France) and the Department of Medicine, Karolinska University Hospital (Stockholm, Sweden). Individual and ethical approval was not mandatory as patient data and samples were used anonymously.
  • the anti-phospholipase-A2-receptor ChLIA (EUROIMMUN GmbHtechnik Labordiagnostika AG, Lubeck, Germany) is based on magnetic beads coated with recombinant human phospholipase-A2-receptor (EUROIMMUN, product number LM 1254-10010 G), that was expressed in human embryonic kidney cells and purified as described previously. 6 The assay was performed fully automatically on a random-access analyzer (EUROIMMUN).
  • All assay reagents were contained in a reagent cartridge (LS 1254-10010 G), including phospho- lipase-A2-receptor-coated beads, acridinium ester-conjugated anti-human IgG secondary antibodies (tracer, LK 0711-10010 G), sample buffer and diluent (LL 9511-10010).
  • sample buffer and beads were transferred into a cuvette and patient sample was added at a dilution of 1:40. After 10 min at 37 °C, unbound antibodies were removed by repeated magnetic force-mediated sedimentation and washing of the beads.
  • Acridinium ester-conjugated anti-human IgG was then added and allowed to bind to the immobilized antibodies for 10 min at 37 °C.
  • the beads were sedimented and washed to remove unbound conjugate, followed by the addition of alkaline hydrogen peroxide to trigger the emission of light.
  • the luminescence output from this reaction which is directly proportional to the amount of anti-phospholipase-A2-receptor bound to the antigen-coated beads, was measured luminometrically in relative light units (RLU) over 10 sec.
  • RLU relative light units
  • the anti-phospholipase-A2-receptor ELISA and RC-IFA were per formed and evaluated as described before using the manufacturer’s cut-off values. 1.3. Statistics
  • ChLIA Clinical sensitivity and specificity were assessed in 155 biopsy-proven pMN patients and 154 disease controls, respectively.
  • the ChLIA was capable of detecting anti-phospholipase-A2- receptor autoantibodies in 16 additional patients compared to ELISA and one additional patient compared to RC-IFA.
  • the ChLIA demonstrated a higher sensitivity (83.9%) for diagnosing pMN than ELISA (73.5%) and RC-IFA (83.2%).
  • Specificity was equally high, ranging between 99.4% (ChLIA) and 100% (ELISA, RC-IFA). Only one control sample (MCD) yielded discrepant qualitative results, showing anti-phospholipase-A2-receptor reactivity exclusively by ChLIA with antibody levels only marginally above the cut-off (Table 2, Figure 1).
  • ChLIA and RC-IFA outperformed the ELISA in terms of the maximum sum of sensitivity and specificity and with regard to sensitivity at pre-defined specificities (Table 3, Fig. 4).
  • the present study investigated the diagnostic performance of a novel anti-phospholipase- A2-receptor ChLIA in comparison with the established ELISA and RC-IFA.
  • the clinical sensitivity of the ChLIA exceeded that of ELISA and RC-IFA by 10.4% and 0.7%, respective ly, at similar specificities (>99%).
  • the antiphospholipase-A2-receptor-positive rates detected by ChLIA (83.9%), ELISA (73.5%) and RC-IFA (83.2%) were equal to or higher than the prevalence data reported for different methods, such as Western blot (53.0-81.7%), RC-IFA (48.0-82.3%), ELISA (50.0-71 8%) 6 , addressable laser bead immunoassay (51.5-66.9%) 17 ' 18 and fluoroimmunoassay (71.0-89.7%).
  • Example 2 Determining the chemiluminescence background in healthy blood donors
  • Example 3 Increasing the sensitivity by considering increases well below the cut off value
  • the concentration of phospholipase-A2-receptor autoantibody was monitored over time using both ELISA and ChLIA in a cohort comprising 60 patients who had been in remission following treatment of MN, but would later one go into relapse.
  • a control group comprised seven patients with persisting proteinuria, but no increased levels of antibodies.
  • a cut off value of 10 CU/ml was disclosed as recommended by the manufacturer.
  • linearity could be detected in the range below 6 RU/ml (linear ELISA range) if the concentrations were detected using chemiluminescence, including the range between 0 to 10 CU/ml, the latter being the ChLIA cut off.
  • ChLIA has a linear range broader than the one reported for ELISA.
  • Example 5 Comparison of an assay detecting the concentration of the phospholipase- A2-receptor autoantibody at concentrations below 14 RU/ml compared to the conventional ELISA assay Western blotting
  • HA-tagged recombinant full-length human phospholipase-A2-receptor protein obtained from whole cell lysates of HEK293 cells transfected using the Lipofectamine® system according to the manufacturer’s protocol.
  • the cell lysate containing HA-tagged recombinant full-length human phospholipase-A2- receptor was prepared from approximately 5 million transfected HEK293 cells, which were lysed in 800 pL lysis-buffer (50 mM Tris pH 7.4, 150 mM NaCI, 1 mM EDTA, 1% (v/v) Triton X-100, 1x protease inhibitor), sonicated 3 x 10 sec and incubated for 1 hour at 4 °C at 12 rpm on a lab rotator. The cell debris was removed by centrifugation for 15 min at 4 °C at 14000 g (HERAEUS Fresco 21).
  • Blocking buffer was prepared in a sterile bottle in order to avoid even small amounts of bacterial or fungal contaminations, since due to the long incubation time of the following immunoblotting steps they could have a negative effect on the sensitivity of the experiment. 3.5% (w/v) skimmed milk powder was dissolved in PBS + 0.1% (v/v) Tween-20 and left stirring for 1 hour at room temperature. Then the solution was filtered through a sterile 100 pm cell strainer to remove little milk particles. The final blocking buffer was stored at 4°C until use (maximum 2 days).
  • the PVDF membrane was blocked with 7 ml_ blocking buffer for 1-2 hours at room tempera ture on a vertical shaker at 17 rpm in a container.
  • the speed and volume ensured an optimal floating without bumping on the edges of the container, which would lead to false signals on the membrane brinks and increase the background.
  • the membrane pieces were first numbered with a pencil and then cut within the marker lane using disinfected scissors, resulting in 8 membrane strips which can be analyzed using different MN patient sera.
  • Each strip was quickly dipped into washing buffer (PBS with 0.1% Tween 20) in order to remove excessed milk and then transferred into the MN patient serum (10 mL of 1:100 diluted in 0.05% (w/v) skimmed milk powder in PBS + 0.1% (v/v) Tween-20; if the serum was strongly hemolytic, a 1:200 dilution was used) of interest, which were each propounded in 10 cm Petri dishes.
  • washing buffer PBS with 0.1% Tween 20
  • Figure 1 6 membrane strips were incubated in MN patient serum of interest, one strip into a positive control and one strip into a negative control. The samples were incubated for a minimum of 18-20 hours at 4 °C and 15 rpm on a vertical shaker.
  • the membrane strips were washed 4x with 10-15 ml_ washing buffer for 5 minutes each on a vertical shaker at 15 rpm.
  • the membrane stripes were then transferred to Petri dishes with secondary antibody (5 ml_ of 1:24.000 dilution in blocking buffer) and incubated for 1.5 hours at room temperature on a vertical shaker at 15 rpm. Then the membrane strips were washed 4x with 10-15 ml_ washing buffer for 5 min each on a vertical shaker at 15 rpm.
  • a piece of parafilm (12x10 cm) was placed onto a clean plastic lid in order to generate a hydrophobic area.
  • ECL-Clarity was pre-mixed immediately before use.
  • the lower edge of the membrane strip was quickly dipped onto a paper tower to remove access liquid and then placed on the parafilm area.
  • the resulting re-assembled whole PVDF membrane was covered with 800 pl_ ECL-Clarity and incubated for 5 min in the dark.
  • the individual membrane strips were picked up with tweezers, quickly dipped on a paper towel to remove access liquid and then arranged without air bubbles between the sheets of a transparent plastic bag in order to keep it wet during the imaging process in a Luminescent Detection Imager 600.
  • the final exposure time was dependent on the serum of interest. Precisely, end points of detection were either a well-defined phospholipase-A2-receptor-specific band, a development time of up to 15 minutes or a high background signal, whichever presented first. For MN patient sera not giving any detectable signal, but having low background, the development step was repeated after a 2 min wash in washing buffer with Super Signal West Femto (Biorad) until the same endpoints.
  • Serum samples from eight patients suffering from a relapse following therapy-induced remission were taken at various time points before the relapse.
  • Figs. 6 to 8 depict the prognosis of a relapse of a MN using a sensitive detecting method based on serological determination of phospholipase-A2-receptor autoantibody in three patients after a therapeutically-induced remission, more specifically Western blot, compared to conventional ELISA.
  • phospholipase-A2-receptor antibodies could be detected using Western blotting fifteen months earlier than using ELISA prior to a relapse.
  • detection of the antibody could be performed nine or three months earlier, respectively.

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Abstract

La présente invention concerne l'utilisation d'un anticorps secondaire comprenant un marqueur chimio-luminescent pour augmenter la fiabilité diagnostique d'un dosage comprenant l'étape de détection d'un auto-anticorps contre le récepteur de la phospholipase-A2, et d'un kit A comprenant une perle comprenant un anticorps secondaire qui reconnaît les anticorps de la classe IgG et/ou le récepteur de la phospholipase-A2 ou une variante de celui-ci, l'anticorps et/ou le récepteur de la phospholipase-A2 comprenant un marqueur chimio-luminescent, et un calibreur qui définit une limite de détection inférieure de la chimioluminescence d'au moins 4, de préférence une limite inférieure d'au moins 3, plus préférablement une limite inférieure d'au moins 2 CU/ml, ainsi qu'un ou plusieurs, de préférence la totalité des éléments du groupe comprenant une ou plusieurs solutions de calibreur, un tampon de dilution d'échantillon, une solution de lavage, un témoin positif, un témoin négatif et une solution de déclenchement de la chimio-luminescence.
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