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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • 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
• • 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
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

• the present invention relates to rat anti-PTX3 monoclonal antibodies, hybridomas for producing said antibodies, an improved method for determining the protein PTX3 in a biological fluid and a kit for performing said determination. More particularly, said method, said rat anti-PTX3 monoclonal antibodies and said kit are useful for the early diagnosis of the risk of death in human individuals suffering from cardiovascular and/or cerebrovascular diseases.
• the pentraxins are a group of proteins which include C-reactive protein (CRP) and serum amyloid P (SAP), produced by the liver in response to inflammatory mediators. The levels thereof in the serum increase in response to various stimuli and have been used for monitoring infections, inflammatory conditions and tissue damage.
• CRP C-reactive protein
• SAP serum amyloid P
• PTX3 is a new member of this family which was found in endothelial cells stimulated by interleukin-1 (IL-1).
• IL-1 interleukin-1
• PTX3 a typical long-chain pentraxin, is characterized by a C-terminal region of 203 amino acids which displays homology with the classical pentraxins and by an N-terminal region of 178 amino acids devoid of homology.
• PTX3 is produced in various types of cell, principally in endothelial cells and in mononuclear phagocytes, in response to IL-1 and to tumour necrosis factor (TNF), but not to interleukin-6 (IL-6), Further, PTX3 is produced by monocytes in response to components of mycobacterial cell walls and by unstimulated synoviocytes in patients with rheumatoid arthritis.
• the protein PTX3 was identified as far back as 1992 (Breviario F. et al. "Cloning of a new gene related to C-reactive protein and serum amyloid P component" J. Biol. Chem. 1992, 267: 22190-22197).
• PTX3 protein in infarcted patients are indicative of the risk of death in the three months following the episode (Latini R. et al., "Prognostic significance of the long pentraxin PTX3 in acute myocardial infarction: comparison with C-reactive protein, NT-proBNP and troponin T.” abstract 3091 , Supplement IV, page 680, Circulation 2003, 108 (17),). More particularly, the authors reported that in a representative number of patients with myocardial infarction with elevation of the ST segment the levels of PTX3 protein in the acute phase provide independent information predictive of the risk of death.
• 96-well ELISA plates (Nunc Roskilde, Denmark) were coated with 100 ⁇ l of rat monoclonal antibody NMB4 (as ascites, diluted 1 :5000 in buffer used for the coating) and incubated for one night at 4°C; b) the plates were then thoroughly washed with a Dulbecco phosphate buffer saline containing 0.05% Tween 20 (washing buffer) and 200 ⁇ l of 5% milk powder to block non-specific binding sites; c) after incubation for 2 hours at ambient temperature, the plates were again washed 3 times with washing buffer; d) 50 ⁇ l of standard recombinant human PTX3 (from 100 pg/ml to 10 ng/ml) diluted in RPMI 1640 medium (Seromed, Berlin, Germany) and 2% bovine serum albumin (Sigma Chemicals, St.
• a first disadvantage of the known method consists in the fact that, in the case of the plasma of some patients, the levels of PTX3 determined at different dilutions of the test plasma sample are not proportional to the dilution performed. The inventors have now found that this disadvantage is surprisingly overcome by adding EDTA to the test plasma sample.
• a second disadvantage consists in the fact that the sensitivity of the known method (ca. 200 pg/ml) is not sufficient for determining PTX3 protein in about 5% of normal subjects.
• step a novel monoclonal antibodies
• step g changing the concentration of streptavidin-peroxidase
• step h using a different chromogen
• the invention thus relates to a method for determining the level of PTX3 protein in samples of a biological fluid comprising the following steps: i) 96-well ELISA plates are coated with 100 ⁇ l of a solution containing a rat monoclonal antibody and incubated for one night at 4°C; ii) the plates are then washed with a buffer solution and a solution capable of blocking the non-specific binding sites; iii) after incubation for 2 hours at ambient temperature, the plates are again washed with washing buffer; iv) in duplicate, 50 ⁇ l of standard recombinant human PTX3 diluted in a suitable medium or samples of the biological fluid under test are placed in each well and the plates are incubated for 2 hours at 37°C; v) the plates are washed repeatedly with washing buffer and 100 ⁇ l of 25 ng/ml biotinylated anti-PTX3 rabbit IgG in washing buffer are then added to each well; vi) the plates are incuba
• the rat monoclonal antibody used in step (i) is the antibody obtained from the hybridoma MNB10 (access No. ABC/PD04001) or from the hybridoma Pen-3 (access No. ABC/PD01004);
• the streptavidin-peroxidase used in step (vii) is diluted 1 :8000;
• the chromogenic substrate used in step (viii) is tetramethylbenzidine (TMB).
• TMB tetramethylbenzidine
• the concentration of rat monoclonal antibody in the solution is about 700 ng/ml.
• the solution used in step (i) advantageously consists of a coating buffer solution.
• the said buffer solution contains 15 mM of carbonate buffer and its pH is 9.6.
• the buffer solution used in step (ii) consists of PBS (phosphate buffer saline) + 0.05% of Tween 20.
• the said solution is used in the amount of about 300 ⁇ l/well.
• the solution capable of blocking non-specific binding sites used in step (ii) consists of a 5% solution of milk powder in a buffer solution consisting of PBS + 0.05% of Tween 20.
• the said solution capable of blocking non-specific binding sites is used in the amount of about 300 ⁇ l/well.
• the washing specified in step (iii) is preferably repeated 3 times, each time using about 300 ⁇ l of solution for each well.
• the standard recombinant human PTX3 used is placed in the wells in quantities increasing from 75 pg/ml to 1.2 ng.
• the medium used to dilute the test plasma samples consists of PBS + 2% of BSA (bovine serum albumin) + 0.18% of K 3 - EDTA.
• BSA bovine serum albumin
• K 3 - EDTA K 3 - EDTA.
• the presence of EDTA in this solution is very important since, as already stated, the inventors have found that this makes it possible to obtain PTX3 measurements proportional to the dilution performed.
• the biotinylated anti-TPX3 rabbit IgG used in step (v) is preferably obtained according Muller B. et al., loc. cit.
• the streptavidin-peroxidase used in step (vii) is preferably the horseradish peroxidase-conjugated streptavidin Amdex (RPN 4401 Amersham, Copenhagen, Denmark). .
• the stop solution used in step (ix) is a 1 M solution of H 2 SO 4 .
• the TMB substrate solution used in step (xii) corresponds to the catalogue number 2642KK of the firm Pharmingen.
• the present invention relates to a hybridoma capable of producing an anti-PTX3 rat monoclonal antibody characterized in that the said hybridoma is selected from the group comprising MNB10 [deposited according to the Budapest Treaty at the Advanced Biotechnology Centre (ABC) of Genoa, Italy on 16.04.04, access No. PD04001] and Pen-3 [deposited according to the Budapest Treaty at the Advanced Biotechnology Centre (ABC) of Genoa, Italy on the date 02.08.2001 , access No. PD01004].
• the selection of the aforesaid hybridomas which produce anti-PTX3 rat monoclonal antibodies according to the invention can be carried out by conventional methods such as those for example described in Example 1 below.
• the present invention relates to a specific anti- PTX3 rat monoclonal antibody selected from the group comprising the monoclonal antibodies produced by the aforesaid hybridomas MNB10 and Pen-3.
• the preparation of the specific anti-PTX3 rat monoclonal antibodies from the hybridomas of the present invention is not subject to particular restrictions and can be carried out by conventional methods such as those for example described in Example 2 below.
• the present invention relates to a kit for the determination of the level of PTX3 protein in biological fluids, characterized in that it includes an anti-PTX3 rat monoclonal antibody.
• the determination of the level of PTX3 protein is carried out on the serum of a human individual suffering from a cardiovascular and/or cerebrovascular disease for early diagnosis of their risk of death.
• the aforesaid kit also includes an anti-PTX3 rabbit polyclonal antibody.
• the aforesaid kit also includes purified recombinant
• the aforesaid kit also includes streptavidin conjugated with horseradish peroxidase.
• the aforesaid kit also includes a washing buffer solution.
• the aforesaid kit also includes a diluent for the samples of biological fluid to be assayed.
• the aforesaid kit also includes, as chromogen, a solution of tetramethylbenzidine (TMB).
• TMB tetramethylbenzidine
• a stop solution can also be included in the aforesaid kit.
• Fig.1 below and the examples that follow serve further to illustrate the invention, without however limiting it.
• Fig.1 is a diagram which illustrates the effect of EDTA on the plasma of a patient with cardiac decompensation.
• the dilutions of whole plasma (1 :2, 1 :4, 1 :8 and 1 :16) are shown on the x-axis.
• the optical density is shown on the y-axis.
• the levels of PTX3 protein detectable in the serum of a patient with cardiac decompensation in the whole plasma dilution interval between about 1 :4 (0.25) and about 1 :16 (0.0625) are, in the absence of EDTA, about three times lower than the levels of protein actually present.
• the addition of EDTA makes it possible to obtain levels that essentially correspond to those of the standard curve.
• the spleen of the previously immunised rat is removed under sterile conditions and washed 3 times with 10 ml of DMEM, transferred into a plastic Petri dish containing 3 ml of DMEM medium and disaggregated by means of needles and/or crushed with a syringe piston;
• the cell suspension is transferred to a test tube, made up to 50 ml with DMEM medium and filtered with a 70 ⁇ m screen so as to remove cell aggregates;
• the myeloma must be in exponential growth phase and not plateau. - wash the cells twice with 50 ml of DMEM medium.
• HT-DMEM medium can be replaced with DMEM for hybridomas.
• - Sepharose Protein G 4 fast flow (Pharmacia cat. 17-0618-01): wash the resin 4 times with PBS by decantation or light centrifugations, then dilute it to 10% with PBS + 0.1 % of sodium azide and store it in the refrigerator;
• - glycine - 0.1 M HCI buffer pH 2.8 dissolve 750 mg of glycine in 100 ml of distilled water and adjust to pH 2.8 with 280 ⁇ l of 37% HCI;
• TRIS-HCl buffer pH 8.8 dissolve 18.17 g of TRIS in 100 ml of distilled water and adjust to pH 8.8 with 37% HCI;
• 96-well ELISA plates (Nunc MaxiSorp 446612) were coated with 100 ⁇ l of a coating buffer solution (15 mM carbonate buffer, pH 9.6) containing purified MNB10 antibody (700 ng/ml) and incubated for one night at 4°C;
• a kit for determining the levels of PTX3 in human biological fluids comprises: 1. Microplate: 12 x 8 wells coated with rat anti-PTX3 antibody MNB10. 2. Biotinylated rabbit anti-PTX3 IgG in phosphate buffer solution. 3. Horseradish peroxidase-conjugated streptavidin in phosphate buffer solution. 4. Standards: purified recombinant PTX3 at 2.4, 1 , 0.5, 0.2 and 0.05 ng/ml in buffer solution. 5. Washing buffer solution: phosphate buffer saline (PBS) solution. 6. Diluent (to dilute the human biological fluid under test): 1% bovine serum albumin and 0.19% K3-EDTA in phosphate buffer saline solution.
• PBS phosphate buffer saline

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Cell Biology (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (7)

Applications Claiming Priority (2)

Publications (2)

Family

ID=35242298

Family Applications (1)

Country Status (9)

Cited By (3)

Families Citing this family (8)

Family Cites Families (8)

• 2004
  • 2004-04-29 IT IT000858A patent/ITMI20040858A1/it unknown
• 2005
  • 2005-04-27 WO PCT/EP2005/004637 patent/WO2005106494A2/en not_active Ceased
  • 2005-04-27 CA CA002562895A patent/CA2562895A1/en not_active Abandoned
  • 2005-04-27 US US11/587,528 patent/US7915004B2/en not_active Expired - Fee Related
  • 2005-04-27 KR KR1020067024272A patent/KR101168292B1/ko not_active Expired - Fee Related
  • 2005-04-27 CN CN2005800134880A patent/CN1947018B/zh not_active Expired - Fee Related
  • 2005-04-27 AU AU2005238637A patent/AU2005238637B2/en not_active Ceased
  • 2005-04-27 JP JP2007509981A patent/JP2007534951A/ja active Pending
  • 2005-04-27 EP EP05742234A patent/EP1740956A2/en not_active Withdrawn
• 2011
  • 2011-02-17 JP JP2011032661A patent/JP2011117973A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events