EP2021369A2 - ANTICORPS ET NORME POUR IMMUNOESSAIS DESTINÉS À LA DÉTECTION DES MARQUEURS NT-proBNP ET proBNP - Google Patents

ANTICORPS ET NORME POUR IMMUNOESSAIS DESTINÉS À LA DÉTECTION DES MARQUEURS NT-proBNP ET proBNP

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Publication number
EP2021369A2
EP2021369A2 EP07730785A EP07730785A EP2021369A2 EP 2021369 A2 EP2021369 A2 EP 2021369A2 EP 07730785 A EP07730785 A EP 07730785A EP 07730785 A EP07730785 A EP 07730785A EP 2021369 A2 EP2021369 A2 EP 2021369A2
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Prior art keywords
probnp
antibody
fragment
endogenous
glycosylated
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EP07730785A
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German (de)
English (en)
Inventor
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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Hytest Ltd
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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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EP07730785A 2006-05-26 2007-05-25 ANTICORPS ET NORME POUR IMMUNOESSAIS DESTINÉS À LA DÉTECTION DES MARQUEURS NT-proBNP ET proBNP Withdrawn EP2021369A2 (fr)

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CN102692512A (zh) * 2012-06-26 2012-09-26 南京基蛋生物科技有限公司 NT-proBNP双抗夹心快速检测试剂盒
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11561230B2 (en) 2017-12-28 2023-01-24 Shionogi & Co., Ltd. Reference standard for BNP measurement

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