Classifications

• • 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
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/81—Protease inhibitors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/968—Plasmin, i.e. fibrinolysin

Definitions

• the invention relates to a method for the quantitative determination of plasmin- ⁇ 2-antiplasmin complexes and the use of the determination method as a measure of fibrinolytic changes.
• ⁇ 2-antiplasmin is the most important piasmin inhibitor. His reaction with plasmin results in the formation of an inactive complex composed of one molecule from each of the two components. Two stages are involved in this process; firstly, a reversible complex is formed between the lysine-binding site of the plasmin and the corresponding site in the carboxyl end of the ⁇ 2-antiplasmin molecule. In the second stage, an irreversible complex is formed by cleaving a peptide bond in the inhibito.
• Non-fibrin-specific plasminogen activators such as streptokinase or urokinase, can cause complete consumption of plasmin inhibitors and can be increased due to plasminemia
• plasmin- ⁇ 2-antiplasmin complexes can therefore serve on the one hand as an indicator of generalized plasminemia in hyperfibrinolytic conditions with fibrinogen and ⁇ 2-antiplasmin consumption and possible tendency to bleed, and on the other hand slightly increased plasma levels of plasmin ⁇ 2-antiplasmin complexes indicate continuous thrombus formation and dissolution, as in the case of thrombophilia or PAP complexes in serum, to the entire fibrinolytic potential.
• a major advantage was the development of the double sandwich technique as used in the ELISA systems.
• Various methods have been developed since then; either using an antibody against ⁇ 2-anti-plasmin as the binding antibody and an antibody against that Enzyme for quantification, or vice versa.
• Harpel et al. was the first to publish a sensitive assay with a polyclonal antibody against ⁇ 2-antiplasmin as a binding antibody and POX-labeled Fab fragments against plasminogen as a detection system, whereby the Fab fragments have the advantage over ver intact antibodies offer reduced, non-specific binding of plasminogen and other plasma proteins.
• Other ELISA systems were developed by Holvoet et al. and Mimuro et al.
• DE-Al-41 15 993 discloses a method which relates to the production of a monoclonal antibody BMA PAP6 and the associated hybridoma cell line (BW PAP6), which antibody has a specific affinity for the PAP complex and none, or only one has low affinity for the individual components of this complex.
• BMA PAP6 monoclonal antibody
• BW PAP6 hybridoma cell line
• This antibody was obtained by immunization with PAP complexes cleaved by ammonia treatment.
• this antibody is also only functionally characterized in the ELISA, but not by means of Western blots.
• a test system is therefore proposed here which comprises a specific, other, monoclonal antibody directed against the neoantigen in the PAP complex, which antibody was obtained by immunization with a natural, non-ammonia-treated PAP complex, which antibody also in the Western blot only with the PAP com plex reacts and which antibody is used as a binding antibody.
• a corresponding PAP complex ELISA becomes highly specific and highly sensitive even in the plasma and serum environment.
• the MPW7AP antibody used for the invention is secreted by the associated hybridoma cell line, which is deposited with the Public Health Laboratory Service, European Collection of Animal Cell Cultures, Salisbury, UK, under the number 93031620.
• the cell line is obtained by known methods by fusion of a mouse myeloma cell line (NSO) with spleen cells of a mouse, which was immunized with native PAP complexes.
• NSO mouse myeloma cell line
• the secreted monoclonal antibodies are characterized in that they react specifically in a Western blot only with the PAP complex, but not with the individual components (FIG. 1).
• the test described here is a solid phase enzyme in munassy, in which MPW7AP, a monoclonal antibody specific for the neoantigen of the PAP complex, is adsorbed on plastic microtiter plates.
• MPW7AP a monoclonal antibody specific for the neoantigen of the PAP complex
• MPW2PG POX a peroxidase-labeled monoclonal antibody against the Kringle 1-3 region of the plasminogen part complex.
• Labeling of labeled anti-gene-antibody conjugates is achieved using ABTS, a chromogenic substrate for peroxidase.
• -ELISA reader for 405 nm and 492 nm wavelength (e.g. Anthos-Reader 2001, Zinsser, Austria).
• BSA Serum Albium Bovine, purest
• PBS Phosphate buffered saline
• Dilution buffer 3 (DB3): DB1 + 10% PAP depleted plasma
• Substrate buffer 1.29 citric acid monohydrate, 1.375 g Na2HP ⁇ 4-2H2 ⁇ with distilled water to 100 ml, pH 4.0
• Stop solution 320 mg NaF / 100 ml distilled water; 100 ul / well
• Coating buffers and coated plates are mixed with an antimicrobial substance and therefore stable at 4 ° C.
• Other protein-containing solutions and wash buffers should be freshly made or kept under sterile conditions to prevent microbial growth.
• the wells of an ELISA plate are filled with 100 ⁇ l / well coating solution - ideally using a multi-channel micropipette.
• the plate is then covered with a self-adhesive plastic film and stored at 4 ° C for at least 16 hours or for any length of time.
• the coated plate is emptied and mixed with 100 ⁇ l / well block solution and incubated for 1 h at 37 ° C. in order to block excess reactive groups on the plate surface.
• the plates remain covered with a plastic film for all incubation steps.
• Normal citrate or EDTA plasma can be used, but it should be noted that uninhibited plasmogenogen activators lead to an in vitro formation of PAP complexes. Especially when checking the sale of a thrombolytic therapy, inhibitors should always be added to the blood samples when they are taken (e.g. 2000 KlU / ml aprotinin + 20 M benzamidine final concentration).
• the plasma samples are diluted 1:10 in DB1 for low concentrations and 1: 100 for high concentrations of PAP complexes. 100 ⁇ l / well are required and double determinations are recommended.
• PAP Complex Standard Plasma is dissolved in distilled water and serial dilutions from 1: 100 to 1: 1800 or a zero value are made in DB3 for use with samples with low PAP concentrations and in DB2 for Use for samples with high PAP concentrations.
• 100 ⁇ l / well and double determinations are required.
• the plate is washed three times with approx. 300 ⁇ / well washing buffer, either manually or using an automatic washing device. After each wash, the plate is tapped on an absorbent paper.
• the complexes thus formed are either frozen at -70 ° C or lyophilized at 4 ° C.
• the ELISA measured values of this standard were compared with those of the PAP complexes from the purified components.
• plasmin was freshly prepared from purified glu-plasminogen by incubation with urokinase-Sepharose and, after removal of urokinase-Sepharose, the resulting activity was immediately purified with S-2251 and after 30 min incubation at 37 ° C. with purified ⁇ 2-antiplasmin determined. The differences in the plasmin activity correspond to the amount of PAP complexes formed (FIG. 2).
• the test described is specific for plasmin- ⁇ 2-antiplasmin complexes and gives good plasma recovery, provided the standard was dissolved in PAP-depleted plasma, in the same concentration as the plasma samples to be tested.
• the detection limit is 10 ng / ml in the purified system as well as in plasma.
• the PAP averages are 152 ⁇ 72 ng / ml for citrate and 132 ⁇ 72 ng / ml for EDTA plasma; PAP values are usually elevated in serum samples.
• Thrombolytic therapy gives PAP plasma values of> 800 ng / ml.
• PAP values were still elevated after 12 months (FIG. 3).
• cytopenic purpura2 2.1 ⁇ g / ml
• Standardization is therefore achieved by comparing the PAP complex standard plasma used, in which stable Complexes have formed with PAP complexes that were immediately freshly prepared from purified components.
• Anti-neoantigen monoclonal as binding antibody
• Antiplasminogen monoclonal as detection antibody 152 ⁇ 72 ng / ml> 1 ⁇ g / ml
• This system corresponds to a PAP kit that is commercially available from Teijin.
• Fig. 1 Western blot of 3 monoclonal antibodies which are directed against ⁇ 2-antiplasmin, a component of the PAP complex.
• Antibody ⁇ # 3 ⁇ is the antibody - MPW7AP.
• Fig. 2 Comparison of PAP complexes created in plasma with PAP complexes from purified components.
• the insert shows amidolytic activity of freshly produced plasmin incubated with buffer or an excess of ⁇ 2-antiplasmin. The concentration of complexes formed is calculated from the difference between the two activities, correspondingly inhibited plasmin.
• FIG. 3 PAP complexes in control persons and patients with coronary heart disease.
• Fig. 4 Time course of the PAP complex in the serum of a reactive person.
• Fig. 5 Time course of the generation of PAP complexes in the serum (0-4 hours) before (o), 1 week (•), 2 weeks ( ⁇ 4), after aspirin administration and 1 week after the end of aspirin administration ( ⁇ ) .

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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• 1992
  • 1992-04-14 AT AT0077292A patent/AT398004B/de not_active IP Right Cessation
• 1993
  • 1993-04-14 AU AU38176/93A patent/AU3817693A/en not_active Abandoned
  • 1993-04-14 US US08/157,210 patent/US5587291A/en not_active Expired - Lifetime
  • 1993-04-14 WO PCT/AT1993/000065 patent/WO1993021532A1/de not_active Application Discontinuation
  • 1993-04-14 EP EP93907638A patent/EP0589011A1/de not_active Withdrawn
  • 1993-04-14 JP JP5517841A patent/JPH06508219A/ja active Pending

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