EP4010704A1

EP4010704A1 - An improved autoantibody detection assay

Info

Publication number: EP4010704A1
Application number: EP20749923.7A
Authority: EP
Inventors: Cornelia DÄHNRICH; Sarah KONITZER
Original assignee: Euroimmun Medizinische Labordiagnostika AG
Current assignee: Euroimmun Medizinische Labordiagnostika AG
Priority date: 2019-08-06
Filing date: 2020-08-06
Publication date: 2022-06-15
Also published as: WO2021023836A1

Abstract

The present invention relates to the use of a secondary antibody comprising a chemiluminescent label for increasing the diagnostic reliability of an assay comprising the step detecting 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.

Description

An improved autoantibody detection assay

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 (MN) 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

20-25% of patients) usually have an excellent clinical outcome. On the other end of the disease spectrum, about 20% of patients experience ESRD over a time course of ten years, often despite immunosuppressive therapy. The third group of patients presents with persist ing, in most cases more moderate levels of proteinuria and stable renal function.

It is difficult to tailor a therapy depending on the individual patient’s needs. If a patient belongs to the first group with spontaneous remission, any medication, usually the admin istration of immunosuppressive drugs, will see him exposed to the considerable side effects of such treatment even though no such treatment was necessary as could be concluded with hindsight. By contrast, a patient belonging to group 3 may benefit from the administration of strong immunosuppressive drugs at an early stage of the treatment. Therefore, it has been an accepted clinical strategy to simply wait and watch how the clinical disease activity will develop under supportive therapy and to consider, if required, more stringent therapy options during the follow-up time.

In any event, it would be desirable to recognize as early as possible the disease in its early stages and how the course of the disease will develop. For example, it may be possible to subject a patient without or with mild symptoms to treatment with low-dose immunosuppres sive drugs at an early stage and watch whether he responds positively to such treatment without developing clinical disease activity, thus potentially saving the patient from exposure to harsh immunosuppressive treatment with severe side effects.

The discovery of autoantibodies to phospholipase-A2-receptor has paved the way to a diagnosis of the disease based on serology (Beck, L, Bonegio, R. G., Lambeau, G., Beck, D. M., Powell, D. W., Cummins, T. D., Klein J. B., Salant, D. J. (2009) N. Engl. J. Med. 361(1), 11-21). Autoantibodies to THSD7A may also be detected (US10107810), but the prevalence is lower.

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. It is contemplated that 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, Damoiseaux, 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).

While 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.

In a first aspect, 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.

In a 2^nd aspect, 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.

In a 3^rd aspect, the problem is solved by 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. In a 4^th aspect, 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 immobilized on the bead, f) separating the complex and the immobilized polypeptide or secondary antibody from any excess polypeptide or secondary antibody that is not bound to the bead, g) washing the bead, h) rapidly mixing the bead with a triggering solution initiating emission of chemilumines cence, i) detecting the chemiluminescence in the form of a signal, j) optionally converting the signal to a value in a device-independent unit, and k) optionally comparing the value to a reference value to obtain a diagnostically relevant result, and

L) optionally communicating the value and/or the diagnostically relevant result to the patient or the medical doctor treating the patient who donated the sample, preferably via internet, fax or telephone.

In a preferred embodiment the secondary antibody recognizes class IgG, preferably class lgG4 antibodies. In a preferred embodiment the secondary antibody recognizes human antibodies.

In a preferred embodiment 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.

In a preferred embodiment the ratio of sensitivity to specificity is increased, preferably compared to an ELISA assay.

In a preferred embodiment the sensitivity is increased in samples that are negative accord ing to ELISA assay analysis. In a preferred embodiment the autoantibody binds to a bead, preferably a magnetic bead.

In a preferred embodiment 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.

In a preferred embodiment the increase of the diagnostic reliability is in a high throughput process.

In a preferred embodiment 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. In a preferred embodiment a chemiluminescence signal is detected over a range of 250 to 600 nm, preferably 300 to 500 nm.

In a preferred embodiment at least two samples taken at different time points are processed, wherein the period between the time points is preferably at least one month. In a preferred embodiment 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.

In a 5^th aspect, 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.

In a 6^th aspect, 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. In a 7^th aspect, the problem is solved by 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.

In a 8^th aspect, 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 semi-quantitative value.

In a preferred embodiment, the immunoassay is selected from the group comprising ELISA, Western blot and chemiluminescence. In a preferred embodiment, the autoantibody is bound to a bead coated with a polypeptide comprising phospholipase-A2-receptor or a variant thereof.

In a preferred embodiment, the bead is a magnetic bead. In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, the samples is diluted 1:10 to 1:1000, preferably 1:10 to 1:500. In a preferred embodiment, the value obtained in step d) is converted into an electronic signal and communicated as such to a recipient, preferably via an e-mail. In a 9^th aspect, 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 immobilized on the bead, f) separating the complex and the immobilized polypeptide or secondary antibody from any excess polypeptide or secondary antibody that is not bound to the bead, g) washing the bead, h) rapidly mixing the bead with a triggering solution initiating emission of chemilumines cence, i) detecting the chemiluminescence in the form of a signal, j) optionally converting the signal to a value in a device-independent unit, and k) optionally comparing the value to a reference value to obtain a diagnostically relevant result, and I) optionally communicating the value and/or the diagnostically relevant result to the patient or the medical doctor treating the patient who donated the sample, preferably via internet, fax or telephone. In a preferred embodiment the secondary antibody recognizes class IgG, preferably class lgG4 antibodies.

In a preferred embodiment the secondary antibody recognizes human antibodies.

In a preferred embodiment the ratio of sensitivity to specificity is increased, preferably compared to an ELISA assay. In a preferred embodiment the sensitivity is increased in samples that are negative accord ing to ELISA assay analysis.

In a preferred embodiment the autoantibody binds to a bead, preferably a magnetic bead.

In a preferred embodiment 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. In a preferred embodiment a chemiluminescence signal is detected over a range of 250 to 600 nm, preferably 300 to 500 nm. In a preferred embodiment at least two samples taken at different time points are processed, wherein the period between the time points is preferably at least one month.

In a preferred embodiment 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.

Furthermore, 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.

Furthermore, 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.

Furthermore, 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.

In a preferred embodiment, 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. Analytica chimica acta, 500(1): 279-286). In another preferred embodiment, 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. Preferably, 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. Clin Chem 29: 1474-1479) and luminol or a chemiluminescent derivative thereof such as isoluminol. Such small organic compounds may be coupled to the secondary antibody. In the case of luminol, the trigger solution comprises H₂0₂ at a high pH. In the case of an acridini um ester, a mixture of H₂0₂ and sodium hydroxide is frequently used. The small organic compound is consumed upon emission of the chemiluminescence signal.

In a preferred embodiment, the term “diagnostic reliability” is the ratio of sensitivity and specificity. In a preferred embodiment, the term “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%. In a preferred embodiment, the term “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%.

Various 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. Preferably, 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. For example, 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. Preferably, 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). In another preferred embodiment, lyophilized beads are provided or used. They may be resuspended in incubation buffer before contacting them with the sample.

In a particularly preferred embodiment, 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. For this purpose, 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). 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.

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.

In a preferred embodiment, 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.

Preferably, 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. In addition to the 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 Tween™ 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.

According to the present invention, 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. In a preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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. In another preferred embodiment, 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. In another preferred embodiment, the concentration range of 1 to 20, 2 to 15 and 2 to 10 CU/ml, in particular, is included. In more general terms, 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. In another preferred embodiment, 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. In another preferred embodiment, 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.

In a preferred embodiment, 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. In particular, 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.

In a preferred embodiment, 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. The person skilled in the art is familiar with such compounds and how to attach them to proteins (I Weeks, I Beheshti, F McCapra, A K Campbell, J S Woodhead. (1983) Clin Chem 29: 1474-1479; WO2012/028167). In a preferred embodiment, 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. Preferably the chemiluminescence is detected in a wavelength range that com prises 300 to 500 nm, preferably 250 to 600 nm.

In a preferred embodiment, 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. In a preferred embodiment, the mixing begins less than 10, preferably 6, 5, 4, 3, 2, 1 or 0.5 seconds before the chemilumi nescence is detected. In a preferred embodiment, 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.

The teachings of the present invention may not only be carried out using 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.

In a preferred embodiment, 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. Throughout this application, 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.

When used to carry out the teachings of the present invention, 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. In a preferred embodiment, a lysate comprising mammalian, preferably human kidney cells is used. In a preferred embod iment, 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. In another preferred embodiment, 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. The person skilled in the art is familiar with methods for engineering nucleic acids and polypep tides encoded (for example, described in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning - an introduction, Chapman & Hall) and for producing and purifying native or recombinant polypeptides (for example Handbooks ..Strategies for Protein Purification", ..Antibody Purification", ..Purifying Challenging Proteins" (2009/2010), published by GE Healthcare Life Sciences, and in Burgess, R. R., Deutscher, M. P. (2009), Guide to Protein Purification). In a preferred embodiment, 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.

In a preferred embodiment, 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. Such 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.

The term "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 state of the art compris es various methods that may be used to align two given nucleic acid or amino acid sequenc es and to calculate the degree of identity, see for example Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3^rd edition. In a preferred embodiment, the ClustalW software (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947- 2948) is used using default settings. When designing variants, the location of epitopes reactive with phospholipase-A2-receptor autoantibodies must be considered, which is published in Fresquet, M., Jowitt, T. A., Gummadova, J., et al. (2015) J. Am. Nephrol., 26:302-313 and in Kao, L, Lam, V., Waldman, M. et al. (2015) J. Am. Soc. Nephrol., 26, 291-301. In a preferred embodiment, the variant is a linear, non-folded polypeptide, which is optionally denatured.

In a preferred embodiment, the 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. The person skilled in the art is familiar with methods to modify polypeptides. Any modification is designed such that it does not abolish the biological activity of the variant.

Moreover, 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. In a preferred embodiment, such biological activity is the ability to bind specifically to a phospholipase-A2-receptor autoanti body, as found in a patient suffering from MN. For example, 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. In a preferred embodiment, 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. (2013) (PLOS, 8 (4) e61669), and their results, in particular the epitopes in Table 1 and Fig. 2, may be used to guide the design of variants. Additional guidance and suitable variants may be found in US2019183969 AA and US20170137490. 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. In a preferred embodiment, the term ..diagnosis", as used herein, 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.. In other words, the term “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.

In a preferred embodiment, 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.

In many cases 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. Preferably, such 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. In a more preferred embodiment, 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. In another preferred embodiment, 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. In another preferred embodiment, 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.

In a preferred embodiment, 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. In a preferred embodiment, 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. In another preferred embodiment, 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. In a preferred embodiment, 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. Alternatively, 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.

In a preferred embodiment, 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. In a preferred embodiment, 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. In a preferred embodiment, 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.

In a preferred embodiment, the autoantibody against phospholipase-A2-receptor is detected using chemiluminescence. For this purpose, 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. In a preferred embod iment, 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.

Various assay formats are within the scope of protection. In a preferred embodiment, 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. In another preferred embodiment, 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. In another preferred embodiment, 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.

According to the present invention, a chemiluminescence detection kit is provided 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In another preferred embodiment, 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. Alternatively, 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.

In a preferred embodiment, 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. After the use of the medical device with a patient, 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. Alternatively, from a batch comprising a large number of devices, a small number of devices may be tested for confirming or testing the quality of the entire batch in the sense of quality control, wherein the sample or solution may comprise a known concentration of phospholipase-A2-receptor antibody. The sensitivity of the inventive method based on the detection using chemiluminescence and radioactivity may help detect a deteriorating efficiency or binding activity of the device at an earlier stage. In a preferred embodiment, 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, b. 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, c. optionally a means for removing any sample from the carrier and the detectable means, preferably by washing; d. a detecting device for detecting the presence of the detectable means and converting the results into an electrical signal, preferably a luminometer, and e. optionally a means for receiving the electronical signal from the detecting device and determining if the level of the signal is indicative of an increased likelihood of having or developing MN, by comparing with the level of signal detected in the background or an input reference value obtained with samples from healthy subjects or by com paring the level of signal obtained with one sample with the level of signal obtained with a second sample obtained at a later time point, preferably at least one month later.

In a method, use or device according to the present invention, the linear range for the detection, wherein preferably signals are detected, in CU/ml, comprises the range 3 to 150 CU/ml, preferably 2.5 to 200, more preferably 2 to 250, more preferably 1.5 to 300.

In a preferred embodiment, 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. Analytica chimica acta, 500(1): 279-286). In another preferred embodiment, 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. Preferably, 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. Clin Chem 29: 1474-1479) and luminol or a chemiluminescent derivative thereof such as isoluminol. Such small organic compounds may be coupled to the secondary antibody. In the case of luminol, the trigger solution comprises H₂0₂ at a high pH. In the case of an acridinium ester, a mixture of H₂0₂ and sodium hydrox ide is frequently used. The small organic compound is consumed upon emission of the chemiluminescence signal.

In a preferred embodiment, 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.

For the definition and determination of CU/ml values according to the present invention, calibrators CAL1 (Product number: LC 1254-10110 G) and CAL2 (Product number LC 1254- 10210 G) from EUROIMMUN Medizinische Labordiagnostika AG are preferably used, more preferably on a 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. They include 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) and negative control (LC 1254-20910 G). If the 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 chemiluminescence assay, 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. As no international reference serum exists for PLA2R autoantibodies, a variety of assays are reported in the state of the art, the results of which cannot be directly compared. However, the ELISA by EUROIMMUN Medizinische Labordiagnostika AG (Lubeck, Germany) is the most frequently used assay and is commercially available (“Anti-PLA2R-ELISA”, Product number EA 1254-9601 G), hence all absolute concentration values referred to throughout this document in relative units (RU, “RE” in German publications) are determined using this assay according to the manufacturer’s instructions. The assay is straightforward to use and may be used to carry out the invention or to determine whether an embodiment is within the scope of protection of the present invention. If the 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):

MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLEN

CKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMIT

GPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQY

NHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICY

QFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGW

QWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDH

EIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEF

LVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEG

HWKVKNCEERLFYICKKAGHVLSDAESGCQEGWERHGGFCYKIDTVLRSFDQASSGYYCP

PALVTITNRFEQAFITSLISSVVKM KDSYFWIALQDQNDTGEYTWKPVGQKPEPVQYTHWN

THQPRYSGGCVAMRGRHPLGRWEVKHCRHFKAMSLCKQPVENQEKAEYEERWPFHPCY

LDWESEPGLASCFKVFHSEKVLMKRTWREAEAFCEEFGAHLASFAHIEEENFVNELLHSKF

NWTEERQFWIGFNKRNPLNAGSWEWSDRTPVVSSFLDNTYFGEDARNCAVYKANKTLLPL

HCGSKREWICKIPRDVKPKIPFWYQYDVPWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDL

LTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVN

NQSQRCGFISSITGLWGSEECSVSMPSICKRKKVWLIEKKKDTPKQHGTCPKGWLYFNYKC

LLLNIPKDPSSWKNWTHAQHFCAEEGGTLVAIESEVEQAFITMNLFGQTTSVWIGLQNDDY

ETWLNGKPVVYSNWSPFDIINIPSHNTTEVQKHIPLCALLSSNPNFHFTGKWYFEDCGKEGY

GFVCEKMQDTSGHGVNTSDMYPMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSIT

DQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFAD

SNGRWHSTACESFLQGAICHVPPETRQSEHPELCSETSIPWI KFKSNCYSFSTVLDSMSFE

AAHEFCKKEGSNLLTIKDEAENAFLLEELFAFGSSVQMVWLNAQFDGNSK

SEQ ID N02 (HA-tagged human phospholipase-A2-receptor):

MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLEN

CKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMIT

GPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQY

NHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICY

QFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGW

QWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDH

EIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEF

LVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEG

HWKVKNCEERLFYICKKAGHVLSDAESGCQEGWERHGGFCYKIDTVLRSFDQASSGYYCP

PALVTITNRFEQAFITSLISSVVKM KDSYFWIALQDQNDTGEYTWKPVGQKPEPVQYTHWN

THQPRYSGGCVAMRGRHPLGRWEVKHCRHFKAMSLCKQPVENQEKAEYEERWPFHPCY

LDWESEPGLASCFKVFHSEKVLMKRTWREAEAFCE EFGAHLASFAHIEEENFVNELLHSKF NWTEERQFWIGFNKRNPLNAGSWEWSDRTPVVSSFLDNTYFGEDARNCAVYKANKTLLPL

HCGSKREWICKIPRDVKPKIPFWYQYDVPWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDL

LTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVN

NQSQRCGFISSITGLWGSEECSVSMPSICKRKKVWLIEKKKDTPKQHGTCPKGWLYFNYKC

LLLNIPKDPSSWKNWTHAQHFCAEEGGTLVAIESEVEQAFITMNLFGQTTSVWIGLQNDDY

ETWLNGKPVVYSNWSPFDIINIPSHNTTEVQKHIPLCALLSSNPNFHFTGKWYFEDCGKEGY

GFVCEKMQDTSGHGVNTSDMYPMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSIT

DQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFAD

SNGRWHSTACESFLQGAICHVPPETRQSEHPELCSETSIPWI KFKSNCYSFSTVLDSMSFE

AAHEFCKKEGSNLLTIKDEAENAFLLEELFAFGSSVQMVWLNAQFDGNNETIKWFD GTPTD

QSNWGIRKPDTDYFKPHHCVALRIPEGLWQLSPCQEKKGFICKMEADIHTAEALPEKGPSH

SIIPLAVVLTLIVIVAICTLSFCIYKHNGGFFRRLAGFRNPYYPATNFSTVYLEENILISDLEKSD

QYPYDVPDYA

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.

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. 4. Assay comparison using receiver operating characteristics (ROC) curve analysis for the discrimination between pMN patients (n=155) and disease controls (n=154). The diagonal line indicates no discrimination (area under the curve: 0.5). Fig. 5 shows the results of chemiluminescence measurements of three samples from MN patients. The concentration of the original samples was detected by ELISA.

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:

1. Materials and Methods 1.1. Patients and samples The study included 155 serum samples from pMN patients referred to the Department of Nephrology and Dialysis at Tenon Hospital (Paris, France). The clinical diagnosis of pMN was supported by histopathology of kidney biopsy in the absence of associations suggestive of secondary MN. Moreover, 154 disease control sera were collected from patients with other biopsy-proven glomerular diseases and systemic autoimmune disorders (Table 1).

Table 1. Patient characteristics.

Primary MN (pMN) 155 39/114^a 55 (18-85)^b

Disease controls 154 110/44 42 (18-88)

Secondary MN (sMN) 6 3/3 55 (41-61)

IgA nephro-pathy (IgAN) 10 3/7 47 (20-85)

Focal segmental glomerular sclerosis (FSGS) 10 2/8 54 (26-84)

Membranoprolifierative glomerulonephritis (MPGN) 10 4/6 56 (23-88) Minimal change disease (MCD) 17 10/7 42 (18-87)

Lupus nephritis (LN) class l-IV 33 29/4 36 (18-79)

Lupus nephritis (LN) class V^c 34 28/6 37 (18-62)

Systemic lupus erythematosus (SLE) 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.

1.2. Immunoassays The anti-phospholipase-A2-receptor ChLIA (EUROIMMUN Medizinische 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). Within the device, 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. Using a predefined lot-specific master curve and integrating the results of two calibrators, the system generated a standard curve adapted to the device in use. Based on this standard curve, the results were automati cally converted from RLU into chemiluminescent units per milliliter (CU/ml). In accordance with the manufacturer’s recommendations, results ³10 CU/ml were considered as positive.

The anti-phospholipase-A2-receptor ELISA and RC-IFA (both EUROIMMUN) were per formed and evaluated as described before using the manufacturer’s cut-off values. 1.3. Statistics

Data were evaluated statistically using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). Confidence intervals (95% Cl) were calculated according to the modified Wald method. To examine the discriminatory ability of the assays, receiver-operating characteris- tics (ROC) curve analysis was carried out. Cohen’s kappa test was performed to analyze the agreement between portions, with kappa (K) values corresponding to almost perfect (0.81- 1.00), substantial (0.61-0.80), moderate (0.41-0.60), fair (0.21-0.40), slight (0.01-0.20), and no (£0) agreement. Spearman’s rank correlation test was used to determine the degree of correlation between assays. P-values <0.05 were considered significant.

2. Results

2.1. Diagnostic performance characteristics of ChLIA, ELISA and RC-IFA

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. Thus, 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).

Table 2. Clinical sensitivity and specificity of the anti-phospholipase-A2-receptor ChLIA, ELISA and RC-IFA. pMN 155 130 114 129 sMN 6 0 0 0

IgAN 10 0 0 0

FSGS 10 0 0 0

MPGN 10 0 0 0

MCD 17 1 0 0

LN l-V 33 0 0 0 LN V 34 0 0 0

SLE 34 0 0 0 a Cut-off recommended by the manufacturer. b Borderline results (³14 to <20 RU/ml) were considered as negative.

Among the 41 pMN samples that tested negative by ELISA, anti-phospholipase-A2-receptor reactivity was detectable by ChLIA and RC-IFA in 16 (39.0%) and 15 (36.6%) cases, respectively, the majority yielding results in the low to moderate positive range (Table 3).

Table 3. Reactivity in anti-phospholipase-A2-receptor ChLIA and RC-IFA among 41 pMN samples that tested negative by anti- phospholipase-A2-receptor ELISA.

1 < 2 2.179 Negative

2 < 2 2.212 Negative

3 < 2 2.254 Negative

4 < 2 2.301 Negative

5 < 2 2.303 Negative

6 < 2 2.394 Negative

7 < 2 2.416 Negative

8 < 2 2.512 Negative

9 < 2 2.520 Negative¹⁵

10 3.564 2.613 Negative 11 < 2 2.628 Negative 12 < 2 2.684 Negative

13 < 2 2.754 Negative

14 < 2 2.807 Negative

15 < 2 2.836 Negative

16 < 2 3.840 Negative

17 < 2 4.406 Negative

18 < 2 4.932 Negative

19 4.313 6.731 Negative

20 2.441 9.849 Negative 21 3.058 22 6.004

23 5.522

24 9.683

25 < 2 2.875 Negative

26 < 2 5.698 Negative

27 < 2 5.910 Negative

28 2.853 6.278 Negative

29 2.225 7.930 Negative

30 2.631

31 6.138

N positive 0 16 15 ^a Borderline results (³14 to <20 RU/ml) were considered as negative. ^b Sample positive by RC-IFA for autoantibodies against thrombospondin type 1 domain containing 7A (THSD7A), a podocyte membrane antigen that has been identified as second target of autoantibodies in 2-5% of pMN cases (Tomas NM, Hoxha E, Reinicke AT, et al. Autoantibodies against thrombospondin type 1 domain-containing 7A induce MN. J Clin Invest 2016;126:2519-2532).

ROC curve analysis revealed high areas under the curve for ChLIA (0.899), ELISA (0.927) and RC-IFA (0.916), indicating similar discrimination between pMN patients and disease controls. 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). 2.2. Correlation between ChLIA and ELISA

High overall concordance was found between qualitative anti-phospholipase-A2-receptor results obtained by ChLIA and ELISA, as reflected by an agreement of 94.5% (95% Cl: 91.3- 96.6%) and a k-value of 0.885 (95% Cl: 0.833-0.938). 114 samples (all pMN) were positive and 178 (25 pMN, 153 controls) negative by both methods (Figure 2.). 17 samples (16 pMN, one MCD) yielded discrepant results, i.e. all of them reacted positively by ChLIA, while ELISA reactivity was in the borderline range in four cases and negative in 13 cases. Spear man’s rank correlation analysis revealed a significant correlation between both assays (r=0.978, 95% Cl: 0.969-0.984, P0.001; Figure 3A). 2.3. Correlation between ChLIA and RC-IFA

The anti-phospholipase-A2-receptor ChLIA and RC-IFA yielded concordant results in 99.4% (95% Cl: 97.5-100%) cases, with a k-value of 0.987 (95% Cl: 0.968-1.000), indicating almost perfect agreement. 129 samples (all pMN) were positive and 178 (25 pMN, 153 controls) negative by both methods (Figure 2). There were only two samples (one pMN, one MCD) with divergent qualitative results, both showing low positive ChLIA reactivity while RC-IFA was negative. The Spearman’s rank coefficient indicated strong correlation between ChLIA results and RC-IFA titers (r=0.894, 95% Cl: 0.856-0.923, P0.001 ; Figure 3B).

3. Discussion

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

Using the methodology described in Example 1, samples from a cohort of 202 blood donors were examined. An average of 3.80 ± 2.40 CU/ml (average ± standard deviation) was detected, well below the cut-off of 10 CU/ml. Almost 25% of the blood donors had a value between 4.0 and 10.0 CU/ml, below the cut-off, but above the average.

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.

Two or three time points, each typically with a distance of several weeks to months, were taken. The ChLIA measurements were carried out as described in Example 1 , the ELISA was carried out using the commercial product by EUROIMMUN Medizinische Labordiagnos- tika AG. However, the cut off of 2 RU/ml proposed by Timmermanns et al. was used for the ELISA rather than the cut off of 20 RU/ml according to the manufacturer’s instructions. In 7 out of 60 patients, the (re-)detection of phospholipase-A2-receptor autoantibody in the above cut off range was preceded by an increase in the autoantibody below the ChLIA cut off range of 10 CU/ml, typically between 2 and 9 CU/ml. No such increase could be detected in the control group. In three of the 7 patients, the increase below the cut off could not be detected by ELISA, since all values obtained before the detection were below the ELISA cut off of 2 RU/ml. In three other cases, the first value by ELISA was below 2 RU/ml and the second in the range of 2 and 3 RU/ml, which is still easy to miss and well below the linear ELISA range, which, according to the manufacturer, is reliably linear only from 6 RU/ml on. Overall, detection of the autoantibody concentration by chemiluminescence at a concentra tion range including values well below the chemiluminescence cut off indicated the relapse in several patients well before the autoantibody could be detected either by ChLIA or ELISA at values above the respective cut off value. Example 4: Increased analytical reliability as shown by linearity between antibody concentrations and chemiluminescence units well below the cut off value.

A cut off value of 10 CU/ml was disclosed as recommended by the manufacturer. We prepared a series of diluted solutions comprising autoantibodies to phospholipase-A2- receptor from three independent patient samples and detected the concentration using the methodology as described in Example 1. The original concentrations were detected by ELISA.

Surprisingly a 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.

Overall, the 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 (SEQ ID N02) 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).

4-15% Mini-PROTEAN^® TGX™ Precast Protein Gels from Biorad were used to separate proteins. The protein transfer was performed according to the manufacturer’s instructions.

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. In general the following setup was used (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.

After incubation with human MN serum 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.

Buffer Recipes

All buffers were prepared in Aqua B. Braun distilled water.

Lysis-buffer (home made):

50 mM Tris (Base) pH 7.4 150 mM NaCI 1 mM EDTA 1% (v/v) Triton X-100 1x Proteaseinhibor 5x Laemmli, non-reducing:

300 mM Tris HCI pH 6.8 50% (v/v) Glycerin 10% (w/v) SDS A trace of Bromophenolblau

SDS-PAGE running buffer:

1x TG SDS buffer 1x transfer buffer (home made):

25 mM Tris (Base)

192 M Glycine 20% (v/v) Methanol Blocking buffer: 3.5% (w/v) skimmed milk powder in PBS

0.1% (v/v) Tween-20

Secondary Antibody

Devices Patients:

Serum samples from eight patients suffering from a relapse following therapy-induced remission were taken at various time points before the relapse.

Results:

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.

More specifically, in the case of patient 1, phospholipase-A2-receptor antibodies could be detected using Western blotting fifteen months earlier than using ELISA prior to a relapse. In the case of patient 2 and 3, detection of the antibody could be performed nine or three months earlier, respectively.

A group of five patients after a therapeutically induced remission who did not go through a relapse was also monitored, but no increase in phospholipase-A2-receptor autoantibody levels could be detected, showing that the increase is associated with an increased risk of a relapse.

In the case of all 3 patients proteinuria was observed as part of a relapse, which followed the detection and concentration increase of phospholipase-A2-receptor autoantibodies.

Comparison of ELISA and Western blotting shows that the latter method and other more sensitive methods may be used to resolve increases in concentration well below 14 RU/ml if optimized for this purpose. For example, in patient 2, the Western blot shows the absence of autoantibody 63 months after the MN diagnosis, but a clear band 9 months later and a noticeably stronger band (approximately at least 100% stronger) another 3 months later. At the same time, there is hardly a change in the phospholipase-A2-receptor autoantibody levels as detected by ELISA. In fact, the ELISA in indicates a small decrease in the concentration between the first two samples, while Western blotting shows that the concentration actually increases.

Claims

1. 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 as say comprising the step detecting in a sample an autoantibody against phospholipase- A2-receptor.

2. A use of a secondary antibody and/or phospholipase-A2-receptor or a variant thereof comprising a chemiluminescent label for the early diagnosis of an autoimmune dis ease, preferably a nephrological autoimmune disease, more preferably MN.

3. A method for detection an autoantibody to phospholipase-A2-receptor, comprising the steps a) contacting a sample from a patient comprising antibodies with a polypeptide comprising phospholipase-A2-receptor or a variant thereof under conditions al- lowing for the formation of a complex comprising the polypeptide and an autoan tibody 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 chemilumines cent label, under conditions allowing for the binding of the secondary antibody or polypeptide to the complex such that it is also immobilized on the bead, f) separating the complex and the immobilized polypeptide or secondary antibody from any excess polypeptide or secondary antibody that is not bound to the bead, g) washing the bead, h) rapidly mixing the bead with a triggering solution initiating emission of chemilu minescence, i) detecting the chemiluminescence in the form of a signal, j) optionally converting the signal to a value in a device-independent unit, and k) optionally comparing the value to a reference value to obtain a diagnostically rel evant result, and

L) optionally communicating the value and/or the diagnostically relevant result to the patient or the medical doctor treating the patient who donated the sample, pref erably via internet, fax or telephone.

4. 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, pref erably 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.

5. The use, method or kit according to any of claims 1 to 4, wherein the secondary antibody recognizes class IgG, preferably class lgG4 antibodies.

6. The use, method or kit according to any of claims 1 to 5, wherein the secondary antibody recognizes human antibodies.

7. The use, method or kit according to any of claims 1 to 6, wherein the chemilumines cence of the chemiluminescent label is detected for 1 to 60 seconds, preferably for 2 to 20 seconds, more preferably for 3 to 15 seconds following initiation of the chemilumi nescent detection reaction.

8. The use according to any of claims 1 to 2, wherein the ratio of sensitivity to specificity is increased, preferably compared to an ELISA assay.

9. The use according to any of claims 1 to 2 or 8, wherein the sensitivity is increased in samples that are negative according to ELISA assay analysis.

10. The use, method or kit according to any of claims 1 to 9, wherein the autoantibody binds to a bead, preferably a magnetic bead.

11. The use, method or kit according to any of claims 1 to 10, wherein the chemilumines cent 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.

12. The use according to any of claims 1 to 2 or 8 to 9, wherein the increase of the diagnostic reliability is in a high throughput process.

13. The use according to any of claims 1 to 2 or 8 to 9 or 12, wherein 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.

14. The use or method according to any of claims 1 to 3 and 5 to 13, wherein at least two samples taken at different time points are processed, wherein the period between the time points is preferably at least one month.

15. The use or method according to claim 1 to 3 and 5 to 14, wherein an increase of at least 2, 3, 4 or 5 CU/ml is detected is a concentration window comprising the range 2.0 to 10 CU/ml.