WO2004074840A1

WO2004074840A1 - Method for the quantitative measurement of ligands in the sample of dissolved binding proteins

Info

Publication number: WO2004074840A1
Application number: PCT/EP2004/001272
Authority: WO
Inventors: Christian J. Strasburger; Martin Bidlingmaier; Zida Wu
Original assignee: Klinikum Der Univeristät München Grosshadern- Innenstadt
Priority date: 2003-02-19
Filing date: 2004-02-11
Publication date: 2004-09-02
Also published as: DE10306992A1

Abstract

The invention relates to a method for the quantitative measurement of ligands in the sample of dissolved or suspended binding proteins by using specific antibodies, antibodies used in said method, medicaments, diagnostic agents, (diagnostic) kits containing said antibodies, the use of said antibodies in quantitative analysis and in selective indications, and methods for the production thereof.

Description

applicant:

Clinic of the University of Munich Großhadern - downtown Lindwurmstr. 2a 80337 Munich

Method for the quantitative measurement of ligands in the sample of binding proteins dissolved

The invention relates to a method for the quantitative determination of ligands in the sample of dissolved or suspended binding proteins using specific antibodies, antibodies usable in the method, medicines, diagnostics and (diagnostic) kits containing these

Antibodies, the use of these antibodies in quantitative analysis and in selected indications for therapeutic purposes, and methods for their production.

The quantitative determination of certain chemical compounds from a sample and especially a physiologically occurring compound in body fluids - such as blood - is an important diagnostic tool in medicine. Many of these physiological compounds, such as hormones and other messenger substances, bind specifically to so-called binding proteins or receptors, which in many cases are themselves dissolved or suspended in the liquid - such as the blood. However, there is, for example, the risk that a complex of the compound to be measured (ligand) and binding protein forms before or during the determination and the ligand is thus withdrawn from a measurement or that the binding protein withdraws the measurement from the labeled ligands added for the purpose of measurement. This often leads to a clear interference of the binding protein in the quantitative measurement and thus in the diagnostics. A particularly striking and important example of the danger of such interference of the binding protein is the quantitative determination of human growth hormone.

Human growth hormone (hGH) is a protein hormone that is secreted by the pituitary gland. In addition to the importance that growth hormone has for the length growth of children and adolescents, it is essential for the regulation of a large number of metabolic processes throughout life. In children and adults there are diseases in which the physiological regulation of growth hormone secretion is disturbed. The growth hormone deficiency and the growth hormone excess should be mentioned in particular. The determination of the concentration of growth hormone in serum samples is an essential diagnostic tool and is therefore carried out in many medical laboratories.

As a rule, so-called immunoassays are used for the analysis of the growth hormone concentration. These are based on the principle of the interaction of specific antibodies with an analyte. Competitive assays are also optionally used (such as radioimmunoassays, RIA), in which labeled growth hormone competes with the growth hormone present in the sample for binding to an antibody and thus produces a signal which is inversely proportional to the concentration of the analyte to be measured. However, sandwich immunoassays are now most frequently used, in which an immobilized specific antibody binds the analyte (“capture antibody”), and a second labeled antibody directed against a different epitope of the analyte then binds the now immobilized analyte. that is proportional to the amount of bound Analyte is. The best-known example of this measurement method is the "enzyme linked immunosorbent assay" (ELISA) with a colorimetric end point since the photometer required for the measurement is regularly available. Other possibilities for signal generation are radioactivity, chemiluminescence or (time-resolved) fluorescence.

All of the above-mentioned assay techniques are based on the principle of high-affinity, specific binding between an antibody directed against a certain hormone - in this case growth hormone - and the hormone molecule in the sample to be analyzed. Comparative studies have now shown that when measuring growth hormone of a sample in different laboratories with different methods, extremely large discrepancies occur for the concentrations determined in this sample (1, 2, 5). This is also shown regularly in the central proficiency tests of the German Society for Clinical Chemistry.

The results of various stimulation tests are used to diagnose a growth hormone deficiency. In these tests, after stimulation of growth hormone secretion, blood is drawn at intervals of 15 to 30 minutes over a period of 1 to 2 hours, and growth hormone secretion is determined from the samples. In two independent tests, the growth hormone concentration does not rise above an arbitrary one, but traditionally - e.g. in pediatrics - at a defined cut-off value of approx. 10 ng / ml, the diagnosis “growth hormone manager is considered very likely and the health insurance companies usually reimburse the following therapy costs. These costs of a - potentially lifelong - therapy with recombinant growth hormone are considerable.

Due to the extreme discrepancies in the growth hormone concentration, which is determined using different methods, one Uniform, correct diagnosis is unusually difficult: reaching the cut-off value in a stimulation test depends essentially on the laboratory in which the samples of a particular patient are measured or on the method used there. This has considerable economic effects, but also consequences that directly affect the patient: Depending on the measurement method, individual patients can be subjected to hormone replacement therapy unnecessarily, while it is withheld from other patients who are actually affected.

According to current knowledge, various factors contribute to this extreme discrepancy of the measured values, but all of them have a specific effect on the above-mentioned interaction between antigen (growth hormone) and antibody: growth hormone that is produced by the human pituitary gland is not a completely homogeneous substance, but consists of different isoforms. Precisely because of the high specificity, in particular of the monoclonal antibodies, for a precisely definable segment (epitope) on the surface of the growth hormone molecule, differences in their reactivity with the various isoforms of the hormone can now be expected. Therefore, different portions of this isoform spectrum are measured in the different assays depending on the epitope specificity of the antibodies used. For this reason too, the type of calibrator (standards) used in the respective assay plays a decisive role: The "International Reference Preparation 80/505", which is still used, is a preparation extracted from human pituitary glands, which also consists of various isoforms, while the newer "International Reference Preparation 88/624" is of recombinant origin and consists in pure form of the 22,000 dalton major isoform of growth hormone. The main disruptive factor for the measurement of growth hormone to which the invention described here relates is the presence of a high-affinity growth hormone binding protein (hGH binding protein, hGHBP) in human serum (2-4). The effect of this binding protein on the measurement result can lead to false high or false low values depending on the assay principle used (competitive assay or sandwich assay). In the competitive assay, on the one hand, labeled hGH (as a so-called “tracer”) can be bound by the binding protein and thus withdrawn from the assay mixture, which leads to incorrectly high concentrations. On the other hand, the binding protein can also sterically interact with the specific antibody with the growth hormone molecules from a serum sample In contrast, this steric hindrance to the interaction between antibody and hormone in sandwich immunoassay usually leads to false low results because less hormone or fewer detection antibodies are bound this disturbing factor, errors in the measurement results under certain physiological or pathological conditions which are associated with a change in the hGHBP concentration in the blood, such as, for example, pregnancies in the course of which there is an increase in the hGHBP is coming.

The problem of the variability of hGH measurements and the resulting problems in diagnosis have been discussed in specialist circles for years. Approaches to solution relate, on the one hand, to standardization of the calibrator to be used, and, on the other hand, to attempt to produce a homogeneous assay, at least with regard to the measured hGH isoform, by selecting antibodies which mainly recognize the 22,000 Dalton main isoform of hGH. So far, however, no approaches to solve the problem of interference by hGHBP have been published. In the case of other hormones and other endogenous substances, ex-tests are carried out before analysis, for example in an immunoassay. traction method used to eliminate the binding proteins, but this means a significant additional methodological effort and can lead to considerable measurement inaccuracies. Overall, however, there is still no satisfactory solution to the problem of soluble binding proteins in samples to be analyzed in the prior art.

It is therefore an object of the invention described below to switch off the interference of binding proteins which have been dissolved or suspended in the sample in quantitative measurements of their ligands in a simple manner. This is done by means of a technique with the aid of which these test systems, in particular all currently existing assays for growth hormone, can be made less susceptible to the interfering factor of the binding protein which is always present in, for example, serum samples, such as hGHBP.

The invention therefore relates to a method for the quantitative determination of a ligand of a binding protein in a sample containing ligand and binding protein in dissolved or suspended form, in which the sample to be measured has an antibody A against the binding protein which is directed against the binding site of the ligand on the binding protein is added.

It was found that the binding of the antibody A specifically directed against the binding protein to the binding protein exactly where the ligand binds leads to a displacement of the growth hormone molecules (ligand) from their binding to hGHBP and thereby also a new binding of the ligand prevented. This displacement is, of course, the greater the greater the amount of antibody A added, especially in relation to the binding protein, so that usually a large excess of antibody is favorable. The "released" growth hormone can now be measured quantitatively without steric inhibition by the binding protein. Since the "anti-binding protein Antibody "A itself does not interfere with the" anti-ligand antibodies "B possibly used for the measurement of the ligand (for example hGH), the need for an upstream removal of the antibody A / binding protein complexes from the sample is also eliminated. With the exception of the addition of antibody A and a possible preincubation, the sample can advantageously be used exactly as described by the manufacturer for the particular assay.

Binding protein in the sense of this invention is to be understood to mean all proteins which can bind a ligand to be measured here with high affinity. The binding proteins relevant here are mostly soluble or are present in the sample as a suspension. Examples are certain soluble receptors or, for example, hormone binding protein, such as growth hormone binding protein, which occurs in the cytosol or blood.

“Ligand” in the sense of this invention is to be understood as meaning compounds which bind to a binding protein with high affinity and whose concentration can be measured in certain assays and, for example, is also measured for medical purposes. Examples are messenger substances such as hormones, transmitters, for example. Meurotransmitter, βi cellular signal peptides or proteins, cylokines, chemokines, lymphokines etc.

For the purposes of this invention, “sample” is to be understood as meaning any type of solution to be examined, but in particular solutions of medically relevant substances, such as blood, lymph, serum, urine, liquor, also in a form prepared for sample processing.

For the purposes of this invention, “in dissolved or suspended form” means any form of solution of the binding protein or of the ligand in the broader sense in the solvent. This also includes situations in which, for example, the binding protein is on the edge or beyond the point of failure , but is still part of the solution. "Antibodies" in the sense of this invention are vertebrates or artificially produced proteins or possibly other structures which bind with high affinity to a certain surface confirmation (epitope) of an antigen, that is to say another molecule, preferably mono- or polyclonal (partial) structures of immunoglobulins (eg IgG, IgA, IgD, IgE) or also polyclonal monospecific antibodies Typically, such antibodies contain at least the variable part of immunoglobulins, possibly also at least one domain of the constant part of immunoglobulins (ab) ₂ - fragments of antibodies in the sense of the present invention.

An antibody against the binding protein which is directed against the binding site of the ligand on the binding protein means that the antibody, as a binding epitope, specifically binds the binding site of the ligand on the binding protein with high affinity.

“Quantitative determination” is generally understood to mean any type of measurement of the amount of a dissolved analyte in a sample known to the person skilled in the art. Explicitly, this means, for example, quantification using chromatographic methods with a moving standard and, in particular, using the high affinity interaction between antibody and ligand (Antigen) quantification by competitive assays or binding assays, such as a sandwich assay also in ELISA format It is a particular advantage of this invention that the method of antibody addition presented with practically any known - especially commercially available - test kit or test system for quantitative analysis of the respective analyte (ligand) can be combined with little effort. It is particularly preferred if, in the method according to the invention, the antibody A is added before or during, preferably before, the quantitative determination and / or is incubated with the sample.

Incubation is understood to mean a reaction condition in which reaction partners, here antibody A and binding protein and secondarily also the ligand, can react with one another. The incubation is usually limited in time, here for example 6, 12, 18 or 24 h. Incubation is to be understood here in particular to mean the pre-incubation - for example over 6, 12, 18 or 24 h - which takes place before the start of the quantitative measurement (for example with a commercially available test kit).

It is also preferred if the sample measured in the method according to the invention contains body fluid, preferably human body fluid, in particular blood or human blood.

Body fluid means any fluid obtained from the body of a vertebra, in particular a mammal, in particular a human. In the case of humans, this would be, for example, blood, urine or lymph, but also cytosolic preparations from human cells.

In a further preferred embodiment of the method according to the invention, the ligand is a physiological ligand of the physiological binding protein.

The method according to the invention is also useful if ligand and / or binding protein has been added (exogenously) from the outside, for example after injection or other absorption of recombinant hGH in patients or test subjects. This makes it possible to use a method according to the invention, for example for analyzing samples if there is a suspicion of ping (eg in high-performance sports) with hGH or other hormones (examples of ligands in the sense of the present invention) to suspect doping after quantifying, for example, the hGH concentration in the sample (without falsifying influences of the binding protein (s) in question) to exclude or justify. The binding proteins also interfere in pharmacokinetic analyzes, so that the artificially added hGH is also bound in this regard, and results in, for example, hGH concentration are falsified by the presence of binding protein. A procedure according to the invention can also remedy this in these cases.

A physiological ligand is to be understood as a ligand in the sense given above, which occurs in the body of a vertebrate, in particular in a body fluid, without being added from outside. The same applies to the physiological binding protein, which is also the natural binding protein of the physiological ligand that occurs under physiological conditions.

It is further preferred if the binding protein used in the method according to the invention is soluble, preferably a soluble receptor or a hormone binding protein, in particular a hormone binding protein.

In a preferred embodiment of the method according to the invention, the ligand is a peptidic compound and / or a hormone, preferably a peptidic hormone, in particular a growth hormone.

Peptide means any compound whose components are predominantly linked to one another via a peptide bond R1-NH-C (O) -R2. Hormone is a chemical messenger that acts at a distance from its place of synthesis and release. Hormones are preferably produced by endocrine glands, for example the pituitary gland, the gonads or the epiphysis. Examples are growth hormone or luteinizing hormone LTH, insulin, melatonin, glucagon, gastrin, angiotensin, substance P, interleukins, vasopressin, endorphins, enkephalins, relaxax or the atrionatriuretic factor. Hormone binding proteins are binding proteins (see above) that bind affine hormones.

In a particularly preferred embodiment of the method according to the invention, the ligand is human growth hormone (hGH) and the binding protein human growth hormone binding protein (hGHBP) and the antibody A is against the binding site of the hGH on the hGHBP - judges.

It is also particularly preferred if the antibody A used in the method according to the invention is a monoclonal antibody or a single-chain antibody, preferably a monoclonal antibody. It is particularly preferred if the antibody A used is ZMC1. ZMC1 is a monoclonal antibody which is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP) and was optimized within the scope of the invention to achieve the object. This was received on January 21, 2003 by the applicant under the number DSM ACC2582 (reference number assigned by the depositor: ZMC1) in accordance with the Budapest Treaty at the DSMZ (German Collection for Microorganisms and Cell Cultures GmbH) in Braunschweig (Germany, Maschenroder Weg 1b , 38124 Braunschweig) in viable form.

In this context, monoclonal means in particular the product of an artificial construct in which an antibody-producing cell (B cell) is fused with an immortalized cancer cell (hybridoma), resulting in a hybridoma cell. Specific antibodies can be obtained from this, all of which are directed exclusively to one epitope.

It is further preferred if, in the method according to the invention, the quantitative determination of the ligand is carried out using the binding of the ligand as antigen to a, preferably monoclonal, antibody B. In one embodiment, it is advantageous if the quantitative determination is carried out using a competitive binding test, preferably a “radio-immunoassay” (RIA). It is also just as favorable if the quantitative determination by measuring a function of the concentration of the measurement parameters to be measured increasing ligand, preferably by an enzyme-linked immunosorbent assay (ELISA) and / or by an appropriate sandwich assay.

In a further preferred embodiment of the method according to the invention, the binding protein is not separated from the sample to be measured before the quantitative determination. This is understood in particular to mean that, in the present invention, it is unnecessary to extract the binding protein from the sample before the quantitative analysis thereof, as must be done with other hormones in the prior art. Practically any form of extraction is conceivable in which the ligand to be measured - such as the hormone - remains in the sample while the binding protein is being separated, for example by precipitation and / or filtration with certain molecular weight cut-off limits, chromatographic methods such as Affinity chromatography or HPLC, dialysis for certain ligand sizes etc. But this can be dispensed with preferably when using the method according to the invention, since neither antibody A nor the complex of antibody A and binding protein binds to the ligand to be measured, and thus, by the choice of antibody A. the measurement solution is no longer hindered or adulterated, even if the complexed binding protein remains in the sample. The use of high-affinity Antibodies B for quantitative measurement does not change this either, since these bind to the ligand to be measured and not to Antibody A, the binding protein or the complex of both.

This advantage occurs in particular in a particularly favorable and preferred method according to the invention, in which the antibody A is added such that the antibody A is present in the sample after the addition in a higher concentration than the binding protein, preferably in an at least 50%, in particular at least 100%, preferably at least 200%, in particular at least 400% higher concentration.

It is precisely this addition of antibody A in excess to the binding protein that is particularly favorable since, with sufficient excess of antibody A, both ligands are completely displaced from the binding protein and thus become measurable, and the binding protein is quantitatively compensated and therefore no longer interferes with the determination.

The person skilled in the art can determine the exact, correct and sufficient amount of specific antibody A that must or can be added to the sample for quantitative measurement of the ligand, which is as interference-free as possible, at most, at most, in a few simple preliminary experiments. The optimal addition naturally depends on the amount of binding protein in the sample and possibly the quantitative test system used as well as possibly on the amount of ligand to be measured.

The particularly preferred form of the method according to the invention is a method for the quantitative determination of hGH using the affine binding of hGH as an antigen to a monoclonal antibody. by B in a sample containing human blood, in which hGH and hGHBP are contained in dissolved or suspended form, in which the sample to be measured is antibody A directed against the binding site of hGH on hGHBP, preferably the deposited monoclonal antibody ZMC 1 , in a clear excess over the amount of hGHBP before or during, preferably before, the quantitative determination of the hGH by means of antibody B is added and incubated.

The invention prevents the interference of the growth hormone binding protein by adding a specific excess of a specific, preferably monoclonal antibody, specifically produced against the growth hormone binding protein and characterized by specific experiments, to the sample to be measured. This monoclonal antibody (ZMC 1) was selected on the basis of its particularly preferred property that it binds to the hGHBP molecule exactly where the growth hormone molecule also binds. As a result, if the antibody is added in excess, the growth hormone molecules are displaced from their binding to hGHBP. The thus released ^{_S3, β} growth hormone can now without steric inhibition by the binding protein in any immuno assay are measured for growth hormone. Since the “anti-binding protein antibody” itself does not interfere with the “anti-growth hormone antibodies” used to measure hGH, there is also no need to remove the antibody-hGHBP complexes from the sample beforehand. In practice, the special anti-hGHBP antibody ZMC 1 with the hGH-displacing property of the sample is added before the analysis and the sample is used after preincubation, as described for the respective assay by the manufacturer.

Another object of the invention is an antibody A against a physiological binding protein of a physiological ligand dissolved or suspended in body fluids, the antibody A being is directed to the binding site of the ligand on the binding protein.

It is particularly preferred if the antibody A according to the invention is a monoclonal antibody and / or a single-chain antibody, preferably a monoclonal antibody.

In the present sense, however, the term “antibody” encompasses both polyclonal, in particular polyclonal, monospecific antibodies (ie antibodies with different variable regions, but all of which recognize a specific epitope), as well as monoclonal, chimeric antibodies, anti-idiotypic antibodies (directed against antibodies according to the invention ), which are all present in bound or soluble form and can optionally be labeled by “labels” (for example fluorescent markers, gold markers, coupled enzymes), and also fragments of the abovementioned antibodies. In addition to the fragments of antibodies according to the invention in isolation, antibodies according to the invention can also occur in recombinant form as fusion proteins with other (protein) components. Fragments as such or fragments of antibodies according to the invention as constituents of fusion proteins are typically produced by the methods of enzymatic cleavage, protein synthesis or the recombination methods familiar to the person skilled in the art. According to the present invention, the term antibodies means both polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or synthetic antibodies.

The polyclonal antibodies are heterogeneous mixtures of antibody molecules which are produced from sera from animals which have been immunized with an antigen. A monoclonal antibody contains an essentially homogeneous population of anti bodies that are specifically directed against antigens, the antibodies having essentially the same or the same epitope binding sites. The different antibody variants with monospecificity can belong to the immunoglobulin classes described below, so they can be mixtures of different main or subclasses, preferably a homogeneous mixture of IgG antibodies. This homogeneity can be achieved by an additional purification step (immunoprecipitation, chromatography, for example using antibodies directed against IgG).

Monoclonal antibodies can be obtained by methods known in the art (e.g., Koehler and Milstein, Nature, 256, 495-397, (1975); U.S. Patent 4,376,110; Ausübel et al., Harlow and Lane "Antibodies" : Laboratory Manual, Cold Spring, Harbor Laboratory (1988); Ausubel et al., (Eds), 1998, Current Protocols in Moleeular Biology, John Wiley & Sons, New York)). The description contained in the abovementioned references is included as part of the present invention in the disclosure of the present invention.

Antibodies according to the invention which have been genetically engineered can also be produced by methods as described in the abovementioned publications.

Antibodies according to the invention can belong to one of the following immunoglobulin classes: IgG, IgM, IgE, IgA, GILD and possibly a subclass of the aforementioned classes, such as the subclasses of the IgG or their mixtures. IgG and its subclasses such as, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgGM are preferred. The IgG subtypes IgG1 / k or IgG2b / k are particularly preferred. A hybridoma cell clone that produces monoclonal antibodies according to the invention can be cultivated in vitro or in vivo. Large titers of monoclonal antibodies are preferably produced in vivo or in situ. The chimeric antibodies according to the invention are molecules which contain different constituents, and these are derived from different animal species (e.g. antibodies which have a variable region which is derived from a mouse monoclonal antibody and a constant region of which) human immunoglobulin). Chimeric antibodies are preferably used, on the one hand, to reduce the immunogenicity during use and, on the other hand, to increase the yields in production, for example murine monoclonal antibodies give higher yields from hybridoma cell lines, but also lead to higher immunogenicity in humans, so that human / murine chimeric antibodies are preferably used. Chimeric antibodies and methods for their preparation are known from the prior art (Cabilly et al., Proc. Natl. Sei. USA 81: 3273-3277 (1984); Morrison et al. Proc. Natl. Acad. Sei USA 81: 6851-6855 (1984); Boulianne et al. Nature 312 643-646 (1984); Cabilly et al., EP-A-125023; Neuberger et al., Nature 314: 268-270 (1985); Taniguchi et al. , EP-A-171496; Morrion et al., EP-A-173494; Neuberger et al., WO 86/01533; Kudo et al., EP-A-184187; Sahagan et al., J. Immunol. 137: 1986, 1066-1074; Robinson et al., WO 87/02671; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987); Sun et al., Proc. Natl. Acad. Be USA 84: 214218 (1987), Better et al., Science 240: 1041-1043 (1988) and Harlow and Lane, Antibodies: A Laboratory Manual, as cited above, and these references are included in the present disclosure Invention included.

An anti-idiotypic antibody according to the invention is an antibody which recognizes a determinant which is generally associated with the antigen binding site of an antibody according to the invention. By immunizing an animal of the same type and the same genetic type (eg a mouse strain), an anti-idiotypic antibody can be used as the starting point for a monoclonal antibody against which an anti-idiotypic antibody according to the invention is directed. getting produced. The immunized animal will recognize the idiotypic determinants of the immunizing antibody by producing an antibody which is directed against the idiotypic determinants (namely an anti-idiotypic antibody according to the invention) (US Pat. No. 4,699,880). An anti-idiotypic antibody according to the invention can also be used as an immunogen to elicit an immune response in another animal and to produce an anti-idiotypic antibody there. The anti-anti-idiotypic antibody can, but need not, be identical in terms of its epitope construction to the original monoclonal antibody which caused the anti-idiotypic reaction. In this way, by using antibodies directed against idiotypic determinants of a monoclonal antibody, other clones which express antibodies of identical specificity can be identified.

Monoclonal antibodies, which are directed against a dissolved or suspended physiological binding protein of a physiological ligand in body fluids, can be used to bind anti-ϊdiotypic antibodies in corresponding animals, such as. B. the BALB / c mouse. Spleen cells from such an immunized mouse can be used to produce anti-idiotypic hybridoma cell lines that secrete anti-idiotypic monoclonal antibodies. Furthermore, anti-idiotypic monoclonal antibodies can also be coupled to a carrier (KLH, "keyhole limpet hemocyanin") and then used to immunize further BALB / c mice. The sera of these mice then contain anti-anti-idiotypic antibodies which have the binding properties of the original monoclonal antibodies and are specific for a physiological binding protein of a physiological ligand dissolved or suspended in body fluids (see preferred examples below). The anti-idiotypic monoclonal antibodies thus have their own idiotypic epitopes or "idiotopes" which are structurally similar to the epitope to be examined. The term "antibody" is intended to include both intact molecules and fragments thereof. Fragments include all shortened or modified antibody fragments with one or two binding sites complementary to the antigen, such as antibody parts with a binding site formed by light and heavy chains corresponding to the antibody, such as Fv, Fab or F (ab ') ₂ fragments or Single strand fragments, called. Shortened double-strand fragments such as Fv, Fab or F (ab ') 2-Fab and F (ab') ₂ fragments are preferably devoid of an Fc fragment, such as present in an intact antibody, so that they are transported faster in the bloodstream can and have comparatively less non-specific tissue binding than intact antibodies. It is emphasized here that Fab and F (ab ') ₂ fragments of antibodies according to the invention can be used in a method according to the invention as defined by the present invention. Such fragments are typically produced by proteolytic cleavage using enzymes such as. B. papain (for the production of Fab fragments) or pepsin (for the production of F (ab ') ₂ , fragments) can be used, or can be obtained by chemical oxidation or by genetic engineering of the antibody genes.

Furthermore, an antibody A according to the invention can also have further covalently (or not covalently) coupled molecules or groups, for example a fluorescent label or a label, for example a gold label, or specific epitopes that can be recognized by third-party molecules. Furthermore, an antibody A according to the invention can also be bispecific, that is to say it can recognize different epitopes with its two paratopes, preferably two different epitopes of the same protein or peptide (see above), but the structure of the two paratopes may also be different, but the same Tie epitope or at least overlapping areas. The present invention also relates to mixtures of antibodies according to the invention in the abovementioned sense, for example mixtures of monoclonal antibodies or mixtures of monoclonal antibodies with antibody fragments, mixtures of anti-idiotypic antibodies, etc.

It is also preferred if the above. Ligand, at the binding site of which the antibody A according to the invention binds, is a peptide and / or hormone and / or preferably of human origin, may also be directed against fragments of native poly- or oligopeptides.

In a preferred form of the antibody A according to the invention, the antibody A is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP).

It is particularly preferred if the antibody A according to the invention is the antibody ZMC1, for example the antibody DSM ACC2582 deposited with the DSMZ.

Another object of the invention is a medicament containing an antibody A according to the invention and optionally further active ingredients and additives and / or auxiliaries.

This medicinal product is particularly suitable for treating a deficiency state of one of the ligands already mentioned, since the ligand can be released by the antibody A or its binding can be prevented by binding protein, so that, for example, the plasma level of free ligand increases, for example in the event of a desired increase in the plasma titer of hGH.

The hGH-binding binding protein (hGHBP) corresponds to an extracellular fragment of the hGH receptor. According to the invention against hGHBP Binding antibodies which are directed against its binding site for hGH can also bind to the membrane-bound hGH receptor in a manner such that the hGH receptor is blocked for the attachment of hGH. Antibodies A according to the invention can in principle be used therapeutically as growth hormone receptor antagonists. An antibody according to the invention can thus be used to produce a medicament which inhibits the physiological effect of human growth hormone (hGH). Therefore, a medicament containing one or more antibodies A according to the invention (and possibly further auxiliaries or additives) can be used therapeutically in all those diseases in which the effect of hGH is increased in an unphysiological, for example pathological, manner , According to the invention, this also results in the use of corresponding antibodies A according to the invention for the treatment (or for the production of a medicament for the treatment) of all those disorders, diseases or pathophysiologies for which hGH excess is etiological, for example. such a medicament containing at least one antibody A according to the invention or an antibody A according to the invention can be used for the treatment of acromegaly.

By administering antibodies A according to the invention or corresponding medicaments according to the invention, the effect of pathophysiologically increased extracellular growth hormone concentrations can be blocked in this way without lowering the corresponding serum concentrations. The symptoms of pituitary adenomas, which are responsible for an increased secretion of growth hormones, can thus be treated with an antibody according to the invention.

If necessary, therapy for affected patients with pituitary adenoma can be carried out in combination with drugs or active substances that reduce the secretion of hGH. Here can it is, for example, treatment with somatostatin analogs (for example octreotide) and / or dopamine agonists (for example bromocriptine, cabergoline). As an alternative, conventional methods, such as neurosurgical intervention or radiation, can also be used therapeutically in connection with the administration of medicaments according to the invention or antibodies. In addition, other anti-tumor substances (chemotherapy drugs) can also be used in such therapy.

Finally, a medicament according to the invention, containing antibody A according to the invention, can also contain other substances, in particular somatostatin analogs, dopamine antagonists and / or antineoplastic substances, or can be used separately, for example therapeutically combined in a kit.

In addition, a therapeutic combination of antibodies according to the invention with pegvisomant (Deutsches Ärzteblatt, Volume 98, Issue 9, 2001, A 532) is also conceivable, as is a medicament according to the invention containing antibodies A and pegvisomant according to the invention and, if appropriate, further active substances (for example one of the active ingredients mentioned above). In the case of combined therapy, in a pharmaceutical (containing at least one antibody A and pegvisomant according to the invention), the long-term effect of antibody A according to the invention could be combined with the short half-life of the receptor antagonist pegvisomant due to their longer half-life.

Further diseases in which a medicament according to the invention or an antibody according to the invention can be used or could be used to produce a medicament are in particular diabetes mellitus (for example with regard to the retinopathy mediated indirectly by hGH in diabetes mellitus) or also as antitumor agent in hGH-dependent tumors. An antibody according to the invention will typically be in the form of a lyophilized powder which contains between 0.5 mg and 100 mg of antibody A according to the invention and further additives, for example glycine, manitol and / or sodium phosphate monohydrate. This lyophilized powder is provided in a suitable aqueous solution and then administered, for example subcutaneously, once or several times a day.

In principle, the pharmaceuticals according to the invention can be administered as liquid pharmaceutical forms, in particular in the form of injection solutions.

Suitable additives and / or auxiliary substances are e.g. Solvents or diluents, stabilizers, suspending agents, buffer substances, preservatives, as well as dyes, fillers and / or binders. The choice of excipients and the amounts to be used depend on whether the drug is to be administered parenterally, intrvasally, intravenously, intraperitoneally or intramuscularly. Preparations in the form of suspensions and solutions as well as easily reconstitutable dry preparations are suitable for all parenteral applications.

Another object of the invention is a diagnostic agent containing at least one antibody according to the invention and optionally additives and / or auxiliaries.

A diagnostic is understood to mean a preparation or aids with the help of which, for example, a specific illness can be diagnosed.

The invention furthermore relates to a kit comprising, separated from one another, at least one first preparation comprising an antibody according to the invention and a finished test assay which functions on the basis of an antigen / antibody reaction for the quantitative determination of a ligand which serves as an antigen in this test. A kit is to be understood as a jointly occurring form of different components in a packaging form. Here it is in particular a diagnostic kit that contains the various components necessary for the quantitative analysis of a ligand.

Preferred is a kit in which the first preparation contains an antibody which is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP) and / or in addition to the first preparation and the one based on of an antigen / antibody reaction functioning finished test assay for the quantitative determination of a ligand also contains a preparation for calibration.

A particularly preferred embodiment is a kit according to the invention, in which the first preparation contains the antibody ZMC1 (deposited above) and / or the preparation for calibration “international reference preparation 88/624” or “international reference preparation”

80/505 ⁸⁸ , preferably “International Reference Preparation 88/624” and / or the antibody in the enclosed finished test assay, which functions on the basis of an antigen / antibody reaction, for the quantitative determination of a ligand (hGH), preferably monoclonal, antibody B to the 22 kDa major isoform of hGH.

Another object of the invention is the use of an antibody for, preferably quantitative, determination of a physiological ligand of a physiological binding protein.

Another object of the invention is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of growth disorders. Another object of the invention is a method for producing an antibody according to the invention, ie an antibody which is directed against a binding protein for a ligand, with the following steps: (a) immunization of animals with recombinantly produced binding protein, (b) isolation of Animal immune cells, (c) fusion with myeloma cell lines to hybridoma cell cultures, (d) selection of clones with high specificity for the binding protein. The immunization can be carried out in all animals which are suitable for such purposes, for example in mice, rabbits, pigs, horses, etc. To avoid clones according to the invention which produce antibodies against the binding protein (for example GHBP) (which produce antibodies, that are specifically directed against the ligand binding skins of the binding protein), suitable media (eg wells of microtiter plates, polystyrene beads, plastic tubes) are coated with one (unselected) anti-GHBP capture antibody in one process step (e), and in one Process step (f), the binding protein (eg GHBP) and subsequently the ligand (eg hGH) are added. The ligand should be labeled (for example by biotinylation, label (radioactive label, fluorescent marker, enzyme marker (sea radical peroxidase etc.)) or be detectable via an appropriate anti-ligand antibody to determine the property of the ligand on the binding protein in the container coated with catch antibody. Accordingly, alternative methods are, for example, radioactivity, chemiluminescence, colorimetric methods or enzyme reaction. After a washing step, such "wells" or "beads" can be identified that have no signal after ligand addition. In this case, the capture antibody blocks the ligand binding site, the binding site is therefore no longer available for ligand binding, and the capture antibody interferes with the ligand.

Finally, an antibody according to the invention can be identified by competitive binding assays. For this purpose, as described above, coated, however, each “well” with an anti-binding protein antibody that does not specifically recognize the ligand binding site. After adding binding protein, a potentially interesting antibody (with the property of specifically recognizing the ligand binding site) is marked in each “well” Ligands added. The signal intensity of the ligand in the “well” decreases with increasing concentration of the ligand binding site-specific antibody. In this way, antibodies according to the invention (for example against GHBP) can be selected.

As an alternative to the production with the aid of hybridomas, the so-called “phage display” methods (Morphosys or Cambridge Antibody Technologies) can also be used to generate antibodies according to the invention and then to select them, as described above.

In the following section, the invention is further explained by means of exemplary embodiments, without being restricted thereto.

Examples and illustration:

Illustration:

Figure 1 shows the results of the investigation in embodiment example 2. The results show that the addition of the antibody according to the invention reduces the dose-dependent influence of the negative interference that hGHBP has on the results of the hGH measurement (false low values in the sandwich assays tested) can cancel.

embodiments Example 1:

Production of Antibody A (directed against hGHBP)

More than 20 Balb / c mice were repeatedly immunized with recombinantly produced hGHBP using the generally known method (see package insert Titermax®). When a high titer against hGHBP was reached, the spleen was removed from the animals, and according to the method described by Koehler and Milstein (Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 1975, Aug. 7; 256 (5517): 495- 7) described method produced by fusion with a mouse myeloma cell line hybridoma cell cultures. Clones which produced monoclonal antibodies of high affinity and specificity for hGHBP were also selected according to generally known methods (limited dilution, screening of the hybrid cell culture supernatants with labeled antigen).

Most of the selected clones were directed against those epitopes on the hGHBP molecule which are outside the binding site for hGH. In order to select antibodies A according to the invention which are directed against an epitope inside the binding site (only one such mAb is for the applicable according to the invention), one of the many antibodies that bind hGHBP outside the binding site was used first. This was identified by applying all monoclonal antibodies against GHBP (as many as possible), for example in separate wells of a microtiter plate (polystyrene plates with a highly adsorptive surface) as capture antibodies (alternatively, other methods, for example coated polystyrene beads, coated plastic tubes etc., are used). Then recombinant GHBP was added, which binds to the coating antibody - in an initially still unknown orientation (binding depending on the epitope inside or outside the hormone-receptor interaction site). Finally labeled (in our case biotinylated) growth hormone was added, which can now only bind to the GHBP molecules whose binding site is freely accessible (ie, where the coating antibody does not interfere with the growth hormone binding). Bound antibodies were measured by adding streptavidin-europium, which binds to bound biotinylated hGH and can be measured in a fluorometer (time-resolved fluorescence after addition of enhancement solution). Alternatively, this step can also be carried out using the analog methods (radioactivity, enzyme reaction / colorimetric method or chemiluminescence).

In order to find an antibody which is directed against the binding site, a microtiter plate was coated with one of the above-selected methods against GHBP, which does not interfere with the binding of hGH. Addition of GHBP caused the GHBP molecules to be bound in a directional manner, that is to say with a freely accessible binding site for hGH. Now biotinylated hGH was added and at the same time all other monoclonal antibodies against GHBP (of course each antibody in a separate well of the microtiter plate). If an antibody were to be directed against the binding site, it should hinder the binding of biotinylatedβm hGH depending on the concentration, which could be recognized by a decrease in signal intensity (detection method in our case, time-resolved fluorescence (streptavidin-europium). Attempted to confirm this, the reverse experiment was attempted to displace biotinylated antibodies against GHBP by adding unlabeled hGH, which is possible if the biotinylated antibody competes with the unlabelled hGH for binding to the receptor binding site, ie the epitope of the mAb within the hGH / hGH receptor interaction site lies. Example 2:

Evidence of the effectiveness of an antibody according to the invention and the associated improvement in general quantitative measurement methods in the case of interfering binding protein

Sheep serum - which, in contrast to the sera of other species, does not contain any high-affinity growth hormone binding protein and thus releases the optimal "control medium" - was mixed with various concentrations of growth hormone (preparation 80/505, final concentration 0.3 / 0.6 / 4 and 9 ng / ml) Then aliquots of these sera were mixed with different concentrations of recombinant human GHBP and incubated for 24 hours at 4 ° C. in order to enable the interaction between the added hGH and the added hGHBP with defined concentrations of hGH and defined concentrations of hGHBP The anti-hGHBP antibody ZMC 1 according to the invention and deposited was now added to these samples and incubated for a further 12 hours and then measured in 3 different immunoassays for hGH. The results (see Figure 1) prove that the addition of the antibody according to the invention The negative interference that hGHBP has on the results of the hGH measurement (false low values in the sandwich assays tested) can be reduced or eliminated depending on the dose.

The principle of the method has been successfully tested on three different immunoassays. The results shown above could be confirmed on human serum samples. Depending on the immunoassay, the concentrations of the anti-hGHBP antibody have to be adjusted in order to enable interference-free measurement of hGH. A fundamentally new application of monoclonal antibodies to block interfering binding proteins has thus been established. Literature:

1. Chatelin P, Bouillat B, Cohen R, Sassolas G, Souberbielle JC, Ruitton A et al, Assay of growth hormone levels in human plasma using commercial kits: analysis of some factors influ- encing the results. Acta Pediatr Scand Suppl 1990; 370: 56-61; discussion 62,

2. Ebdrup L, Fisker S, Sorensen HH, Ranke MB, Orskov H. Variety in growth hormone determinations due to use of different immunoassays and to the Interference of growth hormone-binding protein, Horm Res 1999; 51 (SuppM); 20-6,

3. Fisker S, Ebdrup L, Orskov H. Influence of growth hormone binding protein on growth hormone estimation in different immunoassays. Scand J Clin Lab Invest: 1998; 58 (5); 373-81,

4. Fisker S, Orskov H. Factors modifying growth hormone estates in immunoassays. Horm Res 1996; 46 (4-5); 183-7,

5. Jansson C, Boguszewski C, Rosberg S, Carlsson L, Albertsson-Wikland El Growth hormone (GH) assays: Influence of Standard preparations, GH isoforms, assay characteristics, and GH-binding protein. Clin Chem 1997; 43 (6 Pt 1): 950-6.

Claims

claims

1. A method for the quantitative determination of a ligand of a binding protein in a sample containing ligand and binding protein in dissolved or suspended form, characterized in that the sample to be measured has an antibody A against the binding protein which is directed against the binding site of the ligand on the binding protein is added.

2. The method according to claim 1, characterized in that the Antiköφer A is added before or during, preferably before, the quantitative determination and / or is incubated with the sample.

3. The method according to any one of claims 1 or 2, characterized in that the sample contains body fluid, preferably human body fluid, in particular blood or human blood.

4. The method according to any one of claims 1 to 3, characterized in that the ligand is a physiological ligand of the physiological binding protein.

5. The method according to any one of claims 1 to 4, characterized in that the binding protein is soluble, preferably a soluble receptor or a hormone binding protein, in particular a

Hormone binding protein.

6. The method according to any one of claims 1 to 5, characterized in that the ligand is a peptide compound and / or a hormone, preferably a peptide hormone, in particular a

Growth hormone, is.

, Method according to one of claims 1 to 6, characterized in that the ligand is human growth hormone (hGH) and the binding protein is human growth hormone binding protein (hGHBP) and the antibody A against the binding site of the hGH on which hGHBP is directed.

8. The method according to any one of claims 1 to 7, characterized in that the antibody A is a monoclonal antibody or a single-chain antibody, preferably a monoclonal antibody.

9. The method according to any one of claims 1 to 8, characterized in that the antibody A is the monoclonal antibody ZMC 1.

10. The method according to any one of claims 1 to 9, characterized in that the quantitative determination of the ligand under

Utilization of the binding of the ligand as an antigen to a, preferably monoclonal, antibody B takes place.

11. The method according to claim 10, characterized in that the quantitative determination is carried out via a competitive binding test, preferably a "radio-immuno assay" (RIA).

12. The method according to claim 10, characterized in that the quantitative determination is carried out by measuring a measurement parameter increasing depending on the concentration of the ligand to be measured, preferably by an enzyme-linked immunosorbent assay (ELISA) and / or by a sandwich -

Assay.

13. The method according to any one of claims 1 to 12, characterized in that there is no separation of the binding protein from the sample to be measured before the quantitative determination.

14. The method according to any one of claims 1 to 13, characterized in that the antibody A is added so that the antibody A is present in the sample in a higher concentration than the binding protein, preferably in an at least 50%, in particular at least 100 %, preferably at least 200%, in particular at least 400% higher concentration.

15. Antibody A against a physiological binding protein of a physiological ligand dissolved or suspended in body fluids, characterized in that the antibody A against the

Binding site of the ligand is directed to the binding protein.

16. Antibody A according to claim 15, characterized in that the antibody A is a monoclonal antibody and / or a single-chain antibody, preferably a monoclonal antibody.

17. Antibody A according to one of claims 15 or 16, characterized in that the ligand is a peptide and / or hormone and / or preferably of human origin.

18. Antibody A according to one of claims 15 to 17, characterized in that the antibody A is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP).

19. Antibody A according to one of claims 15 to 18, characterized in that the antibody A is the antibody ZMC1.

20. Medicament containing an antibody A according to one of claims 15 to 19 and optionally further active ingredients and additives and / or auxiliaries.

21. Use of an antibody according to any one of claims 15 to 19 or a medicament according to claim 20 for the treatment of diseases, disorders or pathophysiologies which hGH-

Excess, for example, acromegaly.

22. Diagnostic agent containing at least one antibody A according to one of claims 15 to 19 and optionally auxiliary substances.

23. Kit comprising, separated from one another, at least one first preparation comprising an antibody A according to one of claims 15 to 19 and a finished test assay which functions on the basis of an antigen / antibody reaction for the quantitative determination of a ligand which serves as antigen in this test.

24. Kit according to claim 23, characterized in that the first preparation contains an antibody A according to claim 18 and / or in addition to the first preparation and the finished test assay which works on the basis of an antigen / antibody reaction and also for the quantitative determination of a ligand contains a preparation for calibration.

25. Kit according to claim 24, characterized in that the first preparation contains the antibody ZMC1 and / or the preparation for calibration "International reference preparation 88/624" or "International reference preparation 80/505", preferably "Inter- contains national reference preparation 88/624 "and / or the antibody B in the finished test assay, which functions on the basis of an antigen / antibody reaction, for the quantitative determination of a ligand (hGH), preferably a monoclonal antibody against the 22 kDa major isoform of hGH is.

26. Use of an antibody A according to any one of claims 15-19 for, preferably quantitative, determination of a physiological ligand of a physiological binding protein.

27. Use of an antibody A according to claim 18 or 19 for the manufacture of a medicament for the treatment of growth disorders.

28. A method for producing an antibody A according to one of claims 15 to 19, comprising the following steps:

(a) immunization of animals with recombinantly produced binding protein,

(b) isolation of immune cells from the animal, (c) fusion with myeloma cell lines to form hybrido cell cultures,

(d) Selection of clones with high specificity for the binding protein.