WO2007138163A2 - Antibodies and standard for nt-probnp and probnp immunoassays - Google Patents

Antibodies and standard for nt-probnp and probnp immunoassays Download PDF

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WO2007138163A2
WO2007138163A2 PCT/FI2007/050298 FI2007050298W WO2007138163A2 WO 2007138163 A2 WO2007138163 A2 WO 2007138163A2 FI 2007050298 W FI2007050298 W FI 2007050298W WO 2007138163 A2 WO2007138163 A2 WO 2007138163A2
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probnp
antibody
fragment
endogenous
glycosylated
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PCT/FI2007/050298
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French (fr)
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Alexei G. Katrukha
Karina R. Seferyan
Alexander G. Semenov
Natalia N. Tamm
Vladimir L. Filatov
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Hytest Ltd.
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Priority to JP2009511539A priority Critical patent/JP2009538288A/en
Priority to EP07730785A priority patent/EP2021369A2/en
Publication of WO2007138163A2 publication Critical patent/WO2007138163A2/en
Priority to US12/275,324 priority patent/US20090163415A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the present invention provides antibodies to newly discovered forms of proBNP and NT- proBNP. Particular epitopes in these proteins are provided as well. Antibodies specific to these particular epitopes are suitable for precise immunodetection, i.e. determination of the presence and/or quantification of the amount, of both of the proteins in human blood. The novel forms of the proteins are suggested to be utilized as antigens for antibody generation as well as calibrators or immunological standards in different types of immunoassays.
  • proBNP brain natriuretic peptide
  • NT-proBNP N-terminal fragment of proBNP
  • HF heart failure
  • NT-proBNP N-terminal fragment of proBNP
  • BNP belongs to a family of structurally similar peptide hormones. This family includes also atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and urodilatin. BNP and ANP share a wide spectrum of biological properties and both of them are of myocardial origin, while CNP is of endothelial origin. These peptides are characterized by a 17 amino acid ring structure with a disulfide bond between two cystein residues. The ring structure shows high identity level between different natriuretic peptides (11 out of 17 amino acid residues (aar) are identical for all group representatives). BNP molecule is composed of 32 amino acid residues with a disulfide bond located between the residues
  • NT-proBNP and BNP are the products of proteolytic processing of the precursor molecule preproBNP.
  • PreproBNP is composed of 134 aar and is synthesized in cardiac myocytes. Removal of signal peptide (aar 1-26) results in the appearance of proBNP molecule (aar 27-134). Subsequently, proBNP (108 aar) is cleaved by unknown protease forming two peptides - BNP (aar 77-108) and NT-proBNP (aar 1-76). Both BNP (biologically active molecule) and NT-proBNP (physiological activity is not clarified) as well as unprocessed proBNP are secreted into the bloodstream and circulate in human blood.
  • BNP and NT-proBNP measurements in human blood are widely used for evaluation of patients with suspected HF and assessment of severity of the disease.
  • Immunoassays utilize monoclonal as well as polyclonal antibodies, specific to different parts of the human NT-proBNP molecule: epitope 1-13 (4), 65-76 (5), 1-12 and 65-76 (6), 8-29 (Biomedica assay), 1-21 and 39-50 (Roche Elecsys). Recently, new proBNP immunoassay utilizing antibodies specific to the cleavage site of proBNP and BNP-specific antibodies was described (7).
  • ProBNP and NT-proBNP, circulating in human blood are described in literature as polypeptides consisting of 108 and 76 amino acid residues, respectively (molecular masses about 12 and 8.6 kDa).
  • the concentration of proBNP in patient's blood is significantly lower than the concentration of NT-proBNP. According to our recent studies the concentration of proBNP in blood is about 10-20% of that of NT-proBNP.
  • Seidler et al. (8) demonstrated that in gel filtration studies (non-denaturing conditions) NT-proBNP and proBNP have anomalous mobility and their apparent molecular weights are about 30-40 kDa.
  • one aspect of the invention is drawn to an antibody that specifically recognizes an endogenous glycosylated NT-proBNP or proBNP, or a fragment thereof, and which does not recognize a deglycosylated NT-proBNP or proBNP or a fragment thereof, or fragments of such antibodies.
  • the invention provides an antibody that recognizes an endogenous glycosylated NT-proBNP or proBNP, or a fragment thereof, with higher affinity than the antibody or fragment recognizes a corresponding deglycosylated protein, or fragment thereof, of such antibodies.
  • a related aspect of the invention provides an aptamer having the same specificity as an antibody described above.
  • antibodies include an antibody that is a monoclonal antibody or fragment of a monoclonal antibody, as well as an antibody that is a polyclonal antibody or fragment of a polyclonal antibody. Also, the antibody may be a recombinant antibody or a fragment of a recombinant antibody.
  • Another aspect according to the invention provides for the use of an antibody or antibody fragment as described above in a diagnostic immunoassay method for qualitative or quantitative detection of NT-proBNP or proBNP or a fragment thereof.
  • a diagnostic method for assaying NT-proBNP or proBNP or a fragment thereof in a sample of a patient comprising quantitative or semiquantitative determination of the NT-proBNP or proBNP content of the sample using an antibody or an aptamer, each as described herein.
  • the diagnostic method may further comprise preparing a calibration curve using as the standard a preparation of an endogenous glycosylated NT-proBNP or proBNP isolated from vertebrate blood.
  • the diagnostic method may further comprise preparing a standard curve and comparing the value of the NT-proBNP or proBNP content determined to the standard curve.
  • Exemplary diagnostic methods according to the invention are immunoassay methods.
  • diagnostic immunoassay is a sandwich immunoassay method, using a first capture antibody and a second detection antibody.
  • Another aspect of the invention is directed to a diagnostic method for assaying NT- proBNP or proBNP in a sample of a patient, comprising (a) deglycosylating endogenous NT-proBNP or proBNP contained in the sample; and (b) determining the NT-proBNP or proBNP content of the sample using an antibody or an aptamer specific to NT-proBNP or proBNP.
  • Yet another aspect is an NT-proBNP or proBNP standard or calibration preparation com- prising a glycosylated NT-proBNP or proBNP or a fragment thereof.
  • the glycosylated NT-proBNP or proBNP or a fragment thereof is an endogenous NT-proBNP or proBNP; in some embodiments, the NT-proBNP or proBNP is isolated from vertebrate blood.
  • a related aspect of the invention provides an isolated glycosylated recombinant NT-proBNP or proBNP or a fragment thereof.
  • the glycosylated recombinant NT-proBNP or proBNP, or a fragment thereof is glycosylated in vitro.
  • the glycosylated recombinant NT-proBNP or proBNP, or a fragment thereof is produced in a cell culture or in a cell-free translation system.
  • Yet another aspect of the invention is a use of a molecule as described above, as an antigen for producing an antibody having the same specificity as an antibody described above.
  • the invention provides an immunoassay kit for diagnostic assay of NT- proBNP or proBNP or a fragment thereof in a sample of a patient, the kit comprising (a) a monoclonal or polyclonal antibody having the same specificity as an antibody described hereinabove; (b) a detectable label; and (c) a standard or calibrator preparation as described herein.
  • An exemplary kit is an immunoassay kit for diagnostic assay of NT-proBNP or proBNP or a fragment thereof in a sample of a patient, the kit comprising (a) a first monoclonal capture antibody of the same specificity as an antibody described above; (b) a second monoclonal detection antibody of the same specificity as an antibody of claim 1 or 2, wherein the detection antibody is fluorescently labeled; and (c) a standard or calibration preparation according to claim 14.
  • fragments of proteins, antibodies or any other entities as referred to in this specifica- tion mean any structural and/or functional fragments of said entities, retaining the desired activity.
  • Sandwich immunoassay After deglycosylation, monoclonal antibodies specific to the central region of NT-proBNP molecule (antibodies that do not recognize native endogenous antigen) were able to recognize endogenous protein with the same efficiency as the recombinant (non-glycosylated) form (Fig. 6).
  • NT-proBNP and proBNP do not exist as simple polypeptide chains, as was considered before, but as glycoproteins.
  • mono- or polyclonal antibodies for assays designed to detect one or both of the molecules in human blood should either recognize those parts of the molecules that are not affected by glycosylation, or they should recognize the glycosylated part of the molecule.
  • Glycosylated forms of both of the proteins should be used for the preparation of standards and calibrators for such assays.
  • Glycosylated forms of the antigens should be used for animal immunization to obtain antibodies specific to the glycosylated part of the molecule.
  • Fig. 1 illustrates epitope map of NT-proBNP-specific monoclonal antibodies used in the study.
  • Fig. 2 illustrates specificities of different MAbs to endogenous NT-proBNP (antigen from HF patients' plasma).
  • the results are presented as a ratio of signals in plasma to signals with recombinant NT-proBNP standard preparation in different two-site MAbs combinations.
  • One MAb in such combination was able to interact effectively with endogenous NT-proBNP (MAb 24El 1, epitope 67-76 or MAb 13G12, epitope 13-20), whereas another one was out of the set of antibodies, specific to different regions of NT- proBNP molecule.
  • the concentration of recombinant NT-proBNP was the same as that of the endogenous antigen, determined in HF patients' plasma by 15C4-13G12 assay.
  • Fig. 3 shows the calibration curve for the assay 15C4-13G12 and dilution curves for two individual plasma samples.
  • Recombinant NT-proBNP expressed in E. coli, HyTest
  • reconstituted in pooled normal human plasma was used as a calibrator.
  • Fig. 4 illustrates gel filtration studies (Superdex 75 10/300 GL column) of four plasma samples from HF patients.
  • NT-proBNP immunoreactivity in the fractions was quantified by sandwich immunoassay utilizing monoclonal antibodies 15C4 (epitope 63-71) and 13Gl 2 (epitope 13-20) recognizing endogenous NT-proBNP.
  • Fig. 5 illustrates Western blotting studies of affinity-purified endogenous NT-proBNP.
  • Tracks 1, 4 recombinant NT-proBNP (50 ng/track), tracks 2, 5: recombinant proBNP (50 ng/track), tracks 3, 6: endogenous NT-proBNP purified from human plasma (200 ng/track).
  • MAbs 15Fl 1 epipitope 13-24
  • MAb HDl epitopope 31-39
  • Fig. 6 illustrates the specificities of different monoclonal antibodies to endogenous NT- proBNP (black columns) or to endogenous NT-proBNP after deglycosylation - treatment with O-glycosidase and sialidase - (grey columns). The results are represented as ratio of signals (endogenous/recombinant) in different two-site MAb combinations.
  • Three forms of NT-proBNP a) recombinant (non-glycosylated), b) endogenous, extracted from HF human plasma and c) endogenous, extracted from plasma and treated with enzymes in same concentrations were tested by sandwich immunoassays utilizing different monoclonal antibodies.
  • MAbs 24El 1, epitope 67-76 or MAb 13G12, epitope 13-20 One MAb in such immunoassay was specific to the epitope that is not affected by glycosylation (MAbs 24El 1, epitope 67-76 or MAb 13G12, epitope 13-20), whereas the other one was one out of the set of antibodies specific to different regions of NT- proBNP molecule.
  • Fig. 7 illustrates gel filtration studies (Superdex 75 10/300 GL column) of endogenous NT- proBNP extracted from plasma (uniform line) and endogenous NT-proBNP treated with O- glycosidase and sialidase (dotted line).
  • NT-proBNP immunoreactivities in the fractions were quantified by two sandwich immunoassays 15C4-13G12 (A) and 11D1-13G12 ( ⁇ ).
  • Immunoassay 15C4-13G12 is not sensitive to glycosylation.
  • Immunoassay 11D1-13G12 is sensitive to glycosylation.
  • Fig. 8 illustrates the Western blotting studies of affinity-purified NT-proBNP before and after deglycosylation.
  • Tracks 1, 5 recombinant NT-proBNP (E. coli, 50 ng per track); tracks 2, 6: recombinant proBNP (E. coli, 50 ng per track); tracks 3, 7: affinity-purified endogenous NT-proBNP (200 ng per track); tracks 4, 8: affinity-purified endogenous NT- proBNP after deglycosylation (200 ng per track).
  • MAb 15Fl 1 (epitope 13-24) - tracks 1-4 or MAb 1 IDl (epitope 31-39) - tracks 5-8 were used for the antigen immunostaining.
  • Example 1 Preparation and characterization of monoclonal antibodies (MAbs), specific for human NT-proBNP molecule
  • Synthetic peptides corresponding to sequences 1-24, 13-27, 28-45, 46-60 and 61-76 of human NT-proBNP molecule were conjugated to the bovine serum albumin (BSA) and were used for immunization of mice. Conjugation of small peptides with the carrier protein molecule allowed enhancing the immune response of the animals.
  • BSA bovine serum albumin
  • mice Female Balb/c mice, aged between 6-12 weeks were used for immunization.
  • hybridoma cell lines producing monoclonal antibodies (MAbs), specific to NT- proBNP molecule were obtained after hybridization of mouse spleen cells with myeloma SP2/0 cells.
  • Culture supernatants were tested for reactivity to the whole recombinant NT-proBNP molecule (expressed in E. coli) and eighty-four positive cultures were selected for further work. Among those, 14 produced antibodies specific to region 1-24, 24 to region 13-27, 19 to region 28-45, 13 to region 46-60 and 15 to region 61-76. Selected cultures were subcloned twice by limiting dilution, expanded and frozen. The ascitic fluid containing monoclonal antibodies was produced in Balb/C mice.
  • the antibodies were isolated from the ascitic fluid by Protein-A Sepharose (GE Healthcare) affinity chromatography.
  • the isotypes of the purified antibodies were determined by Monoclonal Antibody Isotyping Kit (Pierce). All MAbs were specified as IgG.
  • Precise epitope mapping of all newly generated antibodies was performed using a library of synthetic peptides 1-12, 5-20, 1-24, 13-27, 28-45, 31-39, 34-42, 37-45, 48-56, 50- 58, 52-60, 46-60, 63-71, 65-73, 67-76 and 61-76, containing overlapping sequences.
  • Synthetic peptides were conjugated with a carrier protein (ovalbumine).
  • the plates were coated with peptide conjugates in concentration of 1 ⁇ g/ml (100 ⁇ l per well). After washing, monoclonal antibodies, reconstituted in PBST, were added into the wells.
  • sandwich- type immunofluoroassays were established for the quantification of NT-proBNP in human blood.
  • Such assay is based on the binding of the antigen to the monoclonal antibody adsorbed on the plate surface thus forming first order immune complex, and on the detection of the first order immune complex by another monoclonal antibody labeled with stable europium (III) chelate.
  • Antibodies were preliminarily transferred into 0.9% water solution of NaCl using gel filtration on Sephadex G25 columns (NAP-5).
  • Antibody labeling with stable europium (III) chelate of 2,2',2",2'";-[[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]bis(methyl- enenitrilo)]tetrakis(acetic acid) was conducted by incubation overnight at +4°C in 50 mmol/L Na-carbonate buffer pH 9.8 containing 200-fold molar excess of europium (III) chelate.
  • Labeled antibodies were separated from the unreacted chelate by gel filtration on Sephadex G25 columns (NAP-5) in a buffer containing 0.01mol/L of Tris-HCl pH 7.8, 0.15 mol/L ofNaCl and 0.1% NaN 3 .
  • Example 5 Patients and blood samples
  • Diagnosis of patients with HF was based on symptoms: dyspnea, orthopnea, lung rales and leg edema, and confirmed by echocardiography studies and X-ray examination. The preliminary diagnosis was made by cardiologist and further confirmed by HF expert. Blood samples were collected from patients with left ventricular ejection fractions less than 30% and left ventricle end-systolic volume more than 90 mL. Venous blood was collected into EDTA-containing Vacuette tubes (Greiner Bio-One) and centrifuged at 3000 g (15 minutes, +4°C). Serum samples were obtained from blood collected in plastic tubes, incubated for 30 min at room temperature, and centrifuged at 500Og (30 min, +20 0 C).
  • Plasma and serum samples were stored at -70 0 C prior to use.
  • pooled serum 39 patients with severe HF
  • pooled plasma (10 HF patients) was used as a source of endogenous antigens.
  • a negative (non-HF) control pooled serum or plasma from 10 healthy donors was used.
  • All generated MAbs were tested in two-site MAb combinations (capture and detection) with recombinant NT-proBNP and with pooled serum or plasma from HF patients as a source of endogenous antigens.
  • the capture antibodies in concentration of 10 ⁇ g/ml were placed into EIA plates (100 ⁇ l per well) and incubated in a phosphate saline buffer for 30 minutes at room temperature and gentle shaking.
  • the mixture of antigen (recombinant NT-proBNP, reconstituted in pooled normal human plasma or endogenous antigen from HF plasma or serum, 50 ⁇ l) and detection antibodies (4 ⁇ g/ml, 50 ⁇ l), dissolved in buffer containing 0.05 mol/L Tris-HCl pH 7.7, 0.9% NaCl, 0.01% Tween 20, 0.5% BSA and 0.05% NaN 3 (buffer B) were added to the wells.
  • the plates were incubated for 30 minutes at room temperature and shaking gently, and washed six times with buffer A.
  • the enhancing solution (1.75 mol/L NaSCN, 1 mol/L NaCl, 50 ml/L glycerol, 200 ml/L 1-propanol, 0.005 mol/L Na 2 CO 3 , 0.05 mol/L glycine-NaOH, pH 10.0)
  • the mixture was incubated for 3 minutes at the same conditions.
  • the fluorescence was measured on a Victor 1420 Multilabel Counter.
  • the assay 15C463-7i-13G12i3.2o was selected and used in further studies because it was able to recognize recombinant and native antigens with the same efficiency.
  • Human recombinant NT-proBNP expressed in E. coli was reconstituted in normal human plasma and was used as a calibrator for sandwich IFA.
  • the detection limit was defined as a concentration (measured 20 times in a single run) producing a signal 2 SD above the mean for a calibrator that is free of analyte.
  • Typical calibration curve for recombinant NT- proBNP and serial dilutions of human plasma samples are shown in Fig. 3.
  • the detection limit was 10 ng/L
  • immunoassay was linear in the range of 15 - 100 000 ng/L.
  • the column was calibrated using a set of standard proteins (GE Healthcare): albumin (Mr 67000 Da), ovalbumin (Mr 43000 Da), chymotrypsinogen (Mr 25000 Da), ribonuclease A (Mr 13700 Da) and aprotinin (Mr 6517.5 Da, from Sigma).
  • Recombinant NT-proBNP was reconstituted in pooled plasma from healthy donors before loading onto the Superdex 75 column.
  • Example 10 Western blotting studies of endogenous NT-proBNP
  • NT-proBNP was purified from pooled HF patients' plasma by means of affinity chromatography.
  • affinity matrix a mixture of antibodies, specific to different regions of NT-proBNP molecule (15C4, 24El 1, 18H5, 15Fl 1), was immobilized on the BrCN- activated Sepharose CL-4B (GE Healthcare) according to the standard protocol.
  • CL-4B BrCN- activated Sepharose CL-4B
  • Affinity matrix with immobilized antibodies was washed with 0.1 mol/L glycine, pH 2.0 and then equilibrated with 0.02 mol/L Tris-HCl, pH 7.5, containing 0.15 mol/L of NaCl. Pooled plasma of HF patients was loaded onto anti-NT-proBNP-Sepharose with the flow rate of 1 ml/min at +4°C. Peptides were eluted with a water solution containing 0.1 mol/L of HCl. The eluate was neutralized by 2 mol/L of Tris-HCl. Recovery after affinity chromatography was 88%.
  • the eluate was then loaded onto Sepharose CL-4B with immobilized MAbs that do not interact with NT-proBNP (negative chromatography) to remove proteins that bind nonspecifically to the affinity matrix.
  • NT-proBNP was concentrated by a second round of affinity chromatography.
  • the solution containing NT-proBNP was loaded onto anti-NT-proBNP-Sepharose (10-fold molar excess of antibodies regarding to the NT-proBNP concentration) with the flow rate of 1 ml/min at +4°C.
  • the peptides were eluted by 0.1 mol/L HCl, lyophilized, reconstituted in water and stored under -70 0 C before use.
  • the proBNP contamination of the NT-proBNP preparation was determined by proBNP - specific immunoassay and was found to be less than 9% from total amount of NT-proBNP.
  • NT-proBNP concentration was determined by 15C463-7i-13G12i3.2o immunoassay
  • 50 ng of recombinant NT-proBNP (proBNP) were loaded per track.
  • peptides were transferred onto nitrocellulose membrane (Trans-Blot® Transfer membrane, 0.2 ⁇ m, BioRad). Transfer was performed at constant voltage (100 V) and lasted for 40 minutes. Nonspecific binding was blocked by incubation of the membrane in the 10% solution of non-fat dry milk in PBST. Immunochemical staining of the peptides with NT-proBNP-specific MAbs, conjugated with horseradish peroxidase was performed during 12 hours at +4°C in the 10% solution of non-fat dry milk in PBST. Immune complexes were visualized by incubation in substrate solution, containing diaminobenzidine and nickel chloride.
  • Example 11 Testing of deglycosylated endogenous NT-proBNP in sandwich IFA
  • NT-proBNP extracted from pooled human plasma was treated by deglycosylation enzymes O-glycosidase (S. pneumoniae) and sialidase (A. ureafaciens) (QA-Bio, USA). Treatment was performed in a buffer containing 0.075 mol/L of sodium phosphate, pH 5.0 for 1 hour at +37°C. Water solution, containing 0.075 mol/L of sodium phosphate, pH 5.0 without enzymes was added to the studied peptides as a negative control.
  • O-glycosidase S. pneumoniae
  • sialidase A. ureafaciens
  • NT-proBNP glycosylated, and polysaccharide residues prevent antibodies from interacting with the endogenous antigen. Since antibodies specific to the central part of the NT-proBNP molecule are unable to recognize endogenous antigen, MAbs specific to the other regions not affected by glycosylation should be used in NT-proBNP and proBNP assays.
  • Example 12 Gel-filtration studies of endogenous NT-proBNP before and after deglycosylation
  • NT-proBNP treated and non-treated with the O-glycosidase and sialidase was studied by gel- filtration (GF) method.
  • NT-proBNP extracted from pooled HF patients' plasma, was treated with enzymes and the immunological activity was thereafter determined in fractions in two immunoassays 15C4-13G12 and 11D1-13G12. According to the data presented in Example 11, assay 15C4-13G12 is not sensitive to glycosylation, whereas 11D1-13G12 assay can recognize endogenous NT-proBNP only after removal of carbohydrate moieties.
  • the samples of a) endogenous NT-proBNP, extracted from pooled HF patients' plasma, b) endogenous NT-proBNP, extracted from pooled HF patients' plasma after deglycosylation, c) recombinant NT-proBNP and d) recombinant proBNP in the same concentrations (330 ng/ml) were reconstituted in 150 ⁇ l of 0.1 mol/L sodium phosphate, pH 7.4, containing 0.7 mol/L of NaCl, 0.005 mol/L of EDTA and 5 g/L of bovine serum albumin.
  • the samples were applied onto Superdex 75 10/300 GL gel filtration column equilibrated with 0.1 mol/L sodium phosphate, pH 7.4, containing 0.7 mol/L NaCl and 0.005 mol/L EDTA. Proteins were eluted at a flow rate of 0.7 ml/min and fractions with a volume of 0.5 ml were collected.
  • the NT-proBNP immunological activities in the fractions were measured by sandwich immunoassay 15C5-13G12, utilizing monoclonal antibodies not sensitive to glycosylation and 11D1-13G12 assay that does not interact with glycosylated endogenous NT-proBNP.
  • the column was calibrated using the set of standard proteins: albumin (Mr 67000 Da), ovalbumin (Mr 43000 Da), chymotrypsinogen (Mr 25000 Da), ribonuclease A (Mr 13700 Da) and aprotinin (Mr 6517.5 Da).
  • Recombinant NT-proBNP and proBNP were reconstituted in pooled plasma from healthy donors before loading onto the Superdex 75 column.
  • NT-proBNP Being measured in the 15C4-13G12 immunoassay endogenous NT-proBNP revealed two peaks of immunoreactivity (Fig. 7), the major one with molecular weight of about 28 kDa and the minor one with molecular weight of about 51 kDa. After deglycosylation we observed shift of both peaks of immunological activity towards the proteins with lower molecular masses. The major peak corresponded to the proteins with molecular masses of about 18 kDa and the minor peak to the proteins with molecular masses of about 51 kDa.
  • Example 13 Immunochemical staining of affinity-purified endogenous NT-proBNP (before and after deglycosylation) by NT-proBNP - specific monoclonal antibodies in Western blotting.
  • Electrophoresis and Western blotting procedure were performed in the same way as described in Example 10.
  • MAb 15Fl 1 epitopope 13-24 was used for the protein visualization in the sample before deglycosylation.
  • the major immunological activity was detected as a diffused zone in the area corresponding to the proteins with molecular masses of about 15 kDa and higher.
  • endogenous NT-proBNP was not stained by MAb HDl specific to the region aar 31-39 of NT-proBNP molecule.
  • MAb HDl specific to the region aar 31-39 of NT-proBNP molecule.
  • both of the antibodies were able to detect a peptide with apparent molecular mass about 13 kDa. This band was still a little above the recombinant NT-proBNP band, which could be explained by the fact that deglycosylation was not complete.
  • the endogenous protein cannot be detected in Western blotting experiments by antibodies specific to the central region of NT-proBNP molecule, whereas it becomes "visible" by such antibodies after deglycosylation.
  • NT-proBNP assay with a low detection limit. Scand. J. Clin. Lab. Invest. Suppl. 1999; 230:177-181.
  • proBNP and pro ANP oligomerise through leucine zipper- like coiled-coil motifs. Biochem. Biophys. Res. Commun. 1999; 255:495- 501. 9. Crimmins DL. Human N-terminal proBNP is a monomer. Clin. Chem. 2005; 51:1035-1038.

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Description

Antibodies and Standard for NT-proBNP and proBNP Immunoassays
Field of the Invention
The present invention provides antibodies to newly discovered forms of proBNP and NT- proBNP. Particular epitopes in these proteins are provided as well. Antibodies specific to these particular epitopes are suitable for precise immunodetection, i.e. determination of the presence and/or quantification of the amount, of both of the proteins in human blood. The novel forms of the proteins are suggested to be utilized as antigens for antibody generation as well as calibrators or immunological standards in different types of immunoassays.
Background of the Invention
The pro-form of brain natriuretic peptide (proBNP) as well as BNP and N-terminal fragment of proBNP (NT-proBNP) are recognized markers of heart failure (HF) and left ventricle dysfunction. They are also used for risk stratification in patients with various cardiac pathologies, and therapy monitoring in patients with HF. BNP is a peptide hormone with natriuretic, vasodilatory and renin inhibitory properties (reviewed in (1-3)).
BNP belongs to a family of structurally similar peptide hormones. This family includes also atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and urodilatin. BNP and ANP share a wide spectrum of biological properties and both of them are of myocardial origin, while CNP is of endothelial origin. These peptides are characterized by a 17 amino acid ring structure with a disulfide bond between two cystein residues. The ring structure shows high identity level between different natriuretic peptides (11 out of 17 amino acid residues (aar) are identical for all group representatives). BNP molecule is composed of 32 amino acid residues with a disulfide bond located between the residues
Cysio and CyS26.
NT-proBNP and BNP are the products of proteolytic processing of the precursor molecule preproBNP. PreproBNP is composed of 134 aar and is synthesized in cardiac myocytes. Removal of signal peptide (aar 1-26) results in the appearance of proBNP molecule (aar 27-134). Subsequently, proBNP (108 aar) is cleaved by unknown protease forming two peptides - BNP (aar 77-108) and NT-proBNP (aar 1-76). Both BNP (biologically active molecule) and NT-proBNP (physiological activity is not clarified) as well as unprocessed proBNP are secreted into the bloodstream and circulate in human blood.
It has been established that proBNP synthesis increases in response to mechanical or neurohormonal stimulation of the heart that leads to the increase of BNP and NT-proBNP concentrations in blood. Elevated levels of BNP and NT-proBNP are described for patients with different cardiovascular pathologies - heart failure, left ventricular dysfunction, unstable angina and myocardial infarction. Blood concentration of both analytes in HF patients correlates with severity of the disease. It has been reported that peptide concentrations are already elevated in asymptomatic patients during the very early stage of heart failure (NYHA I stage according to the New York Heart Association classification). Patients with symptoms of severe HF (NYHA class III or IV) and myocardial dysfunction show significantly increased values of BNP. Therefore, BNP and NT-proBNP measurements in human blood are widely used for evaluation of patients with suspected HF and assessment of severity of the disease.
Different immunoassay methods for NT-proBNP measurement in human plasma have been described in literature. Immunoassays utilize monoclonal as well as polyclonal antibodies, specific to different parts of the human NT-proBNP molecule: epitope 1-13 (4), 65-76 (5), 1-12 and 65-76 (6), 8-29 (Biomedica assay), 1-21 and 39-50 (Roche Elecsys). Recently, new proBNP immunoassay utilizing antibodies specific to the cleavage site of proBNP and BNP-specific antibodies was described (7).
There is still no consensus regarding antibodies which should be used in the assays. However, some published data demonstrate that antibodies specific to the central regions of the NT-proBNP molecule are not able to recognize the analyte in human blood. Thus, Hughes (5) demonstrated that rabbit polyclonal antibodies specific to aar 37-49 did not recognize NT-proBNP in patients' blood in contrast to antibodies specific to aar 65-76. The reason of the observed absence of signal was not clear because of the small amount of information available regarding biochemical properties of NT-proBNP circulating in human blood.
Gel filtration (GF) studies in non-denaturating conditions demonstrated that NT-proBNP and proBNP immunoreactivities are represented in fractions of proteins with apparent molecular masses of 3- to 4-fold higher than expected values. Seidler et al. (8) assumed that the differences observed in molecular masses during chromatography in different conditions are due to oligomerization of NT-proBNP and proBNP molecules in human blood. However, this hypothesis was rejected by Crimmins (9) who showed that synthetic NT-proBNP does not form oligomers in vitro. Consequently, so far the form in which NT- proBNP circulates in human blood has not been defined, and there is no explanation to the abnormalities as observed.
However, information about biochemical properties of NT-proBNP in human blood could significantly affect the current approach to NT-proBNP measurement. It is not clear if the existing methods, i.e. those utilizing antibodies specific to the central part of the molecule, are able to determine all NT-proBNP (proBNP) presented in the sample. Therefore, there is a need for precise characterization of the NT-proBNP and proBNP circulating in human blood.
Summary of the Invention
ProBNP and NT-proBNP, circulating in human blood, are described in literature as polypeptides consisting of 108 and 76 amino acid residues, respectively (molecular masses about 12 and 8.6 kDa). The concentration of proBNP in patient's blood is significantly lower than the concentration of NT-proBNP. According to our recent studies the concentration of proBNP in blood is about 10-20% of that of NT-proBNP. Seidler et al. (8) demonstrated that in gel filtration studies (non-denaturing conditions) NT-proBNP and proBNP have anomalous mobility and their apparent molecular weights are about 30-40 kDa. The authors suggested that in human blood both proteins are present in homo- oligomeric forms. In our studies we have demonstrated that in blood both proteins have apparent molecular weight (gel filtration studies, Superdex 75 10/300 GL column) of about 30 kDa (Fig. 4).
We generated a panel of high affinity monoclonal antibodies specific to NT-proBNP (also cross-reacting with proBNP) epitopes, covering the whole sequence of the protein (Fig. 1). All MAbs were generated after immunization of mice with synthetic peptides corresponding to different fragments of the NT-proBNP molecule. All MAbs were tested in two-site combinations (sandwich immunofluorescent assays) with recombinant protein as well as with endogenous protein, isolated from a patient's blood. The studies revealed that antibodies specific to the central part of NT-proBNP (epitopes located in the region 28-60) did not properly recognize the antigen in human blood (Fig. 2). On the other hand, most of the antibodies with epitopes located on peptides 5-27 and 61-76 recognized antigens in human blood with the same efficiency as recombinant proteins.
By means of affinity chromatography (affinity column containing immobilized monoclonal antibodies, specific to different regions of NT-proBNP) we purified endogenous antigen from human blood and analyzed it in Western blotting. NT-proBNP and proBNP molecules were stained by several NT-proBNP-specific monoclonal antibodies (Fig. 5). In the tracks containing endogenous protein none of the tested monoclonal antibodies recognized any protein band with the same molecular mass as recombinant NT-proBNP. The major immunological activity was concentrated on a diffused zone in the area corresponding to the proteins with higher molecular masses (15-70 kDa).
Such wide diversity of NT-proBNP and proBNP forms (Fig. 5) seen in Western blotting studies can be explained by glycosylation of NT-proBNP and proBNP molecules circulating in human blood.
Accordingly, one aspect of the invention is drawn to an antibody that specifically recognizes an endogenous glycosylated NT-proBNP or proBNP, or a fragment thereof, and which does not recognize a deglycosylated NT-proBNP or proBNP or a fragment thereof, or fragments of such antibodies. In addition, the invention provides an antibody that recognizes an endogenous glycosylated NT-proBNP or proBNP, or a fragment thereof, with higher affinity than the antibody or fragment recognizes a corresponding deglycosylated protein, or fragment thereof, of such antibodies. A related aspect of the invention provides an aptamer having the same specificity as an antibody described above. Further the above-described antibodies include an antibody that is a monoclonal antibody or fragment of a monoclonal antibody, as well as an antibody that is a polyclonal antibody or fragment of a polyclonal antibody. Also, the antibody may be a recombinant antibody or a fragment of a recombinant antibody.
Another aspect according to the invention provides for the use of an antibody or antibody fragment as described above in a diagnostic immunoassay method for qualitative or quantitative detection of NT-proBNP or proBNP or a fragment thereof. Related thereto is a diagnostic method for assaying NT-proBNP or proBNP or a fragment thereof in a sample of a patient, comprising quantitative or semiquantitative determination of the NT-proBNP or proBNP content of the sample using an antibody or an aptamer, each as described herein. The diagnostic method may further comprise preparing a calibration curve using as the standard a preparation of an endogenous glycosylated NT-proBNP or proBNP isolated from vertebrate blood. Also, the diagnostic method may further comprise preparing a standard curve and comparing the value of the NT-proBNP or proBNP content determined to the standard curve. Exemplary diagnostic methods according to the invention are immunoassay methods. For example, on diagnostic immunoassay is a sandwich immunoassay method, using a first capture antibody and a second detection antibody.
Another aspect of the invention is directed to a diagnostic method for assaying NT- proBNP or proBNP in a sample of a patient, comprising (a) deglycosylating endogenous NT-proBNP or proBNP contained in the sample; and (b) determining the NT-proBNP or proBNP content of the sample using an antibody or an aptamer specific to NT-proBNP or proBNP.
Yet another aspect is an NT-proBNP or proBNP standard or calibration preparation com- prising a glycosylated NT-proBNP or proBNP or a fragment thereof. In some embodiments, the glycosylated NT-proBNP or proBNP or a fragment thereof is an endogenous NT-proBNP or proBNP; in some embodiments, the NT-proBNP or proBNP is isolated from vertebrate blood. A related aspect of the invention provides an isolated glycosylated recombinant NT-proBNP or proBNP or a fragment thereof. In some embodiments, the glycosylated recombinant NT-proBNP or proBNP, or a fragment thereof, is glycosylated in vitro. In some embodiments, the glycosylated recombinant NT-proBNP or proBNP, or a fragment thereof, is produced in a cell culture or in a cell-free translation system.
Yet another aspect of the invention is a use of a molecule as described above, as an antigen for producing an antibody having the same specificity as an antibody described above.
In another aspect, the invention provides an immunoassay kit for diagnostic assay of NT- proBNP or proBNP or a fragment thereof in a sample of a patient, the kit comprising (a) a monoclonal or polyclonal antibody having the same specificity as an antibody described hereinabove; (b) a detectable label; and (c) a standard or calibrator preparation as described herein. An exemplary kit is an immunoassay kit for diagnostic assay of NT-proBNP or proBNP or a fragment thereof in a sample of a patient, the kit comprising (a) a first monoclonal capture antibody of the same specificity as an antibody described above; (b) a second monoclonal detection antibody of the same specificity as an antibody of claim 1 or 2, wherein the detection antibody is fluorescently labeled; and (c) a standard or calibration preparation according to claim 14.
The fragments of proteins, antibodies or any other entities as referred to in this specifica- tion mean any structural and/or functional fragments of said entities, retaining the desired activity.
Other features and advantages of the present invention will be better understood by reference to the following detailed description, including the Drawings and the Examples.
Detailed Description of the Invention
We treated isolated NT-proBNP and proBNP with deglycosylation enzymes and analyzed protein preparations by means of sandwich immunoassay, gel filtration HPLC and Western blotting. These experiments, described in detail in the Examples below, yielded the following results:
Sandwich immunoassay: After deglycosylation, monoclonal antibodies specific to the central region of NT-proBNP molecule (antibodies that do not recognize native endogenous antigen) were able to recognize endogenous protein with the same efficiency as the recombinant (non-glycosylated) form (Fig. 6).
HPLC gel filtration chromatography: After deglycosylation, a shift of the peak of immunological activity towards the peak of recombinant protein (i.e. towards the proteins with lower molecular mass) was observed (Fig. 7). Western blotting:
1) After deglycosylation, the appearance of the protein band revealing NT-proBNP immunological activity on the same level as recombinant NT-proBNP was observed.
2) Antibodies specific to the central part of the NT-proBNP molecule (antibodies that do not recognize native endogenous antigen) were able to recognize this band
(endogenous protein after deglycosylation) (Fig. 8).
Conclusions
It is clearly disclosed in the present specification that in human blood NT-proBNP and proBNP do not exist as simple polypeptide chains, as was considered before, but as glycoproteins. This means that mono- or polyclonal antibodies for assays designed to detect one or both of the molecules in human blood should either recognize those parts of the molecules that are not affected by glycosylation, or they should recognize the glycosylated part of the molecule.
Glycosylated forms of both of the proteins should be used for the preparation of standards and calibrators for such assays.
Glycosylated forms of the antigens should be used for animal immunization to obtain antibodies specific to the glycosylated part of the molecule.
Brief Description of the Drawings
The accompanying drawings are provided to help further in describing the invention, which drawings are the following:
Fig. 1 illustrates epitope map of NT-proBNP-specific monoclonal antibodies used in the study.
Fig. 2 illustrates specificities of different MAbs to endogenous NT-proBNP (antigen from HF patients' plasma). The results are presented as a ratio of signals in plasma to signals with recombinant NT-proBNP standard preparation in different two-site MAbs combinations. One MAb in such combination was able to interact effectively with endogenous NT-proBNP (MAb 24El 1, epitope 67-76 or MAb 13G12, epitope 13-20), whereas another one was out of the set of antibodies, specific to different regions of NT- proBNP molecule. The concentration of recombinant NT-proBNP was the same as that of the endogenous antigen, determined in HF patients' plasma by 15C4-13G12 assay.
Fig. 3 shows the calibration curve for the assay 15C4-13G12 and dilution curves for two individual plasma samples. Recombinant NT-proBNP (expressed in E. coli, HyTest) reconstituted in pooled normal human plasma was used as a calibrator.
Fig. 4 illustrates gel filtration studies (Superdex 75 10/300 GL column) of four plasma samples from HF patients. NT-proBNP immunoreactivity in the fractions was quantified by sandwich immunoassay utilizing monoclonal antibodies 15C4 (epitope 63-71) and 13Gl 2 (epitope 13-20) recognizing endogenous NT-proBNP.
Fig. 5 illustrates Western blotting studies of affinity-purified endogenous NT-proBNP. Tracks 1, 4: recombinant NT-proBNP (50 ng/track), tracks 2, 5: recombinant proBNP (50 ng/track), tracks 3, 6: endogenous NT-proBNP purified from human plasma (200 ng/track). For immuno staining MAbs 15Fl 1 (epitope 13-24) - tracks 1-3 or MAb HDl (epitope 31-39) - tracks 4-6 were used.
Fig. 6 illustrates the specificities of different monoclonal antibodies to endogenous NT- proBNP (black columns) or to endogenous NT-proBNP after deglycosylation - treatment with O-glycosidase and sialidase - (grey columns). The results are represented as ratio of signals (endogenous/recombinant) in different two-site MAb combinations. Three forms of NT-proBNP a) recombinant (non-glycosylated), b) endogenous, extracted from HF human plasma and c) endogenous, extracted from plasma and treated with enzymes in same concentrations were tested by sandwich immunoassays utilizing different monoclonal antibodies. One MAb in such immunoassay was specific to the epitope that is not affected by glycosylation (MAbs 24El 1, epitope 67-76 or MAb 13G12, epitope 13-20), whereas the other one was one out of the set of antibodies specific to different regions of NT- proBNP molecule.
Fig. 7 illustrates gel filtration studies (Superdex 75 10/300 GL column) of endogenous NT- proBNP extracted from plasma (uniform line) and endogenous NT-proBNP treated with O- glycosidase and sialidase (dotted line). NT-proBNP immunoreactivities in the fractions were quantified by two sandwich immunoassays 15C4-13G12 (A) and 11D1-13G12 (■). Immunoassay 15C4-13G12 is not sensitive to glycosylation. Immunoassay 11D1-13G12 is sensitive to glycosylation.
Fig. 8 illustrates the Western blotting studies of affinity-purified NT-proBNP before and after deglycosylation. Tracks 1, 5: recombinant NT-proBNP (E. coli, 50 ng per track); tracks 2, 6: recombinant proBNP (E. coli, 50 ng per track); tracks 3, 7: affinity-purified endogenous NT-proBNP (200 ng per track); tracks 4, 8: affinity-purified endogenous NT- proBNP after deglycosylation (200 ng per track). MAb 15Fl 1 (epitope 13-24) - tracks 1-4 or MAb 1 IDl (epitope 31-39) - tracks 5-8 were used for the antigen immunostaining.
Experimental
Example 1: Preparation and characterization of monoclonal antibodies (MAbs), specific for human NT-proBNP molecule
Synthetic peptides corresponding to sequences 1-24, 13-27, 28-45, 46-60 and 61-76 of human NT-proBNP molecule (HyTest, Finland) were conjugated to the bovine serum albumin (BSA) and were used for immunization of mice. Conjugation of small peptides with the carrier protein molecule allowed enhancing the immune response of the animals.
Female Balb/c mice, aged between 6-12 weeks were used for immunization.
The hybridoma cell lines producing monoclonal antibodies (MAbs), specific to NT- proBNP molecule were obtained after hybridization of mouse spleen cells with myeloma SP2/0 cells.
Culture supernatants were tested for reactivity to the whole recombinant NT-proBNP molecule (expressed in E. coli) and eighty-four positive cultures were selected for further work. Among those, 14 produced antibodies specific to region 1-24, 24 to region 13-27, 19 to region 28-45, 13 to region 46-60 and 15 to region 61-76. Selected cultures were subcloned twice by limiting dilution, expanded and frozen. The ascitic fluid containing monoclonal antibodies was produced in Balb/C mice.
The antibodies were isolated from the ascitic fluid by Protein-A Sepharose (GE Healthcare) affinity chromatography.
The isotypes of the purified antibodies were determined by Monoclonal Antibody Isotyping Kit (Pierce). All MAbs were specified as IgG.
Example 2: Epitope analysis
Precise epitope mapping of all newly generated antibodies was performed using a library of synthetic peptides 1-12, 5-20, 1-24, 13-27, 28-45, 31-39, 34-42, 37-45, 48-56, 50- 58, 52-60, 46-60, 63-71, 65-73, 67-76 and 61-76, containing overlapping sequences. Synthetic peptides were conjugated with a carrier protein (ovalbumine). The plates were coated with peptide conjugates in concentration of 1 μg/ml (100 μl per well). After washing, monoclonal antibodies, reconstituted in PBST, were added into the wells. After 30-minute incubation at room temperature the plates were washed and HRP-conjugated rabbit anti-mouse Fc-specific polyclonal antibodies were added to each well. After 30- minute incubation the plates were washed with PBST, and color development was achieved with the ø-phenylenediamine substrate system. Absorbance was measured at 492 nm using Victor 1420 Multilabel Counter. Four groups of antibodies with epitopes located in regions 1-12, 5-12, 5-20 and 13-24 were discriminated among MAbs specific to the region 1-24. Two groups of antibodies with epitopes 13-20, 13-24 and 31-39, 34-39 were discriminated among antibodies specific to regions 13-27 and 28-45, respectively. Four groups of antibodies with epitopes 46-56, 48-56, 46-60 and 52-58 were discriminated among antibodies specific to peptide 46-60 and three groups, 63-71, 67-73 and 67-76, among the antibodies specific to peptide 61-76.
Example 3: Development of sandwich immunofluoroassays (IFA) for quantitative measurement of human NT-proBNP
According to the invention sandwich- type immunofluoroassays were established for the quantification of NT-proBNP in human blood. Such assay is based on the binding of the antigen to the monoclonal antibody adsorbed on the plate surface thus forming first order immune complex, and on the detection of the first order immune complex by another monoclonal antibody labeled with stable europium (III) chelate.
Example 4: Conjugation of antibodies with stable europium chelate
Antibodies were preliminarily transferred into 0.9% water solution of NaCl using gel filtration on Sephadex G25 columns (NAP-5). Antibody labeling with stable europium (III) chelate of 2,2',2",2'";-[[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]bis(methyl- enenitrilo)]tetrakis(acetic acid) was conducted by incubation overnight at +4°C in 50 mmol/L Na-carbonate buffer pH 9.8 containing 200-fold molar excess of europium (III) chelate. Labeled antibodies were separated from the unreacted chelate by gel filtration on Sephadex G25 columns (NAP-5) in a buffer containing 0.01mol/L of Tris-HCl pH 7.8, 0.15 mol/L ofNaCl and 0.1% NaN3.
Example 5: Patients and blood samples
Diagnosis of patients with HF was based on symptoms: dyspnea, orthopnea, lung rales and leg edema, and confirmed by echocardiography studies and X-ray examination. The preliminary diagnosis was made by cardiologist and further confirmed by HF expert. Blood samples were collected from patients with left ventricular ejection fractions less than 30% and left ventricle end-systolic volume more than 90 mL. Venous blood was collected into EDTA-containing Vacuette tubes (Greiner Bio-One) and centrifuged at 3000 g (15 minutes, +4°C). Serum samples were obtained from blood collected in plastic tubes, incubated for 30 min at room temperature, and centrifuged at 500Og (30 min, +20 0C). Plasma and serum samples were stored at -700C prior to use. For MAb testing, pooled serum (39 patients with severe HF) or pooled plasma (10 HF patients) was used as a source of endogenous antigens. As a negative (non-HF) control pooled serum or plasma from 10 healthy donors was used.
Example 6: Sandwich IFA
All generated MAbs were tested in two-site MAb combinations (capture and detection) with recombinant NT-proBNP and with pooled serum or plasma from HF patients as a source of endogenous antigens. The capture antibodies in concentration of 10 μg/ml were placed into EIA plates (100 μl per well) and incubated in a phosphate saline buffer for 30 minutes at room temperature and gentle shaking. After twofold washing of the titration plates with a buffer containing 0.01 mol/L Tris-HCl pH 7.8, 0.15 mol/L NaCl, 0.025% Tween 20 and 0.05% NaN3 (buffer A), the mixture of antigen (recombinant NT-proBNP, reconstituted in pooled normal human plasma or endogenous antigen from HF plasma or serum, 50 μl) and detection antibodies (4 μg/ml, 50 μl), dissolved in buffer containing 0.05 mol/L Tris-HCl pH 7.7, 0.9% NaCl, 0.01% Tween 20, 0.5% BSA and 0.05% NaN3 (buffer B) were added to the wells. The plates were incubated for 30 minutes at room temperature and shaking gently, and washed six times with buffer A. After addition of the enhancing solution (1.75 mol/L NaSCN, 1 mol/L NaCl, 50 ml/L glycerol, 200 ml/L 1-propanol, 0.005 mol/L Na2CO3, 0.05 mol/L glycine-NaOH, pH 10.0), the mixture was incubated for 3 minutes at the same conditions. The fluorescence was measured on a Victor 1420 Multilabel Counter.
Example 7: Blood sample testing in different immunoassays
All MAbs with remote epitopes being utilized in 2-site combinations (sandwich immunoassays) were able to recognize recombinant NT-proBNP and proBNP with a low detection limit (10-100 ng/L). At the same time, only few MAb combinations were able to recognize antigen from serum and plasma of HF patients. Only two-site combinations utilizing one MAb specific to region 13-27 and another MAb specific to region 61-76 recognized the endogenous antigen with high sensitivity.
None of the 13 MAbs specific to region 46-60 were able to recognize the endogenous antigen being used in 2-site combinations with any other antibody. When MAbs specific to the very N-terminal region or to region 28-45 were tested in pairs with antibodies recognizing sequence 61-76, the ratio of signals from the endogenous to recombinant peptide was significantly lower than in the assays utilizing MAbs specific to epitope 13-
27. Only MAb 29D12, which is specific to peptide 5-12, elicited high signals upon interaction with either serum or plasma samples being tested in pairs with MAbs specific to peptide 67-76. Results of testing of recombinant and endogenous (pooled plasma of heart failure subjects) forms of NT-proBNP in sandwich immunoassays, utilizing antibodies specific to different regions of NT-proBNP molecule are presented in Fig. 2.
The assay 15C463-7i-13G12i3.2o was selected and used in further studies because it was able to recognize recombinant and native antigens with the same efficiency.
Consequently, we demonstrated that MAbs specific to the central part (region 28-60) of NT-proBNP molecule are almost unable to recognize the antigen in human blood.
Example 8: Characterization of 15C4-13G12 immunoassay
Sandwich immunofluorometric assay utilizing MAb 15C4-13G12 was performed as described in Example 6.
The specificities of antibodies used in the assay were confirmed by Western blotting analysis (data not shown). Both of the antibodies detected recombinant proBNP and NT- proBNP expressed in E. coli.
Human recombinant NT-proBNP expressed in E. coli was reconstituted in normal human plasma and was used as a calibrator for sandwich IFA. The detection limit was defined as a concentration (measured 20 times in a single run) producing a signal 2 SD above the mean for a calibrator that is free of analyte. Typical calibration curve for recombinant NT- proBNP and serial dilutions of human plasma samples are shown in Fig. 3. The detection limit was 10 ng/L, immunoassay was linear in the range of 15 - 100 000 ng/L.
Example 9: Gel filtration studies of recombinant and endogenous NT-proBNP
Seidler et al. (8) demonstrated that in gel filtration studies (in non-denaturing conditions) NT-proBNP and proBNP have anomalous mobility and their apparent molecular weights are about 30-40 kDa. The authors suggested that in human blood both proteins are presented in homo-oligomeric forms. In our studies we carried out gel filtration studies using HF patients' plasma as a source of endogenous NT-proBNP. Individual patient plasma samples were centrifuged at 10 000 g for 10 minutes and super- natants (150 μl) were applied onto Superdex 75 10/300 GL gel filtration column equilibrated with the buffer containing 0.1 mol/L sodium phosphate, pH 7.4, 0.3 mol/L NaCl and 0.005 mol/L EDTA. Proteins were eluted at a flow rate 0.8 ml/min and fractions with a volume of 0.7 ml were collected. Subsequently, the NT-proBNP immunological activity was measured in 15C463-7i-13G12i3.2o sandwich immunoassay.
The column was calibrated using a set of standard proteins (GE Healthcare): albumin (Mr 67000 Da), ovalbumin (Mr 43000 Da), chymotrypsinogen (Mr 25000 Da), ribonuclease A (Mr 13700 Da) and aprotinin (Mr 6517.5 Da, from Sigma). Recombinant NT-proBNP was reconstituted in pooled plasma from healthy donors before loading onto the Superdex 75 column.
Fraction analysis by the NT-proBNP assay revealed one peak of immunoreactivity (about 30 kDa) in human plasma samples (Fig. 4). The position of the NT-proBNP activity maximum did not coincide with those established for recombinant NT-proBNP (about 16 kDa) and recombinant proBNP (about 21 kDa). We thus observed that endogenous NT- proBNP in GF has significantly higher apparent molecular weight than the recombinant protein. The observed differences in apparent molecular weights of endogenous and recombinant proteins could be explained by posttranslational modifications (in the central part of the molecule) of endogenous NT-proBNP. The existence of such modifications could also explain the fact that antibodies specific to the central region of the molecule are not able to recognize the endogenous antigen.
Example 10: Western blotting studies of endogenous NT-proBNP
NT-proBNP was purified from pooled HF patients' plasma by means of affinity chromatography. To obtain the affinity matrix, a mixture of antibodies, specific to different regions of NT-proBNP molecule (15C4, 24El 1, 18H5, 15Fl 1), was immobilized on the BrCN- activated Sepharose CL-4B (GE Healthcare) according to the standard protocol. In the above-described experiments (see Example 8) it was demonstrated that the above- mentioned MAbs were able to recognize the endogenous antigen with high efficiency. Affinity matrix with immobilized antibodies was washed with 0.1 mol/L glycine, pH 2.0 and then equilibrated with 0.02 mol/L Tris-HCl, pH 7.5, containing 0.15 mol/L of NaCl. Pooled plasma of HF patients was loaded onto anti-NT-proBNP-Sepharose with the flow rate of 1 ml/min at +4°C. Peptides were eluted with a water solution containing 0.1 mol/L of HCl. The eluate was neutralized by 2 mol/L of Tris-HCl. Recovery after affinity chromatography was 88%.
The eluate was then loaded onto Sepharose CL-4B with immobilized MAbs that do not interact with NT-proBNP (negative chromatography) to remove proteins that bind nonspecifically to the affinity matrix.
Subsequently, NT-proBNP was concentrated by a second round of affinity chromatography. The solution containing NT-proBNP was loaded onto anti-NT-proBNP-Sepharose (10-fold molar excess of antibodies regarding to the NT-proBNP concentration) with the flow rate of 1 ml/min at +4°C. The peptides were eluted by 0.1 mol/L HCl, lyophilized, reconstituted in water and stored under -700C before use.
The proBNP contamination of the NT-proBNP preparation was determined by proBNP - specific immunoassay and was found to be less than 9% from total amount of NT-proBNP.
Samples of endogenous NT-proBNP as well as recombinant NT-proBNP and proBNP were boiled for 5 minutes in 0.25 mol/L Tris-HCl pH 6.8, 10 ml/L 2-mercaptoethanol, 20 g/L sodium dodecyl sulfate and 100 ml/L glycerol and loaded onto gel for protein separation by means of Tricine electrophoresis as described by Schagger and von Jagow (10). The electrophoresis was performed at constant voltage (150 V) under +4°C for 4 hours, using 16.5% T, 3% C separating gel. 200 ng of NT-proBNP (concentration was determined by 15C463-7i-13G12i3.2o immunoassay) extracted from pooled HF patients' plasma, or 50 ng of recombinant NT-proBNP (proBNP) were loaded per track.
After the electrophoresis the peptides were transferred onto nitrocellulose membrane (Trans-Blot® Transfer membrane, 0.2 μm, BioRad). Transfer was performed at constant voltage (100 V) and lasted for 40 minutes. Nonspecific binding was blocked by incubation of the membrane in the 10% solution of non-fat dry milk in PBST. Immunochemical staining of the peptides with NT-proBNP-specific MAbs, conjugated with horseradish peroxidase was performed during 12 hours at +4°C in the 10% solution of non-fat dry milk in PBST. Immune complexes were visualized by incubation in substrate solution, containing diaminobenzidine and nickel chloride.
It was shown that in the tracks containing endogenous protein none of the tested monoclonal antibodies recognizes any protein band with the same molecular mass as recombinant NT-proBNP (Fig. 5). When tested by MAb 15Fl 1 (epitope 13-24) an immunoreactive protein was detected in several diffused zones in the area corresponding to the proteins with molecular masses of 15 kDa and higher. In contrast to MAb 15Fl 1, endogenous NT-proBNP was not stained by MAb HDl specific to the region aar 31-39. These data are in agreement with the results of IFA described in Example 7, where HDl scarcely detected NT-proBNP in human plasma.
The existence of different diffused NT-proBNP zones stained by MAb 15Fl 1 in Western blotting studies could be explained by glycosylation of NT-proBNP molecules circulating in human blood.
Example 11: Testing of deglycosylated endogenous NT-proBNP in sandwich IFA
NT-proBNP extracted from pooled human plasma was treated by deglycosylation enzymes O-glycosidase (S. pneumoniae) and sialidase (A. ureafaciens) (QA-Bio, USA). Treatment was performed in a buffer containing 0.075 mol/L of sodium phosphate, pH 5.0 for 1 hour at +37°C. Water solution, containing 0.075 mol/L of sodium phosphate, pH 5.0 without enzymes was added to the studied peptides as a negative control.
After deglycosylation endogenous NT-proBNP was tested in immunoassays utilizing MAbs specific to different parts of the molecule as described in Example 7.
Immunological activities measured in assays utilizing MAbs specific to the central part of the molecule increased significantly after deglycosylation (Fig. 6). In the assays utilizing MAbs 5D328-45, HDl31 39, 5E23i_39, 16E634-39 the signal increased 7.5-10 fold. For MAbs 15D748-56, and I6DIO48-56 specific to the region 46-56 of NT-proBNP the immunological activity increased about 50 fold. Results in Fig. 6 are presented as a signal ratio of endogenous (treated or non-treated)/recombinant NT-proBNP (%), where the ratio of signals endogenous deglycosylated/recombinant in the assay utilizing MAbs 15C4-13G12 was taken as 100%.
Consequently, we have shown that the central portion of the NT-proBNP molecule is glycosylated, and polysaccharide residues prevent antibodies from interacting with the endogenous antigen. Since antibodies specific to the central part of the NT-proBNP molecule are unable to recognize endogenous antigen, MAbs specific to the other regions not affected by glycosylation should be used in NT-proBNP and proBNP assays.
Example 12: Gel-filtration studies of endogenous NT-proBNP before and after deglycosylation
NT-proBNP treated and non-treated with the O-glycosidase and sialidase was studied by gel- filtration (GF) method. NT-proBNP, extracted from pooled HF patients' plasma, was treated with enzymes and the immunological activity was thereafter determined in fractions in two immunoassays 15C4-13G12 and 11D1-13G12. According to the data presented in Example 11, assay 15C4-13G12 is not sensitive to glycosylation, whereas 11D1-13G12 assay can recognize endogenous NT-proBNP only after removal of carbohydrate moieties.
The samples of a) endogenous NT-proBNP, extracted from pooled HF patients' plasma, b) endogenous NT-proBNP, extracted from pooled HF patients' plasma after deglycosylation, c) recombinant NT-proBNP and d) recombinant proBNP in the same concentrations (330 ng/ml) were reconstituted in 150 μl of 0.1 mol/L sodium phosphate, pH 7.4, containing 0.7 mol/L of NaCl, 0.005 mol/L of EDTA and 5 g/L of bovine serum albumin. The samples were applied onto Superdex 75 10/300 GL gel filtration column equilibrated with 0.1 mol/L sodium phosphate, pH 7.4, containing 0.7 mol/L NaCl and 0.005 mol/L EDTA. Proteins were eluted at a flow rate of 0.7 ml/min and fractions with a volume of 0.5 ml were collected. The NT-proBNP immunological activities in the fractions were measured by sandwich immunoassay 15C5-13G12, utilizing monoclonal antibodies not sensitive to glycosylation and 11D1-13G12 assay that does not interact with glycosylated endogenous NT-proBNP. The column was calibrated using the set of standard proteins: albumin (Mr 67000 Da), ovalbumin (Mr 43000 Da), chymotrypsinogen (Mr 25000 Da), ribonuclease A (Mr 13700 Da) and aprotinin (Mr 6517.5 Da). Recombinant NT-proBNP and proBNP were reconstituted in pooled plasma from healthy donors before loading onto the Superdex 75 column.
Being measured in the 15C4-13G12 immunoassay endogenous NT-proBNP revealed two peaks of immunoreactivity (Fig. 7), the major one with molecular weight of about 28 kDa and the minor one with molecular weight of about 51 kDa. After deglycosylation we observed shift of both peaks of immunological activity towards the proteins with lower molecular masses. The major peak corresponded to the proteins with molecular masses of about 18 kDa and the minor peak to the proteins with molecular masses of about 51 kDa.
Being measured in the 11D1-13G12 immunoassay (utilizes antibody HDl, sensitive to glycosylation), endogenous NT-proBNP gave almost no response, whereas significant response was observed in the case of deglycosylated protein. Profile of immunoreactivity was very similar to the profile, measured by 15C4-13G12 immunoassay.
Example 13: Immunochemical staining of affinity-purified endogenous NT-proBNP (before and after deglycosylation) by NT-proBNP - specific monoclonal antibodies in Western blotting.
Electrophoresis and Western blotting procedure were performed in the same way as described in Example 10.
In this case MAb 15Fl 1 (epitope 13-24) was used for the protein visualization in the sample before deglycosylation. The major immunological activity was detected as a diffused zone in the area corresponding to the proteins with molecular masses of about 15 kDa and higher. In contrast to MAb 15Fl 1, endogenous NT-proBNP was not stained by MAb HDl specific to the region aar 31-39 of NT-proBNP molecule. After deglycosy- lation both of the antibodies were able to detect a peptide with apparent molecular mass about 13 kDa. This band was still a little above the recombinant NT-proBNP band, which could be explained by the fact that deglycosylation was not complete. We thus conclude that the endogenous protein cannot be detected in Western blotting experiments by antibodies specific to the central region of NT-proBNP molecule, whereas it becomes "visible" by such antibodies after deglycosylation.
References
1. Mair J, Hammerer-Lercher A, Puschendorf B. The impact of cardiac natriuretic peptide determination on the diagnosis and management of heart failure. Clin. Chem. Lab. Med. 2001; 39:571-88.
2. Cowie MR, Mendez GF. BNP and congestive heart failure. Prog. Cardiovasc. Dis.
2002; 44:293-321.
3. Pandey KN. Biology of natriuretic peptides and their receptors. Peptides 2005;
26:901-932. 4. Hunt PJ, Espiner EA, Nicholls MG, Richards AM, Yandle TG. The role of the circulation in processing pro-brain natriuretic peptide (proBNP) to amino- terminal BNP and BNP-32. Peptides 1997; 18:1475-1481.
5. Hughes D, Talwar S, Squire IB, Davies JE, Ng LL. An immunolumino metric assay for N-terminal pro-brain natriuretic peptide: development of a test for left ventricular dysfunction. Clin. Sci. (Lond) 1999; 96:373-380.
6. Karl J, Borgya A, Gallusser A, et al. Development of a novel, N-terminal-proBNP
(NT-proBNP) assay with a low detection limit. Scand. J. Clin. Lab. Invest. Suppl. 1999; 230:177-181.
7. Giuliani I, Rieunier F, Larue C, et al. Assay for measurement of intact B-type natriuretic peptide prohormone in blood. Clin. Chem. 2006; 52:1054-1061.
8. Seidler T, Pemberton C, Yandle T, Espiner E, Nicholls G, Richards M. The amino terminal regions of proBNP and pro ANP oligomerise through leucine zipper- like coiled-coil motifs. Biochem. Biophys. Res. Commun. 1999; 255:495- 501. 9. Crimmins DL. Human N-terminal proBNP is a monomer. Clin. Chem. 2005; 51:1035-1038.
10. Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987; 166:368-379.

Claims

Claims:
1. An antibody, which specifically recognizes endogenous glycosylated NT-proBNP or proBNP or a fragment thereof, and which does not recognize a deglycosylated NT-proBNP or pro BNP or a fragment thereof, or a fragment of such an antibody.
2. An antibody, which recognizes endogenous glycosylated NT-proBNP or proBNP or a fragment thereof, with higher affinity than said antibody recognizes a corresponding deglycosylated protein or a fragment thereof, or a fragment of such an antibody.
3. An ap tamer having the same specificity as an antibody according to claim 1 or 2.
4. An antibody according to claim 1 or 2, wherein the antibody is a monoclonal antibody or a fragment of a monoclonal antibody.
5. An antibody according to claim 1 or 2, wherein the antibody is a polyclonal antibody or a fragment of a polyclonal antibody.
6. An antibody according to claim 1 or 2, wherein the antibody is a recombinant antibody or a fragment of a recombinant antibody.
7. Use of an antibody or antibody fragment according to any one of claims 1 to 6 in a diagnostic immunoassay method for qualitative or quantitative detection of NT-proBNP or proBNP or a fragment thereof.
8. A diagnostic method for assaying NT-proBNP or proBNP or a fragment thereof in a sample of a patient, comprising quantitative or semiquantitative determination of the NT- proBNP or proBNP content of the sample using an antibody according to claim 1 or 2 or an ap tamer according to claim 3.
9. A diagnostic method according to claim 8, further comprising preparing a calibration curve using as the standard a preparation of an endogenous glycosylated NT-proBNP or proBNP, isolated from vertebrate blood.
10. A diagnostic method according to claim 9, further comprising preparing a standard curve and comparing the value of the NT-proBNP or proBNP content determined to the standard curve.
11. The diagnostic method according to claim 8, wherein the diagnostic method is an immunoassay method.
12. The diagnostic method according to claim 11, wherein the immunoassay method is a sandwich immunoassay method, using a first capture antibody and a second detection antibody.
13. A diagnostic method for assaying NT-proBNP or proBNP in a sample of a patient, comprising
(a) deglycosylating endogenous NT-proBNP or proBNP contained in the sample, and
(b) determining the NT-proBNP or proBNP content of the sample using an antibody or an aptamer specific to NT-proBNP or proBNP.
14. An NT-proBNP or proBNP standard or calibration preparation comprising a glycosylated NT-proBNP or proBNP or a fragment thereof.
15. An endogenous glycosylated NT-proBNP or proBNP or a fragment thereof isolated from vertebrate blood.
16. A glycosylated recombinant NT-proBNP or proBNP or a fragment thereof.
17. A glycosylated recombinant NT-proBNP or proBNP or a fragment thereof according to claim 16, which is glycosylated in vitro.
18. A glycosylated recombinant NT-proBNP or proBNP or a fragment thereof according to claim 16, which is produced in a cell culture or in a cell- free translation system.
19. Use of a molecule according to any one of claims 15 to 18 as an antigen for producing antibodies having the same specificities as an antibody of claim 1 or 2.
20. An immunoassay kit for diagnostic assay of NT-proBNP or proBNP or a fragment thereof in a sample of a patient, the kit comprising
(a) a monoclonal or polyclonal antibody having the same specificity as an antibody of claim 1 or 2,
(b) a detectable label, and
(c) a standard or calibrator preparation according to claim 14.
21. An immunoassay kit for diagnostic assay of NT-proBNP or proBNP or a fragment thereof in a sample of a patient, the kit comprising
(a) a first monoclonal capture antibody having the same specificity as an antibody of claim 1 or 2,
(b) a second monoclonal detection antibody of the same specificity as an antibody of claim 1 or 2, wherein the detection antibody is fiuorescently labeled, and (c) a standard or calibrator preparation according to claim 14.
PCT/FI2007/050298 2006-05-26 2007-05-25 Antibodies and standard for nt-probnp and probnp immunoassays WO2007138163A2 (en)

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