EP1240527A2

EP1240527A2 - Method for determining the biologically effective parathyroid hormone activity in a sample

Info

Publication number: EP1240527A2
Application number: EP00989994A
Authority: EP
Inventors: Franz Paul Armbruster; Albert Missbichler; Heinrich Schmidt-Gayk; Heinz-Jürgen ROTH
Original assignee: Immundiagnostik AG
Current assignee: Immundiagnostik AG
Priority date: 1999-12-17
Filing date: 2000-12-18
Publication date: 2002-09-18
Also published as: US7851163B2; AU2674601A; US20030175802A1; WO2001044818A3; US20110003311A1; WO2001044818A2; DE19961350A1

Abstract

The invention relates to an immunoassay for determining the parathyroid hormone activity in a sample and to a method for diagnosing, finding the causes for and treating disorders of the calcium metabolism, osteopathies and hyper- or hypoparathyreoses. The parathyroid hormone activity is measured by means of an antibody that binds to an epitope in the area of the receptor binding structure 15 to 22 of the parathyroid hormone, and an antibody that recognizes whether the aminoterminal end 1 to 3 of the parathyroid hormone has remained intact. The inventive assay allows taking into account the antagonist properties of some parathyroid hormone fragments.

Description

METHOD FOR DETERMINING THE BIOLOGICALLY EFFECTIVE PARATHORMONE ACTIVITY IN A SAMPLE

The invention relates to a method for determining the parathyroid activity in a sample. The invention particularly relates to the immunological determination of the activity of parathyroid hormone (PTH) and its fragments in a body fluid for the diagnosis, finding of causes and treatment of calcium metabolism disorders, osteopathies and hyper- or hypoparathyroidism.

Human parathyroid hormone (see SWISS-PROT: P01 270, PTHY-HUMAN) is a linear peptide of 84 amino acids (MW 9500 Da) of the parathyroid gland (Glandulae parathyroideae). It increases the calcium and the phosphate content of the blood and is involved in the mobilization of the extracellular hydroxyapatite of the bones. The PTH content of the blood is therefore an important diagnostic parameter in patients with disturbed calcium metabolism and its knowledge is necessary to clarify 1) the presence and extent of hyper- or hypoparathyroidism, 2) the quantification of osteoblast activity, 3) the quantification of osteoclast activity , 4) securing the supply of vitamin D and active vitamin D metabolites, 5) the assessment of an aluminum load, 6) the assessment of a possible estrogen deficiency in postmenopausal dialysis patients, 6) the necessary steroid or cyclosporine administration after kidney transplants, 7) the need for treatment and the prevention of bone marrow changes, uremic conditions and chronic kidney failure.

The determination of the PTH activity effective in the blood is problematic since the peptide hormone is rapidly broken down into active and inactive fragments in the circulation and by the liver, for example by cleavage in the range from amino acids 34 to 37. In addition, the physiological concentration of intact human is PTH (hPTH) in the blood plasma only at about 1 to 5 pmol / L.

In the prior art, the determination of intact hPTH (1-84) is carried out by the immunological detection of two widely separated epitopes on the peptide. However, there are still many patients with eight to ten times higher

maintain intact PTH (1-84) in the blood and low-normal bone-specific alkaline phosphatase (Ostase), i.e. free of symptoms of excessive PTH activity. This is explained by incorrect measurements or a PTH resistance of the osteoblasts, for example by a genetically reduced expression of PTH receptors. The determination of the effective PTH activity is further complicated by the fact that some PTH fragments have an activity of comparable intact hPTH (1-84) (see EP-A 0 349 545), other PTH fragments in turn have an antagonistic effect (see LePage et al (1 998), Clinicai Chemistry, 44: 4, pp805-809; Schmidt-Gayk et al. (1 999) Osteologie forum, 5, pp48-58, and references). It is thus known that the fragments hPTH (3-34) and hPTH (7-34) inhibit the effects of the PTH (Suva et al. (1 987) Science, 237, 893ff; EP 0 451 867).

WO 96/1 0041 teaches that antibodies against amino terminal epitopes of PTH recognize biologically active PTH and that if the amino terminal amino acids serine and valine are lost, the activity is lost.

Mägerlein et al describe in Pharm. -Forsch. / Drug Res. 48 (l), pp1 97-204 (1 998) used an immunoenzymometric assay to determine the concentration of injected PTH fragment 1-37 in pharmacokinetic studies in dogs. The measuring range of the assay ranges from 100 to 4 pmol / L and ends above the physiological PTH levels normally present in the serum.

The true biologically active PTH activity in a sample can currently, if at all, only be determined by the effect on a cellular system such as, for example, pheochromocytoma cells (PC-1 2).

It is an object of the invention to provide a method for determining the total effective parathyroid activity in a sample.

This object is achieved by a method according to claim 1. Further advantageous embodiments of the method are described in the dependent claims. The invention also relates to a test system and a method for diagnosing and the extent of hypo- or hyperparathyroidism and finding the cause of calcium metabolism disorders, osteopathies, kidney failure and diseases. which result from a disturbed homeostasis in the calcium and phosphate content of the blood.

The method according to the invention for determining the biologically active PTH activity in a sample is characterized by the steps: (i) reacting the sample with an antibody which binds an epitope on the PTH which lies in the region of the binding structure to the PTH receptor; (ii) reacting the sample with an antibody that recognizes an epitope formed by terminal amino acids 1 to 3 of the PTH; (iii) determining the amount of molecules recognized by both antibodies; and (iv) calculating the biologically active PTH activity in the sample.

The aforementioned antibodies preferably recognize epitopes of human PTH. The antibody against the receptor binding structure preferably binds an epitope in which at least one of the amino acids 15 to 22 of the human PTH is involved. The invention also includes a method in which a first antibody is bound to a solid phase. The second antibody can carry a label or be conjugated to an enzyme such as alkaline phosphatase or peroxidase. According to the invention, the method is carried out in an immunoassay known per se, preferably in an ELISA, IEMA, ILMA or LIA. The two antibodies are preferably bound to the PTH in the presence of 0.05 to 0.1% by weight of a mild detergent, such as Tween ™ -20 or Triton ™ X-100, so that the amino-terminal epitope PTH (1 -3 ) to the PTH receptor binding structure is prevented or in order to keep the amino terminal epitope PTH (1 -3) accessible for the binding of the antibody. Since both the receptor binding structure and the amino-terminal epitope PTH (1 -3) are necessary for the biological activity of PTH, these two structures can presumably interact with one another. The presence of a mild detergent can significantly improve the sensitivity and accuracy of the assay. In one embodiment of the method, a known aliquot of the sample is also reacted with antibodies which bind to the PTH sequences between amino acids 4 to 14 and 15 to 37. The number of molecules in a sample that bind antibodies against the epitope PTH (1-3) and the receptor binding structure of the PTH and the number of molecules that bind antibodies against the PTH regions 4 to 14 and 15 to 37, are then related to each other and the biologically active PTH activity is determined.

The invention further relates to a diagnostic system for determining the PTH activity in a sample, which is characterized by antibodies which specifically recognize the PTH epitope with amino acids 1 to 3 and antibodies which bind to the region of the PTH receptor binding structure. In one embodiment, the diagnostic system also has antibodies that bind specifically to the region between amino acids 4 and 14. In another embodiment, the system includes antibodies that bind to the portion between amino acids 24 and 37 of the parathyroid hormone or to the central region (53 to 68) or the C-terminal (53 to 84) region of the peptide hormone.

In contrast to previous immunoassays, the method according to the invention takes into account the PTH activity of the non-intact PTH fragments and that apparently intact PTH is biologically inactive if the last or the last two amino-terminal amino acids of the peptide hormone are missing. The antagonistic activity of some PTH fragments is also included in the determination, since not only the agonistic but also the antagonistic PTH fragments can be determined in the method. The antagonistic activity is derived from the excess of

PTH receptor binding structure to intact PTH amino terminus, i.e. from the number of PTH fragments, which contain the binding structure for the PTH receptor, but have no intact amino terminal end. PTH fragments that are neither

Receptor binding structure with amino acids 15 to 22 still have an intact amino-terminal epitope PTH (1 -3) have no agonistic or antagonistic

Activity.

Conventional immunological assays for so-called "truely intact" hPTH (1-84) or hPTHd -37) give false high activity values, since physiological activity is only concluded from the presence of functionally insignificant epitopes. They do not note that correct folding or binding to the PTH receptor is dependent on an intact amino terminus with the amino acids H ₂ N-Ser ¹ -Val ² . However, the fragments hPTH (7-84), hPTH (3-84) or hPTH (4-37), which are often determined as "active" PTH in conventional assays, have no or anta- gonistic activity. Antibody-based assays against the mid-regional (53-68) or C-terminal (53-84) section, on the other hand, overlook biologically active PTH fragments of the hPTH (1-37), hPTH (1-32-36) or hPTH type (1 -38).

Activities are also incorrectly determined with immunoassays based on antibodies against the amino-terminal region of the PTH or against peptides from the hPTH (1-37) sequence, since on the one hand the antagonistic activities of various fragments are not recorded and on the other hand no positive detection the biologically active structural unit. With polyclonal antibodies or antisera against the amino-terminal peptide with amino acids 1 to 5 or 6, even partially antagonistic PTH fragments are recognized as active PTH fragments.

The method according to the invention thus provides the doctor with a PTH activity value which describes the physiological effect of the PTH fragments. There are patients with hyperparathyroidism who have normal or reduced levels of intact hPTH (1-84) in the serum. The hyperparathyroidism can then be based on the fact that these patients secrete the likewise active PTH fragments hPTH (1-37) and hPTH (1-38) into the bloodstream, so that they suffer from excessive PTH activity and its consequences.

Further advantages, features and embodiments of the invention result from the examples below and the attached drawings. It shows:

1 shows a schematic diagram of the method according to the invention;

2 shows a comparison of the amino terminal PTH sequences and the receptor binding structure of humans, cattle, pigs and dogs;

3 shows an analysis of the binding epitopes of monoclonal antibodies against the receptor binding site of the PTH and their cross-reaction with neighboring ones

PTH fragments;

4a, b an analysis of the cross-reaction with different hPTH fragments in an immunoenzymometric assay; 5 shows a standard curve of the LIA assay according to the invention for determining the effective PTH activity in a sample.

See Figure 1 with the principle of the method according to the invention. The antibodies against the amino-terminal epitope of human PTH with the amino acids H _j N-Ser ¹ - Val ² -Ser ³ are obtained by eliciting an immune reaction against one of the following peptides.

SEQ No. 1

H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu ⁴ -lle ⁵ -Gln ⁶ -Leu ⁷ -Met ⁸ -His ⁹ -Asn ^{l 0} -0H

SEQ No. 2:

H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu ⁴ -lle ⁵ -Gln ⁶ -Leu ⁷ -Met ⁸ -His ⁹ -0H

SEQ No. 3: H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu ⁴ -lle ⁵ -Gln ⁶ -Leu ⁷ -Met ⁸ -OH

SEQ No. 4: H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu ⁴ -lle -Gln ⁶ -Leu ⁷ -OH

SEQ No. 5: H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu ⁴ -lle ⁵ -Gln ⁶ -OH

SEQ No. 6:

H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu ⁴ -lle ⁵ -OH

These peptides are synthesized in a known manner, preferably on a lysine framework on a Wang resin. For this purpose, Fmoc-L-lysine (Fmoc) -OH is first coupled to a C-terminal amino acid bound to Wang resin in three coupling cycles. By splitting off the protective groups with piperidine, eight free amino functions are obtained, on which the amino-terminal sequence of the human parathyroid hormone can be synthesized. The Wang resin with the amino-terminal PTH amino acid sequence can then be injected directly - preferably together with complete Freund's adjuvant - into an animal for immunization, for example into a mouse, rat, rabbit or goat. The amino-terminal PTH peptide can also be cleaved off before immunization, isolated and coupled to a carrier protein such as ovalbumin, bovine albumin, thyroglobulin or hemocyanin, for example with dicyclohexylcarbodiimide. After several booster immunizations, the immunoglobulin fraction is isolated, which has an antibody titer against the amino-terminal PTH peptide.

Antibodies which recognize an epitope of the first two amino-terminal amino acids of the hPTH are regularly obtained with the above-mentioned amino-terminal peptides. A fraction with antibodies pure against the amino-terminal epitope hPTH (1-3) can be obtained by affinity chromatography, an hPTH peptide with an intact amino-terminal end being coupled to a solid phase, for example on Wang resin or polystyrene beads. The antibodies directed against the amino terminus PTH (1-3) are bound to it, washed and eluted. The eluate is finally clarified via a second column in which the hPTH peptide coupled to the solid phase has no intact amino terminal epitope, for example as in the peptide sequences SEQ No. 7 and SEQ No. 8 below.

SEQ No. 7 H ₂ N-Val ² -Ser ³ -GIu -lle ⁵ -Gln ⁶ -Leu ⁷ -Met ⁸ -His ⁹ -Asn ¹⁰ - carrier

SEQ No. 8 H ₂ N-Ser ³ -Glu -Ile ⁵ -Gln ^β -Leu ⁷ -Met ⁸ -His ⁹ -Asn ¹⁰ - carrier

In a manner known per se, monoclonal antibodies against the amino-terminal epitope of PTH can also be obtained in this way. However, polyclonal antisera against the N-terminal hPTH epitope are preferred. It is understood that the steps of the purification described above are interchanged and the PTH peptides used to purify and clarify the serum may be longer or shorter. Only the amino-terminal epitope hPTH (1 -3) must be intact in one case. For the production of the second antibody against the sequence of the receptor binding site of the PTH, intact hPTH (1-84) or biologically active hPTH (1-37) is synthesized, coupled to a carrier peptide and with complete Freund's adjuvant in an animal, for example in a mouse. Rat, rabbit or goat, injected.

SEQ No. 9

Ser ¹ -Val ² -Ser ³ -Glu -Ile ⁵ -Gln ⁶ -Leu ⁷ -Met ⁸ -His ⁹ -Asn ^l0 -Leu ^{1 l} -Gly ¹² -Lys ^{l 3} -His ^l4 -Leu ¹⁵ -Asn ¹⁶ - Ser ¹⁷ -Met ^l8 -Glu ¹⁹ -Arg ²⁰ -Val ² -Glu ²² -Trp ²³ -Leu ²⁴ -Arg ²⁵ -Lys ²⁶ -Lys ²⁷ -Leu ²⁸ -Gln ²⁹ -Asp ³⁰ -Val ³¹ - His ³² -Asn ³³ - Phe ³⁴ -Val ³⁵ -Ala ³⁶ -Leu ³⁷

A coupling to a carrier peptide is not absolutely necessary for the generation of an immune reaction, since the PTH sequences between humans, cattle, pigs and rats vary in particular in the region of the receptor binding site between positions 15 and 22 and are therefore antigenic (see FIG. 2) , The bovine PTH in positions 1 (Ala), 7 (Phe) and 1 6 (Ser), for example, differs from the human PTH (1-37) fragment, rat PTH in positions 1 (Ala), 1 6 (Ala), 1 8 (Val) and 36 (Ser), porcines PTH in positions 1 6 (Ser) and 1 8 (Leu) as well as canines PTH in positions 7 (Ser) and 1 6 (Ser) (Heinrich G . et al (1 984) J. Biol. Chem. 259: 3320-3329; Kronenberg HM et al (1 979) Proc. Natl. Acad. Sci. USA 76: 4981 -4985; Schmelzer H.-J., et al. (1 987) Nucleic Acids Res. 1 5: 6740-6740; Rosol T et al. (1 995) Gene 1 60: 241 -243). Coupling to a carrier is recommended for immunization, since the peptides mentioned can not only be immunogenic but also biologically active.

HPTHd -37) is synthesized as described in Example 2 of WO 91/06564. The aforementioned peptides and their derivatives can also be prepared by gene expression in a suitable prokaryotic or eukaryotic host organism and purified by chromatography. In addition to the aforementioned peptide, the peptides hPTH (1-32), hPTH (1-33), hPTH (1-34), hPTH (1-38) and other biologically active hPTH fragments for the production of antibodies against the receptor binding sequence of the hPTH.

After several immunization boosters with purified hPTH peptide or fragment, the immunoglobulin fraction is then isolated from the serum of the immunized animal and tested for binding to the hPTH receptor site. The following heptapeptide hPTH (1 5-22)

SEQ NO. 9 Leu ¹⁵ -Asn ¹⁶ -Ser ¹⁷ -Met ¹⁸ -Glu ¹⁹ -Arg ²⁰ -Val ^2l -Glu ²² - carrier

synthesized on a macroscopic support such as polystyrene beads or polystyrene pins and antibodies which specifically bind the receptor binding structure of the hPTH isolated by affinity chromatography. With the help of the peptide pin technique, monoclonal antibodies against the receptor binding structure of the PTH can also be fished and clones whose antibodies recognize the receptor binding structure can be isolated. It is understood that instead of the above Heptapeptide can also be used as a hexapeptide or little longer sequences from the receptor binding site.

With the antibodies against the N-terminal epitope of hPTH (1 -3) and the receptor binding structure hPTH (1 5-22), antibodies are available which detect the functionally active structure.

According to the invention, the immunoassay is carried out on the active structure of the hPTH under slightly denaturing conditions, so that both epitopes in the unfolded peptide are free for binding to the antibodies. This is done by the otherwise unusual addition of a detergent such as Tween-20 or Triton-X-100 in the binding buffer. The detergents are preferably added to the binding buffer in an amount of 0.01 to 1% by weight, particularly preferably in an amount of 0.1 to 0.3% by weight.

Antagonistic hPTH fragments are characterized by a PTH receptor binding site and the absence of an intact amino terminal epitope hPTH (1 -3). The antagonistic activity of a sample is therefore proportional to the number of PTH fragments which contain the receptor binding site between positions 1 5 and 22, but which are not bound by antibodies against the amino-terminal epitope hPTH (1 -3). The antagonistic activity can thus be determined from the difference between the number of molecules which are recognized by the antibody double against the receptor binding site and the amino terminus hPTH (1 -3) and the Number of fragments recognized by the antibody double against the receptor binding structure (or an N-terminal epitope between amino acids 24 and 37) and an epitope in the region of amino acids 4 and 1 4. In practice, the latter size is equal to the number of hPTH fragments recognized by the antibody double against sections hPTH (9-1 8) and hPTH {24-34), but not by antibodies against hPTH (1- 3) epitope.

The antibodies against the hPTH (4-14) region are obtained when the antibodies against the hPTH (1 -3) epitope are purified. They are contained in the antibody fraction which bind to the amino-terminal peptide hPTH (2-10) with an incomplete hPTH-end epitope (see above). Such antibodies can also be made specifically, e.g. as in WO 96/1 0041 or in Tampe et al. (J. Immunoassay (1,992), 1 3 (1), pp. 1 -1 3). So that the antibodies do not interfere with each other, it can be advantageous here to select two antibodies with the binding sites on the hPTH (1-37) somewhat further apart, for example as indicated above as an alternative. If the binding sites are too far apart or too far C-terminal of the receptor binding site between positions 15 and 22, there is a risk that non-inhibiting hPTH fragments are also detected as antagonists.

The immunoassay of biological PTH activity according to the invention can moreover be combined with existing PTH immunoassays. In some diagnoses, it may be appropriate not only to measure the biological activity of the hPTH, but also the amount and distribution of the hPTH molecules of a certain length. That is, it may be beneficial to have an hPTH assay that uses antibodies with mid-regional (53-68) or C-terminal (53-84) specificity in addition to antibodies to the amino terminus hPTH (1 -3). With such a PTH assay, the effective PTH activity can then be related to individual PTH molecules of a certain length. This explains in particular the cause of certain hyper and hypoparathyroidisms. According to the invention, the PTH activity assay also includes the additional determination of hPTH molecules with an intact carboxy terminus and antibodies against the central region or C-terminal region of the hPTH. The method for determining the activity of PTH and its fragments in a sample can be implemented as EIA, ELISA, RIA, IRMA, LIA or ILMA, FIA or IFMA, as a manual test system or, preferably, in a version adapted to automata in liquid phase or solid-phase technique.

EXAMPLES

Example 1 Antibodies to the hPTH (1-3) epitope

First, a triple lysine branch was made on a Wang resin. For this purpose, Fmoc-L-lysine (Fmoc) -OH was bound to C-terminal alanine, bound to Wang resin, in three coupling cycles. The Fmoc groups were then split off with 20% piperidine and 2% 1,8-diazabicyc! O [5,4,0] undec-7-ene (DBU) in NN-dimethylformamide, NN-dimethylacetamide. The synthesis of the N-terminal hexapeptide hPTH (1-6) then took place on the free amino groups in a manner known per se by solid-phase synthesis and coupling of the C-terminal amino acids with the aid of dicyclohexylcarbodiimide as coupling agent. The peptide

H ₂ N-Ser ¹ -Val ² -Ser ³ -Glu -ll ⁵ -Gln ⁶ - (3 x Lys) -Ala carrier

was then cleaved from the carrier and deblocked at room temperature by treating with trifluoroacetic acid containing 5% scavenger, water, ethanediol and phenol for 30 to 90 minutes. The peptide was washed with trifluoroacetic acid, precipitated from aqueous solution with tert-butyl methyl ether. The crude product was passed through a C 1 8 reverse phase column (10 m, buffer A: 0.01 N HCl in water; buffer B: 20% isopropanol, 30% methanol, 50% water, 0.01 N HCl ; Gradient: 1 0-80% precipitated in 60 minutes) purified by chromatography.

1 mg of purified peptide hPTH (1-6) was then coupled in aqueous solution at room temperature to 10 mg of bovine serum albumin using the carbodiimide method and the coupled peptide was precipitated from the aqueous solution with isopropanol. The peptide was taken up in PBS and aliquoted. For the initial immunization, 1 25 μg of peptide-carrier conjugate was dissolved in 250 μl of PBS, emulsified with a double volume of complete Freund's adjuvant and the emulsion was subcutaneously injected into a rabbit at various locations in several units. After 2 and 4 weeks, further booster injections with peptide and incomplete Freund's adjuvant followed.

After 6 weeks, 50 ml of blood were taken and antibodies against the amino-terminal hPTH (1 -3) epitope were obtained by sequential affinity chromatography by binding to protein A, binding to the peptide hPTH (1 -6) and clarifying an hPTH (2- 1 0) peptide column. In the last step mentioned, antibodies against hPTH (3-6) were also obtained after further elution, which can be used to detect antagonistic PTH fragments.

Example 2 Antibodies against the receptor binding structure

HPTHd -37) was synthesized as described in Example 2 of WO 91/06564. For the immunization, 25 μg peptide hPTH (1-37) bound to Wang resin in 50 μl PBS was injected intraperitoneally into a mouse. After 2 and 4 weeks there was a booster with 50 μl peptide hPTH (1-37) on Wang resin. Monoclonal antibody clones were produced in a manner known per se and the various pooled clones on hPTH (1 5-22) heptapeptide, synthesized on polystyrene pins (see Tampe et al., J. Immunassay (1 992), 1 3 (1), pp. 1 -1 3), tested. In four clones, the binding epitopes with hPTHd -1 0), hPTH (9-1 8), hPTHd 6-24) and hPTH (24-37) were mapped and examined for possible cross-reactivity. FIG. 3 shows an analysis of the monoclonal antibodies against the receptor binding site of the PTH and their cross-reaction with the other PTH fragments.

Example 3 Examination of cross-reactivity to non-active fragments

Synthetic hPTH (1-84) (Bachern AG) and various hPTH fragments with and without an intact amino-terminal end epitope were dissolved in equal amounts of serum and the resulting PTH activities or the recovery of the biologically active PTH fragments in an immunoenzymometric assay (IEMA ) certainly. As The primary antibody was the monoclonal antibody mAK 1 3 / C631 5 against the receptor binding site and bound to the solid phase. The secondary antibody was rabbit polyclonal anti-hPTH (1 -3) IgG. The color reaction was carried out using peroxidase-conjugated goat anti-rabbit IgG and the ABTS system from Röche Diagnostics, Mannheim (ABTS = 2,2'-azino-di- [3-ethylbenzthiazoline sulfonate). The absorbance at 405 nm was measured.

Figures 4a and b show the cross-reaction with different hPTH fragments in two different experiments. The results show that PTH fragments without the last two amino terminal amino acids are not recognized in the test and that all active PTH fragments essentially form a uniform family of curves. Differences are based on differing degrees of purity of the preparations, weighing inaccuracies and the difficulty in determining the exact amount of peptides.

Example 3 Antibodies to hPTH (4-14), hPTH (7-14) and hPTH (24-37)

There were two goat and mouse polyclonal antisera and two monoclonal antibodies which recognize the hPTH region between amino acids 7 and 14, as described by Tampe et al. (J. Immunoassay (1,992) 13 (1), pp1-1 3). The antibodies against the hPTH (24-37) and the hPTH (4-1 4) region were produced as described in WO 96/1 0041.

Example 4 Determination of the effective PTH activity in a sample

In order to lower the detection limit, the concentration ratios were optimized and alkaline phosphatase was used as a detection system in connection with luminescence methods. If peroxidase and a luminescence method are blown away, pseudoperoxidase present in the blood plasma can disadvantageously raise the detection limit. (/ ^' ) Coating a microtiter plate with monoclonal antibody against the

P TH receptor binding structure, 1.0 μg MAb 77/78 (subclone against receptor binding site hPTH (1 5-22)) was added to the wells of a microtiter plate, dissolved in 250 μl 60 mM NaHC0 ₃ , pH 9.6, and incubated the plate overnight at 4 ° C. The MAb solution in the wells was removed and each well was washed five times with 200 μl washing buffer (PBS, pH 7.4 with 0.05% Tween-20). Then 250 ul assay buffer was added to each well. For the assay buffer, 5 g casein was dissolved in 100 ml 0.1 N NaOH and made up to 1 L volume with PBS, pH 7.4, with 0.1% by weight Triton ™ -X 100. The solution was boiled for one hour, the volume was made up to one liter with distilled water, the pH was adjusted to 7.4 and 0.1 g of thimerosal was added to avoid microbial growth. The wells in the microtiter plate were incubated for one hour at room temperature with assay buffer, then the assay buffer was removed and each well was washed five times with 200 μl wash buffer.

(ii) Sample preparation

Venous blood was collected in EDTA tubes (approx. 1.5 to 2 mg K ₂ EDTA per

Milliliters of blood), shaken, and after a clotting time of 1 0 to 1 5 minutes centrifuged at 1 800 x G 1 0 minutes. The plasma supernatant was diluted 1: 1 with assay buffer and stored at -20 ° C until the test. If the samples contained more than 800 pmol / L PTH, the supernatant was diluted 1: 10 with assay buffer. Before being used in the ELISA, the samples were centrifuged briefly at maximum speed.

(iii) Binding of intact PTH (1-84) and PTH fragments with receptor

Binding structure In each case 100 μl of diluted EDTA serum supernatant in assay buffer was incubated for half an hour at room temperature in the prepared wells with shaking. The solutions were then removed from the wells and the wells were washed five times with 200 μl of washing buffer each.

(iv) Determination of the binding of active PTH

In each case, 100 μl of affinity-purified rabbit anti-PTH (1 -3 epitope) antiserum (1: 10 000 diluted in assay buffer with 3% (w / v) PEG 6000) were added to the wells and incubated for one hour in the dark and with shaking at room temperature. The solutions were removed from the wells and each well was washed five times with 200 ul wash buffer. The quantitative determination was carried out with 100 μl anti-rabbit IgG, fc-specific, cross-absorbed and conjugated with alkaline phosphatase (diluted 1: 20,000 in washing buffer). It was incubated for one hour at room temperature. The antibody solutions were then removed and each well was washed five times with 200 μl of washing buffer. For the color reaction, 100 μl of Lumi-Phos ™ Plus [ready-to-use solution with 4-methoxy-4- (3-phosphatphenyl) spiro [1,2-dioxetane-3,2'-adamantane] disodium salt and associated enhancer (Lumigen , Michigan, US) in 2-amino-2-methyl-1-propanol buffer (pH 9.6) 1 added to the wells. The luminescence is then measured at 470 nm in a luminometer.

Solutions of PTH (1-84) in assay buffer with the following concentrations were used as standard: 0, 0, 1, 1, 10, 100 and 1000 pmol / L. The standard curve with alkaline phosphatase for the detection reaction shows that this system allows the determination of a few hundredths pMol PTH per liter.

When using peroxidase for the detection reaction, Lumigen ™ PS-1 was used as the substrate. Briefly described, the wells of the microtiter plate were each washed five times with 250 μl wash buffer. Then 50 μl standard or sample was pipetted into the well and finally 50 μl freshly diluted 1: 500 second anti-hPTH (1 -3) antibody. The mixture was incubated overnight at 2 to 8 ° C. with gentle shaking, the contents of the wells were removed and the wells were washed five times with 250 μl of washing buffer. For the detection reaction, 100 μl POD-antibody conjugate (anti-rabbit IgG fc-specific, cross-absorbed and conjugated with peroxidase; diluted 1: 20,000 in washing buffer) was added, incubated for one hour at room temperature with shaking, the solutions removed and the Wells washed five times with 250μl wash buffer. The luminescence reaction was carried out by adding 100 μl of freshly mixed Lumigen ™ PS substrate solution and incubating for five minutes at room temperature in the dark. The relative light units (RLU) were then determined in a luminometer. Because of the in the plasma However, the so-called pseudoperoxidase present had a higher background or a lower sensitivity of the test.

(v) Determination of antagonistic PTH fragments The antagonistic PTH activity potential was determined by coating a microtiter plate with monoclonal antibody against the hPTH region (7-1 4). 1.0 μg of MAb (clone against hPTH (7-14)) was added to the wells of a microtiter plate, dissolved in 250 μl of 60 mM NaHCO ₃ , pH 9.6, and the plate was incubated at 4 ° C. overnight. The MAb solution in the wells was removed and each well was washed five times with 200 μl washing buffer (PBS, pH 7.4 with 0.05% Tween ™ -20). Then 250 ul assay buffer was added to each well. The wells in the microtiter plate were incubated for one hour at room temperature with assay buffer, the assay buffer was removed and each well was washed five times with 200 μl washing buffer. For the binding of antagonistic PTH fragments, in each case 10Oμl diluted EDTA serum supernatant was incubated in the well for half an hour at room temperature with shaking. The solution was removed from the wells and the well was washed five times with 200 ul wash buffer. The bound antagonistic PTH fragments (7-37) were each labeled with 100 μl of affinity-purified rabbit anti-PTH (24-37) antiserum (diluted 1: 10,000 in assay buffer with 3% (w / v) PEG 6000), as stated above, and after washing with washing buffer quantified by adding 1 00 μl anti-rabbit IgG fc-specific, cross-absorbed and conjugated with alkaline phosphatase (diluted 1: 20,000 in washing buffer) and in an analogous manner to the active PTH- Fragment quantified by luminescence.

The molar difference to the biologically active PTH fragments (with amino-terminal PTH (1 -3) epitope) determined in this method is then a measure of the PTH-antagonistic potential in the sample. The antagonistic capacity of the PTH (4-37) fragments can then be influenced by a factor k _ant with the agonistic capacity of the biologically active PTH fragments hPTHd -37) or hPTHd -34), hPTHd -38), hPTHd - 37 + x ) ... hPTH (1-84) can be related.

As a rule, it will suffice if the agonistic and the antagonistic portions of the amino terminal PTH fragments (ie, amino terminal of the amino acid 38) be related 1: 1. For some indications, however, it can be advantageous to separate the individual portions of biologically active hPTH (1-84), hPTH (l-mid) [mid. = PTH fragment with mid-regional specificity 53-68], hPTHd-Cterm) [Cterm = PTH fragment with C-terminal specificity 53-84] with the active hPTH (1-33-38) fragments and the inhibiting hPTH (4th -37) to relate to each other. The physiological hPTH activity can then essentially be determined as follows.

V> TΗ ~ Α \ rt- ₌ k, [hPTH (l-32/138)] + k _x __ £ [hPTH (lX / mid / cterm)] + k ₂ ± [hPTlHl-84)] k, [hPTH (4-25 to 84)]

where k are factors for the agonistic or antagonistic activity of the various PTH fragments and [hPTH (1-37)], [hPTH (1-84)], [hPTH (1-mid.], [hPTHd -Cterm] and [hPTH (4-37) l, the concentrations of the various fragments in the sample. It should be understood that the term includes hPTH (1-33 ~ 38) fragments of 33 to 38 amino acids. in general, the constant k ₂ for the Fragments hPTHd-mid), hPTH (l-Cterm) and hPTH (1-84) be approximately equal to k. An activity constant will have to be determined for each individual PTH fragment if the activity constants k _u k ₂ k ₃ ... should be different. The proportion of fragments with mid-regional and C-terminal hPTH immunoreactivity and an hPTH (1-3) epitope will then have to be determined specifically, in an analogous manner to the agonistic and antagonistic fragments of the amino terminal area.

As a rule, however, the following simplified equation will meet the requirements.

k, [hPTH {l-32 to 38)] + k, [hPTH (l-84) ^' PTH activity = - ² ic, [hPTHXk-25 to 84)]

where k ,, k ₂ and k ₃ can be set to the same approximation. Example 5 Diagnosis of primary hyperparathyroidism

Case study 1

Clarification of the suspicion of hyperparathyroidism in a 57-year-old patient with hypercalcemia. Tumor hypercalcemia was excluded by o-sono, CT thorax, CT abdomen, MRI abdomen, gastroscopy and colonoscopy. The findings hypophosphaturia, slightly increased cAMP excretion, an increased quotient from calcium clearance to creatinine clearance and also the clinical course with increasing calcium values, the nephrocaicinosis with renal insufficiency, typical depressions, recurrent pancreatitis and marginal osteopathy on the femoral neck. In addition, a low PTH level would be expected in tumor hypercalcemia. The relevant laboratory values for plasma were as follows:

PTHrP: tumor-typical PTH

There was therefore no sarcoidosis or vitamin D-mediated hypercalcemia. The values for 25-hydroxy-vitamin D ₃ and 1, 25-vitamin D _{3 were} too low for this. An attempt at therapy with prednisone did not reduce the calcium. In addition, a low PTH level would be expected in vitamin D-mediated hypercalcemia.

The determination of the biological PTH activity with the aid of the assay according to the invention also includes the activities of the fragments PTH (1-34), PTH (1-37), PTH (1-

38) and amino terminus portion of PTH (1-84), including intact PTH (1-84) - but not the PTH fragments PTH (3-37 / 38) and PTH (3-84) - showed excessive PTH- activated vity in the patient's serum (namely 37 pmol, 31 pmol, 22 pmol and 30 pmol bioactive PTH fragments per liter on 4 different days within a month), corresponding to 3 to 4 times the normal PTH activity, so that the diagnosis of primary hyperparathyroidism became laboratory diagnostic. Because of the clearly elevated PTH fragment 1 to 38 in the serum, the secretion of an N-terminal fragment of the PTH was to be assumed, whereas renal insufficiency did not generally lead to an accumulation of N-terminal, but of central-regional and C-terminal fragments of the PTH leads.

Case study 2

Hyperparathyroidism was suspected in a 48-year-old dialysis patient with a calcium metabolic disorder. Sarcoidosis could be excluded due to normal 1,25-hydroxy-vitamin D ₃ levels. The patient was irregularly hypercalcemic. A bone biopsy was performed for the pathology (bone cylinder 3 times 1, 2 cm each). Microscopically, a trabecular bone was visible, which was accompanied in some places by a slight endosteal fibrosis. There were increased signs of absorption on the bone surface, some with active osteoclasts. The osteoblast activity was slightly increased. There was no stronger osteoid formation. The bone was mostly lamellar. Hematopoietically active bone marrow was found in the medullary spaces.

There was apparently an osteopathy of the hyperparathyroidism type. Osteopathy was active. The conversion so far has been minor. There was no evidence of mineralization disorder (osteoidosis, osteomalacia). There was also no evidence of plasmacytoma. Nevertheless, the measured values of intact PTH were not increased. The laboratory values were as follows:

Because of this finding, partial parathyroidectomy was recommended. The immunoassay of the biologically active PTH activity according to the invention showed that there was no overall increased PTH activity either, so that the surgical treatment could be postponed.

Claims

1 . Method for determining the parathyroid hormone activity in a sample, characterized by the steps:

Reacting a sample with an antibody that recognizes an epitope in the region of the receptor binding structure of the parathyroid hormone;

Reacting the sample with an antibody that recognizes an epitope that is formed by the N-terminal amino acids 1 to 3 of the parathyroid hormone and contains the N-terminal amino acid;

Determining the number of molecules recognized by both antibodies; and or

Calculate the effective parathyroid hormone activity from the number of molecules in the sample.

2. The method according to claim 1, wherein an antibody binds to the region between amino acid 15 and 22 of the human parathyroid hormone.

3. The method according to claim 1 or 2, wherein one antibody is bound to a solid phase and the other antibody carries a label.

4. The method according to any one of claims 1 to 3, wherein the binding of the two antibodies takes place in the presence of 0.05 to 0.1% by weight of a light detergent.

5. The method according to any one of claims 1 to 4, wherein a known aliquot of the sample is reacted with two antibodies that bind to the parathyroid hormone sequences between amino acids 4 to 1 4 and 1 5 to 37.

6. The method according to any one of claims 1 to 5, wherein the number of molecules in a sample that are bound by the antibodies against the epitope with amino acids 1 to 3 and the receptor binding site of the parathyroid hormone, and the number of molecules by antibodies against the parathyroid hormone regions between amino acids 4 and 14 or 1 5 to 37 be bound to each other in order to determine the physiologically active parathyroid hormone activity.

6. Diagnostic system for determining the parathyroid activity in a sample, characterized by antibodies that specifically recognize the parathyroid epitope with amino acids 1 to 3 and antibodies that bind to the region of the receptor binding site of the parathyroid hormone.

7. The diagnostic system of claim 8, further including antibodies that specifically bind to the region between amino acids 4 and 14 of the parathyroid hormone.

8. The diagnostic system of claim 6 or 7, further including antibodies that specifically bind to the region between amino acids 24 and 37 of the parathyroid hormone.

9. A method for the diagnosis and extent of hypo- or hyperparathyroidism, characterized in that the method for determining the PTH activity in a sample claimed in claims 1 to 6 is used.

1 0. Method for finding the cause of calcium metabolism disorders, osteopathies, kidney failure and diseases which result from a disturbed homeostasis in the calcium and phosphate content of the blood, characterized in that the method claimed in claims 1 to 6 for

Determination of the PTH activity in a sample is used.