HK1202624B - Adrenomedullin assays and methods for determining mature adrenomedullin - Google Patents

Abstract

Subject of the present invention is an in vitro method for therapy follow-up in septic patients wherein the concentration of mature ADM 1-52 and/or mature ADM 1-52-Gly in a sample of bodily fluid of said septic patient is determined using an assay comprising two binders that bind to two different regions within the region of mature adrenomedullin and/ or adrenomedullin-Gly that is aminoacid 21-52-amid SEQ ID No. 1 or aminoacid 21 -52-Gly SEQ ID No. 2 wherein each of said regions comprises at least 4 or 5 amino acids. Subject of the present invention are further assays and calibration methods.

Description

Assays for adrenomedullin and methods for determining mature adrenomedullin

Subject of the present invention is an in vitro method for therapy follow-up in septic patients, wherein the concentration of mature ADM1-52 and/or mature ADM1-52-Gly in a body fluid sample of said septic patients is determined using an assay comprising two binding moieties that bind to two different regions of the region of mature ADM1-52 and/or mature ADM1-52-Gly, i.e. amino acids 21-52-amino SEQ ID No.1 or amino acids 21-52-Gly SEQ ID No.2, wherein each of said regions comprises at least 4 or 5 amino acids.

The subject of the invention is also a method for determination and calibration.

Adrenomedullin (ADM) peptide, a novel blood pressure lowering peptide comprising 52 amino acids, which has been isolated from human pheochromocytomas, was first described in Kitamura et al (see 1; values based on the reference appendix). In the same year, a cDNA encoding a precursor peptide comprising 185 amino acids and the complete amino acid sequence of this precursor peptide were also described. The precursor peptide, in particular the signal sequence comprising 21 amino acids at the N-terminus, is called "preproendolin" (pre-proADM). pre-proADM comprises 185 amino acids and has the sequence of SEQ ID No: 3. Mature ADM is shown in SEQ ID No.4 and mature ADM-Gly is shown in SEQ ID No. 5.

The peptide Adrenomedullin (ADM) is a peptide comprising 52 amino acids (SEQ ID No:2) and comprises the 95-146 amino acid peptide of pre-proADM, which is formed from pre-proADM by proteolytic cleavage. To date, essentially only a few fragments of the peptide fragments formed in the cleavage of pre-proADM have been characterized more accurately, in particular the physiologically active peptides Adrenomedullin (ADM) and "PAMP", peptides comprising 20 amino acids (22-41) after 21 amino acids of the signal peptide in pre-proADM. Physiologically active subfragments have also been discovered and studied in more detail for ADM and PAMP. The discovery and characterization of ADM in 1993 led to a strong research activity and a tidal publication, these results being summarized recently in various reviews, and in the context of this specification references, in particular articles from the journal "Peptides", are devoted to ADM (Peptides22(2001)), in particular (2) and (3). Another overview is (4). ADM, in particular, can be considered as a multifunctional regulatory peptide in the current scientific research community. It is released into the blood circulation in an inactive form extended by glycine (5). There are also binding proteins (6) that are specific for ADM and may also modulate the action of ADM.

Those physiological effects of ADM and PAMP that are the most important in the current study are those that affect blood pressure. Thus, ADM is a potent vasodilator, and it is likely that its blood pressure lowering effect is associated with a particular peptide fragment in the terminal part of ADMC.

It has also been found that the above-mentioned further physiologically active peptide PAMP formed from pre-proADM likewise shows a blood pressure lowering effect, although it appears that it has a different mechanism of action than ADM (see reviews (3) and (4), and (7), (8) or (9) and (10) above).

It has also been found that the concentration of ADM that can be measured in the blood circulation and other body fluids in many pathological conditions is significantly higher than that found in healthy control populations. Thus, ADM levels are significantly increased, although to varying degrees, in patients with congestive heart failure, myocardial infarction, renal disease, hypertensive disorders, diabetes, acute phase of shock, and sepsis and septic shock. PAMP concentrations also increased in some of the pathological states described, but plasma levels decreased relative to ADM ((3); page 1702).

It is also known that abnormally high ADM concentrations will be observed in sepsis or septic shock (see (3) and (11), (12), (13), (14), and (15)). This finding is associated with the typical hemodynamic changes known as sepsis and other severe syndromes, such as for example the typical phenomenon of the course of SIRS patients.

Although ADM and PAMP are presumed to be formed in the same precursor peptide, pre-proADM (SEQ ID No:3), in which the amino acid sequences corresponding to these peptides are present in equimolar amounts of the local peptide, the concentrations of ADM and PAMP detectable in biological fluids are markedly different. This is without any exception.

Thus, the measurable concentrations of different cleavage products of the same precursor peptide may be different, e.g. because they are the result of different competitive cleavage pathways, e.g. different competitive cleavage pathways leading to fragmentation of different precursor peptides and thus to different cleavage products in different pathological conditions. Some of the local peptides contained in the precursor peptide may be formed as free peptides or may not be formed, and/or different peptides may be formed in different ways and in different amounts. Even if it is considered that only a single cleavage pathway is used for the treatment of the precursor peptide, and thus all cleavage products are derived from the same precursor peptide and have to form themselves in equimolar amounts, the steady state concentrations of the different local peptides and fragments detectable in the biological fluids may be very different, i.e. e.g. when individual individuals are formed at different rates and/or have different individual stability (lifetime) in the respective biological fluids, or if they are removed from the blood circulation based on different cleavage mechanisms and/or at different cleavage rates.

Adrenomedullin plays a key role during the development of sepsis ((16), (17)) and in many acute and chronic diseases ((18), (4)).

ADM is elevated in sepsis and prognosis of the consequences of sepsis ((19), (14), (11)). Sepsis therapy follow-up is a largely unmet clinical need for early monitoring of therapy success or failure.

There is currently no ADM assay available for routine diagnostics. The sensitivity of currently available tests for determining mature ADM is too low. Therefore, high plasma volume is required for analysis. Moreover, currently available assays show stability related to pre-analytical limitations, e.g. stabilization of the sample by aprotinin ((20), (21)). Additionally, some ADM assays require extensive sample preparation prior to measurement (11).

It is an object of the present invention to provide a conventional method suitable as a direct measure of mature ADM, suitable for use in standard automated laboratories and point-of-care technologies.

Surprisingly, the results indicate that such assays can be used for therapy follow-up in septic patients.

Subject of the present invention is an in vitro method for therapy follow-up in septic patients, wherein the concentration of mature ADM1-52 and/or mature ADM1-52-Gly in a body fluid sample of said septic patients is determined using an assay comprising two binding moieties that bind to two different regions of the region of mature ADM1-52 and/or mature ADM1-52-Gly, i.e. amino acids 21-52-amino SEQ ID No.1 or amino acids 21-52-Gly SEQ ID No.2, wherein each of said regions comprises at least 4 or 5 amino acids.

In one embodiment of the invention, the subject is an in vitro method for therapy follow-up in septic patients, wherein one of said binding moieties binds mature ADM and/or the region comprised in the sequence mature ADM 1-52-Gly:

ADM21-32:CTVQKLAHQIYQ(SEQ ID No.6)

and wherein another of said binding moieties binds to a region comprised in the following sequence mature ADM and/or mature ADM 1-52-Gly:

ADM42-52:APRSKISPQGY(SEQ ID No.7)

in an embodiment of the invention, the assay sensitivity of the assay is capable of quantifying ADM in a healthy human, which is <10pg/ml, preferably <40pg/ml and more preferably <70 pg/ml.

In one embodiment of the invention, the binding moiety exhibits a binding affinity for mature ADM and/or mature ADM1-52-Gly of at least 10⁷M^-1Preferably 10⁸M^-1Preferably, the affinity is greater than 10⁹M^-1Most preferably greater than 10¹⁰M^-1. It is known to those skilled in the art that it is contemplated that higher doses of the compound may be used to compensate for the lower affinity, and that such measurements would not be outside the scope of the present invention.

To determine the affinity of the adrenomedullin antibody, the kinetics of binding of adrenomedullin to the immobilized antibody was determined by means of the label-free surface plasmon resonance technique using the Biacore2000 system (GE Healthcare Europe GmbH, Frieberg, Germany). Reversible immobilization of antibodies was performed using anti-mouse Fc antibodies covalently bound to the CM5 sensor surface at high density according to the manufacturing instructions (mouse antibody capture kit, GE Healthcare) (22).

In one embodiment of the invention, the binding moiety is selected from an anti-adrenomedullin antibody, or an anti-ADM antibody fragment that binds ADM, or a non-Ig scaffold that binds adrenomedullin.

Treatment follow-up means that the concentration of mature ADM1-52 (SEQ ID No.4) and/or mature ADM1-52-Gly (SEQ ID No.5) in a sample is determined at least once after the start of treatment, preferably more than once, preferably twice or once a day.

In one embodiment of the invention, it may be referred to as POC-testing (point of care), a testing technique that allows testing in less than 1 hour in proximity to a patient without the need for a fully automated assay system. An example of such a technique is the immunochromatographic test technique.

In one embodiment of the invention, such assays are sandwich immunoassays, utilizing any kind of assay including, but not limited to, enzymatic labeling, chemo-chemistryThe detection technique of the chemiluminescent label or the electrochemiluminescent label is preferably a fully automated assay. In one embodiment of the invention, such assays are enzyme-labeled sandwich assays. Examples of automated or fully automated assays include assays for one of the following systems: rocheAbbottSiemensBrahmsBiomerieuxAlere

A wide variety of immunoassays are known and can be used in the assays and methods of the invention, these include: radioimmunoassay ("RIA"), homogeneous enzyme-multiplied immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), enzyme protein reactivation immunoassay ("ARIS"), dipstick immunoassay, and immunochromatographic assay.

In one embodiment of the invention, at least one of the two binding moieties is labeled to facilitate detection.

Preferred detection methods include various forms of immunoassays such as, for example, Radioimmunoassays (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats such as, for example, immunochromatographic band assays.

In a preferred embodiment, the label is selected from the group consisting of a chemiluminescent label, an enzymatic label, a fluorescent label, and a radioiodinated label.

The assay may be homogeneous or heterogeneous, competitive or noncompetitive. In one embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the molecule to be detected and/or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, such as the surface of a bead, well or other container, chip or strip, and the second antibody is an antibody labeled, for example, with a dye, radioisotope, or reactivating or catalytically activating moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The usual combinations and procedures involving "sandwich assays" are established and known to those skilled in the art (23).

In another embodiment, the assay comprises two capture molecules, preferably both antibodies, present as a dispersion in a liquid reaction mixture, wherein a first label component is attached to the first capture molecule, wherein said first label component is part of a fluorescence-or chemiluminescence quenching or amplification based label system, and a second label component of said identification system is attached to the second capture molecule, such that upon binding of the two capture molecules to the analyte a detectable signal is generated which allows detection of a formed sandwich complex in a solution comprising the sample.

In another embodiment, the labeling system comprises a rare earth cryptate or a rare earth chelate in combination with a fluorescent or chemiluminescent dye, particularly a dye of the cyanine type.

In the context of the present invention, fluorescence-based assays include the use of dyes, for example dyes which may be selected from the group comprising FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein Isothiocyanate (FITC), IRD-700/800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, CY7, Xanthen, 6-carbonyl-2 ', 4', 7 ', 4, 7-Hexafluorofluorescein (HEX), TET, 6-carbonyl-4', 5 '-dichloro-2', 7 '-dimethoxyfluorescein (JOE), N' -tetramethyl-6-carboxyrhodamine (TAMRA), 6-carbonyl-X-Rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (6), RG, Rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, oregon green, coumarins such as umbelliferone, benzimines such as hurst 33258; phenanthridines such as texas red, jatamine, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrine dyes, polymethine dyes, and the like.

In the context of the present invention, chemiluminescence-based assays involve the use of dyes, based on the physical principles of the chemiluminescent species described in (24). The preferred chemiluminescent dye is an acridinium ester.

As referred to herein, an "assay" or "diagnostic assay" may be of any type used in the field of diagnostics. Such an assay may be based on the binding of the analyte to be detected to one or more capture probes having a certain affinity. In terms of the interaction between the capture molecule and the target or molecule of interest, the affinity constant is preferably greater than 10⁸M^-1。

In the context of the present invention, a "binding moiety molecule" is a molecule that can be used to bind a target molecule or molecule of interest, i.e. an analyte, from a sample (i.e. in the context of the inventive PCT and fragments thereof). The binding moiety molecule must therefore be formed into a shape sufficient to specifically bind the target or molecule of interest, both spatially and in terms of surface characteristics such as surface charge, hydrophobicity, hydrophilicity, presence or absence of Lewis donors and/or acceptors. Thus, for example, binding may be mediated by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bonding interactions or a combination of two or more of the above mentioned interactions between the capture molecule and the target or molecule of interest. In the context of the present invention, for example, the binding moiety molecule is selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the binding moiety molecule is an antibody, including fragments thereof having sufficient affinity to the target molecule or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibodies or fragments derived from variant chains thereof having a length of at least 12 amino acids.

The chemiluminescent label may be an acridinium ester label, a steroid label involving isoluminol labeling, or the like.

The enzyme label may be Lactate Dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, etc.

In one embodiment of the invention, at least one of the two binding moieties is bound to a solid phase such as magnetic particles and polystyrene surfaces.

In one embodiment of the invention, the measured concentration of mature ADM1-52 and/or mature ADM1-52-Gly in the sample is in the range of 10-500pg/ml in plasma or blood.

The ADM assay described was used to detect ADM levels of the present invention. The above mentioned values may be different in other ADM measurements, depending on their way of calibration. The above mentioned values should therefore require such different calibration ADM measurements, taking into account calibration differences. Calibration ADM measurements can be correlated and adjusted by going through their normal range (healthy population). Alternatively, commercially available control samples can be used for different calibration adjustments (ICI Diagnostics, berlin, germany). The median of the normal population, determined with the ADM described, was detected to be 24.7 pg/mL.

In one embodiment of the invention, a threshold is applied such that a value above the threshold indicates that the patient is not responding or responds poorly to treatment, and a value below the threshold indicates that the patient is responding to treatment.

In one embodiment of the invention a threshold of 60-80pg/ml, preferably 70pg/ml, is applied.

In one embodiment of the invention, the sample is selected from the group consisting of human citric acid plasma, heparin plasma, EDTA plasma, whole blood.

In one embodiment of the invention, the sample extracted is measured directly without any further sample preparation.

In one embodiment of the invention, the method is performed on a fully automated apparatus. RocheAbbottSiemensBrahmsBiomerieuxAlere

In one embodiment of the invention, mature ADM1-52 and/or mature ADM1-52-Gly is detected in at least two samples, wherein the samples are taken from the septic patient at different time points. The sample may be taken once a day during the treatment period. Such diagnostic protocols that describe the use of other biomarkers, e.g., (25) and (26), may be applied.

In one embodiment of the invention, the sample volume measured is less than or equal to 50. mu.l.

The in vitro method for therapy follow-up in septic patients of the invention may be combined with other clinical and/or laboratory parameters and/or clinical scores, such as for example the Apache2 score, the SOFA score or others, or one or more parameters contained in the score. The variables/parameters can be combined continuously or intermittently using standard statistical tools.

Subject of the present invention is also an assay for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, wherein said sample comprises two binding moieties binding to two different regions of a mature adrenomedullin and/or adrenomedullin-Gly region, amino acids 21-52-amino SEQ ID No.1 or amino acids 21-52-Gly SEQ ID No.2 of mature adrenomedullin, wherein each of said regions comprises at least 4 or 5 amino acids, and wherein said assay is not an artificially coated acridinium ester (coated-tube-akridinium) sandwich assay.

The subject of the invention is also an assay for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, wherein the sample comprises two binding moieties that bind to mature adrenomedullin and/or an adrenomedullin-Gly region, i.e. amino acids 21-52-amino SEQ ID No.1 or amino acids 21-52-Gly SEQ ID No.2 of mature adrenomedullin, two different regions, wherein each of said regions comprises at least 4 or 5 amino acids, and wherein said assay is an artificially coated tubacridone ester (coated-tube-Akridinium ester) sandwich assay, and wherein one of said binding moieties is an antibody that binds to SEQ ID No.4, wherein the other of the binding moieties is an antibody that binds to SEQ ID No.7(APRSKISPQGY-CO-NH 2).

In one embodiment of the invention for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, one of said binding moieties binds to a region comprised in the following sequence of mature ADM:

CTVQKLAHQIYQ(SEQ ID No.6)

and wherein another of said binding moieties binds to a region comprised in the following sequence of mature ADM:

APRSKISPQGY(SEQ ID No.7)

in an embodiment of the present invention for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, the assay sensitivity of said assay is capable of quantifying ADM in healthy humans and is <10pg/ml, preferably <40pg/ml and more preferably <70 pg/ml.

In one embodiment of the present invention for use in an assay for detecting mature adrenomedullin and/or adrenomedullin-Gly in a sample, said binding moiety exhibits a binding affinity for mature ADM and/or mature ADM1-52-Gly of at least 10⁷M^-1Preferably 10⁸M^-1Preferably, the affinity constant is greater than 10⁹M^-1Most preferably greater than 10¹⁰M^-1. It is known to those skilled in the art that it is contemplated that higher doses of the compound may be used to compensate for the lower affinity, and that such measurements would not be outside the scope of the present invention. Binding affinity can be detected as described above.

In one embodiment of the present invention for use in an assay for detecting mature adrenomedullin and/or adrenomedullin-Gly in a sample, said binding moiety is selected from an anti-adrenomedullin antibody, or an anti-ADM antibody fragment binding to ADM, or a non-Ig scaffold binding to adrenomedullin

In one embodiment of the present invention for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, such an assay is a sandwich assay, preferably a fully automated assay. It may be a fully automated or manual ELISA. It may be a so-called PCT test (point of care). Examples of automated or fully automated tests include assays that can be used in one of the following systems: rocheAbbottSiemensBrahmsBiomerieuxAlereExamples of test formats are provided above.

In one embodiment of the invention for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, at least one of the two binding moieties is labeled for detection. Examples of markers are provided above.

In one embodiment of the present invention for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, at least one of said two binding moieties is bound to a solid phase. Examples of solid phases are provided above.

In one embodiment of the present invention for an assay for detecting mature adrenomedullin and/or adrenomedullin-Gly in a sample, said label is selected from the group consisting of a chemiluminescent label, an enzymatic label, a fluorescent label, a radioiodine label.

A further subject of the invention is a kit comprising the test of the invention, wherein the components of the assay may be contained in one or more containers.

Another subject matter of the invention is a method for calibrating the assay of the invention, wherein a binding moiety, preferably an antibody, is used which binds to a region of at least 5 amino acids within mature adrenomedullin and/or adrenomedullin-Gly amino acids 1-16(SEQ ID No. 8). The binding moiety may be an antibody or antibody fragment or a non-Ig scaffold that binds to a region of at least 5 amino acids within mature adrenomedullin and/or adrenomedullin-Gly amino acids 1-16(SEQ ID No. 8).

In one embodiment of the method of the invention for calibrating an assay, the N-terminal antibody or fragment or scaffold recognizes and binds to the N-terminus of mature adrenomedullin and/or adrenomedullin-Gly. In another preferred embodiment, said anti-ADM antibody or anti-adrenomedullin antibody fragment or non-Ig scaffold binds only to a region within the sequence of mature ADM if the N-terminus of ADM is free. In such embodiments, if the sequence is contained within pro-ADM, the anti-ADM antibody or anti-adrenomedullin antibody fragment or non-Ig scaffold does not bind to a region within the sequence of mature ADM.

The antibodies of the invention suitable for use in calibration assays are such binding moieties that meet the adsorption characteristics of ADM. Such binding moieties must also be compatible with the binding moieties used in the detection assay, e.g., in the case of an ELISA, the binding moieties should not interfere with the binding of the labeled binding moiety to the solid phase binding moiety.

The methods and assays are suitable for routine use. In most cases routine applications require that the required sample volume should not exceed 50 μ l. Conventional applications also require that pre-analytical treatment be kept to a minimum or zero (use of conventional samples, such as EDTA plasma, citric acid plasma). The requirements before analysis must be adapted to clinical routine: the minimum analytical stability (> 90% reproduction) should be at least 2 hours at room temperature.

The antibodies of the invention are proteins that specifically bind to an antigen and comprise one or more polypeptides substantially encoded by immunoglobulin genes recognized as immunoglobulin genes include kappa, lambda, α (Ig A), gamma (IgG)₁,IgG₂,IgG₃,IgG₄) The (IgD), (IgE) and mu (IgM) constant region genes, and a myriad of immunoglobulin variable region genes. Full-length immunoglobulin light chains are typically about 25Kd or 214 amino acids in length. Full-length immunoglobulin heavy chains are typically about 50Kd or 446 amino acids in length. Light chain composed of NH₂Variable region genes at the end (approximately 110 amino acids in length) and kappa or lambda constant region genes at the COOH end. Similarly, the heavy chain is encoded by a variable region gene (approximately 116 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is typically a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen and the constant regions mediate effector functionsCan be used. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab and (Fab')₂And bifunctional hybrid antibodies with single chains ((27), (28), (29), (30), (31)). Immunoglobulin light or heavy chain variable regions comprise framework regions interrupted by three hypervariable regions, also known as Complementarity Determining Regions (CDRs) see (32). As shown above, the CDRs are primarily responsible for binding to an epitope of an antigen. An immune complex is an antibody that specifically binds an antigen, such as a monoclonal antibody, a chimeric antibody, a humanized or human antibody, or a functional antibody fragment.

Chimeric antibodies are antibodies whose light and heavy chain genes are typically constructed by genetic engineering from immunoglobulin variable and constant region genes belonging to different classes. For example, variable segments of the mouse monoclonal antibody derived gene may be linked to human constant segments such as κ and γ 1 or γ 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and an invariant or effector domain from a human antibody, although other mammalian species may be used, or the variable region may be generated by molecular techniques. Methods for making chimeric antibodies are well known in the art, see, e.g., (33). A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs immunoglobulins derived from a non-human (such as mouse, rat, or synthetic) source. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, all CDRs are from a donor immunoglobulin in the humanized immunoglobulin. The constant regions need not be present, but if they are present, they must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90%, such as about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. As a donor antibody providing CDRs, a humanized antibody binds to the same antigen. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substituents substituted by amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. Exemplary conservative substituents are, for example, gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by means of genetic engineering (see, for example, (34)). A human antibody is an antibody in which both the light chain and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce triple source hybridoma cells. Human antibodies can also be produced by phage display methods (see, e.g., (35), (36), (37), which is incorporated herein by reference), or from a library of human combinatorial monoclonal antibodies (see the Morphosys website). Human antibodies can also be made by using transgenic animals carrying human immunoglobulin genes (see, e.g., (38) and (39), which are incorporated herein by reference).

Thus, ADM antibodies can have a form known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody of the invention is a recombinantly produced antibody, such as e.g. an IgG, typically a full-length immunoglobulin, or an antibody fragment comprising at least the F variable domain of a heavy and/or light chain, such as e.g. a chemically conjugated antibody (fragment antigen binding), including but not limited to Fab-fragments including Fab miniantibodies, single chain Fab antibodies, monovalent Fab antibodies with an epitope tag, such as e.g. Fab-V5Sx 2; bivalent Fab antibodies (minibodies) dimerized with the CH3 domain; bivalent or multivalent Fab, e.g. formed via multimerization with the aid of heterologous domains, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx 2; f (ab') 2-fragment, scFv fragment, multimeric multivalent or/and multispecific scFv fragment, bivalent and-Or a bispecific diabody,(bispecific T-cell engagers), trifunctional antibodies, multivalent antibodies, e.g. from a different class than G; single domain antibodies, such as nanobodies derived from camelidae or fish immunoglobulins, as well as many others.

In addition to anti-ADM antibodies, other biopolymer scaffolds are well known in the art to complex target molecules and have been used for the generation of high target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins.

In a preferred embodiment, the ADM antibody format is selected from the group consisting of Fv fragments, scFv fragments, Fab fragments, scFab fragments, (Fab)2 fragmented scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group consisting of scFab fragments, Fab fragments, scFv fragments and bioavailability optimized conjugates thereof, such as pegylated fragments. One of the most preferred forms is the scFab form.

non-Ig backbones can be protein backbones and can be used as antibody mimetics because they are capable of binding a ligand or an antigen. The non-Ig scaffold may be selected from tetranectin-based non-Ig scaffolds (e.g. as described in (40)), fibronectin scaffolds (e.g. as described in (41)); lipocalin-based scaffolds (e.g., as described in (42); ubiquitin protein scaffolds (e.g., as described in (43)), transfer scaffolds (e.g., as described in (44)), protein A scaffolds (e.g., as described in (45)), scaffolds based on ankyrin repeat sequences (e.g., as described in (46)), preferably, the micro-protein forms A cystine knot) scaffold (e.g., as described in (47)), FynSH3 domain-based scaffolds (e.g., as described in (48)), EGFR-A-domain-based scaffolds (e.g., as described in (49)), and Kunitz-domain-based scaffolds (e.g., as described in (59)).

In another preferred embodiment, the anti-ADM antibody or antibody fragment that binds ADM is a monospecific antibody. Monospecific antibodies are antibodies that all have avidity for the same antigen. Monoclonal antibodies are monospecific, but may be produced in other ways than by producing them from normal germ cells.

In a preferred embodiment of the invention, the antibodies of the invention may be produced as follows: balb/c mice were immunized with 100. mu.g of ADM-peptide-BSA-conjugate (emulsified in 100. mu.l of complete Freund's adjuvant) on days 0 and 14 and with 50. mu.g on days 21 and 28 (100. mu.l of complete Freund's adjuvant). Animals received 50 μ g of conjugate dissolved in 100 μ l saline, given one intraperitoneal and one intravenous injection, 3 days before the fusion assay.

Splenocytes from immunized mice were fused with 1ml of 50% polyethylene glycol at 37 ℃ for 30 seconds with cells of the myeloma cell line SP 2/0. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [ RPMI1640 medium supplemented with 20% fetal bovine serum and HAT supplement ]. Two weeks later HT medium was used instead of HAT medium for 3 cell passages and then returned to normal cell culture medium.

Cell culture supernatants were initially screened 3 weeks after fusion for antigen-specific IgG antibodies. Test positive minicell cultures were transferred to 24-well plates for proliferation. After retesting, the selected cultures were cloned and recloned using limiting dilution techniques and the isoforms detected (see also (51) and (52)).

Antibodies can be generated by means of phage display according to the following procedure: the human natural antibody gene library HAL7/8 was used to isolate recombinant single chain F-variable domains (scFv) of anti-adrenomedullin peptides. Antibody gene libraries are screened using a screening strategy that involves the use of a peptide containing a biotin tag linked to an adrenomedullin peptide sequence via two different spacers. A mix of screening rounds using non-specific binding antigen and streptavidin-binding antigen was used to minimize the background of non-specific binding moieties. Eluted phage from the third round of screening have been used to passage E.coli (E.coli) strains expressing monoclonal scFv. Supernatants from cultures of these clones have been used directly for antigen ELISA assays. (53) And (54).

Humanization of murine antibodies was performed according to the following procedure: for humanization of antibodies of mouse origin, the antibody sequences were analyzed for structural interactions of the Framework Regions (FR) with the Complementarity Determining Regions (CDRs) and the antigen. Based on structural modeling, appropriate FRs of human origin are picked and mouse CDR sequences are grafted to human FRs. Variations in the amino acid sequence of the CDRs or FRs may be introduced into the restored structural interactions, which can be eliminated by converting the species into FR sequences. This reversion to structural interactions can be achieved by random methods using phage display libraries or via direct methods guided by molecular simulation (55).

Development of antibodies

We developed mouse monoclonal antibodies that bind the N-, middle-, and C-terminal portions of hadms and examined their affinity constants (table 1).

Peptides for immunization

The JPT peptide technology GmbH (Berlin, Germany) provides peptides. Peptides were conjugated to BSA using the Sulfo-SMCC cross-linking method. The crosslinking procedure was performed according to the manufacturer's instructions (Thermo Fisher/Pierce).

Example 1

Production of antibodies and detection of their affinity constants

Mouse antibodies were generated according to the following method: balb/c mice were immunized with 100. mu.g of peptide-BSA-conjugate on days 0 and 14 (emulsified in 100. mu.l of Freund's complete adjuvant) and with 50. mu.g (in 100. mu.l of Freund's incomplete adjuvant) on days 21 and 28. Three days prior to the fusion test, animals received 50 μ g of conjugate dissolved in 100 μ l saline given one intraperitoneal and one intravenous injection.

Splenocytes from immunized mice were fused with 1ml of 50% polyethylene glycol at 37 ℃ for 30 seconds with cells of the myeloma cell line SP 2/0. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [ RPMI1640 medium supplemented with 20% fetal bovine serum and HAT supplement ]. Two weeks later HT medium was used instead of HAT medium for 3 cell passages and then returned to normal cell culture medium.

Cell culture supernatants were initially screened 3 weeks after fusion for antigen-specific IgG antibodies. Test positive minicell cultures were transferred to 24-well plates for proliferation. After retesting, the selected cultures were cloned and recloned using limiting dilution techniques and the isoforms detected. ((51) and (52)).

Table 1:

production of monoclonal antibodies

Antibodies were produced via standard antibody production methods (56) and purified via protein a. Based on SDS gel electrophoresis analysis, the antibody purity was > 95%.

Affinity constant

To determine the affinity of the antibodies for adrenomedullin, the kinetics of binding of adrenomedullin to immobilized antibodies was determined by means of label-free surface plasmon resonance using the Biacore2000 system (GE Healthcare European Co., Frisburg, Germany). According to the manufacturing instructions (mouse antibody capture kit; GE Healthcare), reversible immobilization of antibodies was performed using an anti-mouse Fc antibody covalently bound to the surface of a CM5 sensor at high density. (22).

Labeling procedure (tracer) 100. mu.g (100. mu.l) of antibody (1mg/ml in PBS, pH7.4) were mixed with 10. mu.l acridine NHS-ester (Akridinium NHS-ester) (1mg/ml acetonitrile solution, InVent GmbH, Germany) (57) and incubated at room temperature for 20 min. By passingLabeled CT-H was purified by gel filtration HPLC on SEC400-5(Bio-Rad Laboratories, Inc., USA). Will purifyThe labeled antibody of (1) was diluted in (300mmol/L potassium phosphate, 100mmol/L NaCl,10mmol/L Na-EDTA,5g/L bovine serum albumin, pH 7.0). The final concentration was approximately 800.000 Relative Light Units (RLU)/200. mu.l of labeled compound (approximately 20ng labeled antibody). By using AutoLumatLB953(Berthold Technologies GmbH)&Kg) for detection of acridinium ester (akridinium ester) chemiluminescence.

Solid phase: polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 hours at room temperature) with antibody ((1.5. mu.g antibody/0.3 mL100mmol/L NaCl,50mmol/L TRIS/HCl, pH 7.8.) after blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH7.4, and dried in vacuo.

Calibration material: the complete protease inhibitor Cocktail Tablets (Roche AG) were prepared using 50mM Tris/HCl,250mM NaCl,0, 2% Triton X-100, 0.5% BSA,20tabs/L protease; synthetic human ADM (Bachem, switzerland) was diluted linearly at ph 7.8. The calibrators were stored at-20 ℃ prior to use.

Example 2

Detection of antibody combinations yielding high signal-to-noise ratios

hADM immunoassay:

pipette 50 μ l of sample (or calibrator) into coated tube, and after addition of labeled secondary antibody (200 μ l), incubate tube at room temperature for 2 hours. Unbound tracer was removed by washing 5 times with wash solution (20mM PBS, pH7.4, 0.1% Triton X-100).

Tube-bound chemiluminescence was detected by using LB 953.

All antibodies were used in sandwich immunoassays as coated tubes and labeled antibodies and combined in the following variations (table 2):

incubations were performed as described for the hADM immunoassay below. The results are shown as the ratio of specific signal (10 ng/ml ADM)/background signal (sample without ADM).

Table 2:

surprisingly, we found that MR-ADM and CT-ADM as combined compositions have the highest signal to noise ratio.

Subsequently, we used this antibody-combination for further studies. We used MR-ADM as the immobilized antibody and CT-ADM as the labeled antibody. A typical dose/signal curve is shown in figure 1. The analytical sensitivity determined (mean of 10 cycles, no ADM sample +2SD) was 2pg ADM/ml.

Example 3

Stability of human adrenomedullin:

human ADM was diluted in human citrate plasma (n-5, final concentration 10ng ADM/ml) and incubated at 24 ℃. At selected time points, aliquots were frozen at-20 ℃. Upon thawing, the sample hADM was quantified by immunoassay using the hADM described above.

Table 3 shows the stability of hADM in human plasma at 24 ℃

Surprisingly, the pre-analytical stability of the analyte was high (only a mean 0.9%/hour loss of immune response) when using the antibody in combination with MR-ADM and CT-ADM in a sandwich immunoassay. In contrast, using other assays, plasma half-life was reported to be only 22 minutes (Hinson 2000). Since the time from sampling to analysis is less than 2 hours in hospital routine, the ADM detection method used is suitable for routine diagnosis. It is noteworthy that no unconventional sample additives (such as Aprotinin, (20)) are required to achieve acceptable ADM-immune response stability.

Example 4

Reproducibility of calibrant-preparation

We found the results of making high variation (mean CV 8.5%, see table 4) determined against ADM. This may be due to high absorption of hADM by plastic and glass surfaces (see also (58). this effect can only be slightly mitigated by the addition of detergents (up to 1% TritonX100 or 1% Tween20), proteins (up to 5% BSA), and high ionic strength (up to 1M Nacl), or combinations thereof. surprisingly, if excess anti-ADM antibody (10ug/ml) is added to the calibrant dilution buffer, ADM assay calibrant-to-preparation recovery and reproducibility improves dramatically to < 1% inter-preparation CV (Table 4).

Fortunately, the presence of the N-terminal antibody did not affect the ADM-signal generated by the combination of MR-and C-terminal antibodies (fig. 11).

Table 4:

inter-preparation variation of calibrators.

ADM assay calibrants were prepared as described above with or without 10ug/ml NT-ADM-antibody. The coefficient of variation was from 5 independent preparation runs. The detection calibrators were measured using the ADM described above. s/n-r is the ratio of signal to noise.

For all the following studies, we used the ADM assay, based on calibrators, prepared in the presence of 10ug/ml NT-ADM antibody, and 10ug/ml NT-ADM antibody as a supplement in tracer buffer.

Example 5

Sensitivity of the composition

The goal of determining sensitivity is to completely cover the ADM concentration of healthy individuals.

ADM concentration in healthy individuals:

healthy individuals were measured using the ADM assay (n 100, mean age 56 years). The median value was 24.7pg/ml, the lowest value was 11pg/ml and the 99 th percentile was 43 pg/ml. Because the assay sensitivity was 2pg/ml, 100% of all healthy individuals were detectable using the ADM assay described (see figure 2).

Example 6

Clinical research

101 ED patients meeting sepsis definition (59) were subsequently hospitalized (average 5 day hospitalization) and received standard of care treatment. EDTA-plasma was generated on day 1 (ED presentation) and sampled daily during hospitalization. The time to freeze the sample for the next ADM measurement was less than 4 hours.

Patient characteristics are summarized in table 5

TABLE 5

26.7% of all patients died during hospitalization, which was considered a treatment non-responder, and 73.3% of all patients survived sepsis, which was considered a treatment responder.

66% of all patients with sepsis had abnormal ADM values >43pg/ml (percentile 99), indicating that ADM is not a marker of infection.

Results of clinical study

Initial ADM was highly prognostic.

We correlated initial ADM values to hospital mortality and compared ADM to APACHE2 sepsis score (see (60).) ADM is highly prognostic of sepsis outcome and comparable to APACHE2 score-supplementary information of significance if ADM and APACHE were combined (figure 4).

ADM in therapy monitoring

Patients were treated based on the criteria of care treatment (table 5). The average hospital stay was 5 days. ADM was measured daily in hospitalizations (day 1 ═ time to admission) and correlated with hospitalized mortality (table 6). ADM changes during hospitalization and changes during this period improved by 52% prognostic value from initial Chi on day 5²19.2 to 29.2.

Using a simple threshold model of 70pg/ml ADM, a 68% risk of death was shown for patients with starting ADM concentrations >70pg/ml and still >70pg/ml during hospitalization (non-responder to treatment). Patients with an overall time ADM value <70pg/ml or progressing from >70pg/ml to <70pg/ml had only 11% mortality (good treatment/treatment responder), and patients with an ADM value >70pg/ml and who reduced their ADM concentration to a value <70pg/ml during hospitalization had 0% mortality. None of the patients developed from <70pg/ml to >70pg/ml during hospitalization. The time required to generate responder/non-responder information was approximately 1 day for all patients. Patients who responded to treatment during hospitalization >70pg/ml required approximately 2 days to pass through the ADM to indicate successful treatment.

TABLE 6

Literature reference

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Description of the drawings

FIG. 1 shows a typical hADM dose/response curve using MR-ADM as the solid phase antibody and CT-ADM as the labeled antibody.

FIG. 2: healthy individuals were measured using the ADM assay (n 100, mean age 56 years). The median value was 24.7pg/ml, the lowest value was 11pg/ml and the 99 th percentile was 43 pg/ml. Because the assay sensitivity was 2pg/ml, 100% of all healthy individuals were detectable using the ADM assay described.

FIG. 3: prediction of hospitalized mortality-results from logistic regression.

FIG. 4: prediction of hospitalized mortality-ADM is independent of Apache and provides additional prognostic information.

FIGS. 5 to 10: ADM-kinetics of individual patients.

Fig. 5 and 6: survivors, ED display (day 1) and ADM <70pg/ml during hospitalization. ADM suggests well-treated patients.

Fig. 7 and 8: survivors, ADM was above 70pg/ml at ED display (day one) and decreased to values below 70pg/ml during hospitalization (treatment responders).

Fig. 9 and 10: deceased patients, ADM was above 70pg/ml at ED display (first day) and did not drop to values below 70pg/ml during hospitalization (treatment non-responders).

FIG. 11: ADM assay in the presence and absence of N-terminal antibody. ADM measurements were performed as described above. A) Reference curve B) in the presence of NT-ADM antibody (10. mu.g/ml, 3.33. mu.g/test). Addition of NT-ADM antibody did not affect the ADM assay.

Sequence of

SEQ ID No.1:ADM21-52

CTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2

SEQ ID No.2:ADM21-52-Gly

CTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYG

SEQ ID No.3:PreProADM

MKLVSVALMYLGSLAFLGADTARLDVASEFRKKWNKWALSRGKRELRMSSSYPTGLADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRVKRYRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYGRRRRRSLPEAGPGRTLVSSKPQAHGAPAPPSGSAPHFL

SEQ ID No.4:ADM1-52

YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2

SEQ ID No.5:ADM1-52-Gly

YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYG

SEQ ID No.6:ADM21-32

CTVQKLAHQIYQ

SEQ ID No.7:ADM42-52

APRSKISPQGY

SEQ ID No.8 ADM1-16-Gly (amino acid)

YRQSMNNFQGLRSFGC

Claims (51)

1. Use of two binding moieties in the preparation of a reagent for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, wherein said two binding moieties are selected from anti-adrenomedullin antibodies, or fragments of anti-ADM antibodies that bind ADM, or bind to the non-Ig backbone of adrenomedullin, and bind to two different regions of amino acids 21-52-amino as shown in SEQ ID No.1 or amino acids 21-52-Gly as shown in SEQ ID No.2, wherein each of said regions comprises at least 4 or 5 amino acids, and wherein said reagent is not used in an artificial coated acridinium ester sandwich assay.

2. Use of two binding moieties in the preparation of a reagent for the detection of mature adrenomedullin and/or adrenomedullin-Gly in a sample, wherein the two binding moieties are selected from anti-adrenomedullin antibody, or an anti-ADM antibody fragment binding to ADM, or to the non-Ig scaffold of adrenomedullin, both binding moieties binding to two different regions of amino acids 21-52-amino as shown in SEQ ID No.1 or amino acids 21-52-Gly as shown in SEQ ID No.2, wherein each of said regions comprises at least 4 or 5 amino acids, and wherein said reagents are used in an artificial coated tube acridinium ester sandwich assay, and wherein one of said binding moieties is an antibody that binds to SEQ ID No.6CTVQKLAHQIYQ, wherein the other of the binding moieties is an antibody which binds to SEQ ID No.7APRSKISPQGY-CO-NH 2.

3. Use according to claim 1, wherein one of the binding moieties binds to a region comprised in the sequence of mature ADM as depicted in SEQ ID No.4 and/or mature ADM1-52-Gly and wherein another of the binding moieties binds to a region comprised in the sequence of mature ADM and/or mature ADM1-52-Gly as depicted in SEQ ID No. 5.

4. The use of any one of claims 1-3, wherein the assay sensitivity of the agent is capable of quantifying ADM in a healthy individual at <10 pg/ml.

5. The use of any one of claims 1-3, wherein the assay sensitivity of the agent is capable of quantifying ADM in a healthy individual at <40 pg/ml.

6. The use of any one of claims 1-3, wherein the assay sensitivity of the agent is capable of quantifying ADM in a healthy individual at <70 pg/ml.

7. The use according to any one of claims 1 to 3, wherein the binding moiety exhibits at least 10⁷M^-1Of adrenomedullin.

8. The use of any one of claims 1-3, wherein the binding moiety is selected from an anti-adrenomedullin antibody, or an anti-ADM antibody fragment that binds ADM, or a non-Ig scaffold that binds adrenomedullin.

9. The use of claim 1 or 3, wherein the reagent is for a sandwich assay.

10. The use of claim 9, wherein the reagent is for a fully automated assay.

11. The use of any one of claims 1-3, wherein at least one of the two binding moieties is labeled to facilitate detection.

12. The use of any one of claims 1-3, wherein at least one of the two binding moieties is bound to a solid phase.

13. The use of claim 11, wherein the label is selected from the group consisting of a chemiluminescent label, an enzymatic label, a fluorescent label, and a radioiodinated label.

14. A kit for detecting mature adrenomedullin and/or adrenomedullin-Gly in a sample comprising two binding moieties selected from an anti-adrenomedullin antibody, or an anti-ADM antibody fragment binding to ADM, or a non-Ig scaffold binding to adrenomedullin, binding to two different regions of amino acids 21-52-amino as shown in SEQ ID No.1 or amino acids 21-52-Gly as shown in SEQ ID No.2, wherein each of said regions comprises at least 4 or 5 amino acids, and wherein said kit is not for use in an artificial coated acridinium ester sandwich assay and the components of said kit can be contained in one or more containers.

15. The kit according to claim 14, wherein one of the binding moieties binds to a region comprised in the sequence of mature ADM as depicted in SEQ ID No.4 and/or mature ADM1-52-Gly and wherein another of the binding moieties binds to a region comprised in the sequence of mature ADM and/or mature ADM1-52-Gly as depicted in SEQ ID No. 5.

16. A kit for detecting mature adrenomedullin and/or adrenomedullin-Gly in a sample, comprising two binding moieties selected from anti-adrenomedullin antibody, or an anti-ADM antibody fragment binding to ADM, or to the non-Ig skeleton of adrenomedullin, both binding moieties binding to two different regions of amino acids 21-52-amino as shown in SEQ ID No.1 or amino acids 21-52-Gly as shown in SEQ ID No.2, wherein each of said regions comprises at least 4 or 5 amino acids, and wherein said kit is for an artificially coated acridinium ester sandwich assay, and wherein one of said binding moieties is an antibody that binds to SEQ ID No.6CTVQKLAHQIYQ, wherein the other of the binding moieties is an antibody which binds to SEQ ID No.7APRSKISPQGY-CO-NH 2.

17. The kit of any one of claims 14-16, wherein the assay sensitivity of the kit is capable of quantifying ADM in a healthy individual at <10 pg/ml.

18. The kit of any one of claims 14-16, wherein the assay sensitivity of the kit is capable of quantifying ADM in a healthy individual at <40 pg/ml.

19. The kit of any one of claims 14-16, wherein the assay sensitivity of the kit is capable of quantifying ADM in a healthy individual at <70 pg/ml.

20. The kit of any one of claims 14-16, wherein the binding moiety exhibits at least 10⁷M^-1Of adrenomedullin.

21. The kit of any one of claims 14-16, wherein the binding moiety is selected from an anti-adrenomedullin antibody, or an anti-ADM antibody fragment that binds ADM, or a non-Ig scaffold that binds adrenomedullin.

22. The kit of claim 14 or 15, wherein the kit is for a sandwich assay.

23. The kit of claim 22, wherein the kit is for a fully automated assay.

24. The kit of any one of claims 14-16, wherein at least one of the two binding moieties is labeled to facilitate detection.

25. The kit of claim 24, wherein the label is selected from the group consisting of a chemiluminescent label, an enzymatic label, a fluorescent label, and a radioiodine label.

26. The kit of any one of claims 14-16, wherein at least one of the two binding moieties is bound to a solid phase.

27. The kit of any one of claims 14 to 16, wherein the kit further comprises a binding moiety that binds to mature adrenomedullin and/or a region of at least 5 amino acids within adrenomedullin-Gly amino acids 1 to 16 as set forth in SEQ ID No. 8.

28. The kit of claim 27, wherein the binding moiety recognizes and binds to the N-terminus of mature adrenomedullin and/or adrenomedullin-Gly.

29. Use of a binding moiety for the preparation of a reagent for calibrating a kit of parts according to any one of claims 14 to 28, wherein the binding moiety binds to mature adrenomedullin and/or to a region of at least 5 amino acids within adrenomedullin-Gly amino acids 1 to 16 as set forth in SEQ ID No. 8.

30. The use of claim 29, wherein the binding moiety recognizes and binds to the N-terminus of mature adrenomedullin and/or adrenomedullin-Gly.

31. Use of two binding moieties selected from anti-adrenomedullin antibody, or an anti-ADM antibody fragment binding to ADM, or a non-Ig scaffold binding to adrenomedullin, binding to two different regions of amino acids 21-52-amino as shown in SEQ ID No.1 or amino acids 21-52-Gly as shown in SEQ ID No.2, and wherein each of said regions comprises at least 4 or 5 amino acids, for the manufacture of a kit for the therapeutic follow-up of patients suspected to have sepsis, wherein an assay comprising said two binding moieties is used to determine the concentration of mature ADM1-52 and/or mature ADM1-52-Gly in a body fluid sample of said septic patient.

32. Use according to claim 31, wherein one of the binding moieties binds to a region comprised in the sequence of mature ADM as shown in SEQ ID No.4 and/or mature ADM1-52-Gly and wherein another of the binding moieties binds to a region comprised in the sequence of mature ADM and/or mature ADM1-52-Gly as shown in SEQ ID No. 5.

33. The use of claim 31 or 32, wherein the assay sensitivity of the assay is capable of quantifying ADM in a healthy individual to <10 pg/ml.

34. The use of claim 31 or 32, wherein the assay sensitivity of the assay is capable of quantifying ADM in a healthy individual to <40 pg/ml.

35. The use of claim 31 or 32, wherein the assay sensitivity of the assay is capable of quantifying ADM in a healthy individual to <70 pg/ml.

36. The use according to claim 31 or 32, wherein the binding moiety exhibits at least 10⁷M^-1And/or mature ADM 1-52-Gly.

37. The use of claim 31 or 32, wherein the binding moiety is selected from an anti-adrenomedullin antibody, or an anti-ADM antibody fragment that binds ADM, or a non-Ig scaffold that binds adrenomedullin.

38. The use of claim 31 or 32, wherein the assay is a sandwich assay.

39. The use of claim 38, wherein the assay is a fully automated assay.

40. Use according to claim 31 or 32, wherein at least one of the two binding moieties is labelled to facilitate detection.

41. The use of claim 31 or 32, wherein at least one of the two binding moieties is bound to a solid phase.

42. The use of claim 40, wherein the label is selected from the group consisting of a chemiluminescent label, an enzymatic label, a fluorescent label, and a radioiodinated label.

43. The use of claim 31 or 32, wherein the concentration of mature ADM1-52 and/or mature ADM1-52-Gly measured in a sample is in the range of 10-500 pg/ml.

44. The use of claim 31 or 32, wherein a threshold is applied such that a value above the threshold indicates that the patient is non-responsive or poorly responsive to the treatment and a value below the threshold indicates that the patient is responsive to the treatment.

45. The use of claim 31 or 32, wherein a threshold of 60-80pg/ml is applied.

46. The use of claim 45, wherein a threshold of 70pg/ml is applied.

47. The use of claim 31 or 32, wherein the sample is selected from the group consisting of human citric acid plasma, heparin plasma, EDTA plasma, whole blood.

48. The use of claim 47, wherein the sample taken is measured directly without any further sample preparation.

49. The use of claim 31 or 32, wherein the assay is performed on a fully automated device.

50. The use of claim 31 or 32, wherein mature ADM1-52 and/or mature ADM1-52-Gly is detected in at least two samples, wherein the samples are taken from the septic patient at different time points.

51. The use of claim 31 or 32, wherein the sample is measured in a volume of less than or equal to 50 μ l.