BNP (1-32) epitope and antibodies directed against said epitope
11340239 · 2022-05-24
Assignee
Inventors
- Francois Rieunier (Bois d'Arcy, FR)
- Isabelle Giuliani (Garches, FR)
- Sylvie Villard-Saussine (Issy les Moulineaux, FR)
Cpc classification
G01N33/74
PHYSICS
G01N2800/324
PHYSICS
G01N2333/58
PHYSICS
G01N2800/52
PHYSICS
International classification
G01N33/74
PHYSICS
Abstract
The present invention relates to a polypeptide carrying a human BNP(1-32) epitope according to Formula (I): a.sub.1-R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2-a.sub.2 as well as ligands specific of the FGRKMDR epitope.
Claims
1. A method for measuring, in a biological sample, the concentration of human BNP(1-32) or a derivative of human proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8), comprising: a) contacting the biological sample with a monoclonal antibody produced by the hybridoma deposited on Apr. 13, 2007 at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15, France) under registration number CNCM 1-3746 that is specific of an epitope of the sequence FGRKMDR (SEQ ID NO: 8) under conditions allowing the formation of antigen-ligand complexes, b) detecting any complex which may have formed; c) contacting known concentrations of a multiepitopic calibrator having the following general formula (III):
t.sub.1-E.sub.1-L.sub.1-E.sub.2[-L.sub.k-1-E.sub.k].sub.n-t.sub.2 (III) wherein: n is an integer between 0 and 8; k is an integer between 3 and n+2 when n>0; E.sub.1, E.sub.2, and E.sub.k are different from one another, wherein one of E.sub.1, E.sub.2 and E.sub.k is a peptide having a sequence of R.sub.1-X.sub.1-FGRKMDR(SEQ ID NO: 8)-X.sub.2-R.sub.2, wherein X.sub.1 is absent or present and when present is selected among C and GC; X.sub.2 is absent or present and when present is selected among I and IS; R.sub.1 and R.sub.2, which may be the same or different, present or absent, represent any amino acid or a peptide chain of 2 to 15 amino acids, provided that said polypeptide of formula (III) does not include any portion of human BNP(1-32) of more than 11 amino acids including the sequence GCFGRKMDRIS (SEQ ID NO: 63), and each of the remaining two from E.sub.1, E.sub.2 and E.sub.k independently represents a sequence of 3 to 15 amino acids selected from the sequence of human proBNP(1-108) (SEQ ID NO: 1); t.sub.1 represents a hydrogen atom, an acetyl group, a peptide sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10 N-α acetylated amino acids, a biotinyl or biocytinyl group, a peptide sequence of 1 to 10 amino acids carrying a biotinyl or biocytinyl radical, or a linear amino alkyl (C.sub.1-C.sub.10) carbonyl chain; t.sub.2 represents a hydroxyl radical, an amino radical, a peptide sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10 amino acids carrying a terminal amino group, or a linear or branched amino alkyl (C.sub.1-C.sub.10) carbonyl chain; and L.sub.1 and L.sub.k-1 which may be the same or different, each represents a coupling agent that covalently couples the adjacent peptide chains with said monoclonal antibody; and d) determining the concentration of human BNP(1-32) or a derivative of human proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8) in the sample.
2. A method for measuring, in a biological sample, the concentration of human BNP(1-32) or a derivative of human proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8), comprising: a) contacting the biological sample with at least one monoclonal antibody specific for an epitope of the sequence FGRKMDR (SEQ ID NO: 8) under conditions allowing the formation of antigen-antibody complexes, said monoclonal antibody being produced by the hybridoma deposited on Apr. 13, 2007 at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15, France) under registration number CNCM I-3746; b) detecting any complex which may have formed; and c) determining the concentration of human BNP(1-32) or a derivative of human proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8) in the sample.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1
Peptide Synthesis
(17) Materials and Methods:
(18) Synthetic peptides were prepared by standard methods which are well known to the one skilled in the art. An example of this method is Merrifield synthesis, which is advantageous due to the fact that it can be implemented easily (Merrifield, (1963); R. C. Sheppard (1971); Atherton et al. (1989)). “Pioneer” synthesisers from Perspective, or the “433A” synthesiser from ABI may be used as the automatic synthesiser. The peptides may also be obtained by homogenous phase synthesis.
(19) The following syntheses were carried out in a Pioneer synthesiser using “Fmoc” chemistry (9-fluorenylmethyloxycarbonyl): in each step, the reagents (that is to say the protected amino acid and the coupling activators (TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate)/HOBt (N-hydroxybenzotriazole)) were added in excess (in a “moles of reagent/moles of groups which can be substituted on the resin” ratio=5). At the end of the synthesis process, the peptide was separated from the resin by a trifluoroacetic acid solution (reagent K). The peptide was then precipitated in a cooled ether solution, lyophilised and then subsequently purified by HPLC.
(20) In this way, the inventors synthesised peptides containing the following amino acid sequences:
(21) TABLE-US-00005 SEQ ID NO: 4: Ac-TGCFGRKMDRISTSTAIGCKVLCys-CONH2, SEQ ID NO: 5: 5: Ac-SGCYGRKMDRISTSTAIGCKVL-CysCONH2, SEQ ID NO: 6: Ac-SGCFGRKMDRISSSSGLGCKVL-CysCONH2, SEQ ID NO: 7: Ac-SGCFGRKMDRIATSTAIGCKVL-CysCONH2, SEQ ID NO: 8: Ac-FGRKMDR-CONH2, SEQ ID NO: 9: Ac-GRKMDR-CONH2, SEQ ID NO: 10: Ac-FGRKMD-CONH2, SEQ ID NO: 11: Ac-RKMDRI-CONH2.
Example 2
Immunogen Preparation: Coupling of a Peptide to a Carrier Protein for Immunisation
(22) In order to immunise mice with these peptides, it is necessary to couple said peptides to a carrier protein such as KLH (keyhole limpet haemocyanin), thyroglobulin, or BSA (bovine serum albumin), via different functional groups (thiol, amine, aldehyde, etc.) so as to render the peptide more immunogenic. The coupling reagent used to bind the peptide to the protein may be heterobifunctional or homobifunctional. The most frequently used reagents are BS3, sSMCC, SPDP, glutaraldehyde, etc..
(23) The coupling method used involved the bifunctional sSMCC (Pierce, #22322) molecule, having an NHS ester functional group and a maleimide group as the chemical coupling agent, and KLH (Pierce, #77600) as the carrier protein.
(24) 2-a. KLH Activation
(25) Method:
(26) 20 mg of KLH were solubilised in 2 ml of phosphate buffered saline (20 mM phosphate, 0.9 M NaCl pH 7.2) in order to obtain a final concentration of 10 mg/ml (do not vortex). In parallel, 4 mg of sSMCC were solubilised with 400 μl of water for injection to obtain a final concentration of 10 mg/ml. 2 ml of KLH (20 mg) were subsequently mixed with 200 μl of sSMCC (2 mg), and the mixture was incubated for 1 hour at room temperature (20° C.) whilst being stirred slowly (20 revolutions per min).
(27) 2-b. Desalting the Activated KLH:
(28) Method:
(29) A PD10 Sephadex TM G-25m column (Ge healthcare, USA, ref: 17-0851-01) was equilibrated with phosphate buffered saline (20 mM phosphate, 0.9 M NaCl pH 7.2, 100 mM EDTA). The 2 ml of activated KLH were deposited on the column, and the elution was subsequently started with 3.5 ml of 20 mM PBS buffer supplemented with 0.9 M NaCl, pH 7.2 and 100 mM EDTA; 500 μl fractions were collected. The optical density (OD) was measured at 280 nm for each fraction diluted to 1/25th and the fractions containing the activated KLH were then identified and measured in accordance with the Beer-Lambert law: OD=εCl: wherein OD is the optical density ε=1.499,
C is the concentration and I=1 cm, the concentration of the activated KLH may be determined and is reduced to 7.4 mg/ml in phosphate buffered saline.
(30) 2-c. Coupling of the Peptide to the Activated KLH
(31) Method:
(32) 10 mg of lyophilised peptide were solubilised in 1 ml of Milli-Q water, which was degassed in an ultrasonic disintegrator to obtain a final concentration of 10 mg/ml, and were then mixed with 7.4 mg of activated KLH (i.e. 1 ml of the solution obtained in 2-b.). This mixture was left to incubate for 2 hours at room temperature (20° C.) whilst being stirred slowly (20 rpm). A solution of cysteine at a concentration of 5 mg/ml in a 20 mM PBS buffer+0.9 M NaCl pH 7.2 was subsequently introduced to obtain a final concentration of 1 mM in the peptide/KLH solution, and the entire mixture was left to incubate for 20 minutes at room temperature (20° C.) whilst being stirred slowly (20 rpm).
(33) 2-d. Characterisation of the Coupled Peptide
(34) Method:
(35) The concentration of the coupled peptide was then determined by the Bradford method (Bradford M., Anal. Biochem., 1976; 72: 248-54) as follows: a standard range of from 50 to 1000 μg/mL of KLH was prepared in order to determine the KLH concentration of our sample from the OD at 595 nm. In order to produce this standard range and to carry out this assay, 50 μL of each point of the sample were diluted in 1.5 mL of Coomassie blue (Bio-Rad, #1856210).
(36) Having determined the concentration, PBS was added to the KLH-coupled peptide to bring the concentration of the coupled peptide to 1 mg/mL.
Example 3
Immunisation of Mice and Production of Monoclonal Antibodies
(37) 3-a) Immunisation of Mice:
(38) In order to produce monoclonal antibodies, ten mice (Balb/c strain females, 5 weeks old, ref: SIFE055, Charles Rivers, Mass., USA) were immunised using one of the following peptides:
(39) TABLE-US-00006 (SEQ ID NO: 4) Ac-TGCFGRKMDRISTSTAIGCKVL-Cys-CONH2, (SEQ ID NO: 5) Ac- SGCYGRKMDRISTSTAIGCKVL-CysCONH2,
coupled to KLH in accordance with Example 2 (5 mice for each peptide).
(40) For the first injection, an emulsion of 100 μg of KLH-coupled peptide (at a concentration of 1 mg/ml) diluted to ½ in Freund's complete adjuvant (Sigma, #F-5881) was prepared, and 200 μL of said emulsion (i.e. 100 μg of peptide) were injected subcutaneously into each mouse. At intervals of 20 days, three 200 μL booster shots of an emulsion of KLH-coupled peptide (i.e. 100 μg of peptide) and Freund's incomplete adjuvant (Sigma, #F-5506) were injected subcutaneously, then peritoneally, into each mouse.
(41) 20 days after the last booster shot, and after the antibodies obtained had been assessed by the ELISA method (in accordance with Example 4 described below), the mouse with the greatest reaction against BNP(1-32) was retained in order to undergo hyperimmunisation, in accordance with the following protocol: subcutaneous injection of 200 μL of peptide-KLH at 1 mg/mL diluted to 1/20.sup.th with PBS 45 minute-wait subcutaneous injection of 200 μL of peptide-KLH at 1 mg/mL diluted to 1/20.sup.th with PBS at a site different from the first injection 45 minute-wait subcutaneous injection of 200 μL of peptide-KLH at 1 mg/mL diluted to 1/10.sup.th with PBS at a site different from the previous injections 30 minute-wait intraperitoneal injection of 100 μL of promethazine (2.5% Phenergan, injectable solution, UCB) diluted to 1 mg/mL with PBS 15 minute-wait intraperitoneal injection of 200 μL of peptide-KLH at 1 mg/mL diluted to 3/10.sup.ths with PBS at a site different from the previous injections
(42) After these immunisations, the mouse S2 immunised with the SEQ ID NO: 4 peptide was found to produce an antiserum which was very reactive towards BNP(1-32) when using the protocol for detecting antibodies described below in Example 4. The lymphocytes from the spleen of said mouse were subsequently subjected to lymphocyte fusion, carried out in accordance with the protocol described below in 3b.
(43) 3-b) Production of Monoclonal Antibodies:
(44) Lymphocyte fusion of the spleen cells of the immunised mouse S2 with myeloma SP2 cells (ATCC CRL-1581) was carried out in accordance with Köhler and Milstein's well known protocol (1975) Nature 56:495-497.
(45) The inventors were thus able to produce different hybrid clones. In particular, they obtained a monoclonal antibody, which was given the designation 20G7-15 Mar. 2007 (referred to as “20G7” in the following for convenience). The hybridoma which secretes the 20G7-15 Mar. 2007 (20G7) monoclonal antibody was deposited at the CNCM (French National Collection of Cultures of Microorganisms, Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15, France) with the registration number CNCM I-3746 on Apr. 13, 2007.
(46) It goes without saying that other protocols for obtaining monoclonal antibodies which are well known to the one skilled in the art may be used.
Example 4
Detection of Anti-BNP(1-32) Antibodies to Assess the Response of Mice During Immunisation
(47) 4.1 Materials: The following reagents were used: Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen) BNP(1-32): synthetic peptide (Sigma-Aldrich, USA, #B-5900) or proBNP(1-108) (recombinant protein produced in E. Coli, HyTest, Finland) Tween® 20 (Sigma-Aldrich, USA, #P1379) anti-mouse IgG secondary antibody produced in rabbit and coupled to peroxidase, (Sigma, USA, #A9044) H.sub.2O.sub.2 (0.04% in 0.1 M citrate buffer, pH 4) OPD (ortho-phenylenediamine, Sigma, USA, #P8412) sulphuric acid (H.sub.2SO.sub.4, 4N) serums from mice immunised in Example 3
(48) 4.2 Method and Principle:
(49) An ELISA test was carried out on a solid support to detect the presence of anti-BNP(1-32) antibodies in a mouse serum sample.
(50) Some of the antigen was immobilised by adsorption in the cavities of a 96-well microplate. After the remaining free sites were saturated and blocked, the immune serums were left to incubate, and the antibodies («Ac ») which may have been present bound to the antigen («Ag ») and formed an Ag—Ac complex. This complex was detected using an immunoconjugate (anti-mouse IgG antibody) coupled to an enzyme, which in this case was HRP (horseradish peroxidase), which transforms a colourless substrate into a coloured product which indicates the presence of the desired antibody. The formation of the final coloured product was quantified by carrying out an optical density reading at 490 nm (OD). According to this method, which is well known to the one skilled in the art, the OD obtained indicates the presence (high OD) or absence (low OD) of antibodies in a tested mouse serum sample. There are a number of variants of this test (antigen capture, competition assay . . . ) which are well known to the one skilled in the art. 1) Immobilisation of the antigen on the microplate:
(51) Each antigen, BNP(1-32) or proBNP(1-108), was solubilised in PBS at a final concentration of 0.5 μg/mL and was then immobilised, on the basis of 100 μL per well, on a Maxisorp microplate by being incubated overnight at 4° C. After 3 washes with PBS 0.1% Tween® 20 (PBS-T), the microplate was saturated with a solution (100 μL/well) of 0.1% PBS-T containing 1% milk (semi-skimmed) and was then left to incubate at 37° C. for 1 hour. 2) Immunological detection of the antibodies produced by the mice:
(52) The microplate was washed three times with 0.1% PBS-T. Each serum from previously immunised mice was subsequently diluted tenfold with 0.1% PBS-T containing 0.1% milk (semi-skimmed), then deposited on the basis of 100 μL per well and left to incubate for two hours at 37° C. The microplate was again washed three times with 0.1% PBS-T, then left to incubate for 1 hour at 37° C. in the presence of the conjugate coupled to peroxidase diluted to a 1/3,000.sup.th in 0.1% PBS-T containing 0.1% milk (semi-skimmed) on the basis of 100 μL per well. Finally, the microplate was washed three times with 0.1% PBS-T, then the peroxidase substrate was deposited on the basis of 100 μL per well. The microplate was placed in darkness at room temperature for 20 minutes. The enzymatic reaction was stopped by adding 50 μL of sulphuric acid (H.sub.2SO.sub.4, 4 N) per well, and the OD at 490 nm was subsequently measured in each well.
(53) By using this method for detecting antibodies, the inventors found that the serum from the mouse S2 (immunised with the SEQ ID NO: 4 peptide) was very reactive with BNP(1-32), and proBNP(1-108). Following the lymphocyte fusion which was subsequently carried out between the lymphocytes from said hyperimmune mouse and the Sp2 myeloma, this method also made it possible to identify a hybridoma which produces an important monoclonal antibody: the 20G7 hybridoma, producing the 20G7 monoclonal antibody.
Example 5
Epitopic Characterisation of the 20G7 Monoclonal Antibody
(54) 5.1 Epitopic Characterisation According to the «Spot» Technique
(55) 5.1.1 Materials:
(56) The equipment and reagents are all listed in C. Granier, S. Villard, D. Laune (Mapping and Characterization of Epitopes using the SPOT method. Cells/Cell Biology: A Laboratory Handbook, third edition (Volume 1), chapter 62, editor: Julio Celis, Elsevier, 2005).
(57) 5.1.2 Method:
(58) The “SPOT” or “epitope mapping” method was used to characterise the epitope of the 20G7 monoclonal antibody. This method, described by Frank (Tetrahedron, 1992; 48: 9217-32), allows synthesis on a cellulose membrane of a large number of peptides with sequences predetermined on a functionalised support (aminopolyethyleneglycol-cellulose) and testing of their reactivity towards a soluble ligand, which is, in the present case, the 20G7 antibody.
(59) 5.1.2.1 Peptide Synthesis
(60) The entire peptide synthesis process (amino acid activation, chemical reaction, etc.) is detailed in Molina et al. (Pept Res. 1996, Vol. 9: pp. 151-5), and in C. Granier, S. Villard, D. Laune (Mapping and Characterization of Epitopes using the SPOT method. Cells/Cell Biology: A Laboratory Handbook, third Edition (Volume 1), chapter 62, Editor: Julio Celis, Elsevier, 2005).
(61) The BNP(1-32) sequence was synthesised entirely in the form of overlapping pentadecapeptides (SEQ ID NO: 12 to 20), with an offset of two amino acids:
(62) TABLE-US-00007 SEQ ID NO: 12 SPKMVQGSGCFGRKM SEQ ID NO: 13 KMVQGSGCFGRKMDR SEQ ID NO: 14 VQGSGCFGRKMDRIS SEQ ID NO: 15 GSGCFGRKMDRISSS SEQ ID NO: 16 GCFGRKMDRISSSSG SEQ ID NO: 17 FGRKMDRISSSSGLG SEQ ID NO: 18 RKMDRISSSSGLGCK SEQ ID NO: 19 MDRISSSSGLGCKVL SEQ ID NO: 20 RISSSSGLGCKVLRR
(63) The other following peptides were also synthesised:
(64) TABLE-US-00008 SEQ ID NO: 21 VQGSGCFGR SEQ ID NO: 22 SPKMVQGSGC SEQ ID NO: 23 MDRISSSSGLG SEQ ID NO: 24 RKMDRI SEQ ID NO: 25 RKMDRISS
(65) The BNP(1-32) sequence was also synthesised in the form of overlapping decapeptides (SEQ ID NO: 26 to 48), with an offset of 1 amino acid:
(66) TABLE-US-00009 SEQ ID NO: 26 SPKMVQGSGC SEQ ID NO: 27 PKMVQGSGCF SEQ ID NO: 28 KMVQGSGCFG SEQ ID NO: 29 MVQGSGCFGR SEQ ID NO: 30 VQGSGCFGRK SEQ ID NO: 31 QGSGCFGRKM SEQ ID NO: 32 GSGCFGRKMD SEQ ID NO: 33 SGCFGRKMDR SEQ ID NO: 34 GCFGRKMDRI SEQ ID NO: 35 CFGRKMDRIS SEQ ID NO: 36 FGRKMDRISS SEQ ID NO: 37 GRKMDRISSS SEQ ID NO: 38 RKMDRISSSS SEQ ID NO: 39 KMDRISSSSG SEQ ID NO: 40 MDRISSSSGL SEQ ID NO: 41 DRISSSSGLG SEQ ID NO: 42 RISSSSGLGC SEQ ID NO: 43 ISSSSGLGCK SEQ ID NO: 44 SSSSGLGCKV SEQ ID NO: 45 SSSGLGCKVL SEQ ID NO: 46 SSGLGCKVLR SEQ ID NO: 47 SGLGCKVLRR SEQ ID NO: 48 GLGCKVLRRH
A selection of heptapeptides with an offset of one amino acid were also synthesised (SEQ ID NO: 49 to 53):
(67) TABLE-US-00010 SEQ ID NO: 49 GCFGRKM SEQ ID NO: 50 CFGRKMD SEQ ID NO: 51 FGRKMDR SEQ ID NO: 52 GRKMDRI SEQ ID NO: 53 RKMDRIS
(68) 5.1.2.2 Immunological Test
(69) The followed test for immunoreactivity has been described in detail in Laune et al. (J. Immunol. Methods, 2002, Vol. 267(1), pp. 53-70). In short, the principle was as follows. The membrane was rehydrated by three TBS baths (tris-buffered saline, pH 7.0) with a duration of 10 minutes in each case, and was subsequently saturated by being incubated overnight at room temperature, whilst being stirred, in the presence of 15 ml of a 10% saturation buffer (“blocking buffer”, Roche) and 5% saccharose in TBS 0.1% Tween® 20 (TBS-T). After the membrane was washed three times for 10 minutes with 0.1% TBS-T, the membrane was left to incubate for 90 minutes at 37° C. whilst being stirred, in the presence of the antibody to be tested (20G7 in this case) and the conjugate coupled to alkaline phosphatase diluted with the saturation buffer. After washing the membrane twice with 0.1% TBS-T, then twice with CBS (citrate buffered saline), each bath lasting 10 minutes, the alkaline phosphatase substrate was added and the membrane was incubated at room temperature for 1 to 30 minutes, depending on the speed at which the signal appeared.
(70) 5.1.2.3 Results
(71) In the present case, the BNP(1-32) sequence was synthesised entirely in the form of overlapping pentadecapeptides (SEQ ID NO: 12 to 20), with an offset of two amino acids. As shown in
(72) TABLE-US-00011 SEQ ID NO: 13 K M V Q G S G C F G R K M D R SEQ ID NO: 14 V Q G S G C F G R K M D R I S SEQ ID NO: 15 G S G C F G R K M D R I S S S SEQ ID NO: 16 G C F G R K M D R I S S S S G SEQ ID NO: 17 F G R K M D R I S S S S G L G
(73) To ensure that only this pattern is actually involved in the binding of the antibody to BNP(1-32), shorter peptides (decapeptides (SEQ ID NOs: 26 to 48) and heptapeptides (SEQ ID NOs: 49 to 53) were also synthesised, with an offset of only one amino acid, in order to confirm and validate the epitope. In each experiment, the common peptide sequence identified by 20G7 was F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) (for decapeptides, SEQ ID NO: 33 to 36, and for heptapeptides, SEQ ID NO: 51).
(74) In order to determine which residues are critical and essential for the recognition of the epitope, each residue of the minimal sequence F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) was substituted successively by an alanine (A) in order to assess the involvement of each individual residue in accordance with the “Alascan” method, which is well known and described (Laune et al., above). As shown in
(75) These amino acids in positions 11, 14 and 17 are essential for the recognition of the epitope by the 20G7 monoclonal antibody. These data are the mean values of a plurality (n=4) of repeat experiments. For this reason, it is therefore clear that the F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) epitope, comprising the essential F.sub.11, K.sub.14 and R.sub.17 amino acids, according to the invention, is different from that recognised in the patent application WO2006/88700 which discloses another epitope (R.sub.13(K.sub.14)(M.sub.15)D.sub.16R.sub.17I.sub.18) (SEQ ID NO: 24), the important amino acids of which are R.sub.13, D.sub.16, R.sub.17 and I.sub.18.
(76) In order to achieve a better understanding of the contributory effect of these residues to the binding process of the 20G7 antibody, the F.sub.11, K.sub.14 and R.sub.17 amino acids were substituted by amino acids with closed biochemical properties. For example, F.sub.11 was substituted by other aromatic amino acids (tryptophan and tyrosine). The fact that the sequence composed of these “homologous” amino acids was recognised in the same way by the 20G7 antibody suggests that it is the aromatic nature of the peptide in position 11 that is essential for the antibody binding. With regard to K.sub.14 and R.sub.17, they were both substituted by an arginine and a lysine to study the effect of the lateral chain of the amino acid, and also the presence of a positive charge. It was also found in this case that the substitution was effectively conservative, since binding to 20G7 was retained, thus underlining the importance of the positive charge.
(77) The same does not apply to the 24C5 antibody from HyTest, which has different essential residues.
(78) The VQGSGCFGR, SPKMVQGSGC, MDRISSSSGLG, R.sub.13KMDRI.sub.18 and R.sub.13KMDRISS.sub.20 (SEQ ID NO: 21 to SEQ NO: 25) peptide sequences of human BNP(1-32), which were also synthesised on a membrane, were tested with the 20G7 antibody using the Spot method. Since residues which are essential for the binding of 20G7 were absent, said antibody did not bind to said peptides at all, with the result that 20G7 exhibited a cross reaction of less than 2% with these peptides.
(79) 5.2 Characterisation with Soluble Peptides
(80) In a second step, to ensure that the F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) peptide was really the epitope of the 20G7 antibody, this sequence was synthetized in a soluble form in order to carry out competition assays between this peptide and BNP(1-32). Parallely, in order to confirm the high contribution of the F.sub.11 residue in the binding to the 20G7 antibody, two other additional peptides (one wherein F was substituted by A, and the other wherein F was simply deleted) were also synthetised in a soluble form. Moreover, similar competition assays were carried out with the 24C5 antibody of Hytest to demonstrate that the importance of this residue is specific of the 20G7 antibody.
(81) 1-SEQ ID NO 51: sequence of the native epitope: F.sub.11GRKMDR.sub.17
(82) 2-SEQ ID NO 62: mutated sequence of the epitope: A.sub.11GRKMDR.sub.17
(83) 3-SEQ ID NO 9: sequence deleted of the F.sub.11 residue: G.sub.12RKMDR.sub.17
(84) 5.2.1 Materials Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen) BNP(1-32): synthetic peptide (Sigma-Aldrich, USA, #B-5900) Tween® 20 (Sigma-Aldrich, USA, #P1379) monoclonal 20G7 antibody (Bio-Rad) monoclonal 24C5 antibody (HyTest, Turku, Finland) anti-mouse IgG secondary antibody produced in rabbit and coupled to peroxidase, (Sigma, USA, #A9044) H.sub.2O.sub.2 (0.04% in 0.1 M citrate buffer, pH 4) OPD (ortho-phenylenediamine, Sigma, USA, #P8412) sulphuric acid (H.sub.2SO.sub.4, 4N)
(85) 5.2.2 Methods
(86) The principle of the immunoassay was the same as the one described in 4.2. Briefly, synthetic BNP(1-32) was diluted in PBS buffer to be directly immobilised on a Maxisorp microplate at 0.5 μg/ml. A standard range from 20 to 10,000 ng/ml of each soluble peptide AA11-AA17 (native, mutated or deleted) was prepared in buffer/serum and mixed to 100 μl of monoclonal antibody solution (20G7 or 24C5 antibody), at a final concentration of 0.5 μg/ml in PBS 0.1% Tween® 20 (PBS-T) containing 0.1% milk (semi-skimmed). The binding of the antibody was then detected by an anti-mouse conjugate labelled with peroxydase. The intensity of the response of the 20G7 antibody was compared to the one of monoclonal 24C5 antibody from HyTest. The percentage of inhibition corresponding to the decrease of the recognition of BNP(1-32) in the presence of the soluble peptide was determined for each soluble peptide, in order to determine 1) that the sequence F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) effectively represents the 20G7 antibody epitope and 2) that the F.sub.11 residue was essential for the recognition of BNP(1-32) by 20G7.
(87) 5.2.3 Results
(88)
(89) It is highly remarkable to note the 20G7 antibody of the present invention behaves distinctly from the 24C5 antibody in the recognition of BNP(1-32) in the presence of the soluble AA11-AA17 peptide mutated (SEQ NO:62) or deleted (SEQ NO:9). Adding soluble peptide mutated (A.sub.11GRKMDR.sub.17(SEQ ID NO: 62)) or deleted (G.sub.12RKMDR.sub.17) (SEQ ID NO: 9) does not inhibit the recognition of BNP(1-32) by 20G7, whatever the added peptide concentration (up to 20 μg/ml), whereas a total inhibition is observed when the native peptide (F.sub.11GRKMDR.sub.17 corresponding to the sequence SEQ ID NO: 51) is added. This experiment confirms the importance of the F.sub.11 residue in the binding of the 20G7 antibody to BNP(1-32), on the contrary to the 24C5 antibody.
Example 6
Epitopic Characterisation of Other Anti-BNP(1-32) Monoclonal Antibodies—Comparison with the 20G7 Antibody
(90) 6.1. Materials, Methods and Protocols:
(91) The same nitrocellulose membranes and the same conditions of reactivity as those described in Example 5 were used to study these antibodies.
(92) 6.2. Results:
(93) The inventors thus obtained a series of following characterized monoclonal antibodies: 20G7, 11A8, 17F10, Mab1, Mab2 and Mab3. As shown in Table 1, the other monoclonal antibodies have the same epitope as the monoclonal 20G7 of the present invention, i.e. FGRKMDR (SEQ ID NO: 51), but have different essential amino acids compared to 20G7.
(94) TABLE-US-00012 TABLE 1 Characteristics of the monoclonal antibodies Monoclonal Epitope Essential antibody Immunogen (SEQ ID NO: 51) residues 20G7 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, K and R 11A8 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, G, K and R 17F10 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, K, D and R Mab1 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, G, R and K Mab2 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F and K Mab3 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, G, R and K
(95)
Example 7
Characterisation of the Antibody-Antigen Interaction of the Monoclonal Antibodies by Surface Plasmon Resonance Technology
(96) 7.1. Materials: BIAcore® 2000 & 3000 analyser (Pharmacia, Uppsala, Sweden) BNP(1-32) (synthetic peptide, Sigma, USA, #B-5900) proBNP(1-108) (recombinant protein produced in E. Coli, HyTest, Finland) anti Fc fragment antibodies (Sigma, USA) monoclonal antibodies 20G7, 11A8, 17F10 (Bio-Rad, Marnes la Coquette, France) PBS buffer (phosphate buffered saline), pH 7.4
(97) 7.2. Method:
(98) 7.2.1. Principle:
(99) The BIAcore® 2000 & 3000 analyser (the principle of which is based on the surface plasmon resonance technology (SPR)), was used to define the kinetics and the affinity of the interaction of the 20G7 monoclonal antibody and other monoclonal antibodies with BNP(1-32) or proBNP(1-108). The inventors followed the manufacturer's instructions.
(100) The surface plasmon resonance SPR technique (BIAcore®, Pharmacia) was described in its entirety in Ferrières et al. (2000, FEBS Letters, 479(3): 99-105). A monoclonal antibody was immobilised on a biosensor or a solid surface by using an anti Fc fragment antibody whilst the soluble antigen (BNP(1-32) or proBNP(1-108)) circulated at increasing concentrations (0.001256 to 0.125 μg/ml) in a constant flow on the surface of the biosensor at room temperature. The angle at which the SPR signal is detected is directly proportional to the refractive index of the medium in which the evanescent wave propagates. The variations in the refractive index are expressed in resonance units (RU, where 1000 resonance units correspond to 1 ng of fixed proteins per mm.sup.2 of active area). The quantification of the interaction and the affinity between the antigen and the monoclonal antibody is assessed by calculating the association rate constant (ka) and the dissociation rate constant (kd) by global data processing using the manufacturer's software BIAevaluation (BIAcore®, Pharmacia, Uppsala, Sweden). The equilibrium dissociation constant (KD=kd/ka) in mol/l reflects the affinity of the BNP(1-32) or proBNP(1-108) antigen for the monoclonal antibody.
(101) 7.2.2. Results:
(102) Table 2 shows the characteristics of the interaction between the monoclonal anti-BNP antibodies (including 20G7) and the two recombinant antigens BNP(1-32) and proBNP(1-108).
(103) TABLE-US-00013 TABLE 2 Interactions between various monoclonal anti-BNP antibodies and the BNP(1-32) and proBNP(1-108) antigens. BNP ka (M.sup.-1 s.sup.-1) kd (s.sup.-1) KA (M.sup.-1) KD (M) 20G7 1.40 10.sup.6 2.38 10.sup.−4 5.90 10.sup.9 1.70 10.sup.−10 11A8 8.58 10.sup.5 2.23 10.sup.−3 3.85 10.sup.8 2.59 10.sup.−9 17F10 5.84 10.sup.5 2.82 10.sup.−4 2.07 10.sup.9 4.83 10.sup.−10 ProBNP ka (M.sup.−1 s.sup.−1) kd (s.sup.−1) KA (M.sup.−1) KD (M) 20G7 1.02 10.sup.6 1.74 10.sup.−4 5.90 10.sup.9 1.69 10.sup.−10 11A8 7.63 10.sup.5 1.19 10.sup.−3 6.43 10.sup.8 1.56 10.sup.−9 17F10 9.34 10.sup.5 2.15 10.sup.−4 4.35 10.sup.9 2.30 10.sup.−10
(104) Table 2 summarises the different characteristics (association rate constant (ka) and dissociation rate constant (kd) values allowing the equilibrium dissociation constant (KD in M) of the interaction between BNP(1-32) or proBNP(1-108) and the monoclonal antibodies to be calculated. These results for the interaction confirm the data obtained with the BNP(1-32) and proBNP(1-108) assays inasmuch as the 20G7 monoclonal antibody exhibits an excellent association constant (ka) and a low dissociation constant (kd), allowing it to be characterised by an excellent affinity constant of 1.70.sup.−10 M, identical for BNP(1-32) and proBNP(1-108) (Table 2).
(105) Examples are also provided for other monoclonal antibodies (11A8 and 17F10), the affinity constants of which are in the nanomolar range (2×10.sup.−10 to 9.35×10.sup.−10 M, Table 2).
Example 8
BNP(1-32) Assay Using the 20G7 Monoclonal Antibody
(106) 8.1. Materials: Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen) BNP(1-32) synthetic peptide (Sigma-Aldrich, USA, #B-5900) proBNP(1-108) (recombinant protein produced in E. Coli, HyTest, Finland) Tween® 20 (Sigma-Aldrich, USA, #P1379) L21016 rabbit polyclonal antibody obtained by immunising rabbits with an immunogen targeting the 1-10 region of BNP(1-32), its epitope being the sequence S.sub.1PKMV.sub.5 (SEQ ID NO: 54) of BNP(1-32) 20G7 monoclonal antibody (Bio-Rad) 24C5 and 26E2 monoclonal antibodies (HyTest, Turku, Finland) anti-mouse IgG antibody conjugate produced in rabbit and coupled to peroxidase, (Sigma, USA, #A9044) 0.04% H.sub.2O.sub.2 in a 0.1 M citrate buffer, pH 4 OPD (ortho-phenylenediamine, Sigma, USA, #P8412) sulphuric acid (H.sub.2SO.sub.4, 4N)
(107) 8.2. Method and Principle:
(108) Initially, a standard range of 20 to 10,000 pg/ml of BNP(1-32) was prepared in a buffer/serum from synthetic BNP(1-32).
(109) The assay was based on the sandwich ELISA principle on a microplate, using the L21016 rabbit polyclonal antibody (Bio-Rad) for capture on a solid phase, its epitope being the sequence S.sub.1PKM V.sub.5 of BNP(1-32) fixed by passive adsorption by way of 100 μl of 5 μg/ml solution per well.
(110) 100 μl of monoclonal antibody solution (20G7, 24C5 or 26E2 antibodies) in solution at a concentration of 0.5 μg/ml in PBS 0.1% Tween® 20 (PBS-T) buffer containing 0.1% milk (semi-skimmed) were used as detection reagents. The intensity of the response of 20G7 was thus compared to that of the 24C5 and 26E2 monoclonal antibodies.
(111) Table 3 summarises the results of the analytical assay of BNP(1-32), expressed in optical density (OD) at 490 nm and obtained by said antibodies in the presence of standard concentrations of BNP(1-32).
(112) TABLE-US-00014 TABLE 3 OD values obtained during the analytical assay of BNP(1-32) with different antibodies BNP(1-32) (pg/ml) 20G7 24 C5 26 E2 10,000 3.752 0.077 0.048 5,000 3.056 0.068 0.041 2,500 1.950 0.067 0.035 1,250 1.111 0.056 0.031 625 0.625 0.059 0.029 312.5 0.404 0.063 0.027 156.25 0.180 0.071 0.032 78 0.099 0.052 0.055 39 0.066 0.055 0.032 20 0.031 0.068 0.034 0 0.024 0.071 0.022
(113) It is highly remarkable to note that the two antibodies 24C5 and 26E2 behave quite differently from the 20G7 antibody of the present invention. These results thus confirm that in the latter assay format, 20G7 is much more suitable than the 24C5 and 26E2 antibodies for BNP(1-32) assay.
(114) The standard range shown in
Example 9
Study of the Complementarity of Monoclonal Antibodies in the Sandwich ELISA
(115) 9.1. Materials: Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) primed by the L21016 rabbit polyclonal antibody (Bio-Rad) which recognises the sequence S.sub.1PKM V.sub.5 (SEQ ID NO: 54) of BNP(1-32) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen) Tween®20 (Sigma-Aldrich, USA, #P1379) BNP(1-32) synthetic peptide (Sigma-Aldrich, USA, #B-5900) proBNP(1-108) (recombinant protein produced in E. Coli, HyTest, Finland) 20G7 antibody (directed against the epitope F.sub.11GRKMDR.sub.17 (SEQ ID NO: 8) prepared in different concentrations from 0.001 to 1 μg/ml 50B7 monoclonal antibody (HyTest, Finland) which recognises the C-terminal portion of BNP(1-32) at a single concentration of 0.5 μg/ml anti-mouse IgG secondary antibody produced in rabbit and coupled to peroxidase, (Sigma, USA, #A9044) 0.04% H.sub.2O.sub.2(in 0.1 M citrate buffer, pH 4, Sigma, USA) OPD (ortho-phenylenediamine, Sigma, USA, #P8412) sulphuric acid (H.sub.2SO.sub.4, 4N)
(116) 9.2. Method:
(117) 9.2.1. Principle:
(118) The assay was based on the sandwich ELISA principle on a microplate, using the L21016 rabbit polyclonal antibody (Bio-Rad) for capture on a solid phase, its epitope being the sequence S.sub.1PKMV.sub.5 (SEQ ID NO: 54) of BNP(1-32) fixed by passive adsorption (see Example 8), and a combination of two monoclonal antibodies (the 20G7 monoclonal antibody directed against the epitope F.sub.11GRKMDR.sub.17 (SEQ ID NO: 8) and the 50B7 monoclonal antibody (HyTest, Finland) which targets the C-terminal portion of BNP(1-32)) for detection. However, the 20G7 antibody was used at variable concentrations while the 50B7 monoclonal antibody was used at a constant concentration of 0.5 μg/ml.
(119) The epitopic complementarity of the 20G7 and 50B7 antibodies was studied at variable concentrations of the 20G7 antibody. This format allowed the cooperativity of the two monoclonal antibodies to be assessed in order to improve the BN P(1-32) detection.
(120) 9.2.2. Protocol:
(121) 100 μl of 5 ng/ml BNP(1-32) solution was added into each microplate well, in which the L21016 polyclonal antibody was adsorbed, and was left to incubate for two hours at 37° C. The microplate was washed three times with 0.1% PBS-T, then 100 μl of a mixture containing the 50B7 antibody and one of the 20G7 antibody dilutions were distributed thereon, and it was left to incubate for 2 hours at 37° C. After three washes with 0.1% PBS-T, the peroxidase—rabbit anti-mouse IgG antibody conjugate (diluted to a 1/3,000th with 0.1% PBS-T and containing 0.1% milk (semi-skimmed), on the basis of 100 μl per well, was left to incubate for 1 hour at 37° C. Finally, after 3 washes with 0.1% PBS-T, the H.sub.2O.sub.2+OPD solution was deposited on the basis of 100 μL/well. The microplate was placed in darkness at room temperature for 20 minutes. The enzymatic reaction was stopped by adding 50 μL of sulphuric acid (H.sub.2SO.sub.4, 4 N) per well, and the OD at 490 nm was subsequently measured in each well.
(122) 9.2.3. Results
(123) Table 4 shows the results of the analytical assay of BNP using the 20G7 and 50B7 monoclonal antibodies, expressed in optical density at 490 nm.
(124) TABLE-US-00015 TABLE 4 Cooperativity of the two monoclonal antibodies for detecting BNP(1-32) 20G7 range Optical Density (μg/ml) 490 nm 0.5 3.792 0.1 3.753 0.05 3.747 0.01 3.531 0.005 3.272 0.001 1.948 0 1.296
(125) It was noted that there is synergism between the two antibodies. In other words, it was noted that the effects of the two antibodies were added: in the absence of 20G7, the signal (OD) was limited to 1.296, and the more 20G7 was added, the more the resulting signal (OD) increased.
(126) By using both the 20G7 monoclonal antibody, which targets the F.sub.11GRKMDR.sub.17 (SEQ ID NO: 8) epitope according to the invention (located in the loop of BN P(1-32)), and the 50B7 monoclonal antibody, which targets the C-terminal region of BNP(1-32), BNP(1-32) detection is significantly improved. This demonstrates the cumulative or cooperative contribution of the two monoclonal antibodies used in detection.
(127) This kind of complementarity may also be envisaged between the 20G7 monoclonal antibody and other antibodies which recognise an epitope located in other positions in the BNP(1-32) sequence (principally in the N-terminal position, C-terminal position). The number of antibodies used may also be greater than two as long as no steric hindrance problems are encountered.
Example 10
proBNP(1-108) Assay Using the 20G7 Monoclonal Antibody
(128) 10.1. Materials: BNP(1-32) synthetic peptide (Sigma-Aldrich, USA, #B-5900) proBNP(1-108) (recombinant protein produced in E. Coli, HyTest, Finland). Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) primed with monoclonal antibodies (hinge 76 antibody, for example, from Bio-Rad) which recognises the proBNP(1-108) hinge sequence: epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55) (Giuliani et al., Clin. Chem., 52: 6, 1054-1061, 2006) 20G7 monoclonal antibody (Bio-Rad) coupled to peroxidase 24C5 monoclonal antibody (HyTest, Finland) coupled to peroxidase 26E2 monoclonal antibody (HyTest, Finland) coupled to peroxidase PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen) Tween®20 (Sigma-Aldrich, USA, #P1379)
(129) 10.2. Method:
(130) 10.2.1. Principle of the Analytical Assay of proBNP(1-108):
(131) Initially, a standard range of 20 to 10,000 pg/ml of proBNP(1-108) was prepared in a 0.1% PBS-T buffer from recombinant ProBNP(1-108).
(132) The assay is based on the sandwich ELISA principle on a microplate, using for capture on a solid phase a monoclonal antibody (hinge 76 antibody, for example, from Bio-Rad), and recognising the hinge sequence of proBNP(1-108): epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55) fixed by passive adsorption by way of 100 μl of 0.5 μg/ml solution per well.
(133) 100 μl of monoclonal antibody solution (20G7, 24C5 or 26E2 antibodies) at a concentration of 0.5 μg/ml and coupled to peroxidase in solution in 0.1% PBS-T buffer (containing 0.1% semi-skimmed milk) were used as detection antibodies.
(134) Besides this technical point, the protocol was identical to that of the ELISA in Example 8. The detection characteristics of 20G7 were thus compared with those of the 24C5 and 26E2 monoclonal antibodies.
(135) Table 5 shows the results of the analytical assay of proBNP(1-108) which are expressed in optical density (OD) and were obtained by using said antibodies in the presence of standard concentrations of proBNP(1-108).
(136) 10.3. Results:
(137) TABLE-US-00016 TABLE 5 Analytical assay of proBNP(1-108) with different antibodies ProBNP(1-108) 24 C5 26 E2 (pg/ml) 20G7 (Hytest) (Hytest) 10,000 >4 0.220 0.158 5,000 3.845 0.117 0.041 2,500 3.272 0.069 0.031 1,250 1.955 0.068 0.024 625 0.997 0.070 0.024 312.5 0.512 0.035 0.028 156.25 0.264 0.055 0.027 78 0.126 0.056 0.033 39 0.092 0.056 0.028 20 0.069 0.052 0.028 0 0.045 0.044 0.024
(138) It is highly remarkable to note that the 20G7 antibody of the present invention detects not only BNP(1-32) but also proBNP(1-108). Moreover, in this case too, the two HyTest antibodies behave quite differently from the 20G7 antibody. This confirms the significant benefit of the 20G7 antibody in BNP(1-32) and proBNP(1-108) assays.
(139)
Example 11
proBNP(1-108) and BNP(1-32) Assays Using Other Monoclonal Antibodies Obtained by the Inventors
(140) Table 6 shows the results produced, in accordance with the two ELISA protocols from examples 8 and 10, with the 11A8 and 17F10 monoclonal antibodies.
(141) These labelled monoclonal antibodies used in detection are highly capable of detecting BNP(1-32) and proBNP(1-108) when a rabbit polyclonal (L21016) which targets the .sup.1SPKMV.sup.5 (SEQ ID NO: 54) region or the hinge 76 antibody respectively, are used for capture.
(142) Table 6 shows the results of the analytical assays of proBNP(1-108) and BNP(1-32) which are expressed in optical density and were obtained by said antibodies in the presence of standard concentrations of proBNP(1-108) and BNP(1-32) respectively.
(143) TABLE-US-00017 TABLE 6 Analytical assay of proBNP(1-108) and BNP(1-32) with different monoclonal antibodies BNP(1-32) or proBNP(1- proBNP(1-108) BNP(1-32) 108) (pg/ml) 17F10 11A8 17F10 11A8 10,000 3.769 3.734 3.717 3.693 5000 3.808 3.779 2.712 2.534 2,500 3.453 3.024 1.495 1.481 1,250 2.118 1.406 0.828 0.871 625 0.937 0.618 0.341 0.389 312.5 0.489 0.259 0.172 0.211 156 0.242 0.130 0.095 0.123 78 0.139 0.088 0.070 0.114 39 0.125 0.087 0.059 0.097 20 0.100 0.078 0.059 0.071 0 0.076 0.063 0.059 0.068
(144) It is highly remarkable to note that the 17F10 and 11A8 antibodies of the present invention detect not only BNP(1-32) but also proBNP(1-108).
Example 12
BNP(1-32) Assay and proBNP(1-108) Assay in Subjects with Congestive Heart Failure and in Normal Subjects
(145) 12.1. Samples: 55 EDTA plasmas from subjects with congestive heart failure, who belonged to one of NYHA (New York Heart Association) classes Ito III and had signed a voluntary consent form, originating from a commercial source (ProMedex, NY, USA). The studied population is as follows: 10 patients of NYHA class I, 21 patients of NYHA class II and 24 patients of NYHA class III. 48 EDTA plasmas from normal subjects (healthy volunteers, ProMedex, NY, USA).
(146) 12.2. Materials and Methods for the BNP(1-32) and the proBNP(1-108) Assays
(147) The materials and methods used were identical to those described above in 8.2 for BNP(1-32), and to those described above in 10.2 for the proBNP(1-108).
(148) 12.3 Results
(149) 12.3.1. Results of the BNP(1-32) and the proBNP(1-108) Assays in Patients with Congestive Heart Failure
(150) The BNP(1-32) values obtained from plasmas from patients with congestive heart failure, by means of the BNP(1-32) assay disclosed in 8.2, were found to correlate with those of the proBNP(1-108) assay according to the invention (r.sup.2=0.935,
(151) More in detail, correlations are maintained when the patients are studied according to their NYHA class (r.sup.2=0.997, r.sup.2=0.903, r.sup.2=0.832 respectively for the NYHA classes I, II and III,
(152) Thus, these results with the 20G7 antibody confirm once more the usefulness of the BNP(1-32) or the proBNP(1-108) assay as a marker of congestive heart failure.
(153) These experiments were reproduced with the 24C5 and 26E2 antibodies (HyTest) in a labelled form, but no correlation was observed.
(154) 12.3.2. Results of the BNP(1-32) and the proBNP(1-108) Assays in Healthy Subjects
(155) The proBNP(1-108) values obtained from plasmas from healthy subjects, by means of the proBNP(1-108) assay using the monoclonal hinge 76 antibody in the solid phase and the 20G7 antibody-peroxidase conjugate for detection, were found to be highly correlated (r.sup.2=0.702) with those of the BNP(1-32) assay using the 20G7 antibody in detection (
(156) In conclusion, it is therefore very clear that the 20G7 antibody according to the invention is entirely appropriate for BNP(1-32) and proBNP(1-108) assays in patients suffering from congestive heart failure, by detecting a higher amount of BNP(1-32) and proBNP(1-108) in patients suffering from congestive heart failure than in healthy subjects (Table 7).
(157) TABLE-US-00018 TABLE 7 proBNP(1-108) (pg/ml) BNP(1-32) (pg/ml) Healthy subjects 37 ± 32 227 ± 172 NYHA patients 762 ± 839 1716 ± 1754
Example 13
BNP(1-32) and proBNP(1-108) Assays in Patients Suffering from Renal Failure
(158) 13.1. Samples:
(159) EDTA plasmas from 33 patients with renal failure who has signed a voluntary consent form, originating from Lapeyronie hospital, Montpellier, France.
(160) 13.2. Materials and Methods for the BNP(1-32) and the proBNP(1-108) Assays
(161) The materials and methods used were identical to those described above in 8.2 for BNP(1-32) and in 10.2 for proBNP(1-108).
(162) 13.3 Results
(163) The proBNP(1-108) values obtained from plasmas of patients suffering of renal failure, by means of the proBNP(1-108) assay using the hinge 76 antibody in the solid phase and 20G7 antibody-peroxydase conjugate in detection, were found to be strongly correlated (r.sup.2=0.899) to those of the BNP(1-32) assay using the 20G7 antibody according to the invention (
Example 14
proBNP(1-108) Assay in Patients with Ischemic Stroke
(164) 14.1. Patients Samples: 32 citrated plasma samples from patients with ischemic stroke admitted to the Emergency Department within 3 hours of the Stroke onset were tested. The stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS). 42 citrated plasma samples from apparently healthy blood donor matched by gender and age with the patients from the Stroke population were tested.
All the citrated plasma samples were stored at −80° C. Prior the analysis, the samples were thawed and centrifuged at 3000 g for 15 min at 4° C.
(165) 14.2. Material and Method:
(166) All the samples were tested with the BioPlex™ 2200 proBNP assay (Bio-Rad).
(167) 14.2.1. Principe of the Technology:
(168) The BioPlex™ 2200 combines multiplex, magnetic bead and flow cytometry technologies to provide multi-analyte detection on a fully automated random access platform. Magnetic particles (8 μm diameter, carboxyl-modified surface) are dyed with two fluorophores (classification dyes, CL1 and CL2) which emit at distinct wavelengths and adsorb significantly at 635 nm. The reporter fluorophore, β-phycoerythrin (PE) was chosen for its high molar extinction coefficient, quantum yield, resistance to photobleaching, lack of self-quenching and stability. The detector simultaneously measures light at three wavelengths: the two classification dyes and the reporter dye.
(169) 14.2.2. BioPlex™ 2200 proBNP: Assay Principle
(170) The BioPlex™ 2200 proBNP assay is a two-step sandwich fluorescence immunoassay. In a first step, the BioPlex™ 2200 system combines 50 μL of patient sample, magnetic dyed beads coated with the anti-proBNP(1-108) monoclonal antibody (hinge 76 monoclonal antibody recognizing the epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 58), Bio-Rad) and assay buffer into a reaction vessel. Then, after 11 minutes of incubation and wash cycles, the anti-human BNP monoclonal antibody 20G7 conjugated to phycoerythrin (PE) is added and incubated for 2 minutes. After removal of excess conjugate, the bead mixture is passed through the detector which identifies the dyed beads and the amount of antigens captured on the beads by the fluorescence of PE. After calibration using a set of six distinct calibrators, the three levels of quality controls and patient samples results are expressed in pg/mL.
(171) Two Quality Control beads are also tested with each sample to enhance the integrity of the overall system.
(172) 14.3. Results:
(173) The distributions of the BioPlex™ 2200 proBNP values for the Control and the ischemic stroke populations are shown in Table 8 and
(174) TABLE-US-00019 TABLE 8 BioPlex ™ 2200 proBNP concentrations in ischemic stroke and control citrated plasma samples (minimum, 1.sup.st quartile, median, 31.sup.rd quartile and maximum values). Minimum 1.sup.st Quartile Median 3.sup.rd Quartile Maximum Populations (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) Control population 0 0 1 2 23 (N = 42) (IC95%: 0-2) Ischemic 2 34 71 219 1019 Stroke population (IC95%: 38-145) (N = 32)
(175) The inventors clearly demonstrate that the proBNP(1-108) sandwich assay using the monoclonal antibody 20G7 described in this invention can measure proBNP(1-108) concentrations in patients with stroke.
Example 15
proBNP(1-108) Assay in Patients Suffering from an Acute Coronary Disorder
(176) 15.1. Samples: EDTA plasmas from 27 patients with an acute coronary disorder (with Troponine I plasma mean values reaching 12.5±6.9 ng/ml) originating from a commercial source (ProMedex, NY, USA) EDTA plasmas from 48 healthy subjects (healthy volunteers, ProMedex, NY, USA).
(177) 15.2. Materials and Methods
(178) The materials and methods used for the BNP(1-32) assay were identical to those described above in 8.2, and for the proBNP(1-108) in 10.2.
(179) 15.3 Results
(180) The proBNP(1-108) values obtained from plasmas of patients admitted in emergency and diagnosed for an acute coronary disorder by means of the proBNP(1-108) assay described above, were found to be strongly correlated (r.sup.2=0.956) to those obtained with the BNP(1-32) assay using the 20G7 antibody according to the invention (
(181) It is therefore very clear that the sandwich assay using the 20G7 antibody according to the invention (coupled to peroxidase) allows proBNP(1-108) concentrations, which are proportional to the level of BNP(1-32), to be measured. These results once again prove the usefulness of assaying proBNP(1-108) and BNP(1-32) as markers, in particular as markers of an acute coronary disorder.
Example 16
Assay of proBNP(1-108) in the Glycosylated Form Using the 20G7 Monoclonal Antibody
(182) 16.1. Materials: proBNP(1-108) (recombinant protein produced in E. coli for the non-glycosylated form, and from reprogrammed HEK293 cells for the glycosylated form, HyTest, Finland) Maxisorp™ 96-well flat-bottomed microplate (Nunc, Denmark) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen) Tween®20 (Sigma-Aldrich, USA, #P1379) hinge 76 monoclonal antibody which recognises the hinge sequence of proBNP(1-108): epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55) (Giuliani et al., Clin. Chem., 52: 6, 1054-1061, 2006) 20G7 monoclonal antibody (Bio-Rad) coupled to peroxidase
(183) 16.2. Method:
(184) 16.2.1. Principle of the Analytical Assay of Glycosylated proBNP:
(185) A standard range of 20 to 10,000 pg/ml of glycosylated proBNP(1-108) was prepared in a 0.1% PBS-T buffer. A standard range of 20 to 10,000 pg/ml of non-glycosylated proBNP(1-108) was prepared in the same way in a 0.1% PBS-T buffer.
(186) The assay is based on the sandwich ELISA principle on a microplate, using for capture on a solid phase the monoclonal antibody (hinge 76 antibody, from Bio-Rad), recognising the epitopic sequence RAPR.sub.76S.sub.77P (SEQ ID NO: 55) of proBNP(1-108) and fixed by passive adsorption by way of 100 μl of 0.5 μg/ml solution per well.
(187) 100 μl of a solution of the 20G7 monoclonal antibody according to the invention coupled to peroxidase at a concentration of 0.5 μg/ml, in solution in 0.1% PBS-T containing 1% milk (semi-skimmed), were used as detection reagents. The rest of the protocol was identical to that of the ELISA in Example 10. The detection characteristics of the 20G7 antibody were thus compared for the two forms of proBNP(1-108)—glycosylated and non-glycosylated.
(188) 16.3. Results:
(189) The results shown in Table 9 and
(190) TABLE-US-00020 TABLE 9 optical density values at 490 nm from the assay of each proBNP(1-108) form tested. proBNP non-glycosylated glycosylated range (pg/ml) proBNP(1-108) proBNP(1-108) 10,000 4 3.833 5000 3.807 3.801 2,500 3.829 3.023 1,250 3.256 1.679 625 1.816 0.925 312.5 0.989 0.465 156 0.510 0.268 78 0.221 0.130 39 0.109 0.087 20 0.063 0.041 9 0.037 0.028 0 0.021 0.023
(191) ProBNP(1-108) can be detected just as well in the non-glycosylated form as in the glycosylated form by using 20G7. The signal/background ratio is slightly greater for non-glycosylated proBNP than for its glycosylated form (signal/background ratios of 3 and 1.8 respectively are obtained at 20 pg/ml).
Example 17
Immunoreactivity of proBNP(1-108) in its Glycosylated Form and Implications for the proBNP(1-108) Assay with the Hinge 76 Antibody
(192) 17.1. Materials: ProteOn XPR36 analyser (surface plasmon resonance SPR technology, Bio-Rad, USA) proBNP(1-108) (recombinant protein, HyTest, Finland) in the glycosylated and non-glycosylated forms (13 to 200 nM) anti Fc fragment antibodies (Sigma, USA) monoclonal hinge 76 antibody (Bio-Rad) which recognises the epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55) and is used at a concentration of 30 μg/ml in 0.1% PBS-T PBS buffer (phosphate buffered saline), pH 7.4
(193) 17.2. Method:
(194) 17.2.1. Principle:
(195) The ProteOn XPR36 analyser (the principle of which is based on the surface plasmon resonance technology (SPR)), was used to define the kinetics and the affinity of the interaction of the 20G7 monoclonal antibody and with glycosylated and non-glycosylated proBNP(1-108) forms. The inventors followed the manufacturer's instructions. A monoclonal antibody was immobilised on a biosensor (solid surface) by using an anti Fc fragment antibody whilst the glycosylated or non-glycosylated soluble antigen proBNP(1-108) circulated at increasing concentrations (13 to 200 nM) in a constant flow on the surface of the biosensor at room temperature. The angle at which the SPR signal is detected is directly proportional to the refractive index of the medium in which the evanescent wave propagates. The variations in the refractive index are expressed in resonance units (RU, where 1000 resonance units correspond to 1 ng of fixed proteins per mm.sup.2 of surface area). The quantification of the interactions and the affinity between the antigen and the monoclonal antibody is assessed by calculating the association rate constant (ka) and the dissociation rate constant (kd) by global data processing using the device software (Bio-Rad). The equilibrium dissociation constant (KD=kd/ka) in mol/l reflects the affinity of the glycosylated or non-glycosylated proBNP(1-108) antigen for the monoclonal antibody.
(196) 17.2.2. Results:
(197) Table 10 shows the characteristics of the interaction between the anti-hinge antibody (hinge 76 antibody with the epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55) Bio-Rad) and glycosylated and non-glycosylated proBNP(1-108). Although the affinity constant between the anti-hinge antibody and non-glycosylated proBNP(1-108) was greater (1.73.10.sup.−10) than that for glycosylated proBNP(1-108) (2.35.10.sup.−8), the antibody targeting the hinge region of proBNP(1-108) recognised both the glycosylated and non-glycosylated forms with high affinity.
(198) TABLE-US-00021 TABLE 10 Reactivity of the hinge 76 antibody towards glycosylated and non- glycosylated proBNP(1-108) Hinge 76 antibody ka kd KD = kd/ka (M.sup.−1s.sup.−1) (s.sup.−1) M non-glycosylated proBNP(1-108) 1.12.10.sup.6 1.94.10.sup.−4 1.73.10.sup.−10 glycosylated proBNP(1-108) 1.17.10.sup.5 2.72.10.sup.−3 2.35.10.sup.−8
Example 18
Biepitopic and Triepitopic Calibrators
(199) 18.1. The structure of all of the biepitopic and triepitopic calibrators according to the invention may be linear or branched, provided that the immunoreactivity of the incorporated epitopes is preserved.
(200) The synthesis protocols which may be used to produce these calibrators are those in the field of organic chemistry of peptides well known to the one skilled in the art (in this context, see “Peptide synthesis” in Example 1).
(201) For the epitopes E.sub.2 and E.sub.3, the linear peptide sequences according to the invention may be selected in a non-limiting manner from the group consisting of the following sequences:
(202) TABLE-US-00022 SEQ ID NO: 56: PRSPKMVQG SEQ ID NO: 57: APRSPKMV SEQ ID NO: 58: SGLGCKLV SEQ ID NO: 59: SPKMVQGSG SEQ ID NO: 60: YTLRAPRSPKMVG
(203) 18.2. Examples of Synthesised Epitopes According to the Invention
(204) The inventors synthesised the following calibrators:
(205) 18.2.1. Biepitopic Calibrators:
(206) TABLE-US-00023 CaliproBNP1: (SEQ ID NO: 66 and SEQ ID NO: 64) Ac-YTLRAPRSPKMV-Ahx-SFGRKMDRISS-CONH.sub.2 CaliproBNP2: (SEQ ID NO: 66 and SEQ ID NO: 65) Ac-YTLRAPRSPKMV-Ahx-CFGRKMDRISSSSGLGCK-CONH.sub.2 CaliProBNP3: (SEQ ID NO: 67 and SEQ ID NO: 51) Ac-YTLRAPRSPKMVQG-Ahx-FGRKMDR-CONH.sub.2
(207) These three biepitopic calibrators can be used to calibrate a proBNP(1-108) assay, as described above in 10.2, based on the immobilisation in a solid phase of the monoclonal hinge 76 antibody that recognizes the RAPRSP (SEQ ID NO: 55) (Giuliani et al., supra) and, in detection the monoclonal 20G7 antibody-peroxydase for example.
(208) TABLE-US-00024 (SEQ ID NO: 51 and SEQ ID NO: 68) CaliproBNP4: Ac-FGRKMDR-Ahx-SGLGC*KVLRRH-COOH (SEQ ID NO: 51 and SEQ ID NO: 69) CaliproBNP4b: Ac-FGRKMDR-Ahx-SGLGC*KVLR-CONH.sub.
(209) These two biepitopic calibrators can be used to calibrate a BNP(1-32) assay, based on the immobilisation in a solid phase of the monoclonal antibody directed the C-terminal portion of BNP(1-32), such as for example monoclonal antibodies 50B7 or 50E1 (HyTest, Finland) and, in detection the monoclonal 20G7 antibody-peroxydase for example.
(210) TABLE-US-00025 (SEQ ID NO: 59 and SEQ ID NO: 51) CaliproBNP5: Ac-SPKMVQGSG-Ahx-FGRKMDR-CONH.sub.2
(211) This biepitopic calibrator can be used to calibrate a BNP(1-32) assay, as described above in 8.2, based on the immobilization in a solid phase of the polyclonal antibody L21016 (Bio-Rad) and, in detection the monoclonal 20G7 antibody-peroxydase for example. In the calibrators sequences described above, the structural formula of the Ahx group is NH—(CH.sub.2).sub.5—CO.
(212) The binding group of formula —NH—(CH.sub.2).sub.5—CO— is derived from a well-known coupling agent, amino-hexanoic acid (AHX), which enables to covalently couple two peptide sequences together.
(213) C*=C(Acm)=Cysteine blocked by an acetamidomethyl (protecting group, well-known from the one skilled in the art).
(214) 18.2.2. Triepitopic Calibrators:
(215) TABLE-US-00026 (SEQ ID NO: 66 and SEQ ID NO: 51 and SEQ ID NO: 68) CaliproBNP6: Ac-YTLRAPRSPKMV-Ahx-FGRKMDR-Ahx-SGLGC*KVLRRH-COOH (SEQ ID NO: 66 and SEQ ID NO: 51 and SEQ ID NO: 69) CAliproBNP6b: Ac-YTLRAPRSPKMV-Ahx-FGRKMDR-Ahx-SGLGC*KVLR-CONH.sub.2
(216) These two triepitopic calibrators can be used to calibrate both a proBNP(1-108) and a BNP(1-32) assay, using antibodies such as the monoclonal hinge 76 antibody that recognizes the sequence RAPRSP (SEQ ID NO: 55) (Giuliani et al., supra) and the monoclonal 20G7 antibody of the invention and an antibody with an epitope directed against the C-terminal portion of BNP(1-32) for example.
(217) 18.3. Materials and Methods
(218) To depict the usefulness of these calibrators, results of calibration and stabilities are displayed in two different formats, BioPlex™ assay and ELISA assay. The proBNP(1-108) biepitopes, the CaliproBNP1 and CaliproBNP3 compounds, were tested by means of the BioPlex™ 2200 proBNP assay as described in Example 14. The BNP(1-32) biepitope, the CaliproBNP5 compound, was tested by means of the assay described in Example 8.2 and the triepitope, the CaliproBNP6 compound was tested by means of the assay described in Example 10.2.
(219) 18.4. Protocole
(220) The different compounds were tested at different concentrations diluted in 0.1 M succinate buffer, pH 7.6, containing 5% BSA, 2 mM CaCl.sub.2, 10% antiproteases cocktail (Sigma reference P2714), 0.1% Proclin, 0.095% NaN.sub.3 and 0.1% sodium benzoate.
(221) The CaliproBNP1 calibrator was diluted to 1 μg/mL, 0.2 μg/mL, 0.1 μg/mL, and 0.02 μg/mL, the CaliproBNP3 calibrator was diluted to 1.6 μg/mL, 0.8 μg/mL, 0.3 μg/mL, 0.06 μg/mL, the CaliproBNP5 and CaliproBNP6 calibrators were diluted to from 100 ng/ml to 0.01 ng/ml in the same 0.1 M succinate buffer, pH 7.6.
(222) The compounds stability was studied in accelerated condition (room temperature) compared to synthetic BNP(1-32) (Sigma-Aldrich, Etats-Unis, #B-5900) and recombinant proBNP(1-108) (HyTest Ref. 8PRO8) in the following way:
(223) Recombinant proBNP(1-108), synthetic BNP(1-32) and calibrator peptides were diluted in 0.1 M succinate buffer, pH 7.6 described above. Each solution was divided in 10 tubes placed at room temperature (20° C.±5° C.). Then one tube of each solution was frozen at J0, J+7, J+14, J+21. At J+21, the different solutions were thawed and assayed by means of the BioPlex™ 2200 proBNP assay described in Example 14 or of the immunoassays described in Examples 8.2 and 10.2.
(224) In the ELISA assay, a range of proBNP(1-108), BNP(1-32) and CaliproBNP6 from 25 ng/mL to 0.04 ng/mL was tested. For CaliBNP4 and CaliBNP5, the tested range was from 2 ng/mL to 0.004 ng/mL.
(225) In the BioPlex™ 2200 proBNP assay, the stability of two concentrations of proBNP(1-108), 10 ng/mL and 1 ng/mL, of CaliproBNP1, 2 μg/mL and 0.125 μg/mL and CaliproBNP3, 1 μg/mL and 0.3 μg/mL, were analyzed. In ELISA format, three concentrations of proBNP(1-108) and BNP(1-32), 1.56 ng/mL, 0.78 ng/ml and 0.39 ng/mL, and of CaliproBNP5, 62.5 pg/mL, 31 ng/mL and 15.6 ng/mL, were analysed.
(226) 18.5. Results
(227) 18.5.1 CaliproBNP1 and CaliproBNP3 Calibrators in the BioPlex™ 2200 proBNP Assay
(228) The results of the assay of the CaliproBNP1 and CaliproBNP3 compounds in range of different concentrations are displayed in
(229) In a BioPlex™ 2200 proBNP assay, the CaliproBNP1 and CaliproBNP3 compounds enable to generate increasing signal with the compound concentration. These compounds are therefore usable as calibrators of the proBNP(1-108) assay, once standardized on the proBNP(1-108) molecule.
(230) The results of the accelerated stability test of the recombinant proBNP(1-108) and the CaliproBNP1 and CaliproBNP3 compounds in range of different concentrations are displayed in Table 11.
(231) TABLE-US-00027 TABLE 11 Liquid stability* at room temperature** in buffer*** Concentration expressed by the ratio “signal at J0 + X/signal at J0” Compound (μg/mL) J0 + 7 J0 + 14 J0 + 21 recombinant 0.001 0.79 0.65 0.51 proBNP(1-108) 0.01 0.85 0.72 0.69 CaliproBNP1 0.125 0.93 0.92 0.95 2 0.95 0.93 0.91 CaliproBNP3 0.3 0.91 0.98 1.15 1 0.92 1.02 1.06 *Norme of acceptance of stabilities: to make the stability at day X after J0 acceptable, the ratio signal at J0 + X/signal at J0 has to be equal to 1.00 ± 0.2. **Room temperature: 20° C. ± 5° C. ***0.1M succinate buffer, pH 7.6, containing 5% BSA, 2 mM CaCl.sub.2, 10% antiproteases cocktail (Sigma reference P2714), 0.1% Proclin, 0.095% NaN3 and 0.1%. sodium benzoate.
(232) Biepitopic calibrators CaliproBNP1 and CaliproBNP3 clearly show a higher stability than recombinant proBNP(1-108).
(233) 18.5.2 CaliproBNP5 and CaliproBNP6 Calibrators in Immunoassays Based on the Use of 20G7 Antibody
(234) The results of the test of the CaliproBNP5 and CaliproBNP6 compounds in range of different concentrations are displayed in
(235) In the BNP(1-32) and the proBNP(1-108) assay, the biepitopic compound CaliproBNP5 and the triepitopic compound CaliproBNP6 enable to generate a signal increasing with the compound concentration. These compounds are therefore usable as calibrators of the proBNP(1-108) and the BNP(1-32) assay.
(236) The results of the accelerated stability assay of recombinant proBNP(1-108), BNP(1-32) and the CaliproBNP5 compound, in range of different concentrations are displayed in Table 12.
(237) TABLE-US-00028 TABLE 12 Liquid stability* at room temperature** in buffer*** Concentration expressed by the ratio “signal at J0 + X/signal at J0” Compound (ng/mL) J0 + 7 J0 + 14 J0 + 21 recombinant 1.56 0.58 0.5 0.45 proBNP(1-108) 0.78 1.08 0.82 0.88 0.39 0.71 0.66 0.79 synthetic 1.56 0.71 0.7 0.55 BNP(1-32) 0.78 0.69 0.65 0.53 0.39 0.81 0.7 0.53 CaliproBNP5 0.0625 1.02 1.02 1.1 0.031 1.06 1.01 1.06 0.0156 1.1 1.01 1.10 *Norme of acceptance of stabilities: to make the stability at day X after J0 acceptable, the ratio signal at J0 + X/signal at J0 has to be equal to 1.00 ± 0.2. **Room temperature: 20° C. ± 5° C. ***0.1M succinate buffer, pH 7.6, containing 5% BSA, 2 mM CaCl.sub.2, 10% antiproteases cocktail (Sigma reference P2714), 0.1% Proclin, 0.095% NaN3 and 0.1%. sodium benzoate.
(238) Once again, the CaliproBNP5 biepitopic calibrator displays a higher stability than recombinant proBNP(1-108) and BNP(1-32).
(239) TABLE-US-00029 Summary table of the sequences SEQ ID NO: SEQUENCES 1 HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQES PRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFG RKMDRISSSSGLGCKVLRRH 2 SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH 3 HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQES PRPTGVWKSREVATEGIRGHRKMVLYTLRAPR 4 TGCFGRKMDRISTSTAIGCKVL 5 SGCYGRKMDRISTSTAIGCKVL 6 SGCFGRKMDRISSSSGLGCKVL 7 SGCFGRKMDRIATSTAIGCKVL 8 FGRKMDR 9 GRKMDR 10 FGRKMD 11 RKMDRI 12 SPKMVQGSGCFGRKM 13 KMVQGSGCFGRKMDR 14 VQGSGCFGRKMDRIS 15 GSGCFGRKMDRISSS 16 GCFGRKMDRISSSSG 17 FGRKMDRISSSSGLG 18 RKMDRISSSSGLGCK 19 MDRISSSSGLGCKVL 20 RISSSSGLGCKVLRR 21 VQGSGCFGR 22 SPKMVQGSGC 23 MDRISSSSGLG 24 RKMDRI 25 RKMDRISS 26 SPKMVQGSGC 27 PKMVQGSGCF 28 KMVQGSGCFG 29 MVQGSGCFGR 30 VQGSGCFGRK 31 QGSGCFGRKM 32 GSGCFGRKMD 33 SGCFGRKMDR 34 GCFGRKMDRI 35 CFGRKMDRIS 36 FGRKMDRISS 37 GRKMDRISSS 38 RKMDRISSSS 39 KMDRISSSSG 40 MDRISSSSGL 41 DRISSSSGLG 42 RISSSSGLGC 43 ISSSSGLGCK 44 SSSSGLGCKV 45 SSSGLGCKVL 46 SSGLGCKVLR 47 SGLGCKVLRR 48 GLGCKVLRRH 49 GCFGRKM 50 CFGRKMD 51 FGRKMDR 52 GRKMDRI 53 RKMDRIS 54 SPKMV 55 RAPRSP 56 PRSPKMVQG 57 APRSPKMV 58 SGLGCKVL 59 SPKMVQGSG 60 YTLRAPRSPKMVG 61 FGRKMDRISSSS 62 AGRKMDR 63 GCFGRKMDRIS 64 SFGRKMDRISS 65 CFGRKMDRISSSSGLGCK 66 YTLRAPRSPKMV 67 YTLRAPRSPKMVQG 68 SGLGCKVLRRH 69 SGLGCKVLR