EP1556698A1 - Kits de dosage elisa pour la detection de la collagenase 3 en tant que proenzyme et sous forme activee dans des liquides corporels et des surnageants de cultures cellulaires - Google Patents

Kits de dosage elisa pour la detection de la collagenase 3 en tant que proenzyme et sous forme activee dans des liquides corporels et des surnageants de cultures cellulaires

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Publication number
EP1556698A1
EP1556698A1 EP03753260A EP03753260A EP1556698A1 EP 1556698 A1 EP1556698 A1 EP 1556698A1 EP 03753260 A EP03753260 A EP 03753260A EP 03753260 A EP03753260 A EP 03753260A EP 1556698 A1 EP1556698 A1 EP 1556698A1
Authority
EP
European Patent Office
Prior art keywords
collagenase
procollagenase
monoclonal antibodies
activated
elisa kit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03753260A
Other languages
German (de)
English (en)
Inventor
Matthias Dettloff
Thorsten Stroh
Peter Sveshnikov
Peter Bendzko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
Original Assignee
Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE20213551U external-priority patent/DE20213551U1/de
Application filed by Invitek Gesellschaft fuer Biotechnik und Biodesign mbH filed Critical Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
Publication of EP1556698A1 publication Critical patent/EP1556698A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the invention relates to ELISA kits for the detection of collagenase 3 as a proenzyme and in activated form in body fluids, in particular in human serum and synovial fluid as well as in cell culture supernatants, and monoclonal antibodies that specifically recognize collagenase 3 in latent and activated form.
  • Areas of application are medicine and here in particular diagnostics, in particular the nerlauf diagnosis of inflammatory rheumatic diseases (rheumatoid arthritis), systemic lupus erythematosus (sLE) with organ involvement and tissue proliferation as well as tumor diseases (e.g. breast and colorectal carcinomas).
  • the enzyme-linked immunosorbent assay (ELISA) technique is the current technology standard in clinical laboratories. With this technology i.a. Marker proteins for certain diseases can be determined in patient body fluids.
  • MMPs Matrix metalloprotemases
  • ⁇ agase, H. and Woessner, F. Jr., J. Biol. Chem. 1999, 274, 21491-21494 Based on their preferred substrates and structural features, MMPs can be classified into collagenases, gelatinases, stromelysins and membrane-type metalloproteases.
  • Collagenase 3 (MMP-13) is released by cells as an inactive proenzyme (Prokollagenase 3, Pro-MMP-13) and converted extracellularly into the activated form by the cleavage of a propeptide.
  • procollagenase 3 and activated collagenase 3 are typically undetectable in the differentiated, adult tissue. However, their occurrence is described in connection with a whole series of destructive clinical pictures: In the development of breast carcinomas (Nielsen BS et al., Cancer Res. 2001 61: 7091-7100), rheumatoid arthritis (Westhoff CS et al., Arthritis Rheum. 1999 42: 1517-1527) and osteoarthritis (Shlopov BN et al. Arthritis Rheum. 1997 40: 2065-2074) are the levels of procollagenase 3-mR ⁇ A strongly upregulated in the affected tissue types. These findings make it clear that collagenase 3 is a marker protein of great interest for medical diagnostics.
  • the first test the Biotrak Matrix Metalloproteinase-13 ELISA system, has been validated for the body fluids serum and plasma.
  • the problem with the test is the very low sensitivity.
  • the test is not validated for joint fluid testing and therefore cannot be used for this purpose.
  • the second test system on the market is only intended for use in cell culture and can therefore not be used for the investigation of procollagenase 3 in body fluids.
  • the invention was therefore based on the object of providing an ELISA kit which, in contrast to the tests on the market, is distinguished by a high sensitivity and both for the detection of procollagenase 3 and the activated form of this enzyme in body fluids, is particularly suitable in human serum and synovial fluid, as well as in cell culture supernatants.
  • the invention should also offer the possibility for the first time in Cell culture supernatants and body fluids to determine the quantitative relationship between latent and activated form of this enzyme in a highly specific manner.
  • the ELISA kits according to the invention for the detection of procollagenase 3 and activated collagenase 3 comprise at least: a) a solid support with attached monoclonal antibodies, the sensitive and specific human procollagenase 3 or activated collagenase
  • Collagenase 3 d) a buffer for diluting the samples to be examined; e) a detectably labeled conjugate that binds to collagenase 3; and f) a substrate which permits the detection of the detectably labeled conjugate, the monoclonal antibodies mentioned under a) either preferably being anti-MMP-13 clone M34 (mouse), and particularly preferably monoclonal antibodies which are of the hybridoma with the deposit number
  • PSM ACC 2572 be formed, or preferably anti-MMP-13 clone EE1 (mouse).
  • Either a combination of two components is used as the detectably labeled conjugate, the first component being biotinylated antibodies which bind to collagenase 3; and as a second component around a highly polymeric streptavidin conjugate that binds to the biotinylated antibodies.
  • conjugated antibodies that bind to collagenase 3 can also be used.
  • the antibodies, which act as a conjugate, can be monoclonal and / or polyclonal antibodies.
  • Human recombinant procollagenase 3 which was expressed in eukaryotic cells (Sf9 cells), is used as a standard for the quantitative determination of procollagenase 3 in body fluids and cell culture supernatants. It is either in solution or in freeze-dried form, in which it can be kept for several months without loss of quality. If they are in freeze-dried form, the recombinant procollagenase 3 must first be reconstituted by adding distilled water before use. Human recombinant activated collagenase 3 is made from procollagenase 3 mentioned above with the addition of acetaminophenyl-mercury-acetate (APMA). manufactured and used in the same way.
  • APMA acetaminophenyl-mercury-acetate
  • the buffer provided for the dilution of samples to be examined contains, in addition to blocking and stabilizing substances, sodium citrate among others. Surprisingly, it was found that this reagent is particularly well suited for the preparation of human serum for measuring collagenase 3.
  • the buffer for making a standard series of recombinant procollagenase 3 or activated collagenase 3 for measuring these markers in serum contains human serum.
  • Microtite plates to which the monoclonal antibodies according to the invention are bound are preferably used as solid supports. These microtitre plates are produced so that they can be stored for several months without loss of quality.
  • the invention also relates to monoclonal antibodies which specifically recognize and bind collagenase 3 as a proenzyme or activated enzyme, these monoclonal antibodies from hybridoma cell lines with the deposit number
  • DSM deposit number
  • ACC 2572 are produced or properties such as the monoclonal antibodies from the hybridoma cell line with the accession number [DSM ACC 2572
  • Antibodies from the hybridoma cell line with the accession number [DSM ACC 2572] have, which, however, can be biochemically or molecular biologically modified or synthetic, with the antibody possibly missing parts or parts that are not necessary for the recognition of procollagenase 3, or these parts are replaced by others.
  • procollagenase 3 and of activated collagenase 3 in body fluids is made possible with high sensitivity by the ELISA kits according to the invention and thus makes these potential disease markers more accessible to medical diagnosis.
  • the lower detection limit of the ELIS As is 4 pg procollagenase 3 or 6 pg activated collagenase 3 / ml sample.
  • the standard curve determined during the measurement by examining a human recombinant procollagenase 3 or the activated collagenase 3 allows a rapid calculation of the collagenase content in samples with the aid of the regression function on which the standard course is based.
  • Another decisive advantage is that the ELISA kits can be stored in the refrigerator, which significantly improves handling and user friendliness.
  • the ELISA kits according to the invention for the detection of procollagenase 3 or activated collagenase 3 have an overall shelf life of at least one month for the end user. Production takes place in accordance with the standards of EN 46001 and EN ISO 9001. For the first time, the ELISA kits offer the opportunity to examine synovial fluid.
  • the ELISA kits according to the invention show for the first time that collagenase 3 is a marker for monitoring the course of rheumatoid arthritis, but also of severe cases with organ involvement and tissue proliferation of systemic lupus erythematosus.
  • collagenase 3 is a marker for monitoring the course of rheumatoid arthritis, but also of severe cases with organ involvement and tissue proliferation of systemic lupus erythematosus.
  • an increase in the level of MMP-13 in the serum as demonstrated by the ELISA kit according to the invention, precedes an acute clinical deterioration of the clinical picture.
  • the enzyme cannot be detected in the serum at all times, which is why this marker is primarily suitable for the prognosis of selected diseases, especially for a preventive start of therapy before the patient becomes clinically noticeable
  • the invention also relates to the use of collagenase 3 - as a serological marker for diagnosis and in particular for monitoring the course of inflammatory rheumatic diseases, in particular rheumatoid arthritis and - as a serological marker for diagnostics and in particular for monitoring the course of systemic lupus erythematosus, in particular for prognosis with simultaneous tissue proliferation (tumor formation).
  • Collagenase 3 can also be used as a serological marker for the diagnosis and monitoring of other tumor diseases, in particular of breast carcinomas and colorectal carcinomas. Collagenase 3 can also be used as a serological marker for the diagnosis and monitoring of further diseases in which an increase in collagenase 3 is described.
  • mice were used for immunization.
  • the antigen was prepared as follows: 50 ⁇ g MMP-13 in 100 ⁇ l PBS + 100 ⁇ l 6M urea were presented. 100 ⁇ l of CFA or IFA were added to this solution. The injection was carried out according to the following scheme: Day 0: 50 ⁇ g MMP-13 intraperitoneally in CFA Day 13: 50 ⁇ g intraperitoneally in IFA Day 41: 50 ⁇ g intravenously in 1 ml PBS Day 44: Hybridization of pre-lymphocytes with spleen and SP 2/0 myeloma cells.
  • the plate was washed three times and 100 ⁇ l of anti-mouse IgG (H + L) -POD conjugate was added for a further hour (37 ° C.). After a further five washes, the POD content in the wells was detected with 100 ⁇ l of TMB substrate (20 min, RT). The reaction was stopped with 50 ⁇ l 2 MH 2 SO and the absorption was measured at 450 nm. The hybridoma supernatants positive in the ELISA were cloned and recloned to monoclonality. 5 independent monoclonal antibodies were obtained, from which a total of 12 subclones with partially changed affinities were obtained.
  • a hybridoma cell line which produces the monoclonal antibodies anti-MMP-13 clone M34 (mouse) according to the invention (IgGl), was in the German collection of Microorganisms and cell cultures GmbH (DSM ACC 2572Z) in Braunschweig under the number pSM ACC 2572 [filed on 08.28.02,
  • FIG. 1 The principle of the ELISAs is shown in Figure 1.
  • a series of dilutions is created from the human recombinant collagenase 3 as standard, which contains the recombinant procollagenase 3 (or activated collagenase 3) in the following concentrations: 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 63 pg / ml, 32 pg / ml, 16 pg / ml, 0 pg / ml (start of the dilution series to determine activated collagenase 3: 2000 pg / ml).
  • the standard dilution is prepared using a special buffer system which contains 10% human serum. In each case 100 ⁇ l of these standard dilutions are duplicated in the wells of the microtiter plate, to the monoclonal antibodies from the hybridoma cell line with the
  • the samples to be measured are diluted with the buffer provided for sample preparation. 100 ⁇ l of the diluted samples are then also applied in duplicate. After 120 minutes of incubation on a shaker at room temperature, the microtitre plate is washed four times with the wash buffer and then the remaining liquid is removed by knocking out on paper towels. This is followed by the entry of 100 ⁇ l detection solution 1, which contains biotinylated antibodies, in all wells used on the microtiter plate. These are either polyclonal antibodies or a monoclonal antibody or a cocktail of several monoclonal antibodies.
  • the microtitre plate is again washed four times and knocked out on a paper towel.
  • the detection solution 2 consisting of a streptavidin-peroxidase conjugate and a dilution buffer, is prepared according to the instructions and in turn pipetted 100 ⁇ l into the corresponding wells of the microtiter plate.
  • Another 30 minute incubation follows. The microtiter plate is then washed and knocked out five times, provided with 100 ⁇ l per well of substrate solution (tetramethylbenzidine) and incubated in the dark for 15 minutes.
  • Example 3 Examination of body fluids with the ELISA kit
  • Table 1 Measurement of Pro-MMP-13 and activated MMP-13 with the two InviLISA MMP-13 kits. The table shows the size of the sample population and the number of positive samples (in the respective tests and in total).
  • RA rheumatoid arthritis
  • pSS primary Sjogren's syndrome
  • sLE systemic lupus erythematosus.
  • Table 1 shows the results of these measurements: While in a control group of blood donors only about 2% reacted positively in the tests, 20% of the sera from patients with rheumatoid arthritis showed positive signals.
  • Fig. 2 shows significantly increased active MMP-13 values immediately before the onset of clinical deterioration, which is accompanied by a massive swelling of the right knee joint. The slow clinical improvement follows a decrease in the MMP-13 titer.
  • Fig. 3 like Fig. 2, shows the particular suitability of activated collagenase 3 as a marker for rheumatoid arthritis.
  • the measured MMP-13 values were not increased or are below the defined cut-off of 300 pg / ml MMP-13.
  • the patient in question was X-rayed in November 1998, with the wrists classified as Grade 1 by Larsen.
  • the patient suffered a massive flare-up which was accompanied by measured, greatly increased MMP-13 values.
  • control x-rays showed a progressive destruction of the wrists (Larsen grade 2) and an beginning destruction of the ankles (Larsen grade 1).
  • Fig. 4 shows two exemplary course measurements of patients with systemic lupus erythematosus (sLE). While even sera from patients with severe disease courses showed no or only marginally increased MMP-13 values (Fig. 4 A, sLE with renal involvement, very severe symptoms), sera from sLE patients with tissue proliferation showed increased values of activated MMP-13 (Fig. 4 B, sLE with renal involvement, mebran proliferating glomerulo-nephritis type IV a, severe symptoms). These measurements indicate that MMP-13 can be an indicator of tumor growth.
  • Step 1 Incubation of standards or samples on the titre plate. specific
  • Step 2 Detection of the bound collagenase 3 (MMP-13) with biotinylated antibody (duration: 90 minutes)
  • Step 3 addition of streptavidin-peroxidase conjugate (duration: 30 minutes)
  • Step 4 color development after addition of TMB substrate (duration : 15 minutes)
  • FIG. 2 Measurement of activated MMP-13 in the serum of a patient with rheumatoid arthritis (Larsen III, DAS score> 3.8). No elevated Pro-MMP-13 values were detectable in the samples. The increase in the MMP-13 values in the serum immediately at the time of a relapse and the subsequent decrease in the values during the remission is evident (arrows).
  • Figure 3 Measurement of Pro-MMP-13 (black bars) and activated MMP-13 (gray bars) in the serum of a patient with rheumatoid arthritis.
  • RA rheumatoid arthritis
  • St. stage
  • HG wrist
  • FG ankle.
  • Figure 4 Measurement of Pro-MMP-13 black bars) or activated MMP-13 (gray bars) in sera from two patients with sLE.
  • A sLE patient with renal involvement, neplirotic syndrome, very severe symptoms.
  • B sLE patient with renal involvement, severe symptoms, proliferating glomerulo-nephritis type IV a. Explanations in the text.

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  • Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

Kits de dosage ELISA pour la détection de la procollagénase 3 et de la collagénase 3 activée dans des liquides corporels, en particulier dans le sérum et les liquides articulaires de l'homme, ainsi que dans des surnageants de cultures cellulaires, et anticorps monoclonaux qui reconnaissent spécifiquement ces antigènes. Le kit de dosage ELISA comporte, dans des emballages séparés, au moins (a) un support solide sur lequel sont fixés des anticorps monoclonaux qui se lient de manière sensible et spécifique à la procollagénase 3 ou à la collagénase 3 activée humaines, (b) de la procollagénase 3 ou de la collagénase 3 activée humaines recombinées en tant que norme pour la détermination quantitative de cette enzyme, (c) un tampon pour la production d'une série standard de la collagénase 3 recombinée, (d) un tampon pour diluer les liquides corporels à étudier, (e) un conjugué marqué aux fins de détection, qui se lie à la collagénase 3 et (f) un substrat qui permet la visualisation du conjugué marqué aux fins de détection. Pour les anticorps monoclonaux cités sous (a), il s'agit de préférence d'anticorps monoclonaux qui sont formés de l'hybridome ayant le numéro d'enregistrement DSM ACC 2572. Les domaines d'utilisation dudit kit sont la médecine et en particulier le diagnostic médical, plus précisément le contrôle de l'évolution de l'arthrite rhumatoïde, du lupus érythémateux disséminé ainsi que des maladies tumorales.
EP03753260A 2002-08-29 2003-08-27 Kits de dosage elisa pour la detection de la collagenase 3 en tant que proenzyme et sous forme activee dans des liquides corporels et des surnageants de cultures cellulaires Withdrawn EP1556698A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE20213551U DE20213551U1 (de) 2002-08-29 2002-08-29 Elisa-Kit zum Nachweis von Prokollagenase 3 in Körperflüssigkeiten und Zellkulturüberständen
DE20213551U 2002-08-29
DE10314404 2003-03-28
DE10314404 2003-03-28
PCT/DE2003/002864 WO2004021007A1 (fr) 2002-08-29 2003-08-27 Kits de dosage elisa pour la detection de la collagenase 3 en tant que proenzyme et sous forme activee dans des liquides corporels et des surnageants de cultures cellulaires

Publications (1)

Publication Number Publication Date
EP1556698A1 true EP1556698A1 (fr) 2005-07-27

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EP03753260A Withdrawn EP1556698A1 (fr) 2002-08-29 2003-08-27 Kits de dosage elisa pour la detection de la collagenase 3 en tant que proenzyme et sous forme activee dans des liquides corporels et des surnageants de cultures cellulaires

Country Status (4)

Country Link
US (1) US20060105400A1 (fr)
EP (1) EP1556698A1 (fr)
AU (1) AU2003271513A1 (fr)
WO (1) WO2004021007A1 (fr)

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
CN103424546B (zh) * 2012-05-18 2015-03-11 苏州红冠庄国药股份有限公司 鹿皮胶中所含生物活性成分的特异性鉴定方法
RU2532352C2 (ru) * 2012-06-28 2014-11-10 Федеральное государственное бюджетное учреждение науки Институт биохимии имени А.Н. Баха РАН Российской академии наук (ИНБИ РАН) Способ проведения иммунохроматографического анализа для серодиагностики

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Publication number Priority date Publication date Assignee Title
WO1998029560A1 (fr) * 1996-12-26 1998-07-09 Fuji Yakuhin Kogyo Kabushiki Kaisha Anticorps monoclonal contre la collagenase 3 et procede de dosage immunologique utilisant cet anticorps
US6143506A (en) * 1998-08-13 2000-11-07 The Research Foundation Of State Of Ny Diagnostic method for detection of periodontitis or peri-implantitis
DE19913428A1 (de) * 1999-03-25 2000-09-28 Max Delbrueck Centrum Verwendung von Kollagenase 3 zum Nachweis von destruktiven Gelenkerkrankungen, insbesondere zur Prognose des Krankheitsverlaufs und zur genetischen Prädisposition der Rheumatoiden Arthritis (RA)
FI990888A0 (fi) * 1999-04-20 1999-04-20 Medix Biochemica Ab Oy Menetelmä ja testikittejä respiratorisen alueen tulehduksen läsnäolon ja vaikeusasteen arvioimiseksi

Non-Patent Citations (2)

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See also references of WO2004021007A1 *

Also Published As

Publication number Publication date
AU2003271513A1 (en) 2004-03-19
US20060105400A1 (en) 2006-05-18
WO2004021007A1 (fr) 2004-03-11

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