WO2021097684A1 - 新的抗可溶性cd14亚型抗体及其应用 - Google Patents

新的抗可溶性cd14亚型抗体及其应用 Download PDF

Info

Publication number
WO2021097684A1
WO2021097684A1 PCT/CN2019/119536 CN2019119536W WO2021097684A1 WO 2021097684 A1 WO2021097684 A1 WO 2021097684A1 CN 2019119536 W CN2019119536 W CN 2019119536W WO 2021097684 A1 WO2021097684 A1 WO 2021097684A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence number
cdr1
cdr3
cdr2
amino acid
Prior art date
Application number
PCT/CN2019/119536
Other languages
English (en)
French (fr)
Inventor
于丽娜
李可
何建文
Original Assignee
深圳迈瑞生物医疗电子股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳迈瑞生物医疗电子股份有限公司 filed Critical 深圳迈瑞生物医疗电子股份有限公司
Priority to EP19953573.3A priority Critical patent/EP4059517A4/en
Priority to PCT/CN2019/119536 priority patent/WO2021097684A1/zh
Priority to CN201980097405.2A priority patent/CN113939314B/zh
Priority to CN202210524748.6A priority patent/CN115232211A/zh
Publication of WO2021097684A1 publication Critical patent/WO2021097684A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This application relates to an anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof, a kit for detecting soluble CD14 subtype, a method for detecting soluble CD14 subtype, and corresponding applications.
  • Sepsis is a life-threatening organ dysfunction caused by host response imbalance caused by infection.
  • the literature reports that the incidence of sepsis is increasing year by year, and the fatality rate remains high, which has seriously threatened human life and health.
  • early diagnosis is an effective way to improve the survival rate of patients with sepsis.
  • blood culture is the "gold standard” for diagnosis, but the positive rate of blood culture is low and the culture time is long. It is urgent to find highly specific and sensitive biomarkers for early sepsis. Diagnose and predict accurately.
  • the diagnostic criteria for sepsis are also constantly updated.
  • the latest diagnostic criteria for sepsis (Sepsis 3.0) emphasizes infection and the loss of control of the body's response and organ functional damage.
  • Sepsis 3.0 reflects the progress of clinical research and diagnosis, as well as the complexity of sepsis and the importance of clinical diagnosis.
  • biomarkers include interleukin 6 (IL-6), C reactive protein (CRP) and procalcitonin (PCT).
  • IL-6 interleukin 6
  • CRP C reactive protein
  • PCT procalcitonin
  • CRP is an acute-phase protein synthesized by the liver. It is highly sensitive to bacterial infections, but has low specificity, long half-life, and no clear correlation with the progression of sepsis.
  • PCT is widely used in the diagnosis of infection and sepsis. PCT tends not to increase in non-bacterial infections. PCT is often not significantly increased in certain types of infections, such as skin and soft tissue infections. PCT is not suitable for judging the prognosis of perioperative abdominal infection and septic shock infection. In addition, in some non-infectious diseases, there will be an increase in PCT. Although PCT has been shown to have a clear correlation with infection, it is prone to false positive diagnoses in cases such as trauma, surgery, and burns. Therefore, PCT is not a perfect marker for the diagnosis of infection and sepsis. It is not reliable to use PCT alone to diagnose infection and sepsis.
  • CD14 is a high-affinity receptor of lipopolysaccharide-lipopolysaccharide binding protein complex, which is anchored on the cell surface with glycosylphosphatidylinositol.
  • CD14 mainly exists on the membrane surface of monocytes and macrophages.
  • the existing forms of CD14 include membrane-bound CD14 (mCD14) and soluble CD14 (sCD14).
  • mCD14 membrane-bound CD14
  • sCD14 soluble CD14
  • Membrane-bound CD14 is mainly expressed on the surface of monocytes/macrophages. Soluble CD14 is distributed in plasma. It is the part of LPS-LBF-CD14 complex that releases human blood after CD14 falls off the cell membrane. It mainly mediates the response of endothelial cells and epithelial cells to LPS.
  • the soluble CD14 subtype (sCD14-ST or Presepsin for short) is a new biological indicator of sepsis. It has potential application value in the diagnosis and prognosis of sepsis and the use of antibacterial drugs.
  • the research hotspot of sepsis markers in recent years. Shirakawa et al. established a rabbit endotoxin shock model and a cecal ligation and perforation model (CLP model) and found that the former did not cause an increase in the concentration of Presepsin, while the CLP model increased significantly. Therefore, it is believed that the increase in Presepsin is caused by cell phagocytosis caused by bacterial infection, rather than physiological anti-inflammatory.
  • Presepsin is the N-terminal fragment of sCD14 that is cleaved by cathepsin D and other proteases in plasma, and its relative molecular weight is about 13kDa. At present, Presepsin is considered to be highly sensitive and specific in assessing the severity and prognosis of sepsis, and can accurately guide the application of antimicrobial agents for sepsis.
  • an object of the present application is to provide an anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof, which is suitable for detecting the soluble CD14 subtype in a sample.
  • another object of the present application is to provide a kit for detecting soluble CD14 subtype, a method for detecting soluble CD14 subtype, and corresponding applications.
  • the first aspect of this application relates to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope composed of the amino acid sequence of SEQ ID No. 42, and the anti-soluble CD14 subtype single Cloned antibodies or antigen-binding antibody fragments include:
  • VH CDR1 X 1 X 2 X 3 MX 4 ;
  • VH CDR2 YIX 5 X 6 ADX 7 ;
  • VH CDR3 X 8 X 9 X 10 AX 11 ;
  • VL Light chain variable region
  • CDR complementarity determining region
  • VL CDR1 KX 12 X 13 X 14 N;
  • VL CDR2 LX 15 X 16 ;
  • VL CDR3 VX 17 X 18 X 19 ;
  • X 1 to X 19 are one or more amino acid sequences described as the following options:
  • X 1 any amino acid
  • X 2 F or V;
  • X 3 A, E or K;
  • X 4 A or L;
  • X 5 SYMGS, SSGSS, AYTMY, TYSGS or SSKSS;
  • X 6 GAYY, TIYY, AKYY, TKAS or SNLA;
  • X 7 AKLG, TVKG, SYTA, MTKG or YGNT;
  • X 12 YYAS, SSAT, SSQS or SGSS;
  • X 13 AAKL, LLAT, LLYS or KAAS;
  • X 14 YRNIKL, TWAKGN, NGKTYL or YTNGAL;
  • X 15 VQS, KTS, QTS or VSK;
  • X 16 LAS, LDS, LTA or DSK
  • X 17 G, A, S or Q;
  • X 18 GTH, GNT, GTA or TAI;
  • X 19 FITA, FPRT, YGHV or NYGH.
  • the second aspect of the application relates to a kit for detecting a soluble CD14 subtype
  • the kit for detecting a soluble CD14 subtype at least comprises the anti-soluble CD14 subtype monoclonal antibody or an antigen thereof according to the first aspect of the application Binding antibody fragments.
  • the third aspect of the present application relates to a method for detecting soluble CD14 subtypes, the method comprising:
  • the sample to be tested and the solid phase support coated with capture antibody are mixed, so that the capture antibody coated on the solid phase support binds to the soluble CD14 subtype in the sample to be tested, wherein the capture antibody is according to the present application.
  • a detection substrate is added to the above-mentioned washed sandwich complex to detect the concentration of soluble CD14 subtypes in the sample.
  • the fourth aspect of the application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof of the first aspect of the application in the preparation of an analytical reagent for assessing whether a patient suffers from sepsis, wherein
  • the anti-soluble CD14 subtype monoclonal antibody or its antigen-binding antibody fragment of the first aspect of the present application detects the concentration of the soluble CD14 subtype in the blood sample of the patient, and the concentration of the soluble CD14 subtype is relative to the reference value. Elevated levels are associated with an increased likelihood of patients suffering from sepsis.
  • the fifth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or its antigen-binding antibody fragment of the first aspect of the present application in the preparation of diagnostic reagents for assessing the prognosis of patients with suspected sepsis, wherein
  • the anti-soluble CD14 subtype monoclonal antibody or its antigen-binding antibody fragment of the first aspect of the present application detects the concentration of the soluble CD14 subtype in the blood sample of a sepsis patient, and the concentration of the soluble CD14 subtype is relatively An increase in the reference value is associated with an increased risk of death in patients with suspected sepsis.
  • inventions of the present application also relate to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope composed of the amino acid sequence of SEQ ID No. 42, including:
  • VH containing the VH CDR1 composed of the amino acid sequence of SEQ ID NO: 2 VH CDR2 composed of the amino acid sequence of SEQ ID NO: 12, VH CDR3 composed of the amino acid sequence of SEQ ID NO: 20, and the VH containing the amino acid sequence of SEQ ID NO: 22
  • the embodiment of the application also relates to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope composed of the amino acid sequence of SEQ ID No. 42, wherein the soluble CD14 subtype
  • the capture antibody is an anti-soluble CD14 subtype monoclonal antibody or its antigen-binding antibody fragment secreted by the hybridoma cell strain with the deposit number of CGMCC NO.18536.
  • the anti-soluble CD14 subtype monoclonal antibodies and antigen-binding antibody fragments provided in the examples of the present application have high affinity for the soluble CD14 subtype, and therefore can accurately and quantitatively detect the concentration of the soluble CD14 subtype in the sample.
  • the anti-soluble CD14 subtype monoclonal antibodies and antigen-binding antibody fragments provided in the examples of the present application have high sensitivity for the concentration determination of soluble CD14 subtypes.
  • the examples of this application can perform high-specificity determinations in the double antibody sandwich method, that is, the anti-soluble CD14 subtype monoclonal antibodies and antigen-binding antibody fragments provided in the examples of this application only interact with human blood.
  • the existing soluble CD14 subtype specifically binds, but does not specifically bind to the high molecular weight sCD14 therein.
  • Figure 1 shows the prokaryotic expression protein SDS-PAGE and Western Blot
  • Figure 2 shows the eukaryotic expression protein SDS-PAGE and Western Blot
  • Figure 3 shows SDS-PAGE and Western Blot of anti-soluble CD14 subtype polyclonal antibody
  • Figure 4 shows a standard curve of a kit for detecting soluble CD14 subtypes
  • Figure 5 shows the ROC curve diagram of PCT and the kit of the embodiments of the present application for assessing whether a patient suffers from sepsis;
  • Fig. 6 shows a survival rate curve diagram of the kit of an embodiment of the present application for evaluating the prognosis of suspected sepsis
  • Fig. 7 shows a ROC curve chart of PCT and the kit of the examples of the present application for evaluating the prognosis of suspected sepsis.
  • the so-called “specifically recognize the epitope composed of the amino acid sequence of SEQ ID No. 42” means that the antibody claimed in this application will correspond to SEQ ID No in the sequence of the soluble CD14 subtype.
  • the sequence of the .42 amino acid sequence is used as an epitope for specific recognition.
  • antibodies sometimes also refer to antibodies or antigen-binding antibody fragments thereof, and antigen-binding antibody fragments refer to antibodies that specifically recognize the epitope composed of the amino acid sequence of SEQ ID No. 42 Among the partial fragments, the fragments have the same antigen binding properties as the original antibody.
  • soluble CD14 refers to human soluble CD14.
  • detection can be used interchangeably with terms such as “determination”, “quantitative”, and “analysis”, which means to include quantitative and qualitative determination.
  • the detection in this application is preferably performed in vitro.
  • ROC receiver operating characteristic
  • ROC curve refers to the curve obtained by dividing the diagnostic test result into several critical points, with the sensitivity corresponding to each critical point as the ordinate and the specificity as the abscissa.
  • ROC curve is an effective tool for comprehensive and accurate evaluation of diagnostic tests.
  • Another function of the ROC curve is to determine the optimal threshold for detection.
  • ROC curve method to determine the critical point In most cases, select the point on the curve as close to the upper left as possible, and combine the professional situation to determine the critical point as the best.
  • the first aspect of this application claims an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope composed of the amino acid sequence of SEQ ID No. 42, and the anti-soluble CD14 subtype Monoclonal antibodies or antigen-binding antibody fragments include:
  • VH CDR1 X 1 X 2 X 3 MX 4 ;
  • VH CDR2 YIX 5 X 6 ADX 7 ;
  • VH CDR3 X 8 X 9 X 10 AX 11 ;
  • VL Light chain variable region
  • CDR complementarity determining region
  • VL CDR1 KX 12 X 13 X 14 N;
  • VL CDR2 LX 15 X 16 ;
  • VL CDR3 VX 17 X 18 X 19 ;
  • X 1 to X 19 are one or more amino acid sequences described as options below (see Table 1 to Table 6).
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragments thereof include VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1 , VH CDR2 and VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are selected from the amino acid sequences described in Table 7 (see Table 7).
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragments thereof include VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1 , VH CDR2 and VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are selected from the amino acid sequences recorded in Table 8 (see Table 8).
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragments thereof include VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 are as follows Any one of 1) to 62):
  • VH CDR1 is composed of sequence number 1
  • VH CDR2 is composed of sequence number 10
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 1
  • VH CDR2 is sequence number 11
  • VH CDR3 is sequence number 17
  • VL CDR1 is sequence number 26
  • VL CDR2 is sequence number 28
  • VL CDR3 is composed of amino acid sequences of sequence number 41;
  • VH CDR1 is composed of sequence number 1
  • VH CDR2 is sequence number 12
  • VH CDR3 is sequence number 17
  • VL CDR1 is sequence number 21
  • VL CDR2 is sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 33;
  • VH CDR1 is composed of sequence number 1
  • VH CDR2 is sequence number 12
  • VH CDR3 is sequence number 14
  • VL CDR1 is sequence number 26
  • VL CDR2 is sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 1
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 20
  • VL CDR1 is sequence number 22
  • VL CDR2 is sequence number 30, and VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 17
  • VL CDR1 is composed of sequence number 23
  • VL CDR2 is composed of sequence number 30, and VL CDR3 is composed of amino acid sequences of sequence number 35;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 26
  • VL CDR2 is composed of sequence number 31
  • VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 8
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 24
  • VL CDR2 is composed of sequence number 30, and VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 8
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 23
  • VL CDR2 is composed of sequence number 29, and VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 8
  • VH CDR3 is composed of sequence number 17
  • VL CDR1 is composed of sequence number 24
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 9
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 10
  • VH CDR3 is composed of sequence number 19
  • VL CDR1 is composed of sequence number 24
  • VL CDR2 is composed of sequence number 29, and VL CDR3 is composed of amino acid sequences of sequence number 35;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 11
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 26
  • VL CDR2 is composed of sequence number 29
  • VL CDR3 is composed of amino acid sequences of sequence number 36;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 11
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 26
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 11
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 26
  • VL CDR2 is composed of sequence number 27
  • VL CDR3 is composed of amino acid sequences of sequence number 41;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 20
  • VL CDR1 is composed of sequence number 22
  • VL CDR2 is composed of sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of the sequence number 2
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 29
  • VL CDR3 is composed of amino acid sequences of sequence number 35;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 28, and VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 13
  • VH CDR3 is composed of sequence number 15
  • VL CDR1 is composed of sequence number 23
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 13
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 22
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 2
  • VH CDR2 is composed of sequence number 13
  • VH CDR3 is composed of sequence number 17
  • VL CDR1 is composed of sequence number 22
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 36;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 28, and VL CDR3 is composed of the amino acid sequences of sequence number 41;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is sequence number 7
  • VH CDR3 is sequence number 19
  • VL CDR1 is sequence number 23
  • VL CDR2 is sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 8
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 22
  • VL CDR2 is composed of sequence number 28
  • VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 8
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 22
  • VL CDR2 is composed of sequence number 31
  • VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 8
  • VH CDR3 is composed of sequence number 19
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 9
  • VH CDR3 is composed of sequence number 17
  • VL CDR1 is composed of sequence number 23
  • VL CDR2 is composed of sequence number 29
  • VL CDR3 is composed of amino acid sequences of sequence number 36;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 9
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of sequence number 29
  • VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is sequence number 10
  • VH CDR3 is sequence number 19
  • VL CDR1 is sequence number 22
  • VL CDR2 is sequence number 32
  • VL CDR3 is composed of the amino acid sequences of sequence number 41;
  • VH CDR1 is composed of sequence number 3
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 15
  • VL CDR1 is composed of sequence number 24
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 15
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 28, and VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 23
  • VL CDR2 is composed of sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 33;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is composed of sequence number 10
  • VH CDR3 is composed of sequence number 19
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is composed of sequence number 10
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 24
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is sequence number 12
  • VH CDR3 is sequence number 16
  • VL CDR1 is sequence number 23
  • VL CDR2 is sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 33;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 24
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 4
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 16
  • VL CDR1 is sequence number 22
  • VL CDR2 is sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 7
  • VH CDR3 is sequence number 17
  • VL CDR1 is sequence number 23
  • VL CDR2 is sequence number 30, and VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 7
  • VH CDR3 is sequence number 20
  • VL CDR1 is sequence number 21
  • VL CDR2 is sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 8
  • VH CDR3 is sequence number 18
  • VL CDR1 is sequence number 21
  • VL CDR2 is sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 33;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 10
  • VH CDR3 is sequence number 18
  • VL CDR1 is sequence number 21
  • VL CDR2 is sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 33;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 10
  • VH CDR3 is sequence number 15
  • VL CDR1 is sequence number 21
  • VL CDR2 is sequence number 28, and VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 10
  • VH CDR3 is sequence number 19
  • VL CDR1 is sequence number 26
  • VL CDR2 is sequence number 28, and VL CDR3 is composed of amino acid sequences of sequence number 40;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is composed of sequence number 11
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 33;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 11
  • VH CDR3 is sequence number 16
  • VL CDR1 is sequence number 23
  • VL CDR2 is sequence number 31
  • VL CDR3 is composed of amino acid sequences of sequence number 36;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 11
  • VH CDR3 is sequence number 17
  • VL CDR1 is sequence number 23
  • VL CDR2 is sequence number 28
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is composed of sequence number 11
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 29
  • VL CDR3 is composed of amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 17
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 37;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 12
  • VH CDR3 is sequence number 19
  • VL CDR1 is sequence number 26
  • VL CDR2 is sequence number 31, and VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is composed of sequence number 12
  • VH CDR3 is composed of sequence number 16
  • VL CDR1 is composed of sequence number 23
  • VL CDR2 is composed of sequence number 27, and VL CDR3 is composed of amino acid sequences of sequence number 34;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 12
  • VH CDR3 is sequence number 19
  • VL CDR1 is sequence number 24
  • VL CDR2 is sequence number 31, and VL CDR3 is composed of the amino acid sequences of sequence number 38;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 15
  • VL CDR1 is sequence number 26
  • VL CDR2 is sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 15
  • VL CDR1 is sequence number 26
  • VL CDR2 is sequence number 28
  • VL CDR3 is sequence number 33.
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 18
  • VL CDR1 is sequence number 21
  • VL CDR2 is sequence number 30
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 5
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 20
  • VL CDR1 is sequence number 25
  • VL CDR2 is sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 6
  • VH CDR2 is composed of sequence number 7
  • VH CDR3 is composed of sequence number 18
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 6
  • VH CDR2 is composed of sequence number 10
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 25
  • VL CDR2 is composed of sequence number 32
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 6
  • VH CDR2 is sequence number 11
  • VH CDR3 is sequence number 18
  • VL CDR1 is sequence number 22
  • VL CDR2 is sequence number 29
  • VL CDR3 is composed of amino acid sequences of sequence number 39;
  • VH CDR1 is composed of sequence number 6
  • VH CDR2 is composed of sequence number 11
  • VH CDR3 is composed of sequence number 14
  • VL CDR1 is composed of sequence number 22
  • VL CDR2 is composed of sequence number 30, and VL CDR3 is composed of the amino acid sequences of sequence number 39; or
  • VH CDR1 is composed of sequence number 6
  • VH CDR2 is sequence number 13
  • VH CDR3 is sequence number 20
  • VL CDR1 is sequence number 22
  • VL CDR2 is sequence number 30
  • VL CDR3 is sequence number 38.
  • VH containing the VH CDR1 composed of the amino acid sequence of SEQ ID NO: 2 VH CDR2 composed of the amino acid sequence of SEQ ID NO: 12, VH CDR3 composed of the amino acid sequence of SEQ ID NO: 20, and the VH containing the amino acid sequence of SEQ ID NO: 22
  • the soluble CD14 monoclonal antibody or antigen-binding antibody fragment thereof is an anti-soluble CD14 subtype monoclonal antibody secreted by a hybridoma cell line with the deposit number of CGMCC NO.18536 Antibodies or antigen-binding antibody fragments thereof.
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof does not specifically bind to high molecular weight soluble CD14.
  • the antigen-binding antibody fragment can be selected from Fab, Fab', F(ab')2, single-chain antibody (scFV), dimerized V region (dimeric antibody) ), disulfide bond stability V region (dsFv), sc(Fv) 2, CDR-containing polypeptides, polypeptides containing heavy chain variable regions, and antigen-binding antibodies in the group consisting of polypeptides containing light chain variable regions Fragment.
  • the second aspect of the present application claims a kit for detecting soluble CD14 subtypes, the kit for detecting soluble CD14 subtypes at least comprises the anti-soluble CD14 subtype monoclonal antibody or its monoclonal antibodies according to the first aspect of the present application Antigen-binding antibody fragments.
  • the kit according to the second aspect of the present application may also include other reagent components, including but not limited to primary antibodies, secondary antibodies, labeled antibodies, labeled enzymes and other labeled substances; chromogenic substrates, fluorescent substrates, chemiluminescent substrates, Specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, washing solutions, standard substances, etc.
  • other reagent components including but not limited to primary antibodies, secondary antibodies, labeled antibodies, labeled enzymes and other labeled substances; chromogenic substrates, fluorescent substrates, chemiluminescent substrates, Specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, washing solutions, standard substances, etc.
  • a kit applied to chemiluminescence enzyme immunoassay may include antibodies, enzyme-labeled antibodies, chemiluminescence substrates, diluents, washing solutions, etc. coated on a solid phase.
  • the antibodies of the embodiments of the present application can also be used to prepare enzyme immunoassay kits, electrochemiluminescence immunoassay kits, immunochromatography kits, etc. based on double antibody sandwich method or competition method.
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the present application is used as a capture antibody, which is coated on a solid-phase carrier and used for capturing The soluble CD14 subtype in the sample to be tested.
  • the solid phase carrier may be selected from magnetic microspheres, chips, test paper, etc., preferably magnetic microspheres, more preferably superparamagnetic magnetic beads.
  • the kit for detecting soluble CD14 subtype further comprises a labeled soluble CD14 subtype detection antibody, and the detection antibody may be an anti-soluble CD14 subtype polyclonal antibody.
  • the signal label of the soluble CD14 subtype detection antibody can be, for example, a chemiluminescent label (such as alkaline phosphatase, luminol, isoluminol, acridinium ester, spicy Root peroxidase), electrochemiluminescent markers (for example, terpyridine ruthenium), quantum dots (for example, gold quantum dots, CdSe quantum dots, ZnCdSe quantum dots, etc.), fluorescent microspheres, etc., or combinations thereof.
  • chemiluminescent label such as alkaline phosphatase, luminol, isoluminol, acridinium ester, spicy Root peroxidase
  • electrochemiluminescent markers for example, terpyridine ruthenium
  • quantum dots for example, gold quantum dots, CdSe quantum dots, ZnCdSe quantum dots, etc.
  • fluorescent microspheres etc., or combinations thereof.
  • a method for detecting a soluble CD14 subtype which method includes contacting the anti-soluble CD14 subtype monoclonal antibody of the first aspect of the application with a sample to be tested.
  • the method for immunologically determining the soluble CD14 subtype in the sample using the antibody or antigen-binding antibody fragment of the first aspect of the application may be, for example, enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA) , Chemiluminescence immunoassay (CLIA), fluorescent antibody method (FAT), fluorescence enzyme immunoassay (FEIA), electrochemiluminescence immunoassay (ECLIA), radioimmunoassay (RIA), immunochromatography, agglutination Law, competition law, etc., but not limited to these methods.
  • ELISA enzyme-linked immunosorbent assay
  • CLIA Chemiluminescence immunoassay
  • FAT fluorescent antibody method
  • FET fluorescence enzyme immunoassay
  • ELIA electrochemiluminescence immunoassay
  • RIA radioimmunoassay
  • immunochromatography agglutination Law, competition law, etc., but not limited
  • ELISA enzyme-linked immunosorbent assay
  • sandwich ELISA refers to the use of antibodies with different antigen recognition sites, one of which is coated on a solid phase, and the other antibody is used to clamp the antigen to be detected, thereby forming an antibody-antigen-antibody complex.
  • chemiluminescence enzyme immunoassay After the antigen in the sample is reacted with the antibody immobilized on the solid phase, it reacts with the enzyme-labeled antibody, and after washing, the chemiluminescence substrate is added for enzymatic reaction, and then the luminescence is measured. strength.
  • the method includes:
  • the sample to be tested and the solid phase support coated with capture antibody are mixed, so that the capture antibody coated on the solid phase support binds to the soluble CD14 subtype in the sample to be tested, wherein the capture antibody is according to the present application.
  • a detection substrate is added to the above-mentioned washed sandwich complex to detect the concentration of soluble CD14 subtypes in the sample.
  • a chemiluminescent substrate is added to the above-mentioned cleaned sandwich complex, and the number of photons generated by the reaction is detected to obtain the chemiluminescence signal value of the sample, and the chemiluminescence signal value is proportional to the concentration of the soluble CD14 subtype.
  • the sample to be tested may be blood, blood components such as whole blood, serum, or plasma, preferably a plasma sample.
  • the sample of the present application is not limited to this, and can also be body fluids such as urine, tissue fluid, lymph fluid, joint fluid, milk, cerebrospinal fluid, pus, saliva, tears, mucus, nasal fluid, sputum, ascites, water, semen, etc.
  • the fourth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the first aspect of the present application in the preparation of an analytical reagent for evaluating whether a patient suffers from sepsis, wherein,
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the first aspect of the present application is used to detect the concentration of the soluble CD14 subtype in the blood sample of the patient, and the concentration of the soluble CD14 subtype is relative to the reference An increase in the level of the value is associated with an increased likelihood of the patient suffering from sepsis.
  • the concentration of the soluble CD14 subtype is compared with the cutoff value to determine whether the concentration of the soluble CD14 subtype is higher than the cutoff value, thereby assessing whether the patient has sepsis.
  • the increase in the concentration of the soluble CD14 subtype relative to the reference value is positively correlated with the increase in the possibility of the patient suffering from sepsis.
  • the concentration of the soluble CD14 subtype in the blood sample of the patient is detected within 72 hours after the patient is suspected of having sepsis.
  • the fifth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the first aspect of the present application in the preparation of analytical reagents for evaluating the prognosis of patients with suspected sepsis, wherein,
  • the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the first aspect of the present application is used to detect the concentration of the soluble CD14 subtype in a blood sample of a patient suspected of sepsis, the soluble CD14 subtype
  • the increase in the level of the concentration relative to the reference value is associated with an increased risk of death in patients with suspected sepsis.
  • an increase in the concentration of the soluble CD14 subtype relative to a reference value is positively correlated with an increase in the risk of death of the suspected sepsis patient.
  • the concentration of the soluble CD14 subtype in the blood sample of the patient is detected within 72 hours after the patient is suspected of having sepsis.
  • Step 1.1 Expression of soluble CD14 subtype immunogen
  • the soluble CD14 subtype immunogen is expressed in a prokaryotic expression system and a eukaryotic expression system respectively. The corresponding process is described in detail below:
  • the pET30a expression vector was selected to construct a plasmid and transformed into E. coli BL21star (DE3) to obtain a prokaryotic expression engineering strain.
  • the recombinant plasmid was induced to express efficiently in E. coli expression system by IPTG (isopropyl thiogalactoside) method.
  • the target protein mainly exists in the inclusion bodies .
  • the target protein is purified by Ni 2+ NTA affinity column.
  • the target protein sequence is Thr Thr Pro Glu Pro Cys Glu Leu Asp Asp Glu Asp Phe Arg Cys Val Cys Asn Phe Ser Glu Pro Gln Pro Asp Trp Ser Glu Ala Phe Gln Cys Val Ser Ala Val Glu Val Glu Ile His Ala Gly Gly Leu Asn Leu Glu Pro Phe Leu Lys Arg Val Asp Ala Asp Ala Asp Pro Arg Gln Tyr Ala (SEQ ID No. 43).
  • the Bradford method was used to determine the concentration of the target protein as 5.42 mg/mL, and SDS-PAGE and Western Blot were used to determine the purity and molecular weight of the target protein.
  • the pcDNA3.4 expression vector was selected to construct the plasmid.
  • the HEK293-6E expression system was used, and the culture conditions were as follows: culture in a serum-free FreeStyle TM 293 Expression Medium (Thermo Fisher Scientific) matrix at 37° C. and 5% CO 2 .
  • the prokaryotic system and the eukaryotic system expressed and purified proteins, namely antigens, obtained in step 1.1 are mixed in a certain proportion, and coupled with the carrier protein keyhole limpet hemocyanin (KLH for short), so as to act as an immunogen for New Zealand white rabbits Immunization is performed as follows: the immunogen is diluted with normal saline, and then mixed with an adjuvant such as Freund's incomplete adjuvant (Sigma-Aldrich) at 1:1. The antigen and adjuvant were completely mixed to form a stable emulsion, which was injected subcutaneously under the skin around the shoulders of New Zealand white rabbits and intramuscularly injected into the back thigh.
  • an adjuvant such as Freund's incomplete adjuvant (Sigma-Aldrich)
  • Each area uses about 1/4 of the immunogen, and the amount of antigen per immunization is about 100 ⁇ g to 500 ⁇ g, a total of four immunizations, and the interval between each immunization is 2 weeks.
  • the antiserum of New Zealand white rabbits was collected and subjected to antigen affinity purification to obtain soluble CD14 subtype polyclonal antibodies.
  • Figure 3 shows the results of SDS-PAGE and Western Blot of anti-soluble CD14 subtype polyclonal antibody.
  • lane M is the protein marker
  • the first lane is polyclonal antibody
  • the second lane is rabbit IgG
  • the first lane is Immunogen (100ng) + purified antibody (1 ⁇ g/mL) + goat anti-rabbit IgG [IRDye800cw] (0.125 ⁇ g/mL)
  • the second lane is immunogen (50ng) + purified antibody (1 ⁇ g/mL) + goat anti-rabbit IgG [IRDye800cw] (0.125 ⁇ g/mL)
  • the third lane is immunogen (100ng) + unimmunized serum + goat anti-rabbit IgG [IRDye800cw] (0.125 ⁇ g/mL)
  • the fourth lane is immunogen (100ng) + goat anti Rabbit IgG [IRDye800cw] (0.125 ⁇ g/mL).
  • the prokaryotic system and the eukaryotic system expressed and purified proteins, namely antigens, obtained in step 1.1 are mixed in a certain proportion, and coupled with the carrier protein keyhole limpet hemocyanin (KLH for short), so as to act as an immunogen for mice as follows Steps for immunization: in the first immunization, the antigen (10 ⁇ g to 50 ⁇ g) is mixed with an adjuvant such as Freund's complete adjuvant (Sigma-Aldrich) at a ratio of 1:1, and then multiple subcutaneous injections are performed. The second immunization was performed 3 weeks later.
  • an adjuvant such as Freund's complete adjuvant (Sigma-Aldrich)
  • the dose of the second immunization was the same as that of the first immunization, and Freund's incomplete adjuvant (Sigma-Aldrich) was added for subcutaneous injection. Three weeks later, the third immunization was performed. The dose of the third immunization was the same as that of the first immunization, but no adjuvant was added. After 3 weeks, a dose of 100 ⁇ g to 500 ⁇ g was used for booster immunization. Three days after the booster immunization, the spleen was taken for fusion.
  • the ELISA method was used to evaluate the valence of the mouse antiserum, and the spleen cells of the animal with the best titers were selected for fusion with myeloma cells to obtain the mother clone cell line with the deposit number CGMCC NO. 18536.
  • various known myeloma cells can be used, for example, P3, NS-1, P3U1, SP2/0 derived from mouse, YB2/0, Y3-Ag1 derived from rat SKO-007 derived from humans, human-mouse hybrid myeloma cells SHM-D33, etc.
  • the SP2/0 of mouse is preferably used in this application.
  • the subsequent experimental procedure is the same as the positive sieve experiment. Based on the intensity of the final luminescence value, it is judged whether the cell supernatant antibody is reactive with the anti-screening substance.
  • Subsequent subcloning is performed after screening the mother clone. Affinity ranking and epitope classification were performed on the subclones. Pair the subclonal supernatant with the polyclonal antibody prepared in step 1.2 to detect the antigen.
  • Table 9-11 summarizes the anti-soluble CD14 subtype monoclonal antibody affinity ranking, epitope classification and matched antibody screening results. According to the affinity ranking and the result of antibody pairing, the antibody with clone number 16D5B2 was screened for subsequent production and purification to obtain mouse monoclonal antibodies.
  • Step 1.4 Determination of the amino acid sequence of the anti-soluble CD14 subtype monoclonal antibody
  • the variable region of the anti-soluble CD14 subtype monoclonal antibody obtained by screening in step 1.3 was sequenced. First, follow The kit operation manual extracts the total RNA of the hybridoma cells, and then according to the PrimeScript TM 1st Strand cDNA Synthesis Kit operation manual, the total RNA is reverse transcribed into cDNA with epitope-specific antisense primers or universal primers. The cDNA sequence synthesized by reverse transcription can be used to obtain the corresponding amino acid sequence of the anti-soluble CD14 subtype monoclonal antibody.
  • the anti-soluble CD14 subtype monoclonal antibody includes, for example, the VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 9.
  • VH CDR2 composed of the amino acid sequence of SEQ ID NO: 16
  • VH CDR3 composed of the amino acid sequence of SEQ ID NO: 16
  • VL CDR1 composed of the amino acid sequence of SEQ ID No. 21
  • VL CDR2 composed of the amino acid sequence of SEQ ID NO: 29
  • the VL composed of the amino acid sequence of No. 38 is the VL of CDR3, wherein the anti-soluble CD14 subtype monoclonal antibody specifically recognizes the epitope composed of the amino acid sequence of SEQ ID No. 42.
  • Example 2 Determination of specific epitope of anti-soluble CD14 subtype monoclonal antibody
  • the method for determining the epitope is not particularly limited.
  • the target protein sequence moves 3 amino acids from the N-terminus to the C-terminus to synthesize a polypeptide, and each peptide chain is composed of 15 amino acids.
  • Composition the polypeptides are labeled with biotin, and are coated on a 96-well plate containing streptavidin through biotin-streptavidin binding; then the antibody to be tested and peroxidase are labeled
  • the goat anti-mouse IgG was added to a 96-well plate; the signal value of the final reaction was used to determine the recognized amino acid sequence of the antibody to be tested.
  • Example 3 Preparation of a kit for detecting soluble CD14 subtype
  • the monoclonal antibody with the clone number 16D5B2 screened in Example 1 was used as a capture antibody and coated on superparamagnetic magnetic beads (Merck, Superparamagnetic beads) surface.
  • the surface of the superparamagnetic magnetic beads is modified with carboxyl groups, and the carboxyl groups on the surface of the superparamagnetic magnetic beads are coupled with the amino group of the monoclonal antibody clone number 16D5B2 under the catalysis of EDC/NHS, thereby preparing the magnetic bead coating.
  • the anti-soluble CD14 subtype polyclonal antibody prepared in step 1.2 of Example 1 was used as the detection antibody, and the detection antibody was labeled with alkaline phosphatase (Roche Life Science).
  • the detection reaction is based on the sandwich method: the sample to be tested (40 ⁇ L), the capture antibody (50 ⁇ L) and the detection antibody (50 ⁇ L) are added to the reaction tube and incubated, where the sample to be tested is plasma.
  • the antigen in the sample to be tested, the soluble CD14 subtype is combined with the capture antibody and the detection antibody to form a sandwich complex.
  • the magnetic field adsorbs the magnetic beads to wash away unbound substances.
  • the luminescent substrate (3-(2-helicaladamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxane Ethane, AMPPD) is decomposed by alkaline phosphatase, a phosphate group is removed, and an unstable intermediate product is generated.
  • This intermediate product generates methyl meta-oxybenzoate anion through intramolecular electron transfer, which is an excited meta-oxybenzene When the methyl formate anion returns from the excited state to the ground state, chemiluminescence is generated.
  • the number of photons generated by the intermediate reaction is measured by a photomultiplier tube, and the number of photons generated is proportional to the concentration of the soluble CD14 subtype in the sample.
  • a series of samples containing different concentrations of soluble CD14 subtypes are tested, and the corresponding luminescence values can be obtained, and then a standard curve can be established, as shown in Figure 4.
  • Example 4 Application of anti-soluble CD14 subtype monoclonal antibodies or antigen-binding antibody fragments in the preparation of analytical reagents for assessing whether a patient suffers from sepsis
  • the soluble CD14 subtype of samples from 141 patients suspected of having sepsis was detected using the kit prepared in Example 3. At the same time, the patient's PCT concentration level was detected, and the diagnostic efficacy of soluble CD14 subtype and PCT on sepsis was compared.
  • the detection threshold of soluble CD14 subtype is 500pg/mL, and its sensitivity and specificity for the diagnosis of sepsis are 0.76 and 0.91, respectively.
  • the AUC areas of the ROC curves of the soluble CD14 subtype and PCT are 0.91 and 0.84, respectively. It is concluded that the soluble CD14 subtype has a higher diagnostic power.
  • Example 5 Application of anti-soluble CD14 subtype monoclonal antibodies or antigen-binding antibody fragments in the preparation of analytical reagents for assessing the prognosis of patients with suspected sepsis
  • the kit prepared in Example 3 was used to detect the concentration of soluble CD14 subtypes in plasma samples of 88 patients with suspected sepsis within 72 hours of admission, and to track the prognosis of patients with suspected sepsis within 30 hours, using Kaplan-Meier analysis method is used to obtain the relationship curve diagram of the hospital's survival rate within 30 days (see Figure 6). For different groups, the survival rate of patients is significantly different. For group III (soluble CD14 subtype concentration ⁇ 1003.97pg/mL), the survival probability of patients is ⁇ 40% (p ⁇ 0.01).
  • the concentration values of PCT and soluble CD14 subtypes within 72 hours of admission were compared with the prognostic effect of survival rate within 30 days of admission.
  • the AUC of PCT detection and soluble CD14 subtype detection are 0.72 and 0.83, respectively. Therefore, soluble CD14 subtype can more accurately predict the survival of patients.
  • the anti-soluble CD14 subtype monoclonal antibody or its antigen-binding antibody fragments of the examples of the application or the kits of the examples of the application have high specificity and sensitivity and are suitable for detecting Measure the soluble CD14 subtypes in the sample, thereby improving the quality and accuracy of the detection of soluble CD14 subtypes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位。还涉及一种用于检测可溶性CD14亚型的试剂盒、一种用于检测可溶性CD14亚型的方法以及相应的应用。

Description

新的抗可溶性CD14亚型抗体及其应用 技术领域
本申请涉及一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段、一种用于检测可溶性CD14亚型的试剂盒、一种用于检测可溶性CD14亚型的方法以及相应的应用。
背景技术
脓毒血症(sepsis)是感染引起宿主反应失调导致的危及生命的器官功能障碍。文献资料报道脓毒血症发病率逐年上升，且病死率居高不下，已严重威胁到人类的生命健康。根据脓毒血症治疗指南，早期诊断是提高脓毒血症患者存活率的有效途径。在脓毒血症的诊断中，血培养是诊断的“金标准”，但血培养的阳性率低且培养时间长，亟需寻找高度特异性和灵敏的生物标志物对脓毒血症进行早期诊断和准确预测。
对于脓毒血症的诊断标准也不断的更新。脓毒血症最新的诊断标准(Sepsis 3.0)强调感染以及其导致的机体的反应失控和器官的功能损伤。虽然对于Sepsis 3.0仍然存在不少争议，但其反应了临床研究和诊断的进步，也反应了脓毒血症的复杂性和临床诊断的重要性。目前在诊断脓毒血症方面，临床广泛的应用的生物标志物有白介素6(Interleukin 6，IL-6)、C反应蛋白(C reactive protein，CRP)和降钙素原(procalcitonin，PCT)。CRP是由肝脏合成的急性期蛋白，对细菌性感染有较强的敏感度，但特异度不高、半衰期长、与脓毒症的病情进展没有明确的相关性。IL-6峰值出现早，可以在临床症状出现之前升高，有利于脓毒症的早期诊断，但是敏感度不高。PCT广泛应用于感染以及脓毒血症的诊断。PCT在非细菌性的感染中往往不会升高。PCT在某些感染类型，如皮肤软组织感染中增高往往不明显。PCT不适用于判断围手术期腹腔感染脓毒性休克感染的预后。此外，在一些非感染性疾病中，也会伴随着PCT的升高。虽然PCT已经证明与感染有明显的相关性，但在如外伤、手术、烧伤等情况下，很容易出现假阳性诊断。因此，PCT并不 是感染以及脓毒血症诊断的完美标志物，仅用PCT来诊断感染和脓毒血症是不可靠的。
CD14是脂多糖-脂多糖结合蛋白复合体的高亲和力受体，以糖基磷脂酰肌醇锚定于细胞表面。CD14主要存在于单核细胞和巨噬细胞膜表面。CD14的存在形式包括膜结合性CD14(mCD14)和可溶性CD14(sCD14)。膜结合性CD14主要表达在单核/巨噬细胞表面。可溶性CD14分布于血浆中，是CD14从细胞膜脱落后释放人血的那部分LPS-LBF-CD14复合物，主要介导内皮细胞和上皮细胞对LPS的应答。
可溶性CD14亚型(soluble CD14 subtype，简称sCD14-ST或Presepsin)是脓毒血症的新生物学指标，在脓毒血症的诊断、预后以及指导抗菌药物的使用方面有着潜在的应用价值，是近几年脓毒血症标志物研究的热点。Shirakawa等通过建立家兔内毒素休克模型和盲肠结扎穿孔模型(CLP模型)发现，前者并未能引起Presepsin浓度升高，而CLP模型显著升高。故认为Presepsin的升高是由细菌感染引起的细胞吞噬引起，而并非生理性抗炎导致。Presepsin是sCD14在血浆中被组织蛋白酶D等蛋白酶切割后N端的片段，其相对分子量约为13kDa。目前Presepsin被认为在评估脓毒症严重程度及预后方面具有高敏感和特异性，并能准确的指导脓毒症抗菌药的应用。
迄今一直在尝试如何对脓毒血症进行高度特异性和灵敏地检测、早期诊断和准确预测。
发明内容
因此，本申请的一个目的是提供一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其适合用于检测样本中的可溶性CD14亚型。此外，本申请的另一目的是提供一种用于检测可溶性CD14亚型的试剂盒、一种用于检测可溶性CD14亚型的方法以及相应的应用。
本申请第一方面涉及一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，该抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括：
(i)重链可变区(VH)互补性决定区(CDR)：
VH CDR1：X 1X 2X 3MX 4；
VH CDR2：YIX 5X 6ADX 7；
VH CDR3：X 8X 9X 10AX 11；
和
(ii)轻链可变区(VL)互补性决定区(CDR)：
VL CDR1：KX 12X 13X 14N；
VL CDR2：LX 15X 16；
VL CDR3：VX 17X 18X 19；
其中X 1至X 19是下列作为选择项记载的1个或多个氨基酸序列：
X 1＝任意一个氨基酸；
X 2＝F或V；X 3＝A、E或K；X 4＝A或L；
X 5＝SYMGS、SSGSS、AYTMY、TYSGS或SSKSS；
X 6＝GAYY、TIYY、AKYY、TKAS或SNLA；
X 7＝AKLG、TVKG、SYTA、MTKG或YGNT；
X 8＝A或Q；X 9＝G或Q；X 10＝Q或F；X 11＝M、Y或V；
X 12＝YYAS、SSAT、SSQS或SGSS；
X 13＝AAKL、LLAT、LLYS或KAAS；
X 14＝YRNIKL、TWAKGN、NGKTYL或YTNGAL；
X 15＝VQS、KTS、QTS或VSK；
X 16＝LAS、LDS、LTA或DSK；
X 17＝G、A、S或Q；
X 18＝GTH、GNT、GTA或TAI；
X 19＝FITA、FPRT、YGHV或NYGH。
本申请第二方面涉及一种用于检测可溶性CD14亚型的试剂盒，该用于检测可溶性CD14亚型的试剂盒至少包含根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
本申请第三方面涉及一种用于检测可溶性CD14亚型的方法，所述方法包括：
将待测样本和包被有捕获抗体的固相支持物混合，使得固相支持物上包被的捕获抗体与待测样本中的可溶性CD14亚型结合，其中所述捕获抗体为根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段；
将上述混合物进行清洗，除去未结合的物质；
在上述经清洗的混合物中加入经标记的检测抗体进行混匀，使得所述检测抗体与所述捕获抗体上结合的可溶性CD14亚型结合，形成夹心复合物；
对上述夹心复合物进行清洗，除去未结合的物质；
在上述经清洗的夹心复合物中加入检测底物，以检测样本中的可溶性CD14亚型的浓度。
本申请第四方面涉及本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估患者是否患有脓毒血症的分析试剂中的应用，其中，使用本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段对患者的血液样本中的可溶性CD14亚型的浓度进行检测，所述可溶性CD14亚型的浓度相对于参考值的水平升高与患者患有脓毒血症的可能性增加相关。
本申请第五方面涉及本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估疑似脓毒血症患者的预后的诊断试剂中的应用，其中，使用本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段对脓毒血症患者的血液样本中的可溶性CD14亚型的浓度进行检测，所述可溶性CD14亚型的浓度相对于参考值的水平升高与疑似脓毒血症患者的死亡风险增加相关。
本申请实施例还涉及一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，包括：
(a)包含由序列编号1的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号17的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号32的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；
(b)包含由序列编号2的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号37的氨基酸序列构成的VL CDR3的VL；
(c)包含由序列编号3的氨基酸序列构成的VH CDR1、由序列编号9 的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号29的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL；
(d)包含由序列编号5的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号18的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号39的氨基酸序列构成的VL CDR3的VL；
(e)包含由序列编号4的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号23的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；或
(f)包含由序列编号6的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL。
本申请实施例还涉及一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，其中所述可溶性CD14亚型捕获抗体为由保藏号为CGMCC NO.18536的杂交瘤细胞株分泌产生的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
本申请实施例所提供的抗可溶性CD14亚型单克隆抗体及其抗原结合性抗体片段具有对可溶性CD14亚型高的亲和性，因此能准确地定量检测样本中的可溶性CD14亚型的浓度。此外，本申请实施例提供的抗可溶性CD14亚型单克隆抗体及其抗原结合性抗体片段对于可溶性CD14亚型的浓度测定具有高灵敏度。此外，本申请实施例能够在双抗体夹心法中进行高特异性测定，也就是说，本申请实施例所提供的抗可溶性CD14亚型单克隆抗体及其抗原结合性抗体片段仅与人血液中存在的可溶性CD14亚型特异性地结合、而不与其中的高分子量sCD14特异性结合。
附图说明
在下文中，参考附图描述本申请的一些实施方式。根据以下详细描述和附图，将进一步理解本申请的目的和优点。附图示出：
图1示出原核表达蛋白SDS-PAGE和Western Blot；
图2示出真核表达蛋白SDS-PAGE和Western Blot；
图3示出抗可溶性CD14亚型多克隆抗体SDS-PAGE和Western Blot；
图4示出用于检测可溶性CD14亚型的试剂盒的标准曲线；
图5示出PCT和本申请实施例的试剂盒用于评估患者是否患有脓毒血症的ROC曲线图；
图6示出本申请实施例的试剂盒用于评估疑似脓毒血症的预后的生存率曲线图；
图7示出PCT和本申请实施例的试剂盒用于评估疑似脓毒血症的预后的ROC曲线图。
具体实施方式
在下文中，将通过具体实施例更详细地描述本申请。然而，这些实施例只是代表性的并且本申请将决不被理解为受这些实施例的限制。
当在本申请的上下文中这些表述、特征、数值或范围结合例如为“大约、基本上、一般而言、至少、最少”等的表述提及时，本申请同样包括准确的或精确的表述、特征、数值或范围等。
在本申请的范围中，所谓“特异性地识别由序列编号SEQ ID No.42的氨基酸序列构成的表位”是指本申请要求保护的抗体将相当于可溶性CD14亚型的序列中SEQ ID No.42的氨基酸序列的序列作为表位而进行特异性识别。
在本申请的范围中，抗体有时也指抗体或其抗原结合性抗体片段，而抗原结合性抗体片段是指在对由SEQ ID No.42的氨基酸序列构成的表位进行特异性识别的抗体的部分片段中，具有与原抗体相同的抗原结合性的片段。
在本申请的范围中，除非另作说明，可溶性CD14均指人可溶性CD14。
在本申请的范围中，术语检测可以与“测定”、“定量”、“分析”等用语相互变换地使用，意指包含定量的和定性的确定。本申请中的检测优选在体外进行。
在本申请的范围中，术语ROC(receiver operating characteristic)曲线是指将诊断试验结果划分为若干临界点，以每个临界点对应的灵敏度为纵坐标，特异度为横坐标，作图得到的曲线。ROC曲线是一种全面、准确评价诊断试验的有效工具。ROC曲线的另一个作用是确定检测的最佳阈值。ROC曲线法确定临界点多数情况下，选择曲线上尽量靠近左上方的点，并结合专业情况，确定临界点为最佳。在应用中，根据ROC曲线，结合各切点的灵敏度和特异度结果，选择曲线上尽量靠近左上方约登指数(Youden index)最大的切点为最佳临界点，从而使试验的灵敏度和特异度均较高，同时误诊率和漏诊率较小。
本申请第一方面请求保护一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，该抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括：
(i)重链可变区(VH)互补性决定区(CDR)：
VH CDR1：X 1X 2X 3MX 4；
VH CDR2：YIX 5X 6ADX 7；
VH CDR3：X 8X 9X 10AX 11；
和
(ii)轻链可变区(VL)互补性决定区(CDR)：
VL CDR1：KX 12X 13X 14N；
VL CDR2：LX 15X 16；
VL CDR3：VX 17X 18X 19；
其中X 1至X 19是下列作为选择项记载的1个或多个氨基酸序列(见表1至表6)。
表1
Figure PCTCN2019119536-appb-000001
表2
Figure PCTCN2019119536-appb-000002
Figure PCTCN2019119536-appb-000003
表3
Figure PCTCN2019119536-appb-000004
表4
Figure PCTCN2019119536-appb-000005
表5
Figure PCTCN2019119536-appb-000006
表6
Figure PCTCN2019119536-appb-000007
在本申请第一方面的一些实施方式中，该抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3，其中VH CDR1、VH CDR2和VH CDR3以 及VL CDR1、VL CDR2和VL CDR3是选自表7中记载的氨基酸序列(见表7)。
表7
Figure PCTCN2019119536-appb-000008
Figure PCTCN2019119536-appb-000009
在本申请第一方面的一些实施方式中，该抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3，其中VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3是选自表8中记载的氨基酸序列(见表8)。
表8
Figure PCTCN2019119536-appb-000010
Figure PCTCN2019119536-appb-000011
在本申请第一方面的一些实施方式中，该抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括VH CDR1、VH CDR2和VH CDR3，以及VL CDR1、VL CDR2和VL CDR3是下述1)至62)中的任意一种：
1)VH CDR1由序列编号1、VH CDR2由序列编号10、VH CDR3由序列编号14、VL CDR1由序列编号21、VL CDR2由序列编号32、VL CDR3由序列编号34的各氨基酸序列构成；
2)VH CDR1由序列编号1、VH CDR2由序列编号11、VH CDR3由序列编号17、VL CDR1由序列编号26、VL CDR2由序列编号28、VL CDR3由序列编号41的各氨基酸序列构成；
3)VH CDR1由序列编号1、VH CDR2由序列编号12、VH CDR3由序列编号17、VL CDR1由序列编号21、VL CDR2由序列编号32、VL CDR3 由序列编号33的各氨基酸序列构成；
4)VH CDR1由序列编号1、VH CDR2由序列编号12、VH CDR3由序列编号14、VL CDR1由序列编号26、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
5)VH CDR1由序列编号1、VH CDR2由序列编号13、VH CDR3由序列编号20、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号40的各氨基酸序列构成；
6)VH CDR1由序列编号2、VH CDR2由序列编号7、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号30、VL CDR3由序列编号35的各氨基酸序列构成；
7)VH CDR1由序列编号2、VH CDR2由序列编号7、VH CDR3由序列编号14、VL CDR1由序列编号26、VL CDR2由序列编号31、VL CDR3由序列编号38的各氨基酸序列构成；
8)VH CDR1由序列编号2、VH CDR2由序列编号8、VH CDR3由序列编号14、VL CDR1由序列编号24、VL CDR2由序列编号30、VL CDR3由序列编号38的各氨基酸序列构成；
9)VH CDR1由序列编号2、VH CDR2由序列编号8、VH CDR3由序列编号18、VL CDR1由序列编号23、VL CDR2由序列编号29、VL CDR3由序列编号40的各氨基酸序列构成；
10)VH CDR1由序列编号2、VH CDR2由序列编号8、VH CDR3由序列编号17、VL CDR1由序列编号24、VL CDR2由序列编号27、VL CDR3由序列编号38的各氨基酸序列构成；
11)VH CDR1由序列编号2、VH CDR2由序列编号9、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号27、VL CDR3由序列编号38的各氨基酸序列构成；
12)VH CDR1由序列编号2、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号24、VL CDR2由序列编号29、VL CDR3由序列编号35的各氨基酸序列构成；
13)VH CDR1由序列编号2、VH CDR2由序列编号11、VH CDR3由序列编号18、VL CDR1由序列编号26、VL CDR2由序列编号29、VL CDR3由序列编号36的各氨基酸序列构成；
14)VH CDR1由序列编号2、VH CDR2由序列编号11、VH CDR3由序列编号14、VL CDR1由序列编号26、VL CDR2由序列编号32、VL CDR3由序列编号34的各氨基酸序列构成；
15)VH CDR1由序列编号2、VH CDR2由序列编号11、VH CDR3由序列编号18、VL CDR1由序列编号26、VL CDR2由序列编号27、VL CDR3由序列编号41的各氨基酸序列构成；
16)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号20、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号37的各氨基酸序列构成；
17)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号29、VL CDR3由序列编号35的各氨基酸序列构成；
18)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号28、VL CDR3由序列编号40的各氨基酸序列构成；
19)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号25、VL CDR2由序列编号30、VL CDR3由序列编号34的各氨基酸序列构成；
20)VH CDR1由序列编号2、VH CDR2由序列编号13、VH CDR3由序列编号15、VL CDR1由序列编号23、VL CDR2由序列编号32、VL CDR3由序列编号34的各氨基酸序列构成；
21)VH CDR1由序列编号2、VH CDR2由序列编号13、VH CDR3由序列编号16、VL CDR1由序列编号22、VL CDR2由序列编号27、VL CDR3由序列编号34的各氨基酸序列构成；
22)VH CDR1由序列编号2、VH CDR2由序列编号13、VH CDR3由序列编号17、VL CDR1由序列编号22、VL CDR2由序列编号32、VL CDR3由序列编号36的各氨基酸序列构成；
23)VH CDR1由序列编号3、VH CDR2由序列编号7、VH CDR3由序列编号16、VL CDR1由序列编号25、VL CDR2由序列编号28、VL CDR3由序列编号41的各氨基酸序列构成；
24)VH CDR1由序列编号3、VH CDR2由序列编号7、VH CDR3由序 列编号19、VL CDR1由序列编号23、VL CDR2由序列编号31、VL CDR3由序列编号34的各氨基酸序列构成；
25)VH CDR1由序列编号3、VH CDR2由序列编号8、VH CDR3由序列编号16、VL CDR1由序列编号22、VL CDR2由序列编号28、VL CDR3由序列编号37的各氨基酸序列构成；
26)VH CDR1由序列编号3、VH CDR2由序列编号8、VH CDR3由序列编号18、VL CDR1由序列编号22、VL CDR2由序列编号31、VL CDR3由序列编号37的各氨基酸序列构成；
27)VH CDR1由序列编号3、VH CDR2由序列编号8、VH CDR3由序列编号19、VL CDR1由序列编号25、VL CDR2由序列编号27、VL CDR3由序列编号37的各氨基酸序列构成；
28)VH CDR1由序列编号3、VH CDR2由序列编号9、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号29、VL CDR3由序列编号36的各氨基酸序列构成；
29)VH CDR1由序列编号3、VH CDR2由序列编号9、VH CDR3由序列编号16、VL CDR1由序列编号21、VL CDR2由序列编号29、VL CDR3由序列编号38的各氨基酸序列构成；
30)VH CDR1由序列编号3、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号22、VL CDR2由序列编号32、VL CDR3由序列编号41的各氨基酸序列构成；
31)VH CDR1由序列编号3、VH CDR2由序列编号12、VH CDR3由序列编号15、VL CDR1由序列编号24、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
32)VH CDR1由序列编号4、VH CDR2由序列编号7、VH CDR3由序列编号15、VL CDR1由序列编号25、VL CDR2由序列编号28、VL CDR3由序列编号37的各氨基酸序列构成；
33)VH CDR1由序列编号4、VH CDR2由序列编号7、VH CDR3由序列编号18、VL CDR1由序列编号23、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
34)VH CDR1由序列编号4、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3 由序列编号40的各氨基酸序列构成；
35)VH CDR1由序列编号4、VH CDR2由序列编号10、VH CDR3由序列编号14、VL CDR1由序列编号24、VL CDR2由序列编号27、VL CDR3由序列编号37的各氨基酸序列构成；
36)VH CDR1由序列编号4、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号23、VL CDR2由序列编号30、VL CDR3由序列编号33的各氨基酸序列构成；
37)VH CDR1由序列编号4、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号24、VL CDR2由序列编号27、VL CDR3由序列编号34的各氨基酸序列构成；
38)VH CDR1由序列编号4、VH CDR2由序列编号13、VH CDR3由序列编号16、VL CDR1由序列编号22、VL CDR2由序列编号32、VL CDR3由序列编号40的各氨基酸序列构成；
39)VH CDR1由序列编号5、VH CDR2由序列编号7、VH CDR3由序列编号14、VL CDR1由序列编号21、VL CDR2由序列编号30、VL CDR3由序列编号40的各氨基酸序列构成；
40)VH CDR1由序列编号5、VH CDR2由序列编号7、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号30、VL CDR3由序列编号34的各氨基酸序列构成；
41)VH CDR1由序列编号5、VH CDR2由序列编号7、VH CDR3由序列编号20、VL CDR1由序列编号21、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；
42)VH CDR1由序列编号5、VH CDR2由序列编号8、VH CDR3由序列编号18、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
43)VH CDR1由序列编号5、VH CDR2由序列编号10、VH CDR3由序列编号18、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
44)VH CDR1由序列编号5、VH CDR2由序列编号10、VH CDR3由序列编号15、VL CDR1由序列编号21、VL CDR2由序列编号28、VL CDR3由序列编号38的各氨基酸序列构成；
45)VH CDR1由序列编号5、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号26、VL CDR2由序列编号28、VL CDR3由序列编号40的各氨基酸序列构成；
46)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号14、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
47)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号16、VL CDR1由序列编号23、VL CDR2由序列编号31、VL CDR3由序列编号36的各氨基酸序列构成；
48)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号28、VL CDR3由序列编号39的各氨基酸序列构成；
49)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号16、VL CDR1由序列编号25、VL CDR2由序列编号29、VL CDR3由序列编号38的各氨基酸序列构成；
50)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号17、VL CDR1由序列编号21、VL CDR2由序列编号27、VL CDR3由序列编号37的各氨基酸序列构成；
51)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号19、VL CDR1由序列编号26、VL CDR2由序列编号31、VL CDR3由序列编号39的各氨基酸序列构成；
52)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号23、VL CDR2由序列编号27、VL CDR3由序列编号34的各氨基酸序列构成；
53)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号19、VL CDR1由序列编号24、VL CDR2由序列编号31、VL CDR3由序列编号38的各氨基酸序列构成；
54)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序列编号15、VL CDR1由序列编号26、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；
55)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序 列编号15、VL CDR1由序列编号26、VL CDR2由序列编号28、VL CDR3由序列编号33的各氨基酸序列构成；
56)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序列编号18、VL CDR1由序列编号21、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；
57)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序列编号20、VL CDR1由序列编号25、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
58)VH CDR1由序列编号6、VH CDR2由序列编号7、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
59)VH CDR1由序列编号6、VH CDR2由序列编号10、VH CDR3由序列编号14、VL CDR1由序列编号25、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
60)VH CDR1由序列编号6、VH CDR2由序列编号11、VH CDR3由序列编号18、VL CDR1由序列编号22、VL CDR2由序列编号29、VL CDR3由序列编号39的各氨基酸序列构成；
61)VH CDR1由序列编号6、VH CDR2由序列编号11、VH CDR3由序列编号14、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；或
62)VH CDR1由序列编号6、VH CDR2由序列编号13、VH CDR3由序列编号20、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号38的各氨基酸序列构成。
在本申请第一方面的一些实施方式中，涉及一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，其包括：
(a)包含由序列编号1的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号17的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号32的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；
(b)包含由序列编号2的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号37的氨基酸序列构成的VL CDR3的VL；
(c)包含由序列编号3的氨基酸序列构成的VH CDR1、由序列编号9的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号29的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL；
(d)包含由序列编号5的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号18的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号39的氨基酸序列构成的VL CDR3的VL；
(e)包含由序列编号4的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号23的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；或
(f)包含由序列编号6的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL。
在本申请第一方面的一些实施方式中，所述可溶性CD14单克隆抗体或其抗原结合性抗体片段为由保藏号为CGMCC NO.18536的杂交瘤细胞株分泌产生的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
在本申请第一方面的一些实施方式中，该抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段不与高分子量可溶性CD14特异性地结合。
在本申请第一方面的一些实施方式中，该抗原结合性抗体片段可以选自 Fab、Fab’、F(ab’)2、单链抗体(scFV)、二聚体化V区(二聚抗体)、二硫键稳定性V区(dsFv)、sc(Fv)2、包含CDR的多肽、包含重链可变区的多肽以及包含轻链可变区的多肽组成的组中的抗原结合性抗体片段。
本申请第二方面请求保护一种用于检测可溶性CD14亚型的试剂盒，该用于检测可溶性CD14亚型的试剂盒至少包含根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
根据本申请第二方面的试剂盒还可以包括其他试剂成分，包括但不限于一次抗体、二次抗体、标记抗体、标记酶等标记物质；显色底物、荧光底物、化学发光底物、生物素-链霉亲和素等特异性结合物质、不溶性载体、阻断剂、稀释液、洗涤液、标准物质等。
例如，应用于化学发光酶免疫测定法(CLEIA)的试剂盒可以包括包被于固相上的抗体、酶标记抗体、化学发光底物、稀释液、洗涤液等。当然，在其他实施例中，本申请实施例的抗体也可应用于制备基于双抗体夹心法或竞争法的酶免疫测定试剂盒、电化学发光免疫测定试剂盒、免疫层析试剂盒等。
在本申请第二方面的一些实施方式中，根据本申请的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段用作捕获抗体，所述捕获抗体包被在固相载体上并用于捕获待测样本中的可溶性CD14亚型。
在本申请第二方面的一些实施方式中，所述固相载体可以选自磁性微球、芯片、试纸等，优选为磁性微球，更优选为超顺磁性磁珠。
在本申请第二方面的一些实施方式中，用于检测可溶性CD14亚型的试剂盒还包含经标记的可溶性CD14亚型检测抗体，该检测抗体可以为抗可溶性CD14亚型多克隆抗体。
在本申请第二方面的一些实施方式中，可溶性CD14亚型检测抗体的信号标记物可以例如是化学发光标记物(例如碱性磷酸酶、鲁米诺、异鲁米诺、吖啶酯、辣根过氧化物酶)、电化学发光标记物(例如三联吡啶钌)、量子点(例如金量子点、CdSe量子点、ZnCdSe量子点等)、荧光微球等，或其组合。
本申请第三方面请请求保护一种用于检测可溶性CD14亚型的方法，该方法包括使本申请第一方面的抗可溶性CD14亚型单克隆抗体与待测样本接触。
采用本申请第一方面的抗体或抗原结合性抗体片段对样本中的可溶性 CD14亚型进行免疫学测定的方法例如可以为酶联免疫吸附测定法(ELISA)、化学发光酶免疫测定法(CLEIA)、化学发光免疫测定法(CLIA)、荧光抗体法(FAT)、荧光酶免疫测定法(FEIA)、电化学发光免疫测定法(ECLIA)、放射免疫测定法(RIA)、免疫层析法、凝集法、竞争法等等，但并不限于这些方法。本申请实施例尤其是适合应用于化学发光酶免疫测定法。
ELISA(enzyme-linked immunosorbent assay)是使用酶标记抗体进行的免疫测定法之一，包括直接法、间接法等，例如夹心ELISA。所谓的夹心ELISA是指：使用抗原识别部位不同的抗体，其中一个抗体包被于固相上，并使用另一抗体来夹持要进行检测的抗原，从而形成抗体-抗原-抗体复合体。
化学发光酶免疫测定法的原理为：使样本中的抗原与固定于固相上的抗体进行反应后，与酶标记抗体进行反应，并在洗涤后加入化学发光底物进行酶反应，然后测定发光强度。
在本申请第三方面的一些实施方式中，所述方法包括：
将待测样本和包被有捕获抗体的固相支持物混合，使得固相支持物上包被的捕获抗体与待测样本中的可溶性CD14亚型结合，其中所述捕获抗体为根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段；
将上述混合物进行清洗，除去未结合的物质；
在上述经清洗的混合物中加入经标记的检测抗体进行混匀，使得所述检测抗体与所述捕获抗体上结合的可溶性CD14亚型结合，形成夹心复合物；
对上述夹心复合物进行清洗，除去未结合的物质；
在上述经清洗的夹心复合物中加入检测底物，以检测样本中的可溶性CD14亚型的浓度。例如，在上述经清洗的夹心复合物中加入化学发光底物，检测反应所产生的光子数，以得到样本的化学发光信号值，该化学发光信号值与可溶性CD14亚型的浓度成正比。
在本申请第三方面的一些实施方式中，待测样本可以是血液、血液组分例如全血、血清或血浆，优选为血浆样本。但本申请的样本不限于此，也可以是尿、组织液、淋巴液、关节液、乳汁、脑脊髓液、脓、唾液、泪液、粘液、鼻水、痰、腹水、用水、***等体液。
本申请第四方面涉及根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估患者是否患有脓毒血症的分析 试剂中的应用，其中，使用根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段对患者的血液样本中的可溶性CD14亚型的浓度进行检测，所述可溶性CD14亚型的浓度相对于参考值的水平升高与患者患有脓毒血症的可能性增加相关。
在本申请第四方面的一些实施方式中，将可溶性CD14亚型的浓度与临界值进行比较，判断可溶性CD14亚型的浓度是否高于临界值，从而评估患者是否患有脓毒血症。所述可溶性CD14亚型的浓度相对于参考值的水平升高与患者患有脓毒血症的可能性增加正相关。
在本申请第四方面的一些实施方式中，在对患者产生患有脓毒血症的怀疑后72小时内检测该患者的血液样本中的可溶性CD14亚型的浓度。
本申请第五方面涉及根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估疑似脓毒血症患者的预后的分析试剂中的应用，其中，使用根据本申请第一方面的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段对疑似脓毒血症患者的血液样本中的可溶性CD14亚型的浓度进行检测，所述可溶性CD14亚型的浓度相对于参考值的水平升高与疑似脓毒血症患者的死亡风险增加相关。
在本申请第五方面的一些实施方式中，所述可溶性CD14亚型的浓度相对于参考值的水平升高与所述疑似脓毒血症患者的死亡风险增加正相关。
在本申请第五方面的一些实施方式中，在对患者产生患有脓毒血症的怀疑后72小时内检测该患者的血液样本中的可溶性CD14亚型的浓度。
以下描述本申请的一些实施例，这些实施例不是为了限制本申请，而是为了更好地说明本申请。
实施例1：抗可溶性CD14亚型抗体的制备
步骤1.1可溶性CD14亚型免疫原的表达
可溶性CD14亚型免疫原分别采用原核表达***和真核表达***进行表达，下面详细描述相应的过程：
(1)采用原核表达***来表达可溶性CD14亚型免疫原
在合成目标DNA序列后，选择pET30a表达载体来构建质粒，并且将其转化为大肠杆菌BL21star(DE3)，从而获得原核表达工程菌株。通过IPTG(异丙基硫代半乳糖苷)法诱导该重组质粒在大肠杆菌表达体系中高效表达。 目标蛋白主要存在于包涵体中，采用Ni 2+ NTA亲和柱纯化，获得目标蛋白，该目标蛋白序列为Thr Thr Pro Glu Pro Cys Glu Leu Asp Asp Glu Asp Phe Arg Cys Val Cys Asn Phe Ser Glu Pro Gln Pro Asp Trp Ser Glu Ala Phe Gln Cys Val Ser Ala Val Glu Val Glu Ile His Ala Gly Gly Leu Asn Leu Glu Pro Phe Leu Lys Arg Val Asp Ala Asp Ala Asp Pro Arg Gln Tyr Ala(SEQ ID No.43)。如图1所示，采用Bradford法测定目标蛋白的浓度为5.42mg/mL，采用SDS-PAGE和Western Blot确定目标蛋白的纯度和分子量。
(2)采用真核表达***来表达可溶性CD14亚型免疫原
在合成目标DNA序列后，选择pcDNA3.4表达载体来构建质粒。采用HEK293-6E表达体系，其培养条件为：在serum-free FreeStyle TM 293 Expression Medium(Thermo Fisher Scientific)基质中在37℃，5％CO 2下进行培养。采用瞬时转染的方法将构建的质粒在293-6E表达体系转染。培养6天后，收集细胞培养上清。将上清液过滤并进亲和纯化。将纯化后的蛋白采用优化后的实验条件进行酶切，并纯化后获得目标蛋白。如图2所示，采用Bradford法测定目标蛋白的浓度为1.55mg/mL，采用SDS-PAGE和Western Blot确定目标蛋白的纯度和分子量。
步骤1.2抗可溶性CD14亚型多克隆抗体的制备
将步骤1.1所获得的原核***和真核***表达纯化的蛋白、即抗原按一定比例混合，并且与载体蛋白钥孔血蓝蛋白(简称KLH)进行偶联，从而作为免疫原对新西兰大白兔按如下步骤进行免疫：用生理盐水稀释免疫原，然后与佐剂例如弗氏不完全佐剂(Sigma-Aldrich)进行1:1混合。抗原和佐剂完全混合形成稳定的乳剂，将该乳剂在新西兰大白兔双肩周围的皮肤下进行皮下注射以及在后大腿进行肌肉注射。每个区域大约用1/4的免疫原，每次免疫的抗原量约100μg至500μg，一共免疫四次，每次免疫间隔为2周。完成第四次免疫后，收集新西兰大白兔的抗血清，并进行抗原亲和纯化，获得可溶性CD14亚型多克隆抗体。在图3中示出抗可溶性CD14亚型多克隆抗体SDS-PAGE和Western Blot的结果。在图3左侧SDS-PAGE中，泳道M为Protein marker，第一泳道为多克隆抗体，第二泳道为兔IgG；在图3右侧Western Blot中，泳道M为Protein marker，第一泳道为免疫原(100ng)+纯化抗体(1μg/mL)+山羊抗兔IgG[IRDye800cw](0.125μg/mL)，第二泳道为免疫原(50ng)+纯化抗体(1μg/mL)+山羊抗兔IgG[IRDye800cw](0.125μg/mL)，第三泳道为 免疫原(100ng)+未免疫血清+山羊抗兔IgG[IRDye800cw](0.125μg/mL)，第四泳道为免疫原(100ng)+山羊抗兔IgG[IRDye800cw](0.125μg/mL)。
步骤1.3抗可溶性CD14亚型单克隆抗体的制备
将步骤1.1所获得的原核***和真核***表达纯化的蛋白、即抗原按一定比例混合，并与载体蛋白钥孔血蓝蛋白(简称KLH)进行偶联，从而作为免疫原对小鼠按如下步骤进行免疫：在初次免疫时将抗原(10μg至50μg)与佐剂例如弗氏完全佐剂(Sigma-Aldrich)按1:1混合，然后进行皮下多点注射。3周后进行第二次免疫，第二次免疫剂量与初次免疫剂量相同，添加弗氏不完全佐剂(Sigma-Aldrich)进行皮下注射。又3周后进行第三次免疫，第三次免疫剂量与初次免疫剂量相同，但不添加佐剂。经3周后，采用剂量为100μg至500μg来进行加强免疫。在加强免疫3天后，取脾进行融合。在第三次免疫之后，采用ELISA的方法评估小鼠的抗血清的价效，选择效价最好的动物的脾细胞与骨髓瘤细胞进行融合，获得母克隆细胞株，保藏号为CGMCC NO.18536。在本申请的范围中，骨髓瘤细胞可以使用公知的各种细胞，例如，来源于小鼠的P3、NS-1、P3U1、SP2/0、来源于大鼠的YB2/0、Y3-Ag1、来源于人的SKO-007、人鼠杂交骨髓瘤细胞SHM-D33等。在本申请中优选使用小鼠的SP2/0。
然后，通过ELISA方法对母克隆进行正筛和反筛(正筛和反筛实验均采用间接ELISA的方法)。
对于正筛实验，用包被缓冲液(PBS缓冲液，pH＝7.4)稀释抗原，配置成浓度为1μg/mL的待包被抗原。将100μL的待包被抗原加入到96孔板内，在室温下孵育1小时。采用PBS缓冲液(pH＝7.4)作为清洗液，去除未反应的抗原。将100μL的封闭液加入到96孔板内，室温孵育1小时，封闭未反应的固相表面。再次用清洗液(PBS缓冲液，pH＝7.4)去除过量的封闭液。然后，加入100μL的待筛选细胞上清，室温孵育30分钟。在采用清洗液洗(PBS缓冲液，pH＝7.4)去除未与固相包被抗原结合的抗体后，添加100μL的标记有过氧化物酶的山羊抗鼠抗体。标记有过氧化物酶的山羊抗鼠抗体与识别固相抗原的鼠抗体结合，并且催化底物液发光。通过发光的强度，判断细胞上清中是否存在可与抗原结合的抗体。
在抗体筛选过程中，通过采用sCD14进行反筛，确定被筛选的克隆与sCD14无交叉反应。对于反筛实验，将包被缓冲液(PBS缓冲液，pH＝7.4) 分别稀释His-tag蛋白和sCD14蛋白，配置成浓度为1μg/mL的待包被物。之后的实验步骤与正筛实验相同。通过最终发光值的强度，判断细胞上清抗体是否与反筛物质有反应性。
在筛选母克隆之后进行后续的亚克隆。对亚克隆进行亲和力排序和表位归类。将亚克隆上清与通过步骤1.2制备的多克隆抗体配对检测抗原。在表9-11中总结了抗可溶性CD14亚型单克隆抗体亲和力排序、表位归类以及配对抗体筛选结果。根据亲和力排序和抗体配对结果，筛选出克隆号为16D5B2的抗体来进行后续的生产和纯化，从而获得鼠单克隆抗体。
表9
Figure PCTCN2019119536-appb-000012
表10
Figure PCTCN2019119536-appb-000013
Figure PCTCN2019119536-appb-000014
表11
Figure PCTCN2019119536-appb-000015
步骤1.4：抗可溶性CD14亚型单克隆抗体氨基酸序列测定
对通过步骤1.3筛选所得到的抗可溶性CD14亚型单克隆抗体的可变区进行测序。首先，按照
Figure PCTCN2019119536-appb-000016
试剂盒操作手册将杂交瘤细胞的总RNA提取出来，然后按照PrimeScript TM 1st Strand cDNA Synthesis Kit的操作手册，用表位特异的反义引物或通用引物将总RNA逆转录成cDNA。通过逆转录合成的cDNA序列可得其对应抗可溶性CD14亚型单克隆抗体氨基酸序列，该抗可溶性CD14亚型单克隆抗体例如包括包含由序列编号3的氨基酸序列构成的VH CDR1、由序列编号9的氨基酸序列构成的VH CDR2和由序列编号16的氨基 酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号29的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL，其中该抗可溶性CD14亚型单克隆抗体特异性地识别由SEQ ID No.42的氨基酸序列构成的表位。
实施例2：抗可溶性CD14亚型单克隆抗体的特异性表位确定
在本申请中，对表位的确定方法没有特别的限定，例如可以通过下方式进行：首先，由目标蛋白序列N端依次向C端移动3个氨基酸合成多肽，每条肽链由15个氨基酸组成；然后，将多肽分别标记有生物素，并且通过生物素-链霉亲和素结合包被于含有链霉亲和素的96孔板上；接着分别将待测抗体以及过氧化物酶标记的山羊抗鼠IgG加入到96孔板中；通过最终反应的信号值确定待测抗体的识别的氨基酸序列。
实施例3：用于检测可溶性CD14亚型的试剂盒的制备
将通过实施例1筛选出的克隆号为16D5B2的单克隆抗体用作捕获抗体，包被于超顺磁性磁珠(Merck，
Figure PCTCN2019119536-appb-000017
超顺磁性磁珠)表面。超顺磁性磁珠表面修饰有羧基，超顺磁性磁珠表面的羧基在EDC/NHS催化下与克隆号为16D5B2的单克隆抗体的氨基偶联，从而制备得到磁珠包被物。将磁珠包被物在50mM的MES(2-吗啉乙磺酸)缓冲液(0.5M NaCl，0.5％BSA，0.05％吐温20，pH＝6.0)中稀释，磁珠包被物浓度为1.0mg/mL。将通过实施例1中步骤1.2中所制备的抗可溶性CD14亚型多克隆抗体作为检测抗体，该检测抗体标记有碱性磷酸酶(Roche Life Science)。将酶标标记物稀释于50mM的MES缓冲液(0.5M NaCl，0.5％BSA，0.05％吐温20，pH＝6.0)，酶标标记物浓度为1μg/mL。
在当前的实施例中，检测反应基于夹心法模式：将待测样本(40μL)、捕获抗体(50μL)和检测抗体(50μL)加入到反应管中孵育，其中待测样本为血浆。待测样本内的抗原、即可溶性CD14亚型与捕获抗体和检测抗体结合，形成夹心复合物。在反应管内孵育反应后，磁场吸附磁珠，从而洗去未结合的物质。
将化学发光底物添加到反应管内，发光底物(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷，AMPPD)被碱性磷酸酶所分解， 脱去一个磷酸基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光。接着，通过光电倍增管对中间反应所产生的光子数进行测量，所产生的光子数与样本中的可溶性CD14亚型的浓度成正比。最后，将一系列含不同浓度的可溶性CD14亚型的样本进行测试，可得到其相应的发光值，进而建立标准曲线，如图4所示。
实施例4：抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估患者是否患有脓毒血症的分析试剂中的应用
用通过实施例3制备的试剂盒对141名疑似患有脓毒血症的患者样本的可溶性CD14亚型进行检测。同时检测患者的PCT浓度水平，并对比可溶性CD14亚型和PCT对脓毒血症的诊断效力。可溶性CD14亚型检测的阈值为500pg/mL，其诊断脓毒血症的灵敏度和特异性分别为0.76和0.91。如图5所示，可溶性CD14亚型和PCT的ROC曲线的AUC面积分别为0.91和0.84。由此得出可溶性CD14亚型具有更高的诊断效力。
实施例5：抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估疑似脓毒血症患者的预后的分析试剂中的应用
用通过实施例3制备的试剂盒对88名疑似脓毒血症患者的入院72小时内的血浆样本可溶性CD14亚型的浓度进行检测，并且追踪疑似脓毒血症患者30内的预后情况，采用Kaplan-Meier分析法得来医院30天内的生存率的关系曲线图(见图6)。对于不同分组，患者的生存率有显著不同，其中对于组Ⅲ(可溶性CD14亚型浓度≥1003.97pg/mL)，患者的生存概率<40％(p<0.01)。
以上述疑似脓毒血症患者88人为对象，利用ROC曲线，比较患者入院72小时内PCT、可溶性CD14亚型的浓度值与患者入院30天内的生存率预后效力。如图7所示，PCT检测和可溶性CD14亚型检测的AUC分别为0.72、0.83，因此，可溶性CD14亚型能够更准确地预测患者的生存状况。
从上面描述的各个实施例可以得出：本申请实施例的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段或本申请实施例的试剂盒具有高度特异性和灵敏度并且适用于检测待测样本中的可溶性CD14亚型，从而提高可溶性CD14亚型检测的质量和精度。
本申请不通过根据实施例进行的描述而限制于此。更确切地说，本申请包括每个新的特征以及特征的每个组合，这尤其是包含在权利要求中的特征的每个组合，即使该特征或者该组合本身未详细地在权利要求中或者实施例中说明时也是如此。

Claims (20)

  1. 一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，其中，
    所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括：
    (i)重链可变区(VH)互补性决定区(CDR)：
    VH CDR1：X 1X 2X 3MX 4；
    VH CDR2：YIX 5X 6ADX 7；
    VH CDR3：X 8X 9X 10AX 11；
    和
    (ii)轻链可变区(VL)互补性决定区(CDR)：
    VL CDR1：KX 12X 13X 14N；
    VL CDR2：LX 15X 16；
    VL CDR3：VX 17X 18X 19；
    其中X 1至X 19是下列作为选择项记载的1个或多个氨基酸序列：
    X 1＝任意一个氨基酸；
    X 2＝F或V；X3＝A、E或K；X4＝A或L；
    X 5＝SYMGS、SSGSS、AYTMY、TYSGS或SSKSS；
    X 6＝GAYY、TIYY、AKYY、TKAS或SNLA；
    X 7＝AKLG、TVKG、SYTA、MTKG或YGNT；
    X 8＝A或Q；X 9＝G或Q；X 10＝Q或F；X 11＝M、Y或V；
    X 12＝YYAS、SSAT、SSQS或SGSS；
    X 13＝AAKL、LLAT、LLYS或KAAS；
    X 14＝YRNIKL、TWAKGN、NGKTYL或YTNGAL；
    X 15＝VQS、KTS、QTS或VSK；
    X 16＝LAS、LDS、LTA或DSK；
    X 17＝G、A、S或Q；
    X 18＝GTH、GNT、GTA或TAI；
    X 19＝FITA、FPRT、YGHV或NYGH。
  2. 根据权利要求1所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其中，
    所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3，
    其中VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和 VL CDR3是下列作为选择项记载的多个氨基酸序列：
    (a)VH CDR1＝SVAML、SVEML、SFAML、SFAMA、SVKML或SFKMA；
    (b)VH CDR2＝YISYMGSTIYYADAKLG、YISSKSSSNLAADAKLG、YISSGSSTIYYADTVKG、YISSGSSTIYYADMTKG、YITYSGSGAYYADYGNT、YISYMGSTIYYADTVKG或YISSKSSTIYYADTVKG；
    (c)VH CDR3＝AGFAY、QQFAY、QGFAY、QGFAM、QQVAY、AGFAM或AGFAY；
    (d)VL CDR1＝KSSQSLLYSNGKTYLN、KYYASAAKLYRNIKLN、KYYASLLATYRNIKLN、KYYASLLATTWAKGNN、KSSQSLLYSTWAKGNN或KSSQSLLYSYRNIKLN；
    (e)VL CDR2＝LVQSLDS、LVSKLTA、LVSKLDS、LVQSDSK、LQTSDSK或LVSKLAS；和
    (f)VL CDR3＝VQGTHNYGH、VGGTHFPRT、VGTAIFPRT、VGGTHFITA、VSGTHFPRT、VQGTHFPRT、VQTAIFPRT、VQGTHYGHV或VQGNTFITA。
  3. 根据权利要求1所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其中，
    所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3，
    其中VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3是下列作为选择项记载的多个氨基酸序列：
    (a)VH CDR1＝SVAML、SVEML、SFAML、SFAMA、SVKML或SFKMA；
    (b)VH CDR2＝YISYMGSTIYYADAKLG、YISSKSSSNLAADAKLG、YISSGSSTIYYADTVKG或YISSGSSTIYYADMTKG；
    (c)VH CDR3＝AGFAY、QQFAY、QGFAY或QGFAM；
    (d)VL CDR1＝KYYASAAKLYRNIKLN、KYYASLLATYRNIKLN、KSSQSLLYSNGKTYLN或KYYASLLATTWAKGNN；
    (e)VL CDR2＝LVQSLDS、LVSKLDS、LVSKLTA或LVQSDSK；和
    (f)VL CDR3＝VQGTHNYGH、VGGTHFPRT、VQGTHFPRT、VGTAIFPRT或VGGTHFITA。
  4. 根据权利要求1至3中任一项所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其中，
    所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段包括VH CDR1、VH CDR2和VH CDR3，以及VL CDR1、VL CDR2和VL CDR3是下 述1)至62)中的任意一种：
    1)VH CDR1由序列编号1、VH CDR2由序列编号10、VH CDR3由序列编号14、VL CDR1由序列编号21、VL CDR2由序列编号32、VL CDR3由序列编号34的各氨基酸序列构成；
    2)VH CDR1由序列编号1、VH CDR2由序列编号11、VH CDR3由序列编号17、VL CDR1由序列编号26、VL CDR2由序列编号28、VL CDR3由序列编号41的各氨基酸序列构成；
    3)VH CDR1由序列编号1、VH CDR2由序列编号12、VH CDR3由序列编号17、VL CDR1由序列编号21、VL CDR2由序列编号32、VL CDR3由序列编号33的各氨基酸序列构成；
    4)VH CDR1由序列编号1、VH CDR2由序列编号12、VH CDR3由序列编号14、VL CDR1由序列编号26、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
    5)VH CDR1由序列编号1、VH CDR2由序列编号13、VH CDR3由序列编号20、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号40的各氨基酸序列构成；
    6)VH CDR1由序列编号2、VH CDR2由序列编号7、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号30、VL CDR3由序列编号35的各氨基酸序列构成；
    7)VH CDR1由序列编号2、VH CDR2由序列编号7、VH CDR3由序列编号14、VL CDR1由序列编号26、VL CDR2由序列编号31、VL CDR3由序列编号38的各氨基酸序列构成；
    8)VH CDR1由序列编号2、VH CDR2由序列编号8、VH CDR3由序列编号14、VL CDR1由序列编号24、VL CDR2由序列编号30、VL CDR3由序列编号38的各氨基酸序列构成；
    9)VH CDR1由序列编号2、VH CDR2由序列编号8、VH CDR3由序列编号18、VL CDR1由序列编号23、VL CDR2由序列编号29、VL CDR3由序列编号40的各氨基酸序列构成；
    10)VH CDR1由序列编号2、VH CDR2由序列编号8、VH CDR3由序列编号17、VL CDR1由序列编号24、VL CDR2由序列编号27、VL CDR3由序列编号38的各氨基酸序列构成；
    11)VH CDR1由序列编号2、VH CDR2由序列编号9、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号27、VL CDR3由序列编号38的各氨基酸序列构成；
    12)VH CDR1由序列编号2、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号24、VL CDR2由序列编号29、VL CDR3由序列编号35的各氨基酸序列构成；
    13)VH CDR1由序列编号2、VH CDR2由序列编号11、VH CDR3由序列编号18、VL CDR1由序列编号26、VL CDR2由序列编号29、VL CDR3由序列编号36的各氨基酸序列构成；
    14)VH CDR1由序列编号2、VH CDR2由序列编号11、VH CDR3由序列编号14、VL CDR1由序列编号26、VL CDR2由序列编号32、VL CDR3由序列编号34的各氨基酸序列构成；
    15)VH CDR1由序列编号2、VH CDR2由序列编号11、VH CDR3由序列编号18、VL CDR1由序列编号26、VL CDR2由序列编号27、VL CDR3由序列编号41的各氨基酸序列构成；
    16)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号20、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号37的各氨基酸序列构成；
    17)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号29、VL CDR3由序列编号35的各氨基酸序列构成；
    18)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号28、VL CDR3由序列编号40的各氨基酸序列构成；
    19)VH CDR1由序列编号2、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号25、VL CDR2由序列编号30、VL CDR3由序列编号34的各氨基酸序列构成；
    20)VH CDR1由序列编号2、VH CDR2由序列编号13、VH CDR3由序列编号15、VL CDR1由序列编号23、VL CDR2由序列编号32、VL CDR3由序列编号34的各氨基酸序列构成；
    21)VH CDR1由序列编号2、VH CDR2由序列编号13、VH CDR3由序列编号16、VL CDR1由序列编号22、VL CDR2由序列编号27、VL CDR3由序列编号34的各氨基酸序列构成；
    22)VH CDR1由序列编号2、VH CDR2由序列编号13、VH CDR3由序列编号17、VL CDR1由序列编号22、VL CDR2由序列编号32、VL CDR3由序列编号36的各氨基酸序列构成；
    23)VH CDR1由序列编号3、VH CDR2由序列编号7、VH CDR3由序 列编号16、VL CDR1由序列编号25、VL CDR2由序列编号28、VL CDR3由序列编号41的各氨基酸序列构成；
    24)VH CDR1由序列编号3、VH CDR2由序列编号7、VH CDR3由序列编号19、VL CDR1由序列编号23、VL CDR2由序列编号31、VL CDR3由序列编号34的各氨基酸序列构成；
    25)VH CDR1由序列编号3、VH CDR2由序列编号8、VH CDR3由序列编号16、VL CDR1由序列编号22、VL CDR2由序列编号28、VL CDR3由序列编号37的各氨基酸序列构成；
    26)VH CDR1由序列编号3、VH CDR2由序列编号8、VH CDR3由序列编号18、VL CDR1由序列编号22、VL CDR2由序列编号31、VL CDR3由序列编号37的各氨基酸序列构成；
    27)VH CDR1由序列编号3、VH CDR2由序列编号8、VH CDR3由序列编号19、VL CDR1由序列编号25、VL CDR2由序列编号27、VL CDR3由序列编号37的各氨基酸序列构成；
    28)VH CDR1由序列编号3、VH CDR2由序列编号9、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号29、VL CDR3由序列编号36的各氨基酸序列构成；
    29)VH CDR1由序列编号3、VH CDR2由序列编号9、VH CDR3由序列编号16、VL CDR1由序列编号21、VL CDR2由序列编号29、VL CDR3由序列编号38的各氨基酸序列构成；
    30)VH CDR1由序列编号3、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号22、VL CDR2由序列编号32、VL CDR3由序列编号41的各氨基酸序列构成；
    31)VH CDR1由序列编号3、VH CDR2由序列编号12、VH CDR3由序列编号15、VL CDR1由序列编号24、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
    32)VH CDR1由序列编号4、VH CDR2由序列编号7、VH CDR3由序列编号15、VL CDR1由序列编号25、VL CDR2由序列编号28、VL CDR3由序列编号37的各氨基酸序列构成；
    33)VH CDR1由序列编号4、VH CDR2由序列编号7、VH CDR3由序列编号18、VL CDR1由序列编号23、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
    34)VH CDR1由序列编号4、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3 由序列编号40的各氨基酸序列构成；
    35)VH CDR1由序列编号4、VH CDR2由序列编号10、VH CDR3由序列编号14、VL CDR1由序列编号24、VL CDR2由序列编号27、VL CDR3由序列编号37的各氨基酸序列构成；
    36)VH CDR1由序列编号4、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号23、VL CDR2由序列编号30、VL CDR3由序列编号33的各氨基酸序列构成；
    37)VH CDR1由序列编号4、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号24、VL CDR2由序列编号27、VL CDR3由序列编号34的各氨基酸序列构成；
    38)VH CDR1由序列编号4、VH CDR2由序列编号13、VH CDR3由序列编号16、VL CDR1由序列编号22、VL CDR2由序列编号32、VL CDR3由序列编号40的各氨基酸序列构成；
    39)VH CDR1由序列编号5、VH CDR2由序列编号7、VH CDR3由序列编号14、VL CDR1由序列编号21、VL CDR2由序列编号30、VL CDR3由序列编号40的各氨基酸序列构成；
    40)VH CDR1由序列编号5、VH CDR2由序列编号7、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号30、VL CDR3由序列编号34的各氨基酸序列构成；
    41)VH CDR1由序列编号5、VH CDR2由序列编号7、VH CDR3由序列编号20、VL CDR1由序列编号21、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；
    42)VH CDR1由序列编号5、VH CDR2由序列编号8、VH CDR3由序列编号18、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
    43)VH CDR1由序列编号5、VH CDR2由序列编号10、VH CDR3由序列编号18、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成。
    44)VH CDR1由序列编号5、VH CDR2由序列编号10、VH CDR3由序列编号15、VL CDR1由序列编号21、VL CDR2由序列编号28、VL CDR3由序列编号38的各氨基酸序列构成；
    45)VH CDR1由序列编号5、VH CDR2由序列编号10、VH CDR3由序列编号19、VL CDR1由序列编号26、VL CDR2由序列编号28、VL CDR3由序列编号40的各氨基酸序列构成；
    46)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号14、VL CDR1由序列编号21、VL CDR2由序列编号31、VL CDR3由序列编号33的各氨基酸序列构成；
    47)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号16、VL CDR1由序列编号23、VL CDR2由序列编号31、VL CDR3由序列编号36的各氨基酸序列构成；
    48)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号17、VL CDR1由序列编号23、VL CDR2由序列编号28、VL CDR3由序列编号39的各氨基酸序列构成；
    49)VH CDR1由序列编号5、VH CDR2由序列编号11、VH CDR3由序列编号16、VL CDR1由序列编号25、VL CDR2由序列编号29、VL CDR3由序列编号38的各氨基酸序列构成；
    50)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号17、VL CDR1由序列编号21、VL CDR2由序列编号27、VL CDR3由序列编号37的各氨基酸序列构成；
    51)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号19、VL CDR1由序列编号26、VL CDR2由序列编号31、VL CDR3由序列编号39的各氨基酸序列构成；
    52)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号16、VL CDR1由序列编号23、VL CDR2由序列编号27、VL CDR3由序列编号34的各氨基酸序列构成；
    53)VH CDR1由序列编号5、VH CDR2由序列编号12、VH CDR3由序列编号19、VL CDR1由序列编号24、VL CDR2由序列编号31、VL CDR3由序列编号38的各氨基酸序列构成；
    54)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序列编号15、VL CDR1由序列编号26、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；
    55)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序列编号15、VL CDR1由序列编号26、VL CDR2由序列编号28、VL CDR3由序列编号33的各氨基酸序列构成；
    56)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序列编号18、VL CDR1由序列编号21、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；
    57)VH CDR1由序列编号5、VH CDR2由序列编号13、VH CDR3由序 列编号20、VL CDR1由序列编号25、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
    58)VH CDR1由序列编号6、VH CDR2由序列编号7、VH CDR3由序列编号18、VL CDR1由序列编号25、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
    59)VH CDR1由序列编号6、VH CDR2由序列编号10、VH CDR3由序列编号14、VL CDR1由序列编号25、VL CDR2由序列编号32、VL CDR3由序列编号39的各氨基酸序列构成；
    60)VH CDR1由序列编号6、VH CDR2由序列编号11、VH CDR3由序列编号18、VL CDR1由序列编号22、VL CDR2由序列编号29、VL CDR3由序列编号39的各氨基酸序列构成；
    61)VH CDR1由序列编号6、VH CDR2由序列编号11、VH CDR3由序列编号14、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号39的各氨基酸序列构成；或
    62)VH CDR1由序列编号6、VH CDR2由序列编号13、VH CDR3由序列编号20、VL CDR1由序列编号22、VL CDR2由序列编号30、VL CDR3由序列编号38的各氨基酸序列构成。
  5. 根据权利要求1所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其包括：
    (a)包含由序列编号1的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号17的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号32的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；
    (b)包含由序列编号2的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号37的氨基酸序列构成的VL CDR3的VL；
    (c)包含由序列编号3的氨基酸序列构成的VH CDR1、由序列编号9的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号29的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL；
    (d)包含由序列编号5的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号18的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号39的氨基酸序列构成的VL CDR3的VL；
    (e)包含由序列编号4的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号23的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；或
    (f)包含由序列编号6的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL。
  6. 根据权利要求1所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其中，所述可溶性CD14单克隆抗体或其抗原结合性抗体片段为由保藏号为CGMCC NO.18536的杂交瘤细胞株分泌产生的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
  7. 根据权利要求1至6中任一项所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，
    其中所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段不与高分子量可溶性CD14特异性地结合。
  8. 根据权利要求1至7中任一项所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，
    其中所述抗原结合性抗体片段是选自Fab、Fab’、F(ab’)2、单链抗体(scFV)、二聚体化V区(二聚抗体)、二硫键稳定性V区(dsFv)、sc(Fv)2、包含CDR的多肽、包含重链可变区的多肽以及包含轻链可变区的多肽组成的组中的抗原结合性抗体片段。
  9. 一种用于检测可溶性CD14亚型的试剂盒，其中，
    所述用于检测可溶性CD14亚型的试剂盒至少包含根据权利要求1至8中任一项所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
  10. 根据权利要求9所述的用于检测可溶性CD14亚型的试剂盒，
    其中所述抗可溶性CD14亚型单克隆抗体或其抗原结合性片段用作捕获抗体，所述捕获抗体包被在固相载体上并用于捕获待测样本中的可溶性CD14亚型。
  11. 根据权利要求10所述的用于检测可溶性CD14亚型的试剂盒，
    其中所述捕获抗体包被于磁性磁珠表面、尤其是超顺磁性磁珠表面。
  12. 根据权利要求10或11所述的用于检测可溶性CD14亚型的试剂盒，
    其中所述用于检测可溶性CD14亚型的试剂盒还包含经标记的检测抗体，所述检测抗体为抗可溶性CD14亚型多克隆抗体。
  13. 一种检测可溶性CD14亚型的方法，包括：
    将待测样本和包被有捕获抗体的固相支持物混合，使得固相支持物上包被的捕获抗体与待测样本中的可溶性CD14亚型结合，其中所述捕获抗体为根据权利要求1至8中任一项所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段；
    将上述混合物进行清洗，除去未结合的物质；
    在上述经清洗的混合物中加入经标记的检测抗体进行混匀，使得所述检测抗体与所述捕获抗体上结合的可溶性CD14亚型结合，形成夹心复合物；
    对上述夹心复合物进行清洗，除去未结合的物质；
    在上述经清洗的夹心复合物中加入检测底物，以检测样本中的可溶性CD14亚型的浓度。
  14. 根据权利要求13所述的方法，所述待测样本为血液样本、尤其是血浆样本。
  15. 根据权利要求1至8中任一项所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段在制备用于评估患者是否患有脓毒血症的分析试剂中的应用，其中，使用所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段对患者的血液样本中的可溶性CD14亚型的浓度进行检测，所述可溶性CD14亚型的浓度相对于参考值的水平升高与患者患有脓毒血症的可能性增加相关。
  16. 根据权利要求15所述的应用，
    其中，在对患者产生患有脓毒血症的怀疑后72小时内检测该患者的血液样本中的可溶性CD14亚型的浓度。
  17. 根据权利要求1至8中任一项所述的抗可溶性CD14亚型单克隆抗体 或其抗原结合性抗体片段在制备用于评估疑似脓毒血症患者的预后的分析试剂中的应用，其中，使用所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段对疑似脓毒血症患者的血液样本中的可溶性CD14亚型的浓度进行检测，所述可溶性CD14亚型的浓度相对于参考值的水平升高与疑似脓毒血症患者的死亡风险增加相关。
  18. 一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，包括：
    (a)包含由序列编号1的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号17的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号32的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；
    (b)包含由序列编号2的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号37的氨基酸序列构成的VL CDR3的VL；
    (c)包含由序列编号3的氨基酸序列构成的VH CDR1、由序列编号9的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号29的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL；
    (d)包含由序列编号5的氨基酸序列构成的VH CDR1、由序列编号13的氨基酸序列构成的VH CDR2和由序列编号18的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号21的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号39的氨基酸序列构成的VL CDR3的VL；
    (e)包含由序列编号4的氨基酸序列构成的VH CDR1、由序列编号12的氨基酸序列构成的VH CDR2和由序列编号16的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号23的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号33的氨基酸序列构成的VL CDR3的VL；或
    (f)包含由序列编号6的氨基酸序列构成的VH CDR1、由序列编号13 的氨基酸序列构成的VH CDR2和由序列编号20的氨基酸序列构成的VH CDR3的VH，以及包含由序列编号22的氨基酸序列构成的VL CDR1、由序列编号30的氨基酸序列构成的VL CDR2和由序列编号38的氨基酸序列构成的VL CDR3的VL。
  19. 根据权利要求18所述的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，
    其中所述抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段不与高分子量可溶性CD14特异性地结合。
  20. 一种抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段，其特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，其中，所述可溶性CD14亚型捕获抗体为由保藏号为CGMCC NO.18536的杂交瘤细胞株分泌产生的抗可溶性CD14亚型单克隆抗体或其抗原结合性抗体片段。
PCT/CN2019/119536 2019-11-19 2019-11-19 新的抗可溶性cd14亚型抗体及其应用 WO2021097684A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP19953573.3A EP4059517A4 (en) 2019-11-19 2019-11-19 NEW SOLUBLE CD14 ANTI-SUBTYPE ANTIBODY AND ITS USE
PCT/CN2019/119536 WO2021097684A1 (zh) 2019-11-19 2019-11-19 新的抗可溶性cd14亚型抗体及其应用
CN201980097405.2A CN113939314B (zh) 2019-11-19 2019-11-19 新的抗可溶性cd14亚型抗体及其应用
CN202210524748.6A CN115232211A (zh) 2019-11-19 2019-11-19 新的抗可溶性cd14亚型抗体及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/119536 WO2021097684A1 (zh) 2019-11-19 2019-11-19 新的抗可溶性cd14亚型抗体及其应用

Publications (1)

Publication Number Publication Date
WO2021097684A1 true WO2021097684A1 (zh) 2021-05-27

Family

ID=75979920

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/119536 WO2021097684A1 (zh) 2019-11-19 2019-11-19 新的抗可溶性cd14亚型抗体及其应用

Country Status (3)

Country Link
EP (1) EP4059517A4 (zh)
CN (2) CN113939314B (zh)
WO (1) WO2021097684A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116444673B (zh) * 2023-06-14 2023-09-15 北京汇智和源生物技术有限公司 人源膜cd14单克隆抗体及其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091478A1 (en) * 2000-11-22 2004-05-13 Shoji Furusako Anti-cd14 monoclonal antibody having effect of inhibiting cd14/tlr binding
WO2014122660A1 (en) * 2013-02-11 2014-08-14 Mor Research Applications Ltd. Cd14 inhibitors as an effective treatment for hcv infection

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004044005A1 (ja) * 2002-11-12 2004-05-27 Mochida Pharmaceutical Co., Ltd. ヒト低分子量ｃｄ１４測定キットおよび抗体
KR101904497B1 (ko) * 2010-01-29 2018-10-04 가부시키가이샤 엘에스아이 메디엔스 인간 ｓＣＤ１４―ＳＴ의 분석 방법
CN106255751B (zh) * 2014-02-26 2020-04-10 持田制药株式会社 新的抗普莱晒谱星抗体
CA2996395A1 (en) * 2015-08-25 2017-03-02 Mochida Pharmaceutical Co., Ltd. Specifically purified anti-presepsin antibody

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091478A1 (en) * 2000-11-22 2004-05-13 Shoji Furusako Anti-cd14 monoclonal antibody having effect of inhibiting cd14/tlr binding
WO2014122660A1 (en) * 2013-02-11 2014-08-14 Mor Research Applications Ltd. Cd14 inhibitors as an effective treatment for hcv infection

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ALVES PATR�CIA TERRA; FUJIMURA PATR�CIA TIEMI; MORAIS L�A DUARTE DA SILVA; GOULART LUIZ RICARDO: "Revisiting the CD14: Epitope mapping by Phage Display", IMMUNOBIOLOGY, URBAN UND FISCHER VERLAG, DE, vol. 219, no. 11, 10 August 2014 (2014-08-10), DE, pages 822 - 829, XP029081965, ISSN: 0171-2985, DOI: 10.1016/j.imbio.2014.07.002 *
FENG QIJIA, XU ZHI, WU GUOMING, ET AL.: "Prediction of B-cell antigen epitope on CD14", SICHUAN MEDICAL JOURNAL, vol. 28, no. 9, 1 January 2007 (2007-01-01), pages 959 - 961, XP055814414, ISSN: 1004-0501, DOI: 10.16252/j.cnki.issn1004-0501-2007.09.003 *
FENG QIJIA: "The Screening of Mimic Peptide of Lipopolysaccharide Binding Protein Binding to CD14 and Its Protective Effects on Endotoxin-induced Acute Lung Injury", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, 30 May 2008 (2008-05-30), XP055814404 *
IWAKI, D. NISHITANI, C. MITSUZAWA, H. HYAKUSHIMA, N. SANO, H. KUROKI, Y.: "The CD14 region spanning amino acids 57-64 is critical for interaction with the extracellular Toll-like receptor 2 domain", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 328, no. 1, 4 March 2005 (2005-03-04), Amsterdam NL, pages 173 - 176, XP004723755, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2004.12.162 *
JIANG QIN, YUMEI FU, LINYI HOU, JIEPING LYU, WENKAI ZHANG: "Study of the Anti-CD14 Monoclonal Antibody on sI CAM-1 and KC Expression of Lung Tissue with Sepsis in Rat", CHINESE JOURNAL OF CLINICIANS, CN, vol. 10, no. 21, 1 November 2016 (2016-11-01), CN, pages 3203 - 3209, XP055814397, ISSN: 1674-0785, DOI: 10.3877/cma.j.issn.1674-0785.2016.21.013 *
See also references of EP4059517A4 *
ZHAO YUANMING, LU SHU-HUA; SHEN AI-HUA: "The Study about Detection of Soluble CD14 Subtype as a Marker of Sepsis", CHINESE JOURNAL OF LABORATORY DIAGNOSIS / ZHONG GUO SHI YAN ZHEN DUAN XUE, vol. 23, no. 5, 1 May 2019 (2019-05-01), pages 804 - 806, XP055814408, ISSN: 1007-4287 *
ZIEGLER-HEITBROCK H W L, ET AL.: "THE ANTIBODY MY4 RECOGNIZES CD14 ON PORCINE MONOCYTES AND MACROPHAGES", SCANDINAVIAN JOURNAL OF IMMUNOLOGY, BLACKWELL SCIENCE PUBL., OXFORD, GB, vol. 40, no. 05, 1 January 1994 (1994-01-01), GB, pages 509 - 514, XP008055051, ISSN: 0300-9475 *

Also Published As

Publication number Publication date
CN115232211A (zh) 2022-10-25
EP4059517A4 (en) 2023-04-05
EP4059517A1 (en) 2022-09-21
CN113939314B (zh) 2022-09-30
CN113939314A (zh) 2022-01-14

Similar Documents

Publication Publication Date Title
US20210333292A1 (en) Method for diagnosing or monitoring kidney function or diagnosing kidney dysfunction
US20110076700A1 (en) Anti-crp antibody and utilization of the same
WO2021097684A1 (zh) 新的抗可溶性cd14亚型抗体及其应用
US20220283182A1 (en) Kit and method for combined detection of pct and presepsin, and use thereof
JP6606552B2 (ja) 特異的に精製された抗プレセプシン抗体
WO2021253335A1 (zh) 抗可溶性cd14亚型抗体、试剂盒及其应用
JP6037043B2 (ja) ＴＲＡＣＰ−５ｂ（酒石酸抵抗性酸性フォスファターゼ５ｂ）に特異的なタンパク定量法
CN111303289A (zh) 抗人Tn型糖基化MUC1抗体及其用途
WO2013084504A1 (ja) 高血圧症の予知方法
WO2023148165A1 (en) Method for diagnosing collagen degradatation associated disease
JP2021156648A (ja) 被験者のスクリ−ニング方法及び鑑別方法
EP3019875A2 (en) Augurin immunoassay
JP5585587B2 (ja) ５．９ｋＤａペプチドの免疫学的測定方法
CN113710699A (zh) Xxiii型胶原蛋白测定

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19953573

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019953573

Country of ref document: EP

Effective date: 20220615

NENP Non-entry into the national phase

Ref country code: DE