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WO2024246048A1 - Détection spécifique de hnl homodimère - Google Patents

Détection spécifique de hnl homodimère Download PDF

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Publication number
WO2024246048A1
WO2024246048A1 PCT/EP2024/064616 EP2024064616W WO2024246048A1 WO 2024246048 A1 WO2024246048 A1 WO 2024246048A1 EP 2024064616 W EP2024064616 W EP 2024064616W WO 2024246048 A1 WO2024246048 A1 WO 2024246048A1
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Prior art keywords
monoclonal antibody
seq
hnl
sample
light chain
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Inventor
Per Venge
Shengyuan Xu
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P&M Venge AB
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P&M Venge AB
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    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the present invention relates to the methods and means for detecting and monitoring bacterial infections as well as monitoring efficiency of an antibacterial treatment. More specifically, the present invention relates to a method for detecting a presence or an absence of homodimeric human neutrophil lipocalin (HNL) in a sample as well as to a method, a kit-of-parts and a device for establishing a presence or an absence of a bacterial infection.
  • HNL homodimeric human neutrophil lipocalin
  • Bacteria can cause a number of conditions and diseases which are a major burden to society. Among such conditions and diseases are sepsis, gastrointestinal tract infections, urinary tract infections, tuberculosis, pneumonia and gonorrhoea.
  • the treatment of bacterial infections largely relies on administration of antibiotics. Incorrect administration of antibiotics, e. g. administration of antibiotics when treating infections which are not caused by bacteria, may lead to that at least some of the bacteria present in the body become drug resistant. These bacteria may spread into the environment and ultimately become multidrug resistant.
  • AMR Bacterial antimicrobial resistance
  • HNL human neutrophil lipocalin
  • HNL is a ubiquitous glycoprotein localised in specific granules of neutrophils. HNL can occur as a 25-kD monomer or a 45-kD disulphide-linked homodimer. Furthermore, a part of HNL can be covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulphide bridge forming a 135-kD complex.
  • gelatinase matrix metalloproteinase 9
  • the amino acid sequence of HNL is shown in SEQ ID NO: 1 corresponding to the protein sequence found in www.uniprot.org/uniprot/P80188, in particular referring to isoform 1.
  • Sensitivity and specificity for the known in the art serum-based assays which employ measurement of the amount of HNL are in the range 75-94 %.
  • the lower figures of the range are obviously not satisfactory for the clinical setting.
  • EP 0756708 Bl relates to the use of HNL for diagnosing inflammation caused by a bacterial infection.
  • Disclosed immunoassays comprise bringing a sample suspected of containing an abnormal level of HNL in contact with an antibody specific for HNL (anti- HNL antibody).
  • anti-HNL antibody means an antibody preparation reacting specifically with HNL, such as in monomeric forms, HNL as included in di-, multi- or heteromeric forms and/or fragments of monomeric HNL exhibiting HNL unique determinants and epitopes.
  • WO 2016/079219 Al relates to a binding agent capable of specifically binding to a polypeptide epitope of HNL as well as to the means and methods for detecting a bacterial infection and discriminating between a bacterial infection and a viral infection.
  • an in vitro method for detecting a presence or an absence of homodimeric human neutrophil lipocalin (HNL) in a sample, wherein the method comprises: i) providing a sample, ii) providing monoclonal antibodies, a first monoclonal antibody and a second monoclonal antibody, for detecting homodimers of HNL, each of the homodimers consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, iii) bringing the sample into contact with the first monoclonal antibody, iv) bringing the sample into contact with the second monoclonal antibody, v) detecting a presence or an absence of homodimeric HNL, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain
  • an in vitro method for establishing a presence or an absence of a bacterial infection, wherein the method comprises: i) providing a sample, ii) providing monoclonal antibodies, a first monoclonal antibody and a second monoclonal antibody, for detecting homodimers of HNL, each of the homodimers consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, iii) bringing the sample into contact with the first monoclonal antibody, iv) bringing the sample into contact with the second monoclonal antibody, v) determining a value corresponding to the amount of homodimeric HNL in the sample, vi) comparing the value with a control value, vii) establishing a presence of a bacterial infection when the value is significantly higher than the control value or establishing an absence of a bacterial infection when the value
  • the steps of providing a sample and providing monoclonal antibodies may be performed in any order in the methods of the present document. Further, the steps of bringing the sample into contact with the first and the second monoclonal antibody, respectively, may be performed in any order, although it is preferred to bring the sample into contact with the first antibody first.
  • kit-of-parts for detecting a presence or an absence of homodimeric HNL in a sample and/or establishing a presence or an absence of a bacterial infection
  • the kit-of-parts comprises one or more of a monoclonal antibody as described herein, and at least one of instructions for use, a buffer and a device, wherein the device is arranged for analysis of a sample.
  • a device for establishing a presence or an absence of a bacterial infection, wherein the device comprises: a first compartment, wherein the first compartment comprises monoclonal antibodies, a first monoclonal antibody and a second monoclonal antibody, for detecting homodimers of HNL, each of the homodimers consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first set of murine complementarity-determining regions (CDRs), the first set of the murine CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6, a second heavy chain CDR (CDRH2), wherein CDR
  • Figure 1 graphically illustrates that employing a method according to the present invention (exemplified as enzyme-linked immunosorbent assay (ELISA) with the first monoclonal antibody MAB3 and the second monoclonal antibody MAB3) specific binding and detection of homodimers of HNL can be achieved.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 2 graphically illustrates that employing a method according to the present invention (exemplified as ELISA with the first monoclonal antibody MAB2 and the second monoclonal antibody MAB2) specific binding to and detection of homodimers of HNL can be achieved.
  • a method according to the present invention exemplified as ELISA with the first monoclonal antibody MAB2 and the second monoclonal antibody MAB2
  • Figure 3 graphically illustrates that a method according to the present invention where the same antibody is used as a capture reagent and a detection reagent cannot be performed with any monoclonal antibody specific to HNL.
  • Figure 3 illustrates that when the antibody MAB5 is used as the first and second monoclonal antibody in a method of the present document, HNL dimers are not detected.
  • Figure 4 diagrammatically illustrates levels of homodimers of HNL in plasma samples obtained from patients suffering from acute infections, such as an acute bacterial infection and an acute viral infection.
  • the levels of homodimers of HNL were detected with help of a method according to the present invention employing the first monoclonal antibody MAB3 and the second monoclonal antibody MAB3.
  • Figure 5 diagrammatically illustrates levels of homodimers of HNL in plasma samples obtained from patients suffering from acute infections, such as a viral infection, bacterial pneumonia, a mycoplasma infection, tonsillitis, a urinary tract infection (UTI), a gastrointestinal (GI) infection, erysipelas and sepsis.
  • acute infections such as a viral infection, bacterial pneumonia, a mycoplasma infection, tonsillitis, a urinary tract infection (UTI), a gastrointestinal (GI) infection, erysipelas and sepsis.
  • the levels of homodimers of HNL were detected with help of a method according to the present invention employing the first monoclonal antibody MAB3 and the second monoclonal antibody MAB3.
  • a first monoclonal antibody includes one or more first monoclonal antibodies
  • a second monoclonal antibody includes one or more second monoclonal antibodies
  • a first monomer includes one or more first monomers
  • a second monomer includes one or more second monomers.
  • first”, “second” and the like are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that each of the terms so used can be substituted with another appropriate term.
  • antibody is understood within the scope of the invention to refer to any protein that can be produced by plasma cells in response to an antigen, i. e. a foreign substance that induces an immune response.
  • An antibody also known as an immunoglobulin, may be exemplified by a peptide comprising natural and/or modified amino acids which permit the peptide to bind to the sequences of the herein described regions of HNL.
  • the term “monoclonal antibody” is understood within the scope of the invention to refer to an antibody, as that term is defined herein, obtained from a population of substantially homogeneous antibodies, i. e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations or alternative post-translational modifications that may be present in minor numbers.
  • the monoclonal antibodies may be produced by hybridoma cells or with help of a recombinant DNA technique.
  • Non-limiting examples of monoclonal antibodies include murine, rabbit, rat, chicken, chimeric, humanised or human monoclonal antibodies as well as functional fragments thereof, i. e. the fragments exhibiting the desired biological activity.
  • Human monoclonal antibodies may be isolated using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci.
  • the monoclonal antibody or a functional fragment thereof may be linked to e. g. a fluorescent moiety, a radioactive moiety, a chromogenic substrate and the like.
  • the first monoclonal antibody may alternatively be denoted a “capturing agent”, “capturing reagent”, “capture agent” and the like.
  • the second monoclonal antibody may be denoted a “detecting agent”, “detecting reagent”, “detection reagent” and the like.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kD) and one “heavy” chain (about 50-70 kD).
  • the N- terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the term “variable light chain” (VL) refers to a variable region of the light chain.
  • variable heavy chain” (VH) refers to a variable region of the heavy chain.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for interactions with other components of the immune system.
  • CDR complementarity-determining region
  • VH CDR3 is located in the variable domain of the heavy chain of the antibody
  • VL CDR1 is located in the variable domain of the light chain of the antibody.
  • sequences of the framework regions are relatively conserved within a species.
  • epitope is understood within the scope of the invention to refer to a part, or a region, of an antigen which an antibody binds to.
  • An epitope typically includes 5-10 amino acids in a unique spatial conformation.
  • an antibody recognises a sequence of contiguous amino acids of an antigen primary structure of the antigen
  • the antibody is said to have a linear epitope.
  • an antibody recognises non-contiguous amino acids juxtaposed by tertiary folding of the antigen then the antibody is said to have a conformational epitope, also known as a discontinuous epitope.
  • Linear epitopes are typically retained on exposure to denaturing solvents, whereas conformational epitopes are typically lost on treatment with denaturing solvents.
  • Methods of determining spatial conformation of epitopes include x-ray crystallography and 2-dimensional nuclear magnetic resonance.
  • binding is understood within the scope of the invention to refer to the ability of the molecule to recognise and bind to a particular target molecule, such as while discriminating against other molecules that are similar in structure and/or composition.
  • the first and second monoclonal antibody of the present document are able to bind specifically to HNL in monomeric and dimeric form.
  • sensitivity is a measure of how well a test can identify true positives
  • specificity is a measure of how well a test can identify true negatives.
  • Sensitivity is calculated by TP/(TP+FN), wherein TP are “true positives”, FN - “false negatives”.
  • Specificity is calculated by TN/(TN+FP), wherein TN - “true negatives” and FP - “false positives”.
  • a method for detecting a presence or an absence of homodimeric human neutrophil lipocalin (HNL) in a sample, wherein the method comprises: providing monoclonal antibodies, a first monoclonal antibody and a second monoclonal antibody, for detecting homodimers of HNL, each of the homodimers consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, bringing a sample into contact with the first monoclonal antibody, bringing the sample into contact with the second monoclonal antibody, detecting a presence or an absence of homodimeric HNL, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first set of murine complementarity-determining regions (CDRs), the first set of
  • the sample may be a bodily fluid.
  • the sample may be derived from a bodily fluid.
  • the bodily fluid may be at least one of blood, plasma, serum, urine, CSF, bone marrow, saliva, sputum, intestinal fluid and another bodily fluid.
  • the sample may also include liquid preparations obtained in various steps during the purification of HNL from its native sources or recombinant production systems.
  • the sample may include at least one of faeces and a liquid preparation thereof.
  • a plasma sample may be used.
  • the sample may be obtained from an animal, such as a mammal, such as a human.
  • the sample may be obtained from a subject suspected to have at least one of a bacterial infection and a viral infection.
  • the bringing of the sample into contact with the first monoclonal antibody may be preceded by bringing the sample into contact with the second monoclonal antibody.
  • the sample may be first incubated in the presence of the first monoclonal antibody in a designated space, then the second monoclonal antibody may be added to the designated space.
  • the incubating the sample in the presence of the first monoclonal antibody may be conducted under conditions that allow binding of the first monoclonal antibody to the first monomer of the homodimer of HNL.
  • the time may be allowed for the second monoclonal antibody to bind to the second monomer of the homodimer of HNL, the second monomer being a monomer of the homodimer to which the first monoclonal antibody has not been bound.
  • the method according to the present invention allows not only for specific detection of HNL but for the specific detection of homodimeric HNL. Since it is the homodimeric HNL that is associated with the presence of a bacterial infection, the method according to the present invention allows for more accurate and reliable detection of a presence or an absence of a bacterial infection compared to what can be provided with known in the art methods.
  • the amino acids of the first monomer to which the first monoclonal antibody binds and the amino acids of the second monomer to which the second monoclonal antibody binds may be the similar.
  • the above CDRs may be configured to bind to an epitope within amino acids 145 to 154 of the first monomer or the second monomer, as defined in SEQ ID NO: 1.
  • the amino acids of the first monomer and those of the second monomer to which the above CDRs may be configured to bind may be determined by X-ray crystallography.
  • the “binding” may be construed as a binding with an affinity of about 75 %, more than about 80 %, more than about 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 % 98 %, 99% or 100 % as assessed e. g. by any one of competitive ELISA, SPR (Biacore) and another suitable method known in the art.
  • the affinity of the binding refers to the strength of the interaction between the two molecules, which is determined by factors such as the shape, charge, and hydrophobicity of the binding sites.
  • amino acid sequence of the heavy chain variable region may comprise a sequence as depicted in SEQ ID NO: 5, and/or the amino acid sequence of the light chain variable region may comprise a sequence as depicted in SEQ ID NO: 4.
  • amino acid sequence of the heavy chain variable region may comprise a sequence as depicted in SEQ ID NO: 3, and/or the amino acid sequence of the light chain variable region may comprise a sequence as depicted in SEQ ID NO: 2.
  • the amino acid sequence of at least one of the heavy chain variable region and the light chain variable region of at least one of the first monoclonal antibody and the second monoclonal antibody may be mutated at a single position or multiple positions.
  • Examples of amino acid residues that may be mutated are those that are within a framework region, at least 10% surface exposed and within 4.5 A of a CDR residue.
  • the amino acid residues to be mutated may be selected by visual inspection of a 3 -dimensional structural model of amino acids in proximity to a CDR and/or to one or more selected amino acid residues of a framework region.
  • At least one amino acid residue of the CDR sequences or the heavy and/or light chain variable region of the above monoclonal antibody may be substituted for another amino acid residue, deleted or at least one amino acid residue may be added to an amino acid sequence.
  • the substitutions, additions and/or deletions may be made anywhere in the amino acid sequence, provided the specific binding activity to HNL is maintained.
  • For each variable region/ CDR sequence at least one, two, three, four, five, six or more substitutions, additions and/or deletions may be made, provided the specific binding activity of the above monoclonal antibody to HNL is maintained.
  • the specific binding activity refers to the ability of an antibody to bind specifically to an HNL molecule with a high degree of selectivity and affinity.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising the first set of the murine CDRs, wherein the heavy chain comprises a sequence as depicted in SEQ ID NO: 5.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising the first set of the murine CDRs, wherein the heavy chain comprises a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % of identity to SEQ ID NO: 5.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising the first set of the murine CDRs, wherein the heavy chain comprises a sequence as depicted in SEQ ID NO: 3.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising the first set of the murine CDRs, wherein the heavy chain comprises a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % of identity to SEQ ID NO: 3.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising the second set of the murine CDRs, wherein the light chain comprises a sequence as depicted in SEQ ID NO: 4.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising the second set of the murine CDRs, wherein the light chain comprises a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % of identity to SEQ ID NO: 4.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising the second set of the murine CDRs, wherein the light chain comprises a sequence as depicted in SEQ ID NO: 2.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising the second set of the murine CDRs, wherein the light chain comprises a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % of identity to SEQ ID NO: 2.
  • a monoclonal antibody as defined in the present document also encompasses monoclonal antibodies having
  • the heavy chain of each of the first monoclonal antibody and the second monoclonal antibody may comprise a heavy chain variable region sequence as depicted in SEQ ID NO: 5, and the light chain of each of the first monoclonal antibody and the second monoclonal antibody may comprise a light chain variable region sequence as depicted in SEQ ID NO: 4.
  • the heavy chain of each of the first monoclonal antibody and the second monoclonal antibody may comprise a heavy chain variable region sequence as depicted in SEQ ID NO: 3, and the light chain of each of the first monoclonal antibody and the second monoclonal antibody may comprise a light chain variable region sequence as depicted in SEQ ID NO: 2.
  • amino acid sequence of at least one of the heavy chain constant region and the light chain constant region of the above monoclonal antibody may be mutated at a single position or multiple positions, as described above, provided the specific binding activity of the above monoclonal antibody is maintained.
  • the amino acid sequence of the first monoclonal antibody may be identical to that of Anti-HNL MAB2 or Anti-HNL MAB3.
  • the amino acid sequence of the second monoclonal antibody may be identical to that of MAB2 or Anti-HNL MAB3.
  • the amino acid sequence of the first monoclonal antibody may be identical to that of the second monoclonal antibody.
  • the first monoclonal antibody may have the amino acid sequence identical to that of Anti-HNL MAB2, whereas the second monoclonal antibody may have the amino acid sequence identical to that Anti-HNL MAB2.
  • the first monoclonal antibody may have the amino acid sequence identical to that of Anti-HNL MAB3, whereas the second monoclonal antibody may have the amino acid sequence identical to that of Anti-HNL MAB3.
  • the amino acid sequences of the light chain variable region of Anti-HNL MAB2 is the following: DIVLTQSTSSLSVSLGDRVTINCRASQDISNYLNWYQEKPDGTVKLLIYFTSRLHSGVPSRF SGSGSGTDYSLTITNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRADAAPTV (see also WO 2016/079219 Al).
  • the amino acid sequence of CDRL1 of Anti-HNL MAB2 is the following: RASQDISNYLN.
  • the amino acid sequence of CDRL2 of Anti-HNL MAB2 is the following: FTSRLHS.
  • the amino acid sequence of CDRL3 of Anti-HNL MAB2 is the following: QQGNTLPWT.
  • the amino acid sequences of CDRL1, CDRL2, CDRL3 of Anti-HNL MAB2 are the same as those depicted as SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively.
  • the amino acid sequence of the heavy chain variable region of Anti-HNL MAB2 is the following: EVQLEESGPGLVAPSQSLSITCTISGFSLTSYGIHWLRQPPGKDLEWLVVIWGDGSTTSNS ALKSRLSISKDNSKSQVFFKMSGLQTDDTAIYYCARHRYSDYHAMDYWGPGTSVTVS (see also WO 2016/079219 Al).
  • the amino acid sequence of CDRH1 of Anti-HNL MAB2 is the following: GFSLTSYGIH.
  • the amino acid sequence of CDRH2 of Anti-HNL MAB2 is the following: VIWGDGSTTSNSALKS.
  • the amino acid sequence of CDRH3 of Anti-HNL MAB2 is the following: HRYSDYHAMDY.
  • amino acid sequences of CDRH1, CDRH2, CDRH3 of Anti-HNL MAB2 are the same as those depicted as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the amino acid sequence of the light chain variable region of Anti-HNL MAB3 is the following: DIVLTQTTSSLSVSLGDRVTINCRASQDISNYLNWYQEKPDGTVKLLIYFTSRLHSGVPSRF SGSGSGTDYSLTITNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRADAAPTV (see also WO 2016/079219 Al).
  • the amino acid sequence of CDRL1 of Anti-HNL MAB3 is the following: RASQDISNYLN.
  • the amino acid sequence of CDRL2 of Anti-HNL MAB3 is the following: FTSRLHS.
  • the amino acid sequence of CDRL3 of Anti-HNL MAB3 is the following: QQGNTLPWT.
  • the amino acid sequences of CDRL1, CDRL2, CDRL3 of Anti-HNL MAB3 are the same as those depicted as SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively.
  • the amino acid sequence of the heavy chain variable region of Anti-HNL MAB3 is the following (see also WO 2016/079219 Al): EVKLQESGPGLVAPSQSLSITCTISGFSLTSYGIHWLRQPPGKDLEWLWIWGDGSTTSNS ALKSRLSISKDNSKSQVFFKMSGLQTDDTAIYYCARHRYSDYHAMDYWGPGTSVTVSS.
  • the above amino acid sequence is the same as that depicted as SEQ ID NO: 5.
  • the amino acid sequence of CDRH1 of Anti-HNL MAB3 is the following: GFSLTSYGIH.
  • the amino acid sequence of CDRH2 of Anti-HNL MAB3 is the following: VIWGDGSTTSNSALKS.
  • the amino acid sequence of CDRH3 of Anti-HNL MAB3 is the following: HRYSDYHAMDY.
  • amino acid sequences of CDRH1, CDRH2, CDRH3 of Anti-HNL MAB3 are the same as those depicted as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • At least one of the first monoclonal antibody and the second monoclonal antibody may be a humanised antibody.
  • the first monoclonal antibody may be immobilised on a solid support and thereby being configured to bind to the first monomer (of a homodimer of HNL), while the second monoclonal antibody may be in solution and thereby being configured to bind to the second monomer (of the homodimer of HNL), i. e. the monomer that has not been bound by the first monoclonal antibody immobilised on the solid support.
  • the first monoclonal antibody may function as a capture reagent
  • the second monoclonal antibody may function as a detection reagent.
  • the first monoclonal antibody may be configured to bind to the first monomer in a way that is different from that described above.
  • the second monoclonal antibody may be configured in bind to the second monomer in a way that is different from that described above.
  • the above method may further comprise determining the amount of homodimeric HNL in the sample.
  • the amount of homodimeric HNL in the sample may be determined by measuring the amount of the immunocomplexes formed between the above monoclonal antibodies and homodimeric HNL present in the sample. Detection of the immunocomplexes formed may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and ELISA, more specifically the double monoclonal antibody sandwich immunoassay technique, as well as immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using e. g.
  • colloidal gold, enzyme or radioisotope labels include Western blots, precipitation reactions, agglutination assays (e. g. gel agglutination assays and hemagglutination assays), immunofluorescence assays, protein A assays and immunoelectrophoresis assays.
  • the above monoclonal antibody may be either labelled or unlabelled.
  • the reporter group may be any suitable reporter group known in the art, including radioisotope, fluorescent group (e. g. fluorescein or rhodamine), luminescent group, enzyme, biotin and dye particles.
  • Labels that can be indirectly detected by their production of a detectable reaction product include various enzymes, such as alkaline phosphatase, horseradish peroxidase, p-galactosidase, xanthine oxidase, saccharide oxidase, e. g. glucose oxidase, and luciferases.
  • the enzyme can cleave an appropriate substrate to form a coloured reaction product or a fluorescent reaction product.
  • the monoclonal antibody may be detected by a third antibody being able to bind to the first and/or second antibody, the third antibody in turn being labelled as described herein.
  • a third antibody is used for detection, it is configured to bind to the monoclonal antibody used as the detecting agent (see elsewhere herein).
  • only the one of the first and second antibodies is labelled.
  • the unlabelled monoclonal antibody may be immobilised on a solid support for use as a “capture agent” (or “capture reagent”) that captures the homodimeric HNL contained within a sample.
  • the solid support may be any material (known to a person skilled in the art) to which the antibody may be attachable.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or another suitable membrane.
  • the support may be a tube, a bead, a particle or a disc.
  • the support may be of glass, fiberglass, latex or a plastic material, such as polyethylene, polypropylene, polystyrene, polyvinylchloride, or a porous matrix.
  • the support may also be a magnetic particle or a fibre optic sensor.
  • immobilisation refers to both non-covalent association, such as adsorption, and a covalent attachment (which may be a direct linkage between the antibody and a functional group on the support or may be a linkage by way of a cross-linking agent). Immobilisation by adsorption to a well in a microtiter plate or to a membrane is contemplated.
  • adsorption may be achieved by contacting the above monoclonal antibody, in a suitable buffer, with the solid support for a suitable amount of time.
  • the suitable duration of the contact depends on the temperature but is typically between about 1 hour and about 1 day.
  • contacting a well of a plastic microtiter plate (including polystyrene or polyvinylchloride) with an amount of the anti-HNL antibody ranging from about 10 ng to about 10 pg, from about 100 ng to about 1 pg is sufficient to immobilise an adequate amount of the monoclonal antibody. Following immobilisation, the remaining protein binding sites on the support may be blocked.
  • any suitable blocking agent known to a person skilled in the art including bovine serum albumin, TweenTM 20 (Sigma Chemical Co., St. Louis, Mo.), heat-inactivated normal goat serum (NGS) or BLOTTO (buffered solution of non-fat dry milk, which also contains a preservative, salts and an antifoaming agent) can be used.
  • the support may be incubated with the sample, i. e. a sample suspected to contain homodimers of HNL.
  • the sample can be applied neat or advantageously can be diluted e. g. in a buffered solution which contains a small amount (0.1 %-5.0 % by weight) of protein, such as BSA, NGS or BLOTTO.
  • the first monoclonal antibody may be used as a capture reagent and is immobilised on a surface, while the second monoclonal antibody is used as a detection reagent and is labelled with a label or alternatively detected using a third antibody, which is labelled.
  • An appropriate duration of contact between the sample and the monoclonal antibody is a duration of time that may be sufficient to detect the presence of an immunocomplex formed between the monoclonal antibody and homodimeric HNL present in the sample, the monoclonal antibody being immunospecific to homodimers of HNL.
  • the incubation time may be sufficient to achieve a level of binding that is at least about 95% of that achieved at the equilibrium between the bound monoclonal antibody and the unbound monoclonal antibody.
  • the time necessary to achieve the equilibrium may be readily determined by monitoring the level of binding that occurs over a period of time. At room temperature an incubation time of about 10 to 30 minutes may be generally sufficient.
  • Unbound constituents of the sample may be removed by washing with an appropriate buffer, such as PBS containing 0.1 % TweenTM 20.
  • an appropriate buffer such as PBS containing 0.1 % TweenTM 20.
  • a “detection reagent” i. e. the second monoclonal antibody that binds to the second monomer of a homodimer of HNL
  • a “detection reagent” i. e. the second monoclonal antibody that binds to the second monomer of a homodimer of HNL
  • the capture reagent i.e. the first monoclonal antibody
  • the detection reagent may be directly labelled and comprise at least a first detectable label or a “reporter” molecule.
  • the detection reagent i.e. the second monoclonal antibody
  • the second monoclonal antibody may be an unlabelled anti-HNL antibody.
  • This unlabelled second monoclonal antibody may then be detected by its binding to a third, labelled antibody or a labelled reagent.
  • the third antibody may be a labelled anti-murine immunoglobulin antibody.
  • the second monoclonal antibody is a rabbit immunoglobulin
  • the third antibody may be a labelled anti-rabbit immunoglobulin antibody.
  • the detection reagent i.e. the second monoclonal antibody
  • the detection reagent may be incubated with the immunocomplex formed by the “capture reagent” (i.e. the first monoclonal antibody) and the homodimeric HNL contained within the sample for a suitable amount of time sufficient to detect the bound detection reagent.
  • An appropriate incubation time may be determined by monitoring the level of binding that occurs over a period of time.
  • the detection reagent that remains unbound may be removed, and the amount of immunocomplexes formed between homodimeric HNL, the capture reagent and the detection reagent may then be measured using a suitable assay and a suitable analytical instrument.
  • a person skilled in the art would be able to choose such assay and such analytical instrument.
  • the value corresponding to the amount of immunocomplexes formed between homodimeric HNL, the capture reagent and the detection reagent shall be at least two-fold greater than the control value.
  • the control value may be obtained by measuring the same for a sample obtained from an individual with a normal amount of homodimeric HNL.
  • the sample and the detection reagent may be contacted simultaneously with the capture agent, rather than sequentially added.
  • the sample and the detection reagent may be pre-incubated together in a designated space before adding the capture reagent to the designated space.
  • the determining of the amount of homodimeric HNL in the sample can be used in a variety of diagnostic, prognostic and monitoring methods, including methods of diagnosing a homodimeric HNL-related disorder, methods of differentiating an inflammatory disease from a non-inflammatory disease and methods of monitoring efficiency of an antibacterial treatment, e. g. for the purpose of tailoring further administration of the antibacterial treatment.
  • a method for establishing a presence or an absence of a bacterial infection comprises: providing monoclonal antibodies, a first monoclonal antibody and a second monoclonal antibody, for detecting homodimers of HNL, each of the homodimers consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, bringing the sample into contact with the first monoclonal antibody, bringing the sample into contact with the second monoclonal antibody determining a value corresponding to the amount of homodimeric HNL in the sample, comparing the value with a control value, establishing a presence of a bacterial infection when the value is significantly higher than the control value or establishing an absence of a bacterial infection when the value is significantly lower than the control value, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a
  • a method for establishing a presence or an absence of inflammation caused by and/or associated with a bacterial infection. Details regarding the first and second monoclonal antibodies and the bringing into contact and incubation of the sample and the monoclonal antibodies are as given elsewhere herein.
  • the level of homodimeric HNL in the sample above a certain threshold, or a control value is correlated with the presence of an homodimeric HNL-related disorder, while a level of homodimeric HNL in the sample below said threshold, or the control value, is indicative of low likelihood of the presence of the HNL-related disorder.
  • a level of homodimeric HNL above a certain threshold, a control value is correlated with the presence of a bacterial infection, while a level homodimeric HNL below said threshold, or the control value, is indicative of low likelihood of the presence of the bacterial infection.
  • a threshold that differentiates most of the normal population from most of the diseased population can be determined using any well-known in the art method.
  • end point values for negative (an absence of the condition), uncertain and positive (a presence of the condition) results can be determined from the data.
  • a normal range indicative of a negative result
  • a range indicative of a positive result can be determined, the range including values of most of the diseased population but excluding almost all those of the normal population.
  • the above method for establishing a presence or an absence of a bacterial infection may further include comparing the concentration of at least one determinant, such as a peptide, protein or isoform thereof, in the sample to a statistically validated threshold for the at least one determinant.
  • a determinant such as a peptide, protein or isoform thereof
  • Determinant may be understood as a polypeptide whose amount under the disease condition is different from that under the normal condition.
  • Exemplary determinants may include TRAIL, IL1RA, IP 10, Mac-2BP, B2M, BCA-1, CHI3L1, Eotaxin, ILla, MCP, CD62L, VEGFR2, CHP, CMPK2, COROIC, EIF2AK2, ISG15, RPL22L1, RTN3, CD112, CD134, CD182, CD231, CD235A, CD335, CD337, CD45, CD49D, CD66A/C/D/E, CD73, CD84, EGFR, GPR162, HLA-A/B/C, ITGAM, NRG1, RAP IB, SELI, SPINT2, SSEA1, IgG non-specific bound molecules, IL1, 1-TAC, TNFR1, ABTB1, ADIPOR1, ARHGDIB, ARPC2, ATP6V0B, Clorf83, CD15, CES1, COROIA, CRP, CSDA, EIF4B, EPSTI1, GAS 7, HERC5, IFI6, KI
  • HGNC Human Genome Organization Naming Committee
  • NCBI National Center for Biotechnology Information
  • Determinants may also encompass non-polypeptide factors, non-blood borne factors or non-analyte physiological markers of health status, such as clinical parameters as well as traditional laboratory risk factors.
  • determinants may include non-polypeptide features (i. e. non-polypeptide determinants) such as neutrophil % (abbreviated Neu (%)), lymphocyte % (abbreviated Lym (%)), monocyte % (abbreviated Mon (%)), absolute neutrophil count (abbreviated ANC) and absolute lymphocyte count (abbreviated ALC), white blood count (abbreviated WBC), age, gender and maximal temperature (i. e. maximal core body temperature since the initial appearance of symptoms).
  • Determinants may also include any calculated indices produced mathematically or combinations of any one of the foregoing measurements, including temporal trends and differences.
  • determinants used in the above method may include at least one determinant that is a polypeptide and at least one non-polypeptide feature.
  • the above method may be used for monitoring effectiveness or non-effectiveness of a therapy with an antibacterial substance, such as an antibiotic.
  • Such therapy may be administered for treating a bacterial infection in case of e. g. sepsis.
  • Such use may comprise repeating the above method at least twice, wherein at the first point in time the sample may be obtained prior to initiation of the therapy or part way through the therapy and at the second point in time the sample may be obtained at the completion of the therapy or part way through the therapy after the first point in time.
  • a lesser amount of homodimeric HNL in the sample at the second point in time than that at the first point in time shall be indicative of the effectiveness of the therapy.
  • a pre-activator substance may be used as neutrophil activator, i.e. a substrate for activating neutrophils.
  • the preactivator substance may be an N-formyl peptide, preferably a tri-peptide fMLP.
  • protein A may be used.
  • other neutrophil activators may be used.
  • At least one of a lipopolysaccharide (LPS), a platelet-activating factor, an unmethylated CpG oligonucleotide and a tumor necrosis factor (TNF) may be used.
  • fMLP may be used alone or in combination with one or more of the other neutrophil activators mentioned above.
  • the above methods according to the first and second aspects, respectively, may be used for distinguishing between a bacterial infection and a non-bacterial infection, such as a viral infection, between a mixed infection (i. e. a co-infection with bacteria and virus) and a viral infection.
  • a non-bacterial infection such as a viral infection
  • a mixed infection i. e. a co-infection with bacteria and virus
  • a viral infection may be any one of sepsis, a gastrointestinal tract infection, a urinary tract infection, a tuberculosis, a pneumonia and a gonorrhoea.
  • the sample may be first brough into contact with the second monoclonal antibody before being brought into contact with the first monoclonal antibody.
  • the methods of the present document may comprise a step of providing a sample that is to be brought into contact with the first monoclonal antibody or the second monoclonal antibody. The methods of the present document are performed in vitro.
  • kits-of-parts for detecting a presence or an absence of homodimeric HNL in a sample and/or establishing a presence or an absence of a bacterial infection
  • the kit-of-parts comprises one or more of a monoclonal antibody as defined herein, , and one or more of instructions for use, a buffer and a device, wherein the device is arranged for analysis of a sample.
  • said kit-of- part comprises a first monoclonal antibody and a second monoclonal antibody as defined herein.
  • the kit-of-parts may only comprise one antibody, which can be used as the first and second monoclonal antibody in a method of the present document.
  • the sample may be derived from at least one of blood, plasma, serum, urine, CSF, bone marrow, saliva, sputum, intestinal fluid, another bodily fluid, faeces and a liquid preparation thereof.
  • the kit-of-parts may further include an enzyme, or the second monoclonal antibody may be labelled with an enzyme for use as a signal-generating means.
  • the kit-of-parts may further include a substrate for the enzyme and a substrate precursor that provides a detectable chromophore or fluorophore.
  • the above buffer of the kit-of-parts may include at least one of a block buffer and a lysis buffer.
  • At least one of the reagents of the kit-of-parts may be provided in a powder form, e. g. as lyophilised reagent.
  • kit-of-parts may be pre-attached to a solid support or may be applied to a surface of the solid support when the kit is in use.
  • the signal-generating means may come pre-associated with the monoclonal antibody described herein.
  • the signal-generating means may require being combined prior to use with at least one of the buffer, an antibody-enzyme conjugate, an enzyme substrate and the like.
  • the solid phase surface may be in a form of a tube, a bead, a microtiter plate, a microsphere or another form suitable for immobilising at least one of proteins, peptides and polypeptides.
  • the kit-of-parts may further comprise at least one agent for detecting and measuring other biological parameters, e. g.
  • Containers for use in the kit-of-parts may typically comprise at least one of a vial, a test tube, a flask, a bottle, a syringe and another suitable container.
  • a device for establishing a presence or an absence of a bacterial infection, wherein the device comprises: a first compartment, wherein the first compartment comprises monoclonal antibodies, a first monoclonal antibody and a second monoclonal antibody, for detecting homodimers of HNL, each of the homodimers consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first set of murine complementarity-determining regions (CDRs), the first set of the murine CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO:
  • the device may be a test strip with immobilised monoclonal antibodies on at least a part of its surface, particles carrying immobilised monoclonal antibodies on at least a part of its surface.
  • the surface may be three-dimensional, e. g. particles with a partially porous surface (layer) may carry the above monoclonal antibodies. It is important that the device comprises a contact area that can be exposed to the sample.
  • the device may be connected to a computer or an apparatus allowing analysis and measurement of the characteristics of the interaction between the monoclonal antibodies disclosed herein and the HNL homodimers present in the sample.
  • the interaction may be characterised using a signal that is specifically generated upon binding of a monoclonal antibody disclosed herein to a homodimer of HNL (no such signal is generated in a control set-up, wherein no above interaction occurs).
  • the signal can be at least one of a physical signal, a chemical signal and a biological signal. Examples of the signal include a chromogenic signal, a fluorogenic signal, a spectroscopically measurable signal, a change in ionization, a change in conductivity and the like.
  • the device may further comprise a binding agent or an antibody for detecting one or more of the above determinants.
  • the device may involve magnetic separation of the analytes of interest or the like prior to their detection.
  • the device may be any one of a point-of-care device, a test strip, a device for a radioimmunoassay, a device ELISA, a device for a “sandwich” immunoassay, a device for a immunoradiometric assay, a device for a gel diffusion precipitation assay, a device for a immunodiffusion assay, a device for an in situ immunoassay using colloidal gold, enzyme or radioisotope labels, a device for a Western blot, a device for a precipitation assay, a device for a gel agglutination assay, a device for a hemagglutination assay, a device for a immunofluorescence assay, a device for a protein A assay, a device for a immunoelectrophoresis assay and a device for a detection of analytes that involve magnetic separation of the analytes. It is appreciated that the above-mentioned embodiments of
  • Anti-HNL MAB2 and Anti-HNL MAB3 were produced by the standard hybridoma technique by the fusion of spleen cells from HNL dimer immunised Balb/c mice with Sp2/0 mouse myeloma cells, followed by cloning. Supernatants were screened for activity against purified HNL with help of ELISA. Selected hybridomas were expanded and antibodies purified from these were of the IgGl subtype.
  • each of Anti-HNL MAB2 and Anti-HNL MAB3 includes amino acids 145-154 as shown in SEQ ID No: 26 of WO 2016/079219. Additionally, the epitope of Anti- HNL MAB2 includes amino acids 83-88 as shown in SEQ ID No: 26 of WO 2016/079219. Proof-of-concept
  • Figure 3 shows the results of performing the method of the present document using a first and second monoclonal antibody (MAB5) not having the CDR sequences of the first and second monoclonal antibodies as defined herein.
  • MAB5 monoclonal antibody
  • Figure 3 shows the results of performing the method of the present document using a first and second monoclonal antibody (MAB5) not having the CDR sequences of the first and second monoclonal antibodies as defined herein.
  • MAB5 as both first monoclonal antibody and the second monoclonal antibody dimeric HNL cannot be detected.
  • MAB1 anti-HNL monoclonal antibody
  • any combination of monoclonal antibodies specific for HNL may be used in a method according to the present document for detecting a presence or an absence of homodimeric human neutrophil lipocalin (HNL) in a sample.
  • HNL human neutrophil lipocalin
  • the patient group consisted of 206 patients, each of which had signs of infection and body temperature of more than 38 °C.
  • the aetiologies of the infections were determined by routine clinical procedures. No patient took immunosuppressive medication.
  • the infection was either bacterial or a viral infection.
  • the bacterial infections included in the study were bacterial pneumonia, a mycoplasma infection, tonsillitis, a urinary tract infection (UTI), a gastrointestinal (GI) infection, erysipelas and sepsis. In the majority of cases the bacterial infection was an acute respiratory infection, such as pneumonia and tonsillitis.
  • HNL MAB3/MAB3 the immunocomplexes between homodimeric HNL and the monoclonal antibodies

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Abstract

La présente divulgation concerne un procédé de détection d'une présence ou d'une absence de lipocaline neutrophile humaine homodimère (HNL) dans un échantillon. La divulgation concerne également un procédé, un kit de pièces et un dispositif permettant d'établir la présence d'une infection bactérienne ou d'une mise hors tension de celle-ci. La présente divulgation permet la détection précise et fiable d'une infection bactérienne et la surveillance de celle-ci ainsi que la surveillance de l'efficacité d'un traitement antibactérien par détection spécifique de HNL homodimère à l'aide d'une combinaison spéciale d'anticorps monoclonaux spécifiques.
PCT/EP2024/064616 2023-06-01 2024-05-28 Détection spécifique de hnl homodimère Pending WO2024246048A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0756708B1 (fr) 1994-04-21 2001-07-04 Pharmacia Diagnostics Ab Utilisation de la lipocaline neutrophile humaine (hnl) comme marqueur diagnostique et preparation d'anticorps anti-hnl
US20130177923A1 (en) * 2007-11-15 2013-07-11 Bioporto Diagnostics A/S Diagnostic use of individual molecular forms of a biomarker
WO2016079219A1 (fr) 2014-11-19 2016-05-26 Koninklijke Philips N.V. Méthode diagnostique faisant appel à la hnl
CN105974128A (zh) * 2016-06-12 2016-09-28 吉林大学 一种人中性粒细胞载脂蛋白同源二聚体的定量装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0756708B1 (fr) 1994-04-21 2001-07-04 Pharmacia Diagnostics Ab Utilisation de la lipocaline neutrophile humaine (hnl) comme marqueur diagnostique et preparation d'anticorps anti-hnl
US20130177923A1 (en) * 2007-11-15 2013-07-11 Bioporto Diagnostics A/S Diagnostic use of individual molecular forms of a biomarker
WO2016079219A1 (fr) 2014-11-19 2016-05-26 Koninklijke Philips N.V. Méthode diagnostique faisant appel à la hnl
CN105974128A (zh) * 2016-06-12 2016-09-28 吉林大学 一种人中性粒细胞载脂蛋白同源二聚体的定量装置

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