DE102004022298A1 - Test kit for enzyme immunoassay identification of human cathepsin K, in blood and other body fluids, has separate packages containing antibodies bonded to micro-titration plates, standard human recombinant cathepsin K and buffers - Google Patents

Abstract

A test kit, for identification of human cathepsin K in blood and other body fluids, has separate packages containing micro-titration plates with bonded antibodies which are sensitive to bonding with specific cathepsin K, human recombinant cathepsin K as a standard for a quantitative register of this enzyme in body fluids, a buffer to produce a standard series of recombinant cathepsin K, a buffer for dilution of the sample, a washing buffer, a marker which bonds with cathepsin K and be detected, and a substrate to give a visible view of the markings.

Description

The The invention relates to an ELISA for the determination of cathepsin K im Blood and other body fluids for the Diagnosis of osteoresorptive processes and osteolytic activity of tumor metastases.

application areas are medicine and especially medical diagnostics.

The enzyme-linked immunosorbent assay (ELISA) technique is current technology standard in clinical laboratories. With this technology can i.a. Marker proteins for certain Diseases in body fluids be determined by patients.

cathepsin K was identified as the osteoclastic protease that was released in the pericellular space (so-called Lakunen), which splits bone collagen and dissolves. In cooperation with the phosphatases, the low pH and probably other factors, the inorganic bone components degrade, the bone is absorbed.

Osteoresorptive Processes are a variety of important and common Underlying diseases. Such include, e.g. Osteoporosis, osteomalacia, Osteodystrophy, Paget's disease and so-called osteolytic metastases from malicious Tumors, e.g. Prostate, breast or lung carcinomas.

biochemical let yourself Bone degradation through calcium and phosphate balance determinations in the blood and urine roughly estimate or there are degradation products of bone collagen in urine or in the Blood determined. To include such e.g. so-called cross-links (NTx, CTx, deoxypridinoline, pyridinoline) or hydroxyproline. The determination of bone-specific acidic Phosphatase in the blood is used by some as a method of detection osteoresorptive activity considered. At the identity and specificity However, the bone-specific phosphatase is in doubt, and The results obtained appear largely dubious and ambiguous.

Of the Invention was therefore the object of a method develop with which the concentration of Cathepsin K, Procathepsin K and cathepsin-K frictions can be determined and that for the diagnosis can be used by osteoresorptive processes.

The Invention is realized according to the claims.

• a) einen festen Träger mit daran gebundenen Antikörpern, die sensitiv und spezifisch Cathepsin K binden;
• b) humanes rekombinantes Cathepsin K als Standard zur quantitativen Bestimmung dieses Enzyms in Körperflüssigkeiten;
• c) einen Puffer zum Herstellen einer Standardreihe des rekombinanten Cathepsin K;
• d) einen Puffer zum Verdünnen der zu untersuchenden Probe;
• e) einen Waschpuffer;
• f) ein detektierbar markiertes Konjugat, das an Cathepsin K bindet;
• g) und ein Substrat, das die Sichtbarmachung des detektierbar markierten Konjugats erlaubt.

The invention relates to a test kit for the determination of cathepsin K in the blood and other body fluids for the diagnosis of osteoresorptive processes and the osteolytic activity of tumor metastases comprising in separate packaging at least:

• a) a solid support with antibodies bound to it, which sensitively and specifically bind cathepsin K;
• b) human recombinant cathepsin K as a standard for the quantitative determination of this enzyme in body fluids;
• c) a buffer for preparing a standard series of the recombinant cathepsin K;
• d) a buffer for diluting the sample to be examined;
• e) a washing buffer;
• f) a detectably labeled conjugate that binds to cathepsin K;
• g) and a substrate that allows visualization of the detectably labeled conjugate.

at the antibodies, the to the solid carrier are preferably monoclonal antibodies which from hybridoma cell lines with accession numbers CCM 7185 and CCM 7186 are produced.

When solid carriers For the most part, microtiter plates are used and detectable labeled conjugate is a conjugated antibody, preferably a polyclonal antibody Antibody, which binds to Cathepsin K.

The standard human recombinant cathepsin K is used in eukaryotic cells and is in solution or lyophilized.

in the The development of the ELISA is described in more detail below.

manufacturing of the human recombinant cathepsin K

DNA:

• DNA sequence: Genbank Accession No. NM_000396 Position 170 ... 1111
• Synthetic gene: No signal sequence, with the N-terminal Activation sequence
• Vector: pQE30

Protein:

• Number of amino acids: A total of 326 - of it 314 ProcathepsinK and 12 vector coded
• Molecular weight: 36.69 kDa
• Amino acid sequence: Underlined amino acids are encoded by the vector:

• Note: Without the activation peptide, cathepsin K 215 amino acids (23.48 kDa).
• Expression of Cathepsin K:
• Expression takes place in the E. coli strain: JM109.
• Cultivation: On the shaker grows the culture for 3h 30min to an OD (550nm) of 0.6. It follows the addition of Inductor IPTG. Expression is stopped after 2 hours. The Culture reaches an OD (550 nm) of 1.8.
• Control expression in 50ml:

The description applies for gel and blot:

The results ( 1 ) illustrate that the protein is not expressed in a soluble form. The band on the blot of the supernatant can be explained by the overflow of contents from the adjacent well.

in the Regarding the occurrence of degradation products were whole Cells dissolved in guanidium and applied to a nickel column.

Isolation and purification of the expressed cathepsin K

Action:

• - Solubilization E. coli cells in 6M Gnd-HCl, 0.1M Tris-HCl pH 8.6
• - centrifugation for the removal of insoluble material
• - Purify on the NiNTA column, equilibration with 6M Gnd-HCl, 0.1M Tris-HCl pH 8.6, elution with 0.5M imidazole in 6M Gnd-HCl, 0.1M Tris-HCl pH 8.6
• - purity control with SDS-PAGE after precipitation with TCA, reduction with MeOH 0.015M and post-purification with gel chromatography Purity control with SDS-PAGE after TCA precipitation

I. Direct dilution of the material

• - Procathepsin K in 6M Gnd-HCl, 0.1M Tris-HCl pH 8.6, suddenly diluted in 0.1M Tris-HCl, 0.5% CHAPS, 1 mM GSSG, pH 8.6 to a final concentration of 10ug / ml and Gnd HCl concentration of 0.1M and then at 4C over Incubated overnight
• - 3 × dialysis against 0.1M Tris-HCl pH 8.6
• - Concentrate in an Amicon stirred cell, cut-off 3kDa
• - centrifugation at 12000 g / 20min
• - Purity control with SDS-PAGE and protein determination after Bradford

II. Gradual elimination of Gnd-HCl by dialysis

• - Procathepsin K in 6M Gnd-HCl, 0.1M Tris-HCl pH 8.6 diluted in 6M Gnd-HCl, 0.1M acetate buffer, 0.015M MeOH, pH 4.0 to a final concentration of 10μg / ml and the pH of the solution became brought to pH 4.0 with concentrated HCl
• - Dialysis against 1M Gnd-HCl, 0.1M acetate pH 4.0
• - Dialysis against 0.5M Gnd-HCl, 0.1M acetate pH 4.0
• - Dialysis against 0.25M Gnd-HCl, 0.1M acetate pH 4.0
• - Dialysis against 0.1M Gnd-HCl, 0.1M acetate pH 4.0
• Dialysis against 0.05M Gnd-HCl, 0.1M acetate pH 4.0
• - 3 × Dialye against 0.1M acetate pH 4.0
• - narrowing in an Amicon stirred cell, cut-off 3kDa
• - centrifugation at 12000 g / 20min
• - purity control with SDS-PAGE, Bradford protein determination

manufacturing of specific antibodies

Either this by direct dilution (I.) as well as by the gradual elimination of Gnd-HCl By dialysis (II :) contained material was used for the immunization of rabbits and mice used.

The Immunization and recovery of polyclonal and monoclonal antibodies was carried out according to known methods.

Provision of specific antibodies:

First of all an immobilization of 5mg of procathepsin K gained by the Stepwise dialysis, at pH 4.0 to 0.5 g POROS AL (Applied Biosystems).

The recovered antibodies were then immunoaffinity purified using this column and used for the development of ELISA. ( 2 )

Development and validation of the ELISA

Optimization of the components and conditions

monoclonal and polyclonal antibodies, which had a high titer, were treated with horseradish peroxidase (horseradish peroxidase, HRP) conjugated using the periodate method. Subsequently were tested different combinations of antibodies. Both the Antibody, which were bound to the microtiter plate as well as the conjugated ones Antibody, were varied. It turned out that the combination of monoclonal Antibody, those from hybridoma cell lines with accession numbers CCM 7185 and CCM 7186 for plate coating the HRP-conjugated polyclonal antibody RAHuCATD the best results supplies.

Of the optimization of the concentration of the coating antibodies, the Conjugate, buffer constituents, incubation times and temperatures, as well as the stability the reagents and components of the test kit according to known methods.

Test procedure:

• 1. 100 μl of the coating solution [Antibody in coating buffer (2 μg / ml)] are added to the wells of the MTP and incubated at 4 ° C for 16 h
• 2. The coating solution is aspirated and the wells are washed 1 x with 350 ul wash buffer.
• 3. It follows the addition of 200 ul blocking solution per well, the incubation at room temperature for 30 min, the suction of the blocking solution and drying the plates.
• 4. 100 μl the standard solutions and the samples diluted 1: 3 with sample buffer are placed in the appropriate Wells pipetted.
• 5. The MTP is incubated for 1 h at room temperature (about 25 ° C) shaking with an orbital MTP shaker at approx. 300 rpm.
• 6. The wells are washed 3 times with 350 μl wash buffer each washed.
• 7. Add 100 μl conjugate solution, 1: 3000 in a stabilizer dilute has been.
• 8. The MTP is incubated for 1 h at room temperature (about 25 ° C) shaking with an orbital MTP shaker at approx. 300 rpm.
• 9. The wells are washed 3 times with 350 μl wash buffer each washed.
• 10. 100 μl the substrate solution is pipetted into each well.
• 11. The MTP is incubated for 10 min at room temperature (about 25 ° C).
• 12. The color development is stopped by adding 100 μl stop solution.
• 13. Optical density is measured with a photometer wavelength of 450 nm.

used material:

• Microtiter plates (MTP): Corning Costar, High Binding
• Coating buffer: 0.1 mol / l carbonate buffer, pH 8.5
• Washing buffer: 0.1 mol / l TBS, pH 7.2 with 0.1% Tween-20
• Blocking solution: TBS, pH 7.2 with 0.5% BSA and 2% sucrose
• Sample buffer: 0.1 mol / l TBS, pH 7.2 with 0.1% Tween-20 and 0.1 % BSA
• Conjugate stabilizer: commercially available
• Substrate solution: commercially available TMB solution
• Stop Solution: 0.2 mol / l sulfuric acid

Standard curve and detection limit:

A representative standard curve constructed using the recombinant human cathepsin K is in 3 shown.

to Creation of the curve were cathepsin K concentrations of 0.1-10 used ng / ml. The lower detection limits are determined using absorbance, which is three standard deviations above the blank, and corresponds to a cathepsin K concentrations of 0.02 ng / ml.

INTRATest variance

Four Serum samples with different cathepsin K concentrations were measured with one examination (8-fold determination). The variance the 8 results per same sample were at all concentrations below 4.5% (Table 1).

Tabelle 1: Intratestvarianz

Table 1: Intratest variance

Intertest variance

Tabelle 2: Intertestvarianz Four serum samples with different cathepsin K concentrations were measured by 6 study. The variance of the 6 results for each same sample was less than 6% at all concentrations (Table 2).

Table 2: Intertest variance

Recovery

Tabelle 3: Wiederfindungsrate Recombinant cathepsin K at concentrations 0.5, 1 and 2 ng / ml was added to four sera of known cathepsin K concentrations and examined. The recovery rate of the added cathepsin K in the serum samples ranged between 82.8% and 97.6% (Table 3)

Table 3: Recovery

linearity

serum samples at various concentrations were diluted and measured. The recovery rate the samples ranged from 88.5% to 110% (Table 4).

Tabelle 4: Linearität

Table 4: Linearity

Specificity of the ELISA

Of the ELISA shows no cross-reactivity with other human cysteine proteinases (cathepsin B, H, L or S) or with the aspartic proteinase cathepsin D. Likewise, no disorder by hemoglobin (5 mg / ml), bilirubin 0.2 mg / ml) and total lipids (10 mg / ml).

stability

The Cathepsin K serum concentrations also remained largely after 7 days Storage at -20 ° C unchanged

cathepsin K concentrations in the serum of normal individuals, patients with proven Ostoeporosis and patients with prostate and breast cancer metastases

embodiment

It 20 normal subjects, 20 patients with osteoporosis and 20 were each 20 patients with metastatic prostate and breast cancer on cathepsin K examined. The measured values leave all three populations clear and sharp (Table 5).

The results are in the 4 to 9 shown graphically.

Claims (11)

Test kit for the determination of cathepsin K in the blood and other body fluids for the diagnosis of osteoretic processes and osteolytic activity of tumor metastases, comprising in separate packaging at least: h) a solid support with antibodies bound thereto which sensitively and specifically bind cathepsin K; i) human recombinant cathepsin K as a standard for the quantitative determination of this enzyme in body fluids; j) a buffer for preparing a standard series of the recombinant cathepsin K; k) a buffer for diluting the sample to be examined; l) a wash buffer; m) a detectably labeled conjugate that binds to cathepsin K; n) and a substrate that allows visualization of the detectably labeled conjugate. Test kit according to claim 1, characterized that it is the antibodies, the to the solid carrier are bound to monoclonal antibodies derived from the hybridoma cell lines with the accession numbers CCM 7185 and CCM 7186. Test kit according to claims 1-2, characterized that as a solid carrier predominantly microtiter plates are used. Test kit according to claims 1-3, characterized that as a detectably labeled conjugate a conjugated antibody used which binds to cathepsin K. Test kit according to claims 1-4, characterized that as a conjugated antibody a polyclonal antibody is used. Test kit according to claims 1-5, characterized that the substances used as conjugates with all the usual Substances can be conjugated preferably with: Horseradish peroxidase, or - alkaline Phosphatase. Test kit according to claims 1-6, characterized that characterized in that the standard used humane recombinant cathepsin K was expressed in eukaryotic cells and in solution or lyophilized. Monoclonal antibodies, The cathepsin K can specifically recognize and bind these monoclonal antibody properties like the monoclonal antibodies from hybridoma cell lines with accession numbers CCM 7185 and CCM 7186. Monoclonal antibodies according to claim 8, wherein the monoclonal antibodies biochemically or molecular biologically changed or be synthetic, being the antibodies if necessary, parts for the detection of cathepsin K are not necessary, in whole or in part missing or these parts are replaced by others. Monoclonal antibodies according to claim 8-9, which from hybridoma cell lines with accession numbers CCM 7185 and CCM 7186 are produced. Hybridoma cell lines with the accession numbers CCM 7185 and CCM 7186