Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/26—Immunoglobulins [IG], 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

• the present invention relates to an antibody against the N-terminal fragment of the precursor of B-type natriuretic peptide (NT-proBNP), a method for detecting NT-proBNP using the antibody, and a composition and kit for use in detecting NT-proBNP.
• NT-proBNP B-type natriuretic peptide
• Heart failure is a disease that progresses due to heart disease and aging, and its incidence continues to increase in an ultra-aging society.
• electrocardiograms, chest X-rays, echocardiograms, and biomarker measurements are used to diagnose heart failure and understand its pathology.
• NT-proBNP N-terminal fragment of B-type natriuretic peptide precursor
• BNP and NT-proBNP are hormones produced by the breakdown of proBNP (B-type natriuretic peptide precursor), a precursor hormone synthesized in the ventricles, and their blood concentrations increase according to the load on the ventricles.
• BNP and NT-proBNP are released into the blood in a 1:1 ratio, and are therefore known as equivalent biomarkers for assisting in the diagnosis of heart failure.
• BNP is an excellent biomarker for heart failure because it relieves stress on the heart through its physiological activity of dilating blood vessels and promoting urinary excretion.
• BNP is only present in plasma, and it has the disadvantage of being less stable.
• NT-proBNP does not have physiological activity, but has a larger molecular weight than BNP, a longer half-life in serum or plasma, and high storage stability, making it more useful in clinical tests.
• NT-proBNP is an N-terminal peptide fragment of proBNP, and usually consists of a sequence of 76 amino acids. It is known that some of the amino acid residues of NT-proBNP have glycans attached (Minamino Naoto et al., Journal of the Japanese Circulation Society, Cardiovascular Specialist, Vol. 25, No. 2, 2017, pp. 261-267 (Non-Patent Document 1)), and in fact, it exists in the blood as various glycan isoforms.
• JP 2007-525427 A describes an immunological detection method using an antibody that recognizes the epitope at positions 42 to 46 of NT-proBNP.
• JP 2005-181304 A describes an immunological sandwich test device that uses an antibody against an epitope portion including positions 1 to 37 of NT-proBNP and an antibody against an epitope portion including positions 38 to 50 of NT-proBNP, and Non-Patent Document 2 (Alexander G. et al., JIFCC, 2016, Vol. 27, No. 3, pp.
• 189-207 describes an immunological detection method that uses a monoclonal antibody against an epitope including positions 22 to 28 or 27 to 31 of NT-proBNP and a monoclonal antibody against an epitope including positions 42 to 46 of NT-proBNP.
• the inventors further investigated immunological detection methods for NT-proBNP and found that conventional detection methods using antibodies against NT-proBNP result in lower measured values in some samples, meaning that detection may be overlooked in some samples, and that, while in theory BNP and NT-proBNP are present in equimolar amounts in the same sample, the amount of NT-proBNP detected by conventional detection methods has a low correlation with the amount of BNP and is not 1:1 equivalent to the amount of BNP, meaning that there are still problems with insufficient detection performance.
• the present invention was made in consideration of the problems with the prior art, and aims to provide an antibody capable of detecting N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP) with high performance, a method for detecting NT-proBNP using the antibody, and a composition and kit for use in detecting NT-proBNP.
• NT-proBNP N-terminal fragment of B-type natriuretic peptide precursor
• the inventors have conducted extensive research to achieve the above-mentioned objective, and have produced a new antibody that is reactive to a region containing the amino acid sequence of positions 64 to 69 of NT-proBNP.
• this antibody was used to detect NT-proBNP from multiple positive specimens, it was found that there existed specimens in which the amount of NT-proBNP detected was significantly different from the amount detected by conventional detection methods (deviating specimens).
• the inventors have found that by using the newly produced antibody, it is possible to detect such diverging specimens, i.e. specimens that were not sufficiently detected by conventional detection methods, without missing any.
• the inventors compared the measured values of NT-proBNP for multiple positive samples (NT-proBNP positive samples) with and without glycosylation treatment, and found that when detected by the conventional detection method, the measured values fluctuated greatly between the two, whereas when detected using the above antibody that shows reactivity to the region containing the amino acid sequence of positions 64 to 69 of NT-proBNP, the fluctuation in the measured values between the two was significantly smaller, and all samples could be detected with high performance without being overlooked. From this, it was confirmed that one of the causes of the decrease in the measured values that occurred in the above-mentioned deviation samples using the conventional detection method was the influence of glycosylation of NT-proBNP.
• the inventors have found that by using the above-mentioned newly created antibody, it is possible to comprehensively detect NT-proBNP, regardless of the presence or absence of glycosylation, that is, to easily detect all glycosylation isoforms without overlooking them without performing treatment such as glycosylation removal, and have completed the present invention.
• the present invention relates to an antibody against NT-proBNP, a method for detecting NT-proBNP using the same, and a composition and kit for use in detecting NT-proBNP, and more specifically relates to the following: [1] An antibody against N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP), which is reactive to a region including the amino acid sequence of positions 64 to 69 of NT-proBNP. [2] The antibody according to [1], which has substantially no reactivity to a region not including the amino acid sequence of positions 64 to 69 of NT-proBNP.
• the present invention has a heavy chain variable region (HV) including the amino acid sequence of SEQ ID NO: 2, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 2, and
• the antibody has a light chain variable region (LV) including the amino acid sequence of SEQ ID NO: 3, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 3.
• the antibody according to any one of [1] to [3].
• [5] A method for immunologically detecting NT-proBNP using the antibody according to any one of [1] to [4].
• [6] An antibody reactive to a region including 5 or more consecutive amino acids in the amino acid sequence of positions 12 to 31 of NT-proBNP, and an antibody reactive to a region including the amino acid sequence of positions 42 to 46 of NT-proBNP.
• kits for immunologically detecting NT-proBNP comprising the antibody according to any one of [1] to [4].
• the present invention makes it possible to provide an antibody that can comprehensively and easily detect N-terminal fragments of the precursor of B-type natriuretic peptide (NT-proBNP) with high performance, in particular an antibody equivalent to BNP, regardless of the presence or absence of glycosylation, a method for detecting NT-proBNP using the antibody, and a composition and kit for use in detecting NT-proBNP.
• NT-proBNP B-type natriuretic peptide
• FIG. 1 shows a correspondence table between the amino acid sequence of HV (heavy chain variable region) and Kabat number position (Kabat).
• FIG. 1 shows a correspondence table between the amino acid sequence of the LV (light chain variable region) and the Kabat number position (Kabat).
• 1 is a graph showing absorbance (Abs.) at 450 nm when Ab3 was reacted with each peptide in the epitope mapping of Test Example 1 (5).
• 1 is a graph showing absorbance (Abs.) at 450 nm when Ab1x was reacted with each peptide in the epitope mapping of Test Example 2(1).
• FIG. 1 is a graph showing absorbance (Abs.) at 450 nm when Ab1y was reacted with each peptide in the epitope mapping of Test Example 2(1).
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in samples detected with Ab2-Ab1x and the NT-proBNP concentration (pg/mL) in samples detected with Ab2-Ab1y in Test Example 2(1).
• FIG. 1 is a graph showing absorbance (Abs.) at 450 nm when Ab1y was reacted with each peptide in the epitope mapping of Test Example 2(1).
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in samples detected with Ab2-Ab1x and the NT-proBNP concentration (pg/mL) in samples detected with Ab2-Ab1y in Test Example 2(1).
• 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in a sample detected with Ab2-Ab1x in Test Example 2(1) and the NT-proBNP concentration (pg/mL) in a sample detected using the Elecsys (registered trademark, the same applies below) reagent (Elecsys).
• 1 is a graph showing absorbance (Abs.) at 450 nm when Ab2 was reacted with each peptide in the epitope mapping of Test Example 2(1).
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab2-Ab1 in Test Example 2(3) and the NT-proBNP concentration (pg/mL) in plasma samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab3-Ab1 in Test Example 2(3) and the NT-proBNP concentration (pg/mL) in plasma samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab3-Ab1 in Test Example 2(3) and the NT-proBNP concentration (pg/mL) in plasma samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab1-Ab2-Ab3 in Test Example 2(3) and the NT-proBNP concentration (pg/mL) in plasma samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in serum samples detected with Ab2-Ab1 in Test Example 3 and the NT-proBNP concentration (pg/mL) in serum samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in serum samples detected with Ab2-Ab1 in Test Example 3 and the NT-proBNP concentration (pg/mL) in serum samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in serum samples detected with Ab3-Ab1 in Test Example 3 and the NT-proBNP concentration (pg/mL) in serum samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in serum samples detected with Ab1-Ab2-Ab3 in Test Example 3 and the NT-proBNP concentration (pg/mL) in serum samples detected with the Elecsys reagent.
• FIG. 1 is a correlation diagram showing the relationship between the NT-proBNP concentration (pg/mL) in serum samples detected with Ab1-Ab2-Ab3 in Test Example 3 and the NT-proBNP concentration (pg/mL) in serum samples detected with the Elecsys reagent.
• FIG. 1 is a graph showing the NT-proBNP concentration (pg/mL) in each plasma sample with (Enz(+)) and without (Enz(-)) deglycosylated treatment, detected with Ab2-Ab1 in Test Example 4(1).
• FIG. 1 is a graph showing the NT-proBNP concentration (pg/mL) in each plasma sample with (Enz(+)) and without (Enz(-)) deglycosylated treatment, detected with Ab3-Ab1 in Test Example 4(1).
• FIG. 1 is a graph showing the NT-proBNP concentration (pg/mL) in each serum sample with (Enz(+)) and without (Enz(-)) deglycosylated treatment, detected with Ab2-Ab1 in Test Example 4(2).
• FIG. 1 is a graph showing the NT-proBNP concentration (pg/mL) in each serum sample with (Enz(+)) and without (Enz(-)) deglycosylated treatment, detected with Ab3-Ab1 in Test Example 4(2).
• FIG. 13 is a correlation diagram showing the relationship between the common logarithm of the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab3-Ab1y in Test Example 5(2) and the common logarithm of the BNP concentration (pg/mL) (log(BNP)).
• FIG. 13 is a correlation diagram showing the relationship between the common logarithm of the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab3-Ab1y in Test Example 5(2) and the common logarithm of the BNP concentration (pg/mL) (log(BNP)).
• FIG. 13 is a correlation diagram showing the relationship between the common logarithm of the NT-proBNP concentration (pg/mL) in plasma samples detected with Ab2-Ab1y in Test Example 5(2) and the common logarithm of the BNP concentration (pg/mL) (log(BNP)).
• FIG. 1 is a correlation diagram showing the relationship between the common logarithm of the NT-proBNP concentration (pg/mL) in a plasma sample detected with the Elecsys reagent in Test Example 5 (2) and the common logarithm of the BNP concentration (pg/mL) (log(BNP)).
• the present invention provides an antibody against NT-proBNP, which is reactive with a region including the amino acid sequence at positions 64 to 69 of NT-proBNP.
• NT-proBNP refers to N-terminal pro-brain natriuretic peptide, and is also referred to as "N-terminal fragment of BNP precursor” or "N-terminal fragment of brain natriuretic peptide precursor".
• NT-proBNP is a protein consisting of the amino acid sequence of the N-terminal side of B-type natriuretic peptide (BNP) separated from the C-terminal side of proBNP (B-type natriuretic peptide precursor), which is a precursor hormone synthesized in the ventricles, and generally contains the amino acid sequence of positions 1 to 76 of proBNP if derived from humans.
• NT-proBNP is preferably derived from humans, and human NT-proBNP is typically identified as having a UniProt Accession No. : A protein containing the amino acid sequence from positions 1 to 76 of the amino acid sequence described in P16860 (the amino acid sequence described in SEQ ID NO: 1).
• the DNA sequence of a gene mutates naturally (i.e., non-artificially) due to mutations or the like, and the amino acid sequence of the protein encoded thereby is also altered accordingly. Therefore, the NT-proBNP detected by the antibody of the present invention is not limited to proteins consisting of the above-mentioned typical amino acid sequences, but includes such natural mutants, as long as it contains the following region to which the antibody shows reactivity.
• the amino acid sequence of NT-proBNP according to the present invention includes an amino acid sequence having a homology (preferably identity) of 70% or more, preferably 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 1 and including the amino acid sequence of positions 64 to 69 below; an amino acid sequence in which one or several amino acids (70 amino acids or less, preferably 45 amino acids or less, 25 amino acids or less, 20 amino acids or less, 10 amino acids or less, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid) have been substituted, deleted, added, and/or inserted in the amino acid sequence shown in SEQ ID NO: 1 and including the amino acid sequence of positions 64 to 69 below.
• a homology preferably identity
• positions of amino acids indicated as “64th position” and “69th position” indicate the number of amino acid residues from the N-terminus in the amino acid sequence of NT-proBNP (typically, the amino acid sequence shown in SEQ ID NO: 1).
• sequence "amino acid sequence of positions X to Y of NT-proBNP (X, Y: numbers 1 to 76)" includes sequences consisting of amino acids corresponding to this sequence.
• a sequence consisting of amino acids that "corresponds" to a specific amino acid sequence refers to an amino acid sequence that is aligned with the control amino acid sequence (i.e., for example, the amino acid sequence from positions 64 to 69 of the amino acid sequence set forth in SEQ ID NO: 1) when the amino acid sequences are aligned using amino acid sequence analysis software (e.g., GENETYX-MAC, Sequencher, etc.) or ClustalW, etc. (for example, parameters: default values (i.e., initial settings)).
• amino acid sequence analysis software e.g., GENETYX-MAC, Sequencher, etc.
• ClustalW etc.
• the individual amino acids may be amino acids that have chemically similar amino acid side chains (e.g., amino acids with hydroxyl groups (serine, threonine), amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan), etc.), but it is more preferable that they are the same amino acid.
• amino acids with hydroxyl groups e.g., amino acids with hydroxyl groups (serine, threonine), amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan), etc.
• the subject detected by the antibody of the present invention includes those containing the region exhibiting the following reactivity, and may be, for example, a part of the amino acid sequence of the NT-proBNP, specifically, one or several amino acids at the N-terminus and/or C-terminus, one or several amino acids added to the N-terminus and/or C-terminus of the amino acid sequence of the NT-proBNP, proBNP, BNP, or degradation products thereof, containing all or a part of the amino acid sequence of the NT-proBNP.
• antibody includes not only complete antibodies, but also functional fragments thereof.
• functional fragment means a part (partial fragment) of a complete antibody that exhibits reactivity with NT-proBNP, and specific examples include Fab, F(ab')2, Fab', variable region fragment (Fv), disulfide-linked Fv, single chain Fv (scFv), sc(Fv)2, diabodies, and polymers thereof.
• Fab means a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain, and can be obtained, for example, by papain digestion of an antibody or by recombinant methods.
• F(ab')2 means a divalent antigen-binding fragment of an immunoglobulin consisting of both light chains and parts of both heavy chains, and can be obtained, for example, by pepsin digestion of an antibody or by recombinant methods.
• Fab' can be obtained, for example, by reducing F(ab')2, and differs from Fab in that it does not contain the hinge portion of an antibody.
• variable region fragment refers to the smallest antibody fragment that has a complete antigen recognition and binding site.
• Fv is a dimer in which the heavy chain variable region and the light chain variable region are strongly linked by non-covalent bonds.
• Single-chain Fv contains the heavy chain variable region and the light chain variable region of an antibody, and these regions are present in a single polypeptide chain.
• sc(Fv)2 is a single chain formed by linking two heavy chain variable regions and two light chain variable regions with a linker or the like.
• “Diabody” is a small antibody fragment that has two antigen binding sites, and this fragment contains a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, and each region forms a pair with a complementary region of another chain.
• the term "antibody” includes all classes and subclasses of immunoglobulins, and also includes polyclonal and monoclonal antibodies.
• a “polyclonal antibody” refers to an antibody preparation containing different antibodies against different epitopes
• a “monoclonal antibody” refers to an antibody (including the functional fragments) obtained from a population of substantially homogeneous antibodies.
• the antibody of the present invention is preferably a monoclonal antibody.
• the antibody of the present invention may be any antibody that exhibits the following reactivity with NT-proBNP, and there are no particular limitations on its origin, type, shape, etc. Specific examples include antibodies derived from humans, antibodies derived from non-human animals (e.g., rabbit antibodies, mouse antibodies, rat antibodies, camel antibodies), chimeric antibodies, humanized antibodies, and functional fragments of these antibodies.
• the antibody of the present invention is reactive to a region containing the amino acid sequence from positions 64 to 69 of NT-proBNP.
• it is an antibody that has an epitope (an antigenic determinant present in an antigen, a site on an antigen to which an antigen-binding domain in an antibody binds) that is at least a region consisting of six consecutive amino acids from positions 64 to 69 of NT-proBNP.
• an antibody "shows reactivity" with a certain region in an amino acid sequence means that the presence of the region in NT-proBNP causes the antibody to show binding activity (affinity) and/or specificity to the antigen NT-proBNP.
• the reactivity can be evaluated by a person skilled in the art using a known immunological method or a method similar thereto, for example, by the method shown in the test example described below.
• a plurality of peptides consisting of 10 to 15 amino acids including the amino acid sequence of the target region (for example, the amino acid sequence of positions 64 to 69 of the amino acid sequence shown in SEQ ID NO: 1) and a part or all of the amino acid sequence before and after the target region in the amino acid sequence of NT-proBNP are prepared so as to include a shift of one amino acid at a time, and the target antibody is reacted and captured with this as a solid-phase peptide, for example.
• the formed peptide-antibody complex can then be detected by a method (for example, a sandwich method) using a labeled antibody or the like (for example, a secondary antibody bound to a labeled substance: if the target antibody is derived from a mouse, HRP-labeled anti-mouse IgG, etc.).
• a method for example, a sandwich method
• a labeled antibody or the like for example, a secondary antibody bound to a labeled substance: if the target antibody is derived from a mouse, HRP-labeled anti-mouse IgG, etc.
• the target antibody when used as the labeled antibody (more preferably, for example, in the case of the method of Test Example 1 (5) below), the target antibody can be evaluated as "reactive" to a region consisting of an amino acid sequence common to peptides that show an absorbance of 80% or more of the maximum value of 1.5 or more, more preferably 2.0 or more, and even more preferably 2.5 or more at a wavelength of 450 nm throughout all peptides.
• the antibody when the maximum absorbance at a wavelength of 450 nm is 0.5 or less, more preferably 0.3 or less, and even more preferably 0.1 or less for a peptide containing the amino acid sequence of the target region, the antibody can be evaluated as "substantially unreactive" to the region.
• the antibody of the present invention is preferably substantially unreactive to a region that does not contain the amino acid sequence at positions 64 to 69 of NT-proBNP (the six consecutive amino acids at positions 64 to 69), and more preferably is substantially unreactive to a region that contains the amino acid sequence before position 63 or after position 70 of NT-proBNP but does not contain any amino acids in the amino acid sequence at positions 64 to 69.
• antibodies of the present invention include antibodies having the following variable regions: A heavy chain variable region (HV) comprising the amino acid sequence set forth in SEQ ID NO: 2, and An example of such an antibody is an antibody having a light chain variable region (LV) comprising the amino acid sequence set forth in SEQ ID NO:3.
• HV heavy chain variable region
• LV light chain variable region
• the present invention provides an antibody having the following CDRs: heavy chain complementarity determining regions (HV CDRs) 1 to 3 determined from a heavy chain variable region (HV) comprising the amino acid sequence set forth in SEQ ID NO: 2; Also provided is an antibody that possesses light chain complementarity determining regions (LV CDRs) 1-3 determined from a light chain variable region (LV) that comprises the amino acid sequence set forth in SEQ ID NO:3.
• HV CDRs heavy chain complementarity determining regions 1 to 3 determined from a heavy chain variable region (HV) comprising the amino acid sequence set forth in SEQ ID NO: 2
• LV CDRs light chain complementarity determining regions
• CDRs determined by the Kabat numbering method include antibodies having the following CDRs: The amino acid sequence set forth in SEQ ID NO: 4 is retained as HV CDR1; the amino acid sequence set forth in SEQ ID NO: 5 is retained as HV CDR2; the amino acid sequence set forth in SEQ ID NO: 6 is retained as HV CDR3; and An example of such an antibody is one that has the amino acid sequence set forth in SEQ ID NO: 7 as LV CDR1, the amino acid sequence set forth in SEQ ID NO: 8 as LV CDR2, and the amino acid sequence set forth in SEQ ID NO: 9 as LV CDR3.
• HV CDR1 is the amino acid sequence of Kabat number positions 31 to 35 in the amino acid sequence described in SEQ ID NO: 2
• HV CDR2 is the amino acid sequence of Kabat number positions 50 to 65 in the amino acid sequence described in SEQ ID NO: 2
• HV CDR3 is the amino acid sequence of Kabat number positions 95 to 102 in the amino acid sequence described in SEQ ID NO: 2.
• a correspondence table between HV amino acid sequences and Kabat number positions (Kabat) is shown in Figure 1.
• LV CDR1 is the amino acid sequence of positions 24 to 34 in the Kabat number position of the amino acid sequence described in SEQ ID NO: 3
• LV CDR2 is the amino acid sequence of positions 50 to 56 in the Kabat number position of the amino acid sequence described in SEQ ID NO: 3
• LV CDR3 is the amino acid sequence of positions 89 to 97 in the Kabat number position of the amino acid sequence described in SEQ ID NO: 3.
• a correspondence table between the LV amino acid sequence and the Kabat number position (Kabat) is shown in Figure 2.
• the antibody of the present invention may be not only an antibody that has a variable region and/or CDR consisting of such a specific amino acid sequence, but also an antibody whose amino acid sequence has been modified without reducing the desired activity (reactivity to NT-proBNP). Modifications to the amino acid sequence include, for example, substitution, deletion, addition, and/or insertion of one or several amino acids in the amino acid sequence.
• the site where the amino acid sequence is modified may be the constant region of the heavy or light chain of the antibody, or may be the variable region (frame region (FR) and CDR) as long as the reactivity is equivalent to that of the antibody before modification.
• Modification of amino acids other than CDR is thought to have a relatively small effect on the reactivity with the antigen (NT-proBNP), but a method of screening antibodies with enhanced affinity to the antigen by modifying amino acids in the CDR is also known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, etc.).
• the number of amino acids modified in the variable region i.e., the variable region, when it is stated that "one or several amino acids have been substituted, deleted, added, and/or inserted", "several” preferably refers to 25 amino acids or less, more preferably 20 amino acids or less, even more preferably 10 amino acids or less, 5 amino acids or less, or 3 amino acids or less (e.g., 2 amino acids or less, 1 amino acid or less). Furthermore, from the viewpoint of having little effect on reactivity with the antigen, it is preferable that all such modifications are made outside the CDR, i.e., in the FR.
• the CDR complementarity determining region
• “several” preferably refers to 5 amino acids or less, more preferably 3 amino acids or less (e.g., 2 amino acids or less, 1 amino acid or less).
• the modification of the amino acid sequence is preferably a conservative substitution.
• conservative substitution means substitution with another amino acid residue having a chemically similar side chain.
• Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs.
• acidic amino acids (aspartic acid and glutamic acid); basic amino acids (lysine, arginine, histidine); and neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxyl groups (serine, threonine), amino acids containing sulfur (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).
• amino acids with hydrocarbon chains glycine, alanine, valine, leucine, isoleucine, proline
• amino acids with hydroxyl groups (serine, threonine)
• amino acids containing sulfur (cysteine, methionine)
• amino acids with amide groups (asparagine, glutamine), amino acids with imino groups
• Antibodies having a variable region containing an amino acid sequence that has 80% or more homology at the amino acid sequence level with the variable region consisting of the specific amino acid sequence described above after modification are also included in the antibodies of the present invention, so long as they have the same reactivity as the antibody before modification.
• Such homology may be at least 80%, but is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more).
• the identity is at least 80%, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more).
• the homology and identity of amino acid sequences can be determined, for example, using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990, parameters: default). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993).
• the term "having equivalent reactivity" in relation to an antibody means that, for example, the reactivity to NT-proBNP is equivalent (e.g., 70% or more, preferably 80% or more, more preferably 90% or more) to that of a subject antibody (typically, an antibody having the amino acid sequence set forth in SEQ ID NO: 4 as HV CDR1, the amino acid sequence set forth in SEQ ID NO: 5 as HV CDR2, the amino acid sequence set forth in SEQ ID NO: 6 as HV CDR3, the amino acid sequence set forth in SEQ ID NO: 7 as LV CDR1, the amino acid sequence set forth in SEQ ID NO: 8 as LV CDR2, and the amino acid sequence set forth in SEQ ID NO: 9 as LV CDR3).
• a subject antibody typically, an antibody having the amino acid sequence set forth in SEQ ID NO: 4 as HV CDR1, the amino acid sequence set forth in SEQ ID NO: 5 as HV CDR2, the amino acid sequence set forth in SEQ ID NO: 6 as HV C
• the constant region other than the variable region of the antibody of the present invention is not particularly limited.
• the antibody of the present invention may be a functional fragment as described above, in which case the constant region may be a part of the fragment or may not have a constant region.
• the antibody of the present invention can be prepared by a person skilled in the art using a conventionally known method or a method similar thereto, by producing an antibody that is reactive to a region (epitope) containing the amino acid sequence of positions 64 to 69 of NT-proBNP. In this case, whether or not the antibody is reactive to the epitope can be evaluated by the method described above.
• the antibody of the present invention can be produced by the hybridoma method or the recombinant DNA method.
• a representative example of the hybridoma method is the method of Kohler and Milstein (Kohler & Milstein, Nature, 256: 495 (1975)).
• the antibody-producing cells used in the cell fusion step in the hybridoma method are spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with an antigen (NT-proBNP, partial peptides thereof, proteins fused with Fc protein, etc., or cells expressing these, etc.).
• antibody-producing cells obtained by allowing the antigen to act in a culture medium on the above-mentioned cells or lymphocytes, etc., previously isolated from non-immunized animals.
• myeloma cells used in the cell fusion step various known cell lines can be used.
• the antibody-producing cells and myeloma cells may be of different animal species origin, as long as they are fusible, but are preferably of the same animal species origin.
• Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then a hybridoma producing a monoclonal antibody specific to NT-proBNP can be obtained by screening.
• Monoclonal antibodies against NT-proBNP can be obtained in a substantially pure and homogenous form by culturing the hybridoma and isolating and purifying the antibody from within the hybridoma or from the culture medium. Alternatively, the antibody can be obtained from the ascites of a mammal to which the hybridoma has been administered.
• the recombinant DNA method involves incorporating synthetic DNA encoding the antibody of the present invention or DNA cloned from the hybridoma into an appropriate vector, which is then introduced into host cells (e.g., mammalian cell lines such as HEK cells, E. coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 WILEY; P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme A. M. et al., Eur. J. Biochem. 192: 767-775 (1990)).
• host cells e.g., mammalian cell lines such as HEK cells, E. coli, yeast cells, insect cells, plant cells, etc.
• host cells e.g., mammalian cell lines such as HEK cells, E. coli, yeast
• DNA encoding the heavy chain or the light chain may be separately incorporated into an expression vector to transform a host cell, or DNA encoding the heavy chain and the light chain may be incorporated into a single expression vector to transform a host cell.
• nucleotide sequence encoding the heavy chain variable region (HV) of the antibody of the present invention a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 is shown in SEQ ID NO: 10, and as a typical example of a nucleotide sequence encoding the light chain variable region (LV) of the antibody of the present invention, a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 is shown in SEQ ID NO: 11, but the present invention is not limited thereto, and each sequence may independently correspond to, for example, an antibody in which the amino acid sequence has been modified, or may be codon-optimized, etc.
• the antibody of the present invention can be obtained in a substantially pure and homogenous form by culturing the host cells and isolating and purifying it from within the host cells or from the culture medium.
• the antibody can be isolated and purified using methods that are commonly used for purifying polypeptides.
• a transgenic animal production technique is used to produce a transgenic animal (cow, goat, sheep, pig, etc.) incorporating an antibody gene, it is possible to obtain a large amount of monoclonal antibodies derived from the antibody gene from the milk of the transgenic animal.
• the present invention can also provide DNA encoding the antibody of the present invention, a vector containing the DNA, and a host cell harboring the DNA, as well as a method for producing the antibody, the method comprising the steps of culturing the host cell and recovering the antibody.
• the method for detecting NT-proBNP of the present invention is an immunological method for detecting NT-proBNP, which is a method for immunologically detecting NT-proBNP in a sample using the above-mentioned antibody against NT-proBNP of the present invention (hereinafter, sometimes simply referred to as the "detection method of the present invention").
• sample to be subjected to the detection method of the present invention is not particularly limited as long as it is a sample in which the target NT-proBNP to be detected may be present, but examples include specimens such as body fluids isolated from a living body (blood (plasma, serum, whole blood), lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, nasal mucus, etc.) and tissues, with blood being preferred, and plasma or serum being more preferred. Furthermore, the sample may be diluted or suspended in a diluent as necessary.
• the diluent examples include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer.
• the sample may be subjected to pretreatment such as a sugar chain removal treatment (e.g., enzyme treatment, treatment by alkaline ⁇ -elimination method, treatment by anhydrous hydrazine decomposition method), but the detection method of the present invention can detect NT-proBNP regardless of the presence or absence of sugar chains, so NT-proBNP can be easily detected without the sugar chain removal treatment, and the sugar chain removal treatment is not essential.
• a sugar chain removal treatment e.g., enzyme treatment, treatment by alkaline ⁇ -elimination method, treatment by anhydrous hydrazine decomposition method
• the detection method of the present invention is an immunological detection method in which the sample is contacted with the antibody of the present invention, and if NT-proBNP is present in the sample, NT-proBNP in the sample is detected based on an immune complex between NT-proBNP and the antibody formed by an antigen-antibody reaction.
• immunological detection methods include, but are not limited to, the EIA method (enzyme immunoassay) using an enzyme as a labeling substance, the ELISA method, which is one aspect of the EIA method, the CLIA method (chemiluminescent immunoassay) using a chemiluminescent compound as a labeling substance, the RIA method (radioimmunoassay) using a radioisotope as a labeling substance, and combinations of these (e.g., CLEIA method (chemiluminescent enzyme immunoassay), BLEIA method (bioluminescent enzyme immunoassay)); immunochromatography; and immunoagglutination methods (latex agglutination, gold colloid agglutination, etc.) that measure by detecting agglutination.
• the detection method of the present invention may be a non-competitive detection method or a competitive detection method.
• the labeling substance can be appropriately selected from those generally used in immunological detection methods and methods similar thereto, and examples thereof include enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), ⁇ -galactosidase ( ⁇ -gal), and luciferase (e.g., firefly luciferase); fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); radioisotopes such as 123I ; particles such as latex particles and gold colloid particles; avidin, biotin, and the like.
• enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), ⁇ -galactosidase ( ⁇ -gal), and luciferase (e.g., firefly
• the detection method of the present invention it is preferable to detect the presence or absence of NT-proBNP in a sample by detecting the presence or absence of a signal corresponding to the labeling substance.
• the "signal” includes coloration (color development), quenching, reflected light, luminescence, fluorescence, radiation from radioisotopes, etc., and includes signals that can be confirmed with the naked eye as well as those that can be confirmed by a detection method or device according to the type of signal.
• various signals (luminescence, fluorescence, etc.) generated by the enzyme-substrate reaction can be detected by adding a chromogenic substrate (e.g., 3,3',5,5'-tetramethylbenzidine (TMB) or o-phenylenediamine dihydrochloride (OPD) that develops color in the presence of hydrogen peroxide through an oxidation catalytic reaction with HRP), a chemiluminescent substrate (e.g., AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt), luciferin, which emits light through hydrolysis with ALP, or a fluorescent substrate.
• a chromogenic substrate e.g., 3,3',5,5'-tetramethylbenzidine (TMB) or o-phenylenediamine dihydrochloride (OPD) that develops color in the presence of hydrogen per
• the labeling substance may be bound to the antibody of the present invention to directly detect NT-proBNP, or a secondary antibody bound to a labeling substance may be used to indirectly detect NT-proBNP without binding a labeling substance to the antibody of the present invention.
• the "secondary antibody” is an antibody that shows reactivity to an antibody that directly binds to an antigen (the primary antibody, i.e., the antibody of the present invention). Protein G or protein A bound to a labeling substance may also be used instead of a secondary antibody.
• the antibody of the present invention may be used as a capture antibody described below, and the NT-proBNP captured by it may be detected with a detection antibody bound to the labeling substance.
• the binding of the antibody to the labeling substance can be appropriately performed using a conventionally known method or a method similar thereto.
• the antibody and the labeling substance can be directly bound via an active group or the like, or indirectly bound via an oligopeptide or linker or the like, or a biotin-avidin system (for example, an antibody is biotinylated, and an avidinated labeling substance is reacted with the antibody, and the interaction between biotin and avidin is utilized to bind the labeling substance to the antibody) can be used.
• the detection method of the present invention a sandwich method such as the CLEIA method is preferred from the viewpoint of tending to have higher sensitivity.
• the detection target substance (NT-proBNP) is captured by a capture antibody fixed (solid-phased) on a solid phase, which is then recognized by a detection antibody bound (labeled) to a labeling substance to form a complex of capture antibody-detection target substance-detection antibody, and after B/F separation (washing), the complex is detected according to the type of labeling substance.
• the detection antibody is made to recognize the detection target substance, and while performing B/F separation, the detection target substance is captured by the capture antibody, and detection according to the type of labeling substance may be performed.
• the solid phase may be any suitable solid phase that is generally used in immunological assays and similar assays, such as magnetic particles, latex particles, or other particles, plastic plates, or fibrous materials such as nitrocellulose fibers.
• the antibody may be directly fixed to the solid phase by a known method such as physical adsorption method, chemical binding method, or a combination of these, or it may be indirectly fixed by the above-mentioned secondary antibody, protein G, protein A, biotin-avidin system, etc.
• the antibody of the present invention is used as at least one of the capture antibody and the detection antibody.
• the other antibody may be an antibody against NT-proBNP, but is preferably an antibody that does not compete with the antibody of the present invention in binding to NT-proBNP (more preferably, an antibody that does not recognize the amino acid sequence at positions 64 to 69 of NT-proBNP, and even more preferably, an antibody that does not recognize any amino acid in the amino acid sequence at positions 64 to 69 of NT-proBNP).
• the other antibody may be a monoclonal antibody or a polyclonal antibody, but from the viewpoint of supply stability, a monoclonal antibody is preferable.
• Either the antibody of the present invention or the other antibody may be a capture antibody or a detection antibody.
• antibodies to be combined with the antibody of the present invention include: An antibody that is reactive to a region containing 5 or more consecutive amino acids in the amino acid sequence of positions 12 to 31 of NT-proBNP (sometimes referred to as "Ab1" in this specification), and an antibody that is reactive to a region containing the amino acid sequence of positions 42 to 46 of NT-proBNP (sometimes referred to as "Ab2" in this specification),
• the antibody may include at least one antibody selected from the group consisting of:
• Ab1 is reactive to a region containing 5 or more consecutive amino acids in the amino acid sequence from positions 12 to 31 of NT-proBNP.
• the "region containing 5 or more consecutive amino acids” is more preferably a "region containing 7 or more consecutive amino acids", and is even more preferably the amino acid sequence from positions 17 to 26 of NT-proBNP, the amino acid sequence from positions 12 to 23 of NT-proBNP, or the amino acid sequence from positions 25 to 31 of NT-proBNP.
• Ab1 is preferably substantially unreactive to a region that does not contain 5 or more consecutive amino acids (preferably 7 or more) in the amino acid sequence from positions 12 to 31 of NT-proBNP, and more preferably a region that contains the amino acid sequence from positions 11 to 32 to 12 of NT-proBNP but does not contain any amino acid in the amino acid sequence from positions 12 to 31.
• the regions in which Ab1 is reactive and unreactive can be evaluated by the above-mentioned method.
• Ab1 can be produced by a person skilled in the art using a conventionally known method or a method similar thereto.
• Conventionally known antibodies or commercially available antibodies may also be used as appropriate.
• Examples of the commercially available antibodies include Murine Monoclonal AB 12-31 manufactured by Roche Diagnostics KK, NT-proBNP (27-31) Monoclonal Antibody manufactured by Bios Pacific, and NT-proBNP (13-27) Monoclonal Antibody manufactured by Bios Pacific, but are not limited to these.
• Ab2 is reactive to a region containing the amino acid sequence at positions 42 to 46 of NT-proBNP, i.e., a region consisting of 5 consecutive amino acids at positions 42 to 46 of NT-proBNP. It is also preferable that Ab2 is substantially unreactive to a region that does not contain the amino acid sequence at positions 42 to 46 of NT-proBNP, and more preferably, a region that contains the amino acid sequence before position 41 or after position 47 of NT-proBNP but does not contain any amino acids in the amino acid sequence at positions 42 to 46.
• the regions where Ab2 is reactive and unreactive can also be evaluated by the above-mentioned method.
• Ab2 can be produced by a person skilled in the art using a conventionally known method or a method similar thereto.
• Conventionally known antibodies or commercially available antibodies may also be used as appropriate.
• Examples of the commercially available antibodies include, but are not limited to, Sheep Monoclonal AB 42-46 manufactured by Roche Diagnostics KK and NT-proBNP (42-46) Monoclonal Antibody manufactured by Bios Pacific.
• the detection method of the present invention it is preferable to use a combination of the antibody of the present invention and Ab1, from the viewpoint of enabling more sensitive detection of NT-proBNP regardless of the presence or absence of glycosylation, and it is particularly preferable to use all three types of antibodies, namely, the antibody of the present invention and both Ab1 and Ab2, from the viewpoint of enabling particularly sensitive detection.
• Any of the antibody of the present invention and Ab1 and/or Ab2 may be a capture antibody or a detection antibody.
• Ab1 is used as a detection antibody
• the antibody of the present invention and Ab2 are used as capture antibodies (preferably, the antibody of the present invention and Ab2 are each bound to a separate solid phase (e.g., magnetic particles)), and a signal corresponding to the type of labeling substance bound to the detection antibody can be detected.
• the amount of NT-proBNP in the sample can be determined by examining where the actual measured value is located on a standard curve created based on the measured values of a standard specimen of known concentration.
• the detection method of the present invention can also be a method for testing heart failure or related diseases for which NT-proBNP is known as a biomarker. Furthermore, the method for obtaining data on the amount of NT-proBNP (e.g., the presence or absence of detection of NT-proBNP, the amount of NT-proBNP in a sample) using the detection method of the present invention can also be described as a method for collecting data on the amount of NT-proBNP described above for diagnosis by a physician, a method for presenting the data to a physician, or a method for assisting a physician in diagnosis.
• NT-proBNP e.g., the presence or absence of detection of NT-proBNP, the amount of NT-proBNP in a sample
• the method for testing heart failure or a disease related thereto includes, for example, An example of such a method includes a detection step of detecting the amount of NT-proBNP in a biological sample separated from a subject, and a determination step of comparing the amount of NT-proBNP detected in the detection step with a reference amount to determine the degree of heart failure or a disease related thereto in the subject.
• examples of "diseases related to heart failure” include myocardial infarction, angina pectoris, cardiomyopathy, arrhythmia, atrial fibrillation, valvular disease, etc.
• the subject may be a human, and the biological sample may be the same as the above-mentioned sample, preferably plasma or serum.
• the detection step may be the above-mentioned detection method of the present invention.
• the amount of NT-proBNP detected in the detection step may be not only an absolute amount, but also a relative amount (for example, a relative amount to the amount of NT-proBNP detected in a subject from the past or before treatment, or in a biological sample isolated from a healthy subject).
• the degree of heart failure or a disease related thereto is determined, and "determining the degree” includes selecting subjects who are likely to suffer from heart failure or a disease related thereto. In addition, the “determining the degree” also includes determining whether or not a subject suffers from heart failure or a disease related thereto, and includes not only distinguishing between healthy subjects, but also distinguishing between subjects suffering from diseases other than heart failure or a disease related thereto.
• the "reference amount" to be compared is set appropriately depending on the purpose of the test, the subject, etc., and is not particularly limited, but for example, a person skilled in the art can set it as a so-called cutoff value based on the above detection method and statistical analysis method. Furthermore, a person skilled in the art can appropriately determine whether a value is "higher” or “lower” than the reference amount based on statistical analysis methods depending on the purpose of the test, the subject, etc.
• composition for detecting NT-proBNP of the present invention is a composition for immunologically detecting NT-proBNP, which contains the above-mentioned antibody against NT-proBNP of the present invention (hereinafter, sometimes simply referred to as "the composition of the present invention").
• the composition of the present invention can be preferably used in the above-mentioned method for detecting NT-proBNP of the present invention.
• the antibody contained in the composition of the present invention is as described above, but may be provided in a form immobilized on the solid phase for use in various immunological detection methods. Such solid phases and methods of immobilization on the solid phase are as described above.
• the antibody of the present invention may also be provided in a form bound to the labeling substance in accordance with various immunological detection methods. Such labeling substances and methods of binding to the labeling substance are as described above.
• composition of the present invention may contain, in addition to the antibody of the present invention, other components acceptable as drugs (reagents, etc.).
• other components include, for example, pharmacologically acceptable carriers or diluents (sterile water, physiological saline, vegetable oil, etc.), additives (excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, antiseptics, etc.).
• excipients include lactose, starch, sorbitol, D-mannitol, and sucrose.
• disintegrants include starch, carboxymethylcellulose, and calcium carbonate.
• buffers include phosphates, citrates, and acetates.
• Examples of the emulsifiers include gum arabic, sodium alginate, and tragacanth.
• the suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate.
• examples of the stabilizer include propylene glycol, diethyl sulfite, and ascorbic acid
• examples of the preservative include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben
• examples of the preservative include sodium azide, benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol.
• the NT-proBNP detection kit of the present invention is a kit for immunologically detecting NT-proBNP, which comprises the above-mentioned antibody against NT-proBNP of the present invention (hereinafter, sometimes simply referred to as "kit of the present invention").
• kit of the present invention can be preferably used in the above-mentioned NT-proBNP detection method of the present invention.
• the antibody contained in the kit of the present invention is as described above, but may be provided in the form of the above-mentioned composition of the present invention.
• the kit of the present invention includes, in addition to the antibody of the present invention, An antibody (Ab1) that is reactive to a region including 5 or more consecutive amino acids in the amino acid sequence of positions 12 to 31 of NT-proBNP, and an antibody (Ab2) that is reactive to a region including the amino acid sequence of positions 42 to 46 of NT-proBNP, It is preferable that the antibody further comprises at least one antibody selected from the group consisting of: and it is more preferable that the antibody further comprises both Ab1 and Ab2.
• Ab1 and Ab2 are as described above, including their preferred embodiments, each of them may be provided in a form immobilized on the solid phase for use in various immunological detection methods. Such solid phases and methods of immobilization on the solid phase are as described above.
• Ab1 and Ab2 may be provided in a form bound to the labeling substance, respectively, in accordance with various immunological detection methods.
• Such labeling substances and methods of binding to the labeling substances are also as described above.
• Ab1 and Ab2 may each be a composition containing the respective antibody.
• the composition may independently contain, in addition to the antibody, other components that are acceptable as a drug (reagent, etc.). Examples of the other components include those listed as other components in the composition of the present invention described above.
• the kit of the present invention may further include, for example, a solution or reagent for pre-treating the sample, a diluent for the sample, a substrate necessary for detecting the labeled substance, a reagent for stopping the detection reaction of the labeled substance (a reaction stopper), a positive control (e.g., NT-proBNP, a preparation), a negative control, an isotype control antibody for the antibody of the present invention, etc.
• a reaction stopper e.g., NT-proBNP, a preparation
• a negative control e.g., an isotype control antibody for the antibody of the present invention
• an antibody preparation is an antibody to which the labeled substance is not bound, it can also be combined with a labeled substance that binds to the antibody (e.g., a secondary antibody, protein G, protein A, etc.).
• the kit of the present invention may include instructions for using the kit.
• a carrier molecule maleimide-activated KLH
• the prepared conjugate was intraperitoneally immunized into ddY mice (age 4 weeks, female) and Balb/c mice (age 4 weeks, female), respectively.
• FAC complete Freund's adjuvant
• FFA complete Freund's adjuvant
• FAA incomplete Freund's adjuvant
• mice from which serum with high absorbance was derived were selected as mice with high antibody titers.
• NT-proBNP 500-2000ng/mL
• NT-proBNP 500-2000ng/mL
• a 96-well immunoassay plate manufactured by ThermoFisher
• BSA BSA
• antibodies were purified from the culture supernatant of each cell sample by a general affinity purification method and diluted to prepare antibody samples (200ng/mL).
• the prepared antibody samples were dispensed at 100 ⁇ L/well into the measurement plate and left to stand at 25°C for 1 hour to allow NT-proBNP to capture the antibodies in the sample.
• 100 ⁇ L/well of HRP-labeled anti-mouse IgG 200 ng/mL was added to the measurement plate and allowed to stand at 25° C. for 1 hour.
• a detection reagent OPD tablet, Fujifilm Wako Pure Chemical Industries, Ltd.
• the absorbance (Abs) at a wavelength of 450 nm 10 minutes after the addition was measured to confirm the formation of a complex between NT-proBNP and the antibody in the sample.
• cells derived from samples with high absorbance were selected as cell samples with high antibody titers.
• Each peptide is a 10 amino acid sequence modified with biotin at the end, and is a biotinylated peptide in which the sequence is shifted by one amino acid at a time to the amino acid sequence of 53-62, the amino acid sequence of 54-63, ..., the amino acid sequence of 66-75, and the amino acid sequence of 67-76.
• Each of these peptides (9.6 M) was dispensed into a streptavidin-immobilized 96-well immunoassay plate (manufactured by ThermoFisher) and left to stand at 25°C for 1 hour, and then washed to prepare a measurement plate for epitope analysis on which each of the peptides was immobilized.
• an antibody (Ab3) was purified and diluted from the culture supernatant of the hybridoma that was made into a single clone in (3) above by a general affinity purification method to prepare an antibody sample (500 ng/mL). The prepared antibody sample was dispensed into the measurement plate at 100 ⁇ L/well and left to stand at 25° C.
• Amino acid sequence of antibody The amino acid sequence of the antibody (Ab3, Example) whose epitope region was identified in (5) above was analyzed. That is, first, the hybridoma obtained as a single clone in (3) above was cultured and collected, and mRNA was extracted and Ab3-derived heavy and light chain cDNA was synthesized using reverse transcription PCR in a conventional manner. Next, the amino acid sequence was analyzed by performing sequence analysis of the amplified products of each cDNA, and based on the obtained amino acid sequence, the amino acid sequences of the variable region and the complementarity determining region of Ab3 were identified by the Kabat numbering method as follows.
• HV heavy chain variable region
• HV CDR1 Heavy chain complementarity determining region 1
• HV CDR2 Heavy chain complementarity determining region 2
• HV CDR3 Heavy chain complementarity determining region 3
• SEQ ID NO:6 Amino acid sequence of light chain variable region (LV)...SEQ ID NO:3 Amino acid sequence of light chain complementarity determining region 1 (LV CDR1)...SEQ ID NO:7 Amino acid sequence of light chain complementarity determining region 2 (LV CDR2)...SEQ ID NO:8 Amino acid sequence of light chain complementarity determining region 3 (LV CDR3)...SEQ ID NO:9.
• Test Example 2 Measurement of NT-proBNP-Positive Plasma Samples (1) Antibodies
• Ab1 A monoclonal antibody prepared in the same manner as in Test Example 1 using the amino acid sequence of positions 10 to 35 of the amino acid sequence of NT-proBNP shown in SEQ ID NO: 1 was used as Ab1x.
• NT-proBNP 27-31) Monoclonal Antibody, manufactured by Bios Pacific
• each peptide is a 15-amino acid sequence modified with biotin at the end, and is a biotinylated peptide in which the sequence is shifted by one amino acid at a time, so that the amino acid sequence is from positions 1 to 15, the amino acid sequence is from positions 2 to 16, ..., the amino acid sequence is from positions 31 to 45, and the amino acid sequence is from positions 32 to 46.
• the maximum absorbance measured was 2.52 for Ab1x and 2.06 for Ab1y. Based on the common amino acid sequence between peptides that were confirmed to have absorbance of 80% or more of the maximum value, the epitope region of Ab1x was identified as being between positions 12-23 of NT-proBNP, and the epitope region of Ab1y was identified as being between positions 25-31 of NT-proBNP.
• NT-proBNP detection evaluation was performed in the same manner as in (3) below, except that NT-proBNP-positive plasma samples (number of samples: 50) different from those in (3) below were used as samples.
• the combination of HRP-labeled antibody Ab1x and magnetic particle-sensitized antibody Ab2 (Ab2-Ab1x), or the combination of HRP-labeled antibody Ab1y and magnetic particle-sensitized antibody Ab2 (Ab2-Ab1y) was performed.
• Figure 6 shows the correlation diagram between the NT-proBNP concentration (pg/mL) in the sample detected with Ab2-Ab1x and the NT-proBNP concentration (pg/mL) in the sample detected with Ab2-Ab1y.
• FIG. 7 shows a correlation diagram between the NT-proBNP concentration (pg/mL) in a sample detected with the combination of Ab1x and Ab2 (Ab2-Ab1x) and the NT-proBNP concentration (pg/mL) in the same sample detected with the Elecsys Reagent (Elecsys Reagent NT-proBNP II, manufactured by Roche Diagnostics KK; sometimes referred to as "Elecsys" in this specification).
• the Elecsys Reagent is a combination of a monoclonal antibody against an epitope including positions 27-31 of NT-proBNP and a monoclonal antibody against an epitope including positions 42-46 of NT-proBNP (Non-Patent Document 2). From FIG. 7, it was confirmed that the correlation coefficient (R) between these is R>0.95, which is equivalent to each other.
• Each peptide is a 15 amino acid sequence modified with biotin at the terminal group, and is a biotinylated peptide in which the sequence is shifted by one amino acid at a time, such as the amino acid sequence of positions 28 to 42, the amino acid sequence of positions 29 to 43, ..., the amino acid sequence of positions 47 to 61, and the amino acid sequence of positions 48 to 62.
• the maximum absorbance measured was 3.37. From the common amino acid sequence between peptides that showed an absorbance of 80% or more of the maximum value, the epitope region of the antibody (Ab2) was identified to be certainly positions 42-46 of NT-proBNP.
• the particles were washed three times with PBS and suspended in a storage solution (1% BSA, 0.01% NaN 3 , PBS) so that the final concentration of the magnetic particles was 2.0 mg/mL to prepare a magnetic particle-sensitized antibody solution.
• a storage solution 1% BSA, 0.01% NaN 3 , PBS
• NT-proBNP detection The standard solution for NT-proBNP measurement was a 1% BSA-containing PBS solution of NT-proBNP peptide (Eurofins) prepared by Fmoc synthesis. NT-proBNP-positive plasma samples (number of samples: 70) purchased from Discovery Life Sciences were used as samples.
• the HRP-labeled antibody solution was a 1% BSA-containing PBS solution of HRP-labeled antibodies prepared using each antibody and Peroxidase Labeling Kit-SH (Dojindo Laboratories, Inc.).
• a reaction diluent PBS containing 1% BSA, 100 ⁇ L
• the magnetic particle-sensitized antibody solution 50 ⁇ L was added and stirred at 37°C for 30 minutes.
• 150 ⁇ L of the HRP-labeled antibody solution 150 ⁇ L of the HRP-labeled antibody solution (antibody: 200 ng/mL) was added and stirred at 37°C for 30 minutes to form a sandwich complex of antibody (antibody-sensitized magnetic particles)-antigen in the sample (or antigen in the standard solution)-antibody (HRP-labeled antibody).
• the above measurements were performed using the following combinations of HRP-labeled antibody and magnetic particle-sensitized antibody: a combination of HRP-labeled antibody Ab1 and magnetic particle-sensitized antibody Ab2 (Ab2-Ab1, Comparative Example); a combination of HRP-labeled antibody Ab1 and magnetic particle-sensitized antibody Ab3 (Ab3-Ab1, Example); and a combination of HRP-labeled antibody Ab1 and magnetic particle-sensitized antibody mixed with magnetic particle-sensitized antibody using Ab2 and magnetic particle-sensitized antibody using Ab3 (Ab1-Ab2-Ab3, Example). Note that the mixing ratio of the mixed antibodies was adjusted so that the reactivity to the standard substance was equivalent to that of magnetic particle-sensitized antibody using Ab2 and magnetic particle-sensitized antibody using Ab3.
• Figure 9 shows the results for Ab2-Ab1
• Figure 10 shows the results for Ab3-Ab1
• Figure 11 shows the results for Ab1-Ab2-Ab3.
• the NT-proBNP concentrations (pg/mL) detected by each antibody combination or Elecsys reagent in the ID26 plasma sample (dissociation sample) are shown in Table 1 below.
• NT-proBNP-positive serum specimens The detection and evaluation of NT-proBNP was performed in the same manner as in Test Example 2, except that NT-proBNP-positive serum specimens (number of specimens: 50) purchased from Discovery Life Sciences were used as specimens. Correlation diagrams between the NT-proBNP concentration (pg/mL) in specimens detected with each combination of Ab1, Ab2, and Ab3 and the NT-proBNP concentration (pg/mL) in the same specimens detected with the Elecsys reagent are shown in Figures 12 to 14.
• Figure 12 shows the results of Ab2-Ab1 (Comparative Example)
• Figure 13 shows the results of Ab3-Ab1 (Example)
• Figure 14 shows the results of Ab1-Ab2-Ab3 (Example).
• NT-proBNP concentrations (pg/mL) detected by each antibody combination or Elecsys reagent in the serum sample (deviation sample) of ID15 are shown in Table 2 below.
• NT-proBNP The detection and evaluation of NT-proBNP was performed on the samples (Enz(+)) that had been subjected to the sugar chain removal treatment in the same manner as in Test Example 2, and the NT-proBNP concentration (pg/mL) in each sample was calculated. The measured value was multiplied by the dilution factor.
• Figure 15 shows the results for Ab2-Ab1
• Figure 16 shows the results for Ab3-Ab1
• each NT-proBNP concentration is shown with the value for each plasma sample that had not been subjected to glycan removal treatment set as 100%.
• the average value, median value, standard deviation, maximum value, and minimum value are shown in Table 3 below for each antibody combination, with 100% being set to "1".
• Figure 17 shows the results of Ab2-Ab1
• Figure 18 shows the results of Ab3-Ab1
• each NT-proBNP concentration is shown with the value of the serum sample that was not subjected to sugar chain removal treatment taken as 100%.
• the average value, median value, standard deviation, maximum value, and minimum value are shown in Table 4 below for each antibody combination, with 100% being set as "1".
• Test Example 5 Measurement of BNP-Positive Plasma Samples (1) Antibodies In this test example, Ab3 prepared in Test Example 1, as well as Ab1y and Ab2 used in Test Example 2, were used as antibodies.
• BNP and NT-proBNP are molecules separated from their precursor, proBNP, and are released into the blood in a 1:1 ratio, and are therefore known as equivalent biomarkers for the diagnosis of heart failure. Therefore, to compare the equivalence between the two biomarkers, the correlation between BNP and NT-proBNP was compared and verified.
• the samples used were plasma samples (number of samples: 72) with known BNP concentrations, with BNP concentrations of 100 pg/mL or more as determined by AIA-Pack CL (registered trademark) BNP (manufactured by Tosoh Corporation) and eGFR of 60 or more.
• the standard solution used was a 1% BSA-containing PBS solution of NT-proBNP peptide (manufactured by Eurofins) prepared by Fmoc synthesis.
• the luciferase-labeled antibody solution used was a 1% BSA-containing PBS solution of luciferase-labeled antibody prepared using each antibody and recombinant luciferase (manufactured by Promega).
• a magnetic particle-sensitized antibody solution was prepared in the same manner as in Test Example 2 (2).
• the combinations of luciferase-labeled antibody and magnetic particle-sensitized antibody were set to a combination where the luciferase-labeled antibody was Ab1y and the magnetic particle-sensitized antibody was Ab3 (Ab3-Ab1y, Example), and a combination where the luciferase-labeled antibody was Ab1y and the magnetic particle-sensitized antibody was Ab2 (Ab2-Ab1y, Comparative Example), and measurements were performed in the same manner as in Test Example 2 (3) except that a luciferase substrate (Dojindo Laboratories, Ltd.) was used as the detection reagent, and the NT-proBNP concentration (pg/mL) in each sample was calculated.
• a luciferase substrate Dojindo Laboratories, Ltd.
• the present invention makes it possible to provide an antibody that can comprehensively and easily detect N-terminal fragments of B-type natriuretic peptide precursors (NT-proBNP) with high performance, in particular an antibody equivalent to BNP, regardless of the presence or absence of glycosylation, a method for detecting NT-proBNP using the same, and a composition and kit for use in detecting NT-proBNP.
• NT-proBNP B-type natriuretic peptide precursors

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