Method for the detection of antibodies

Abstract

The invention relates to a method for the detection and quantification of antibodies in a fluid sample combining immunoprecipitation with an immunoassay. The method can, for example, be used to detect and quantify autoantibodies in a body fluid sample.

Description

[0001] METHOD FOR THE DETECTION OF ANTIBODIES

[0002] DESCRIPTION

[0003] The invention relates to a method for the detection of antibodies in a fluid sample. In particular the invention relates to a method for the detection of antibodies, for example autoantibodies, in a body fluid sample.

[0004] Circulating antibodies are classically detected using established methods such as ELISA, Western blot or immunofluorescence. These methods use blood serum or plasma as source of the primary antibodies in the respective assay. This has the disadvantage, however, that signals at low antibody concentrations are interpreted as negative because these do not differ sufficiently from corresponding controls. Recently, circulating antibodies against the protein nephrin were found in patients with Minimal Change Disease (MCD) and Focal Segmental Glomerulosclerosis (FSGS)

[0019] , These antibodies were detected using immunoprecipitation or quantified using a signal-enhanced ELISA. However, experiments performed by the inventors with this ELISA did not lead to a specific detection, but to many false positive signals, possibly due to the very low amount of antibodies in the blood and possibly also due to a low affinity for the antigen. Immunoprecipitation (IP) is used to enrich and subsequently detect antigens. As a qualitative method, IP does not allow for a precise measurement of antibody titers. However, the quantification of the antibody titers is very important for a deeper clinical and pathophysiological understanding of a disease.

[0005] It is an object of the invention to provide a reliable detection of antibodies in a fluid sample, in particular antibodies, for example autoantibodies, in a body fluid sample, for example anti- nephrin autoantibodies in a blood sample. In particular, it is an object of the invention to provide a method for the quantification of antibody titers, for example, autoantibody titers, in a body fluid sample in the context of low antibody titers.

[0006] For solving the problem the invention provides a method for the detection of a target antibody in a fluid sample, comprising the steps of a) incubating the fluid sample with i. an antigen specifically binding to the target antibody, the antigen being tagged with an affinity tag, and ii. an affinity matrix binding the target antibody, to form a target antibody-antigen complex bound to the affinity matrix; b) immunoprecipitating the target antibody / antigen complex bound to the affinity matrix, formed in step a); c) separating the immunoprecipitated target antibody / antigen complex bound to the affinity matrix from the sample; d) eluting the target antibody / antigen complex from the affinity matrix; e) incubating the eluted target antibody / antigen complex with an immobilized binding partner of the affinity tag under conditions allowing the binding of the target antibody / antigen complex to the immobilized binding partner via the affinity tag of the antigen; and f) detecting the target antibody / antigen complex using a detection antibody directed against the antigen or against a secondary antibody directed against the antigen.

[0007] The method of the invention is particularly useful for the detection of antibody titers in a fluid sample, e.g., a body fluid sample, for example a blood serum sample, in the context of low antibody titers. The method of the invention provides an immunoprecipitation / immunoassay (e.g. IP / ELISA) hybrid assay that can be standardized and used to reliably and sensitively detect and quanitify antibodies in a fluid sample. In the method of the invention, a target antibody is immunoprecipitated in a common complex of the target antibody and a target antibody-specific antigen. The antigen is tagged with an affinity tag. The target antibody is thus coimmunoprecipitated with a tagged antigen bound to the antigen. Subsequently, after separation of the immunoprecipitate comprising the complex of the target antibody and the target antibody-specific antigen from the sample, the complex of the target antibody and the target antibody-specific antigen is bound via the affinity tag to an immobilized affinity tag partner that binds the affinity tag and thus the antigen to which it is attached. The affinity tag partner can, for example be immobilized on the bottom of a microtiter well of a microtiter plate. Subsequently, a detection antibody is used to directly or indirectly detect the antigen within the target antibody / antigen complex bound to the immobilized affinity tag partner. In case of a direct detection, the detection antibody, which may, for example, be fluorescence- or enyme- labeled, is directed against the antigen, in case of an indirect detection, the detection antibody is directed against an antibody directed against the antigen. The labeling of the detection antibody can advantageously be used to quantify the target antibody titer via the amount of antigen bound to the target antibody. The target antibody is preferably not detected directly, but only used to immunoprecipitate the complex of the target antibody and the tagged antigen, e.g., via binding to an affinity resin, for example protein A or G resin, via the Fc portion of the target antibody. The method may further comprise washing steps, for example, after separation of the immunoprecipitate from the sample, and / or after binding of the target antibody / antigen complex to the affinity tag partner. The method of the invention can particularly advantageously be used, for example, in the context of kidney diseases involving autoantibodies directed against the glomerulus, i.e., the filtering unit of the kidney, e.g. in kidney diseases with nephrotic syndrom, for example, podocytopathies.

[0008] The term “target antibody” relates to an antibody (immunoglobin) of interest, i.e. an antibody to be detected with the method of the invention. A target antibody can, for example, be an autoantibody associated with an autoimmune disease, e.g. an autoimmune kidney disease, for example a podocytopathy involving autoantibodies.

[0009] The term “autoantibody” refers to an antibody reacting with one or more self-antigens. An autoantibody is an antibody produced by the immune system of an individual, e.g. a human, that is directed against a constituent or constituents of the individuals own tissues or organs, for example, against one or more of the individual’s own proteins. The term “anti-nephrin autoantibody” refers to an autoantibody that is directed to, i.e., specifically binds to nephrin or a nephrin fragment.

[0010] The term “antigen” refers to a substance, for example, a molecule (e.g. a protein), a virus particle, or a bacterium, stimmulating an immune response against that substance.

[0011] The term “autoantigen” refers to a self-antigen, i.e. an antigen produced from own cellular components or products of an individual. Autoantigens are often normal proteins or protein complexes of the own body that are not recognized by the immune system as the body’s own and trigger autoimmune reactions. The term “nephrin-antigen” relates to a nephrin protein or any part, fragment or derivative thereof to which an anti-nephrin autoantibody specifically binds. The term encompasses the term “recombinant nephrin-antigen”, i.e., a biotechnologically synthesized nephrin molecule, nephrin fragment or nephrin derivative. An example of a nephrin-antigen is the ectodomain of nephrin, i.e. the extracellular part of the nephrin membrane protein. A recombinant nephrin- antigen is, for example, the ectodomain of a recombinant nephrin protein.

[0012] The term “nephrin” refers to a transmembrane protein encoded by the gene NPHS1 and expressed in podocytes of the kidney glomerulus that is an essential component of the slit diaphragm, a cell-cell junction of podocytes (see, e.g., [7], [9]). It is necessary for maintaining the slit diaphragm structure and the proper function of the renal filtration barrier. For a reference sequence for human nephrin see the NCBI Reference Sequence NP 004637.1 (NPHS1 mRNA: NM 004646.4). The extracellular domain (ectodomain) of nephrin consists of nine Ig-like domains [4] and a fibronectin type Ill-like domain and comprises the amino acids of positions 23-1055 according to the above referenc sequence. A recombinant ectodomain may comprise, for example, the amino acids 25-1037 according to the above reference sequence.

[0013] The term “affinity tag” refers to a peptide or protein that has a high binding affinity to a specific binding partner, to which it binds specifically and non-covalently. There are many different affinity tags known to the skilled person (see, for example, [6],

[0013] ,

[0015] ,

[0022] ). An affinity tag is attached to a protein of interest, usually covalently bound, for example, to the end N-terminal or C-terminal end of the protein, by biotechnological means, e.g. by fusing the coding sequence of the affinity tag to the coding sequence of the protein of interest, and producing a recombinant fusion protein from the coding sequence. Examples of affinity tags are biotin tags, Strep-tags (e.g. Strep-tag® II, WSHPQFEK, SEQ ID NO: 1; Twin-Strep-tag®, SA-WSHPQFEK- (GGGS)2-GGSA-WSHPQFEK, SEQ ID NO: 2), GST (glutathione S-transf erase) tags, Human influenza hemagglutinin tags (HA-tags), Flag-tags or Poly-Histidin tags (His-Tags). Biotin has a high affinity to avidin or streptavidin, Strep-tags have a high affinity to streptavidin or to recombinant streptavidin variants (Strep-Tactin®), GST has strong binding affinity for GSH (Glutathione), and the polyhistidin-tag (e.g. His6-tag or His8 tag) has high affinity for metal ions like Ni2+or Co2+ions. Together with their respective binding partner, affinity tags form systems that may also be referred to as “affinity binding system” or “coupling systems”. The term “tagged with an affinity tag”, for example, in relation to an antigen, e.g. a protein or peptide antigen, means that the affinity tag is covalently attached to the antigen, for example at one or both of its ends or within the molecule. The termin “biotinylated” refers to the attachment of biotin as an affinity tag. The term “strep-tagged” refers to the attachment of a Strep-tag (e.g. a Strep-tag® II or Twin-Strep-tag®).

[0014] The terms “binding partner of the affinity tag” or “affinity tag binding partner” refer to the ligand specifically binding to the affinity tag. For example, streptavidin is the affinity tag binding partner of the affinity tag biotin or Strep-tag®, Strep-Tactin is the binding partner of the affinity tag Strep-tag ®, Nickel-ions are the binding partner of the affinity tag His-tag etc.

[0015] The term “affinity matrix” refers to a solid phase having attached thereto a ligand specifically and reversibly binding a target protein or target protein class, e.g. immunoglobulins. The target protein can be bound to the ligand either directly or indirectly, e.g., via an affinity tag covalently bound to the target protein. Examples of an affinity matrix are an affinity resin like protein L, protein A and / or protein G resin, or magnetic affinity matrices like protein L, protein A and / or G magnetic beads.

[0016] The term “affinity matrix binding a target antibody” refers to an affinity matrix specifically binding antibodies, or specific types or classes of antibodies, e.g., IgG, for example via the Fc region of an antibody. The term is not to be construed as meaning that the affinity matrix specifically binds a specific target antibody, e.g. a specific anti-nephrin antibody. A term like “affinity matrix binding the anti-nephrin autoantibody” thus refers to an affinity matrix, e.g. affinity resin, binding the anti-nephrin autoantibody by its antibody-binding properties.

[0017] The term “affinity resin” refers to a solid phase consisting of or comprising a resin, e.g. agarose beads, having attached thereto a ligand specifically and reversibly binding a target protein. The target protein can be bound either directly or indirectly, e.g., via an affinity tag covalently bound to the target protein. Examples of an affinity resin are protein L, protein A and / or protein G resins, for example, agarose resins having attached thereto protein L, protein A and / or protein G as ligand. The term “protein L, protein A or protein G resin” is not to be construed as meaning that it only refers to resins comprising a single kind of protein L, A, or G, for example, only protein A. Rather, the terms “protein L, protein A or protein G resin”, “protein L, protein A and / or protein G resin” or “protein L / A / G resin” refer to a resin, e.g. agarose resin, having attached thereto protein L, protein A, or protein G each alone, or any mixture of two or three of the proteins, i.e. any mixture of protein L and protein A, protein L and protein G, and of protein A and protein G. The term includes any resin, e.g. agarose resin, with bound recombinant chimeric protein L and / or protein A and / or protein G. The terms “protein A or protein G resin” or “protein A / G resin” refers to an agarose resin, e.g., agarose beads, having attached thereto protein A and / or protein G as ligand. The term includes agarose resins with bound recombinant chimeric protein A / G.

[0018] The term “protein L, protein A or protein G magnetic beads” refers to magnetic beads with protein L, protein A and / or protein G bound to them. The beads can, for example, consist of a truncated form of recombinant protein A and / or G covalently coupled to a paramagnetic particle. As mentioned above for the term “affinity resin”, the term “protein L, protein A or protein G magnetic beads” refers to magnetic beads with bound protein L, protein A or protein G alone, and to magnetic beads having bound thereto any mixture of two or all three of the proteins.

[0019] The terms “protein L”, “protein A” and “protein G” refer to proteins originally isolated from bacteria of the genus Peptostreptococcus, Staphylococcus and Streptococcus, respectively, being able to bind to mammalian immunoglobulins, in particular IgGs. Protein L binds to immunoglobulins via the light (kappa) chain. Protein A and protein G bind to immunoglobulins via the Fc portion of the immunoglobulin. The terms also encompass recombinant protein L, recombinant protein A and / or recombinant protein G, e.g. protein G from which the albuminbinding portion has been removed, or recombinant chimeric variants of the proteins. The term “protein L / A / G” encompasses protein L alone, protein A alone or protein G alone, mixtures of protein L and protein A, protein L and protein G, protein A and protein G, and mixtures of all three proteins L, A and G. The terms “protein L”, protein A” and "protein G” also encompass chimeric protein L, protein A or protein G variants. The term “protein L / A / G” encompasses the term “protein A / G”. The term “protein A / G” encompasses protein A alone or protein G alone, mixtures of protein A and G, and chimeric protein A / G variants. The term “immunoprecipitation” (IP) refers to an affinity enrichment and / or purification technique based on antibody-antigen interaction, and involves the precipitation of a complex of an antibody and its specific antigen. Immunoprecipitation is often based on a solid phase (e.g. a bead, e.g., an agarose or magnetic bead) with a binding protein (e.g. protein A).

[0020] The terms “immunoassay” or “immunological assay” refer to a biochemical test based on the specific interaction of an antibody with its antigen. An immunoassay may measure, directly or indirectly, the presence or concentration of an antibody or an antigen. In an immunoassay, a suitable means for the generation of a measurable signal indicating the binding is used. Immunoassays may, for example, involve chemically linking antibodies or antigens with some kind of detectable label, e.g. an enzyme catalyzing a color reaction, or a fluorescence label.

[0021] The term “enzyme immunoassay” (EIA) refers to an immunoassay using the catalytic activity of an enzyme to detect and / or quantify an antibody-antigen interaction.

[0022] „ELISA“ (“enzyme-linked immunosorbent assay”) refers to a solid-phase based immunological assay, i.e., an enzyme immunoassay in which one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, e.g. the bottom of a microtiter well, a magnetic particle or a plastic bead (see, e.g., 1).

[0023] The term „quantifying“ means the determination of the quantity, e.g. the concentration, of an analyte, e.g. an autoantibody, in a sample.

[0024] The term "Fc region” ("fragment crystallizable region”) in relation to an antibody refers to the tail (C-terminal) portion of an antibody being composed of two heavy chain portions contributing two or three constant domains, depending on the antibody class. In a naturally occurring antibody, the Fc region is coupled to the antigen binding fragment (Fab).

[0025] The term “kidney disease” refers to a disease or disorder involving a malfunction or dysfunction of the kidney, for example the tubules or the glomerulus. The term encompasses diseases or disorders due to damage and / or malfunctioning of, for example, tubular cells or glomerular cells, e.g. podocytes. The term “nephrotic kidney disease” refers to a kidney disease characterized by the presence of excess protein in the urine, a condition referred to as proteinuria.

[0026] The term “nephrotic syndrome” (NS) refers to a group of clinical syndromes caused by damage of kidney glomeruli, involving, i.a., proteinuria (e.g. >3-3.5 g protein / 24 h, in adults), hypoalbuminemia (e.g. <25-30 g / L), hyperlipidaemia and edema

[0012] , The term “idiopathic nephrotic syndrome” (INS) relates to primary nephrotic syndrome, i.e. NS with no known cause. Examples of diseases or conditions that typically manifest as nephrotic syndrome are INS, Minimal Change Disease (MCD), Primary Focal Segmental Glomerulosclerosis (FSGS) and membranous nephropathy (MN).

[0027] The term “podocytopathies” refers to kidney diseases in which a direct or indirect podocyte injury causes proteinuria or nephrotic syndrom ([8],

[0010] ). Examples of podocytopathies are minimal change disease (MCD), primary focal segmental glomerulosclerosis (FSGS), and membranous nephropathy.

[0028] The term “minimal change disease” (MCD) refers to a kidney disease involving damage of the kidney glomeruli leading to nephrotic syndrom (

[0018] ,

[0021] ) . It is one of the most common causes of idiopathic nephrotic syndrome in children.

[0029] The term “primary focal segmental glomerulosclerosis” (FSGS) is a disorder causing nephrotic syndrom and involving partially (segmentally) sclerosed (scarred) glomeruli (see [5],

[0017] ).

[0030] The term “autoimmune disease” refers to a disease, illness or physical condition resulting from the anomalous response of the adaptive immune system, i.e. of antibodies, against normal components of the own body. Autoimmune diseases often involve the formation of antibodies against autoantigens (self-antigens), e.g. the body’s own proteins. If in such an autoimmune disease the autoantibodies are assumed to be causative, it is named “antibody-mediated autoimmune disease”.

[0031] The term “membranous nephropathy” (MN), also “membranous glomerulonephritis” (MGN) or “membranous glomerulopathy”, refers to an autoimmune disease involving damage of the glomeruli of the kidneys [2, 16], in particular of the kidney podocytes. As used herein the term preferably relates to primary (idiopathic) MN. In idiopathic MN, autoantibodies attack podocyte foot process proteins, such as, for example, the M-type phospholipase A2 receptor (PLA2RI; [3]) and thrombospondin type-1 domain-containing 7 A (THSD7A;

[0014] ). Antigens and autoantibodies associated with membranous nephropathy are, for example, listed in

[0011] ,

[0032] The term “detection antibody” refers to an antibody used in a detection method, in particular in an immunoassay, and carrying a detectable label, e.g. an enzyme label or fluorescence label. A detectable label is any label attached to a molecule, e.g. an antibody, that can be directly or indirectly detected by any technical means, e.g. by measuring a color change, color intensity, radioactivity, fluorescence etc. An enzyme label, for example, may consist of horseradish peroxidase (HRP) that catalyzes a variety of reactions leading to, e.g., a color change that can, for example, be detected colorimetrically. A detection antibody can be a primary antibody, i.e. an antibody directed directly against a target molecule, i.e. selectively binding to an epitope on the target molecule itself, or a secondary antibody, i.e. an antibody directed against (specifically binding to) a primary antibody that specifically binds to an epitope on the target molecule. The term “detection antibody detecting the target antibody / antigen complex using a detection antibody directed against the antigen or against an antibody directed against the antigen” refers to a primary detection antibody directed directly against an antigen, i.e. an antibody specifically binding to an antigen, in a complex of the target antibody and the antigen complex, or to a secondary detection antibody, i.e. a detection antibody directed against a primary antibody that specifically binds the antigen.

[0033] The term “body fluid” refers to a bodily fluid within the body of an organism, e.g. a human. Examples of body fluids are, for example, blood, blood serum, blood plasma, blood plasmapheresate, saliva, urine, lymph, cerebrospinal fluid (CSF), pleural fluid, peritoneal fluid, synovial fluid and pericardial fluid. The term “blood sample” can refer to a whole blood sample, a blood serum sample or a blood plasma sample. The term "blood plasma” includes plasmapheresate.

[0034] In the method of the invention, in step a), a target antibody, an antigen specifically binding to the target antibody, and an affinity matrix binding the target antibody, are incubated under conditions allowing the formation of a complex of a target antibody / antigen complex and an affinity matrix to which the target antibody / antigen complex is reversibly bound via the target antibody. For example, the tagged antigen can first be added to the fluid sample containing the target antibody, to first form a target antibody / antigen complex, and the affinity matrix can subsequently be added to form a complex of the affinity matrix and the target antibody / antigen complex. Alternatively, the affinity matrix can first be added to the fluid sample containing the target antibody, to form a target antibody / affinity matrix complex, i.e. a complex of the affinity matrix and the target antibody, and the tagged antigen is subsequently added to the fluid sample to form a complex of the affinity -matrix-bound target antibody and the antigen. Further, the affinity matrix and the tagged antigen can be added simultaneously to the fluid sample to form the complex of the affinity matrix, the target antibody and the tagged antigen.

[0035] In a preferred embodiment of the method of the invention, the target antibody / antigen complex is bound to the affinity matrix via the Fc region of the target antibody. The affinity matrix may consist of or comprise an affinity resin or a magnetic affinity matrix, e.g. a protein L, protein A or protein G resin or protein L, protein A or protein G magnetic beads. In a preferred embodiment of the invention, the affinity matrix is an affinity resin, more preferably protein A or protein G resin.

[0036] In a preferred embodiment of the invention the affinity matrix comprising the target antibody / antigen complex is separated from the sample. This can, for example, be done by the application of a magnetic field or by centrifugation, depending on the affinity matrix used. Magnetic field separation is preferably used in case of a magnetic affinity matrix, e.g., when using, for example, protein A or protein G magnetic beads. In case of an affinity resin like, for example, protein A or protein G agarose beads, centrifugation is preferably used. Subsequently, the target antibody / antigen complex is eluted from the affinity resin by a suitable means known to the skilled person, e.g. by an elution buffer having a suitable pH or salt concentration for promoting the dissociation of the target antibody / antigen complex from the affinity matrix. In a preferred embodiment, one or more washing step(s) is(are) carried out before the elution step. The washing step(s) can, for example, serve to remove any unbound complexes and molecules from the affinity matrix. In a further preferred embodiment, the method of the invention comprises a step of buffering the eluted target antibody / antigen complex, i.e., contacting the eluted target antibody / antigen complex with a suitable buffer solution.

[0037] The detection step of the method of the invention involves contacting the target antibody / antigen complex bound to the immobilized affinity-tag binding partner with a detection antibody, for example, a primary detection antibody, i.e., a labeled anti-affinity-tag detection antibody, or a secondary detection antibody, i.e., a labeled antibody directed against a primary anti-affinity-tag antibody. In case the detection antibody is a primary antibody directed against the antigen the detection antibody is preferably directed against at least one other epitope on the antigen than the target antibody to allow simultaneous binding of the target antibody and the primary detection antibody to the same antigen molecule. The same applies in case the detection antibody is a secondary antibody binding to a primary antibody binding to the antigen. In this case the primary antibody directed against the antigen is preferably directed against at least one other epitope of the antigen than the target antibody.

[0038] In a preferred embodiment of the method of the invention, the method comprises the step of quantifying the target antibodies present in the sample by measuring a detectable signal produced using the detection antibody. A detectable signal may be any signal that can be detected by known means, e.g., a fluorescence signal or a color signal. The term “produced using the detection antibody” means that the detectable signal is, preferably quantitatively, dependent on the presence of the detection antibody. As an example, the detection antibody may be tagged with a fluorophore generating a fluorescence signal, or with an enzyme catalyzing a reaction leading to the formation of a coloured compound that can, for example, be detected by colorimetry. The detection antibody is thus preferably tagged with a fluorophore, an enzyme or any other molecule or means that directly or indirectly generates a detectable signal. The detectable signal is preferably a fluorescence signal or a color signal, and the detection antibody is thus preferably fluorescence-labeled or enzyme-labeled. As regards the binding properties of any antibodies binding to the antigen for detection and / or quantification purposes, it is again preferred that these antibodies bind to an epitope different from the epitope of the target antibody. In the method of the invention, the eluted target antibody / antigen complex is incubated with an immobilized binding partner of the affinity tag under conditions allowing the binding of the target antibody / antigen complex to the immobilized binding partner via the affinity tag of the antigen. In this step, the eluted target antibody / antigen complex is bound to the immobilized binding partner of the affinity tag. In a preferred embodiment, the binding partner is immobilized on the bottom of a well of a microtiter plate. This can be accomplished by methods known to the skilled person. Suitable microtiter plates, e.g., Strep-Tactin® coated microtiter plates, are also commercially available.

[0039] In a preferred embodiment the method of the invention further comprises one or more washing step(s) after binding of the target antibody / antigen complex to the immobilized binding partner.

[0040] In a particular preferred embodiment, the method of the invention comprises the following steps: a) incubating the fluid sample with i. an antigen specifically binding to the target antibody, the antigen being tagged with an affinity tag, and ii. an affinity matrix binding the target antibody, to form a target antibody / antigen complex bound to the affinity matrix; b) immunoprecipitating the target antibody / antigen complex bound to the affinity matrix, formed in step a); c) separating the immunoprecipitated target antibody / antigen complex bound to the affinity matrix from the sample; cl) washing the separated immunoprecipitated target antibody / antigen complex bound to the affinity matrix with a washing solution; d) eluting the target antibody / antigen complex from the affinity matrix; dl) buffering the eluted target antibody / antigen complex e) incubating the eluted target antibody / antigen complex with an immobilized binding partner of the affinity tag under conditions allowing the binding of the target antibody / antigen complex to the immobilized binding partner via the affinity tag of the antigen; el) washing the bound target antibody / antigen complex with washing solution; and f) detecting, preferably quantifying, the target antibody / antigen complex using a detection antibody directed against the antigen or against a secondary antibody directed against the antigen. In a preferred embodiment of the method of the invention the affinity tag is a biotin tag and the affinity tag binding partner is avidin or streptavidin. It should be noted here that the term “biotin” encompasses recombinant biotin variants (see, e.g. US 11286285 B2), e.g. mutated biotin. The same applies to the terms “avidin” or “streptavidin”. These terms also encompass recombinant avidin or streptavidin variants, e.g. Strep-Tactin®.

[0041] In a preferred embodiment of the method of the invention, the fluid sample is a body fluid sample, for example, a blood serum or plasma sample, a urine sample, or cerebrospinal fluid (CSF) sample, preferably a blood sample, e.g. blood serum or blood plasma sample.

[0042] In a preferred embodiment of the method of the invention the target antibody is an autoantibody, preferably an antibody or autoantibody associated with a kidney disease, more preferably an autoantibody associated with a podocytopathy, more preferably an anti-nephrin autoantibody. In this embodiment, it is preferred that the antigen is an autoantigen associated with a kidney disease, more preferably an autoantigen associated with a podocytopathy, more preferably a nephrin-antigen.

[0043] In a particular preferred embodiment of the method according to the invention, the target antibody is an anti-nephrin autoantibody, the antigen is a nephrin-antigen, and the sample is a blood sample, e.g., a serum or plasma sample, comprising the steps of: a) incubating the blood sample with i. the nephrin-antigen specifically binding to the anti- nephrin autoantibody, the nephrin-antigen being tagged with an affinity tag, preferably a biotin tag, and ii. an affinity matrix binding the anti-nephrin autoantibody, preferably an affinity resin, further preferred a protein A or protein G affinity resin, to form a target antibody / antigen complex bound to the affinity matrix; b) immunoprecipitating an anti-nephrin autoantibody / nephrin-antigen complex bound to the affinity matrix, formed in step a); c) separating the immunoprecipitated anti-nephrin autoantibody / nephrin-antigen complex bound to the affinity matrix from the sample; d) eluting the anti-nephrin autoantibody / nephrin-antigen complex from the affinity matrix, and preferably buffering the eluted target antibody / antigen complex; e) incubating the eluted anti-nephrin autoantibody / nephrin-antigen complex with an immobilized binding partner of the affinity tag, preferably avidin or streptavidin, under conditions allowing the binding of the anti-nephrin autoantibody / nephrin-antigen complex to the immobilized affinity tag binding partner via the affinity tag of the nephrin-antigen; and f) detecting, preferably quantifying, the anti-nephrin autoantibody / nephrin-antigen complex using a detection antibody directed against the nephrin-antigen or against an antibody directed against the nephrin-antigen.

[0044] Preferably, the anti-nephrin autoantibody / nephrin-antigen complex is bound to the affinity matrix, which preferably is an affinity resin, via binding of the Fc region of the autoantibody to the solid phase. As mentioned above, the complex of the affinity matrix, the anti-nephrin autoantibody, and the nephrin-antigen can be formed by first adding either the nephrin antigen or the affinity matrix, or both together to the blood sample.

[0045] In a further preferred embodiment of the method of the invention, the anti-nephrin autoantibody / nephrin-antigen complex is detected, preferably quantified, via an enzyme-labeled primary antibody directed against the nephrin-antigen, or a via an enzyme-labeled secondary antibody directed against a primary antibody directed against the nephrin-antigen.

[0046] In case the detection antibody is a primary antibody directed against the nephrin-antigen the detection antibody is directed against at least one other epitope on the nephrin-antigen than the anti-nephrin target antibody. The same applies in case the detection antibody is a secondary antibody binding to a primary antibody binding to the nephrin-antigen. In this case the primary antibody directed against the nephrin-antigen is directed against at least one other epitope of the antigen than the anti-nephrin target antibody.

[0047] The invention is further illustrated using the attached figures and examples.

[0048] Figure 1. Simplified schematic representation of an embodiment of the method of the invention.

[0049] Figure 2. Quantitative measurement of anti-nephrin antibodies in patients with glomerular diseases and healthy controls by an embodiment of the method of the invention. Microplates were coated with Strep-Tactin, which can bind strep-tagged nephrin. Defined amounts of strep- tagged nephrin were used to generate a standard curve consisting of five calibrators (6.25, 25, 100, 1000, and 2000 relative units [RU] per mL, corresponding to 0.131, 0.525, 2.1, 21, and 42 ng of recombinant human nephrin). Data are presented as mean and SEM from n=4 independent experiments. Anti -nephrin antibody titers in patients with MCD / primary FSGS (open circles), membranous nephropathy (asterisks), and healthy controls (filled circles) using the method of the invention. Data are shown as scatter dot blots with median and 95% confidence intervals. Confidence interval widths were not adjusted for multiplicity. MCD = minimal change disease, FSGS = focal segmental glomerulosclerosis.

[0050] Figure 1 shows a simplified schematic representation of an embodiment of the method of the invention. A sample 1, for example a blood serum or plasma sample, with target antibodies 2, here anti-nephrin autoantibodies, in it, is incubated with an antigen 3, here a nephrin-antigen, under conditions, e.g. as regards the physico-chemical properties of the incubation medium, time, and temperature, suitable for allowing the binding of the antigen 3 to the target antibody 2. The antigen is tagged with an affinity tag 6, e.g. a biotin tag. A complex 5 of the target antibody 2 and antigen 2 is formed. The target antibody / antigen complex 5 is contacted with an affinity matrix 4, here protein A / G agarose beads. The affinity matrix 4 binds the target antibody / antigen complex 5 via the Fc portion of the target antibody 2. The affinity matrix 4 with bound target antibody / antigen complex 5 is precipitated, e.g. via centrifugation, and the antibody / antigen complex 5 is eluted from the affinity matrix 4. Subsequently, the eluted target antibody / antigen complex 5 is added to a microtiter well 8 of a microtiter well plate 9 coated with an affinity tag binding partner 7, for example avidin or streptavidin. The target antibody / antigen complex 5 is incubated, in the microtiter well 8, with the immobilized affinity tag binding partner 7 under conditions allowing the binding of the target antibody / antigen complex 5 to the immobilized affinity tag binding partner 7 via the affinity tag attached to the antigen. In the embodiment shown here, the target antibody / antigen complex 5 is detected, and quantified, by using a primary antibody 10 directed against the antigen 3, and a secondary detection antibody 11 carrying a detectable label 12 directed against the primary antibody. The primary antibody 10 is contacted with the target antibody / antigen complex 5, such that the primary antibody 10 binds to the antigen, and the secondary detection antibody is contacted with the complex of the target antibody / antigen complex 5 and the primary antibody, such that the detection antibody 11 binds to the primary antibody 10. The primary antibody 10, which is directed to the antigen 3, binds another epitope on the antigen 3 than the target antibody. A detectable signal 13 is generated via the detectable label. The detectable label can, for example, be an enzyme, e.g. horseradish peroxidase (HRP), catalyzing a reaction leading to the formation of a color upon addition of a suitable substrate. The relation between target antibody, antigen and detection antibody is preferably 1 :1 : 1.

[0051] EXAMPLES

[0052] In the following the method of the invention is exemplified by applying it to the detection of anti-nephrin autoantibodies. Anti-nephrin antibody titers in patients with anti-nephrin positive Minimal change disease (MCD) or primary focal segmental glomerulosclerosis (FSGS) were detected and quantified using an embodiment of the method of the invention. MCD and FSGS are podocytopathies leading to nephrotic syndrome.

[0053] Serum / plasma samples form from adult patients with biopsy-proven glomerular diseases were first analyzed for anti-nephrin antibodies using direct Western blotting or ELISA. However, detection of circulating anti-nephrin antibodies using direct Western blotting or ELISA was not successful (not shown). Therefore, a standardized IP / ELISA hybrid assay was developed for the detection and preferably quantification of anti-nephrin antibodies in patient serum / plasma.

[0054] Generation of recombinant human and mouse nephrin

[0055] The ectodomain of human nephrin (NCBI reference sequence: NM 004646.4, amino acids A25-G1037) in the eucaryotic expression vector pXLG was provided by EMBL, Hamburg. The construct contained a N-terminal leader sequence (MWWRLWWLLLLLLLLWPMVWA) for cellular secretion into the cell culture medium as well as a C-terminal Twin-Strep®-tag and an 8x polyhistidine-tag for downstream purification and analysis. The ectodomain of murine nephrin (NCBI Reference Sequence: NM_019459.2, amino acids A36-L1052) was cloned into the eucaryotic expression vector pDSG-IBA (IBA Lifesciences) downstream a BM40 leader sequence and contained either a C-terminal 8x polyhistidine-tag or an N-terminal Twin-Strep®- tag. Sequence confirmation was achieved by Sanger sequencing and sequence alignment using Benchling (Biology Software, 2022-2023). The recombinant protein constructs were expressed in human embryonic kidney (HEK) 293-6E cells in 30 ml serum-free medium (Freestyle 293, Gibco)

[0023] , Cells were transfected using 80 pg of polyethylenimine (Polyscience Inc.) in 150 mM NaCl mixed with 25 pg of plasmid DNA in 150 mM NaCl, which was incubated for 30 min and carefully added to cell medium. 24 hours after transfection, the cell medium was supplemented with 0.5 ml 20% tryptone. Seven days after transfection, the cell culture supernatant was harvested and centrifuged at 14,000 g for 10 min. The cell culture supernatant was concentrated using spin concentrators (vivaspin 20 50K, Sartorius), the polyhistidine- tagged protein was purified under native conditions using NiNTA resin (Thermo Fisher Scientific) and 250 mM imidazole for elution, with subsequent buffer exchange to PBS (ZEBA- spin columns, Thermo Fisher Scientific). Protein constructs containing a Twin-Strep®-tag were purified using strep-tactin® resin according to the manufacturer’s instruction (Strep-Tactin XT, IBA-lifescience). Protein quality and sample purity was validated by Western blot and / or Coomassie staining. The protein concentration was determined using a spectrophotometer (Biozym Scientific).

[0056] Quantitative IP / ELISA assay

[0057] To quantify anti-nephrin antibodies a two-step procedure of immunoprecipitation followed by quantification of immunoprecipitated recombinant nephrin was developed. Firstly, 60 pl patient serum / plasma was mixed with 150 ng of recombinant nephrin in radioimmunoprecipitation assay buffer (RIPA buffer, 150 mM NaCl, 1% NP40, 0.5 % deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris pH 7.4), incubated over night at constant rotation at 4 °C and exposed to MabCaptureC™ High Capacity Protein A Resin (Thermo Fisher Scientific). The resin was washed using Pierce™ Spin Columns (Thermo Fisher Scientific) twice in RIPA buffer followed by 4 washes in phosphate buffered saline (PBS) with 0.2% Tween 20 (PBS-T) and once in PBS. Protein elution was achieved by acidification using IgG elution buffer (Thermo Fisher Scientific) at pH 2.8 and neutralization with IM Tris pH 9. Subsequently, immunoprecipitated recombinant Twin-Strep-tagged nephrin was quantified using a Strep- Tactin-coated microplate (IBA Lifesciences GmbH). To do so, wells were incubated with the eluate diluted in post coat buffer (TBS with BSA, Sigma-Aldrich) with 0.05% Tween 20 for 2 hours at 20 °C. Wells were washed four times with TBS-T (Sigma- Aldrich) and incubated with anti-nephrin antibody (polyclonal sheep anti-nephrin antibody, R&D Systems; AF4269, diluted 1 : 1000 in post coat buffer with 0.05% Tween 20) overnight on a rocking platform at 4°C. Wells were washed four times and incubated with 100 pl of HRP-conjugated anti-shlgG (1 : 10 000, Jackson ImmunoResearch) for one hour at 20 °C. Wells were washed again before application of TMB ELISA peroxidase substrate solution (Avia Systems Biology) for 5 minutes at 20 °C, followed by acidification using 100 pl of 1 mol / L FFPCL-solution to stop the substrate reaction. The absorbance at 450 nm was determined using an ELISA reader (EL808, Bio-Tek instruments). Quantification was done in duplicates and random units (RU / ml) were determined in regard to a standard curve of serial dilutions of recombinant human nephrin (Figure 2).

[0058] Figure 2 shows the quantitative measurement of anti-nephrin antibodies in blood samples from patients with glomerular diseases and healthy controls by an embodiment of the method of the invention. Microplates were coated with Strep-Tactin, which can bind strep-tagged nephrin. Defined amounts of strep-tagged nephrin were used to generate a standard curve (not shown) consisting of five calibrators (6.25, 25, 100, 1000, and 2000 relative units [RU] per mL, corresponding to 0.131, 0.525, 2.1, 21, and 42 ng of recombinant human nephrin). Data are presented as mean and SEM from n=4 independent experiments. The figure shows anti-nephrin antibody titers in patients with MCD / primary FSGS (open circles), membranous nephropathy (asterisks), and healthy controls (filled circles) using the method of the invention. Data are shown as scatter dot blots with median and 95% confidence intervals. Confidence interval widths were not adjusted for multiplicity. The data clearly show that anti-nephrin antibody titers can be quantitatively determined with the method of the invention.

[0059] References

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Claims

CLAIMS1. A method for the detection of a target antibody in a fluid sample, comprising the steps of a) incubating the fluid sample with i. an antigen specifically binding to the target antibody, the antigen being tagged with an affinity tag, and ii. an affinity matrix binding the target antibody, to form a target antibody / antigen complex bound to the affinity matrix; b) immunoprecipitating the target antibody / antigen complex bound to the affinity matrix, formed in step a); c) separating the immunoprecipitated target antibody / antigen complex bound to the affinity matrix from the sample; d) eluting the target antibody / antigen complex from the affinity matrix; e) incubating the eluted target antibody / antigen complex with an immobilized binding partner of the affinity tag under conditions allowing the binding of the target antibody / antigen complex to the immobilized binding partner via the affinity tag of the antigen; and f) detecting the target antibody / antigen complex using a detection antibody directed against the antigen or against an antibody directed against the antigen.

2. The method according to claim 1, wherein the target antibody / antigen complex is bound to the affinity matrix via the Fc region of the target antibody.

3. The method according to claim 1 or 2, wherein, in step b), the target antibody / antigen complex bound to the affinity matrix is immunoprecipitated using an affinity resin, more preferably protein A or protein G resin.

4. The method according to one of the preceding claims, comprising the step of quantifying the target antibodies present in the sample by measuring a detectable signal, preferably a fluorescence signal or color signal, produced using the detection antibody.

5. The method according to one of the preceding claims, further comprising a washing step after step c and / or after step e.

6. The method according to one of the preceding claims, wherein the affinity tag is a biotin tag and the affinity tag binding partner is avidin or streptavidin.

7. The method according to one of the preceding claims, wherein the detection antibody is fluorescence-labeled or enzyme-labeled.

8. The method according to one of the preceding claims, wherein the sample is a blood sample, a urine sample, or a cerebrospinal fluid sample, preferably a blood sample.

9. The method according to one of the preceding claims, wherein the target antibody is an autoantibody, preferably an autoantibody associated with a kidney disease, more preferably an autoantibody associated with a podocytopathy, more preferably an anti-nephrin autoantibody.

10. The method according to claim 9, wherein the antigen is an antigen, preferably an autoantigen, associated with a kidney disease, more preferably an autoantigen associated with a podocytopathy, more preferably a nephrin-antigen.

11. The method according to claim 10, wherein the target antibody is an anti-nephrin autoantibody, the antigen is a nephrin-antigen, and the sample is a blood sample, comprising the steps of: a) incubating the blood sample with i. the nephrin-antigen specifically binding to the anti- nephrin autoantibody, the nephrin-antigen being tagged with an affinity tag, preferably a biotin tag, and ii. an affinity matrix binding the anti-nephrin autoantibody, to form a target antibody / antigen complex bound to the affinity matrix; b) immunoprecipitating an anti-nephrin autoantibody / nephrin-antigen complex bound to the affinity matrix, formed in step a); c) separating the immunoprecipitated anti-nephrin autoantibody / nephrin-antigen complex from the sample; d) eluting the anti-nephrin autoantibody / nephrin-antigen complex; e) incubating the eluted anti-nephrin autoantibody / nephrin-antigen complex with an immobilized binding partner of the affinity tag, preferably avidin or streptavidin, underconditions allowing the binding of the anti-nephrin autoantibody / nephrin-antigen complex to the immobilized affinity tag binding partner via the affinity tag of the nephrin-antigen; and f) detecting the anti-nephrin autoantibody / nephrin-antigen complex using a detection antibody directed against the nephrin-antigen or against an antibody directed against the nephrin-antigen.

12. The method according to claim 11, wherein the anti-nephrin autoantibody / nephrin- antigen complex is bound to the affinity matrix, preferably affinity resin, via binding of the Fc region of the autoantibody to the affinity matrix.

13. The method according to one of claims 11 or 12, wherein the anti-nephrin autoantibody / nephrin-antigen complex is detected, preferably quantified, via an enzyme-labeled primary antibody directed against the nephrin-antigen, or a via an enzyme-labeled secondary antibody directed against a primary antibody directed against the nephrin-antigen..

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