WO2021097685A1 - 混合检测PCT和Presepsin的试剂盒、方法以及应用 - Google Patents

混合检测PCT和Presepsin的试剂盒、方法以及应用 Download PDF

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WO2021097685A1
WO2021097685A1 PCT/CN2019/119537 CN2019119537W WO2021097685A1 WO 2021097685 A1 WO2021097685 A1 WO 2021097685A1 CN 2019119537 W CN2019119537 W CN 2019119537W WO 2021097685 A1 WO2021097685 A1 WO 2021097685A1
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amino acid
soluble
subtype
sequence number
seq
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PCT/CN2019/119537
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English (en)
French (fr)
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于丽娜
李可
何建文
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深圳迈瑞生物医疗电子股份有限公司
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Priority to CN201980096880.8A priority Critical patent/CN113892031B/zh
Priority to EP19953055.1A priority patent/EP4060338A4/en
Priority to PCT/CN2019/119537 priority patent/WO2021097685A1/zh
Priority to CN202211454571.3A priority patent/CN116699141A/zh
Publication of WO2021097685A1 publication Critical patent/WO2021097685A1/zh
Priority to US17/747,868 priority patent/US20220283182A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—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
    • G01N33/68—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G01N2333/575—Hormones
    • G01N2333/585—Calcitonins
    • 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
    • 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
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • This application relates to a kit, method and application for the mixed detection of procalcitonin (PCT) and soluble CD14 subtype (sCD14-ST or Presepsin) in a sample to be tested.
  • PCT procalcitonin
  • sCD14-ST soluble CD14 subtype
  • 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.
  • this application provides a kit for the mixed detection of PCT and Presepsin in a test sample and a method for the mixed detection of PCT and Presepsin in a test sample.
  • this application also provides the application of mixed detection of PCT and Presepsin in the test sample in the diagnosis and prognosis of sepsis in patients.
  • the first aspect of this application relates to a kit for the mixed detection of PCT and Presepsin in a sample to be tested, the kit comprising: a capture antibody mixture containing calcitonin coated on a solid phase The original capture antibody and a soluble CD14 subtype capture antibody coated on a solid phase; and a detection antibody mixture containing a labeled procalcitonin detection antibody and a labeled soluble CD14 subtype detection antibody.
  • the second aspect of the application relates to a method for mixed detection of PCT and Presepsin in a sample to be tested, the method comprising:
  • a detection substrate is added to the above-mentioned cleaned sandwich complex to detect the mixed concentration level of procalcitonin and soluble CD14 subtypes in the sample to be tested.
  • the third aspect of this application relates to the application of soluble CD14 subtype antibody and procalcitonin antibody in the preparation of analytical reagents for assessing whether a patient suffers from sepsis, wherein the patient’s sample to be tested is coated in a solid
  • the procalcitonin capture antibody on the phase and the soluble CD14 subtype capture antibody coated on the solid phase are mixed, and then the labeled procalcitonin detection antibody and the labeled soluble CD14 subtype detection antibody are added to detect the
  • the mixed concentration value of procalcitonin and soluble CD14 subtypes in the sample to be tested, and the increase of the mixed concentration level relative to the reference value is related to the increased possibility of the patient suffering from sepsis.
  • the fourth aspect of the application relates to the application of soluble CD14 subtype antibody and procalcitonin antibody in the preparation of analytical reagents for assessing the prognosis of patients with suspected sepsis, wherein the patient’s sample to be tested is coated in a solid
  • the procalcitonin capture antibody on the phase and the soluble CD14 subtype capture antibody coated on the solid phase are mixed, and then the labeled procalcitonin detection antibody and the labeled soluble CD14 subtype detection antibody are added to detect the
  • the mixed concentration value of procalcitonin and soluble CD14 subtype in the sample to be tested, and the increase of the mixed concentration level relative to the reference value is related to the increased risk of death in patients with suspected sepsis.
  • the kit, method, and application of the embodiments of the present application can detect the mixed signal value of PCT and Presepsin in a sample in a reaction system, and the comparison of the mixed signal value with the reference value can be used to evaluate the patient suffering from sepsis.
  • the possibility of symptom and the evaluation of the prognosis of patients with suspected sepsis have a higher diagnostic and prognostic effect than PCT and Presepsin in a separate test sample.
  • due to the mixed detection of PCT and Presepsin in the sample only one test is required, which can reduce the economic and time cost of the test and reduce the amount of blood used. It can also reduce the impact of different reagent production batches on the test results. Conducive to the production of reagents and automated testing of instruments.
  • Figure 1 shows the ROC curve of mixing PCT capture antibody and Presepsin capture antibody in different proportions and mixing PCT detection antibody and Presepsin detection antibody in different proportions to assess whether a patient has sepsis.
  • Figure 2 is an ROC curve diagram used to assess whether a patient has sepsis according to an embodiment of the application.
  • Figure 3 shows the ROC curves of PCT alone, Presepsin alone, and PCT and Presepsin combined to assess whether a patient has sepsis.
  • Fig. 4 is a graph of survival rate used to evaluate the prognosis of sepsis according to an embodiment of the application.
  • Figure 5 shows the ROC curves of PCT alone, Presepsin alone, and PCT and Presepsin combined respectively for assessing the prognosis of sepsis.
  • Figure 6 shows the SDS-PAGE and Western Blot of the prokaryotic expression protein.
  • Figure 7 shows the eukaryotic expression protein SDS-PAGE and Western Blot
  • Figure 8 shows SDS-PAGE and Western Blot of anti-soluble CD14 subtype polyclonal antibody.
  • CRP Interleukin 6
  • CRP C reactive protein
  • PCT procalcitonin
  • 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 unreliable to use PCT to diagnose infection and sepsis.
  • the soluble CD14 subtype (sCD14-ST or Presepsin for short) is a new biological indicator of sepsis, which has potential application value in the diagnosis and prognosis of sepsis and the use of antibacterial drugs.
  • Presepsin is the N-terminal fragment of sCDl4 that is cleaved by cathepsin D and other proteases in plasma, and its relative molecular weight is about 13kDa.
  • 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.
  • Presepsin is considered to have high specificity in assessing the severity and prognosis of sepsis, and can accurately guide the application of antimicrobial agents for sepsis. Nevertheless, Presepsin as a diagnostic marker still has shortcomings in certain infection scenarios.
  • the embodiments of the present application propose a kit, method, and application for the mixed detection of PCT and Presepsin in a sample to be tested with high diagnostic and prognostic efficacy.
  • 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.
  • a kit for the mixed detection of PCT and Presepsin in a sample to be tested is also referred to as a "mixed detection kit”.
  • the first aspect of the present application provides a kit for the mixed detection of PCT and Presepsin in a sample to be tested (mixed detection kit), comprising: a solid phase component, the solid phase component including coating on the solid phase The procalcitonin capture antibody (PCT capture antibody) and the soluble CD14 subtype capture antibody (Presepsin capture antibody) coated on the solid phase; and a labeling component, the labeling component including a labeled procalcitonin Detection antibody (PCT detection antibody) and labeled soluble CD14 subtype detection antibody (Presepsin detection antibody).
  • PCT capture antibody The procalcitonin capture antibody
  • Presepsin capture antibody soluble CD14 subtype capture antibody coated on the solid phase
  • a labeling component the labeling component including a labeled procalcitonin Detection antibody (PCT detection antibody) and labeled soluble CD14 subtype detection antibody (Presepsin detection antibody).
  • the solid phase component is present in the kit in the form of a capture antibody mixture, and the capture antibody mixture is mixed with procalcitonin coated on the solid phase.
  • Capture antibody and soluble CD14 subtype capture antibody coated on the solid phase may also include a procalcitonin capture antibody component coated on the solid phase and a soluble CD14 subtype capture antibody group coated on the solid phase that are separately packaged from each other. Minute.
  • the labeling component is present in the kit in the form of a detection antibody mixture, and the detection antibody mixture is mixed with a labeled procalcitonin detection antibody And labeled soluble CD14 subtype detection antibody.
  • the labeling component may also include a labeled procalcitonin detection antibody and a labeled soluble CD14 subtype detection antibody that are separately packaged from each other.
  • the procalcitonin capture antibody and the soluble CD14 subtype capture antibody are respectively coated on different solid phases.
  • the procalcitonin capture antibody and the soluble CD14 subtype capture antibody can also be co-coated on the same solid phase.
  • the mixing ratio of the PCT capture antibody and the Presepsin capture antibody is determined according to the principle of maximizing the diagnostic efficacy of diagnosing sepsis, that is, the principle of maximizing the AUC of the ROC curve is selected.
  • the mixing ratio of the PCT capture antibody and the Presepsin capture antibody in the solid phase component may be in the range of about 5:1 to about 1:5, preferably in the range of about 3:1 to about 1:5, and more preferably in the range of about 3. In the range of :1 to 1:3, it may be about 3:1 or 1:1 or 1:3, and more preferably it may be about 1:3.
  • the mixing ratio of the PCT detection antibody and the Presepsin detection antibody is determined according to the principle of maximizing the diagnostic efficacy of diagnosing sepsis, that is, the selection is based on the principle of maximizing the AUC of the ROC curve.
  • the mixing ratio of the PCT detection antibody and the Presepsin detection antibody in the labeling component may be in the range of about 5:1 to about 1:5, preferably in the range of about 3:1 to about 1:5, and more preferably in the range of about 3: In the range of 1 to 1:3, it may especially be about 3:1 or 1:1 or 1:3, and more particularly preferably it may be about 1:3.
  • the mixing ratio of PCT capture antibody and Presepsin capture antibody in the solid phase component may be equal to the mixing ratio of PCT detection antibody and Presepsin detection antibody in the labeling component, preferably both are about 1: 3.
  • both the PCT capture antibody and the PCT detection antibody are commercially available, wherein the PCT capture antibody is, for example, Anti-hPCT 4003 SPTN-5 (Medix Biochemica), and the PCT detection antibody is, for example, Anti- hPCT 4051 SPTN-5 (Medix Biochemica).
  • the soluble CD14 subtype capture antibody specifically recognizes an epitope composed of the amino acid sequence of SEQ ID No. 42, the soluble CD14 subtype capture antibody, the soluble CD14 subtype capture antibodies 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 soluble CD14 subtype capture antibody includes VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, and VL CDR1, VL CDR2 and VL CDR3 are selected from the amino acid sequences described in Table 1 (see Table 1).
  • the soluble CD14 subtype capture antibody includes VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, and VL CDR1, VL CDR2 and VL CDR3 are selected from the amino acid sequences recorded in Table 2 (see Table 2).
  • the soluble CD14 subtype capture antibody includes VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 are any of the following 1) to 62) One:
  • 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 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 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 the 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 composed of sequence number 7
  • VH CDR3 is composed of sequence number 19
  • 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 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 composed of sequence number 10
  • VH CDR3 is composed of sequence number 15
  • VL CDR1 is composed of sequence number 21
  • VL CDR2 is composed of 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 composed of amino acid sequences of 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.
  • the soluble CD14 subtype capture antibody includes:
  • 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 Presepsin capture antibody may be derived from the anti-soluble CD14 subtype monoclonal antibody or its antigen-binding antibody fragment secreted and produced by the hybridoma cell line with the deposit number CGMCC NO.18536, and
  • the Presepsin detection antibody can be derived from the anti-soluble CD14 subtype polyclonal antibody secreted by the hybridoma cell line with the deposit number CGMCC NO.18536.
  • the solid phase carrier may be selected from magnetic microspheres, chips, test paper, etc., preferably magnetic microspheres, more preferably superparamagnetic magnetic beads.
  • the PCT detection antibody and the Presepsin detection antibody are labeled with a signal marker
  • the signal marker may be, for example, a chemiluminescent marker (such as alkaline phosphatase, luminol, isoluminol). Connaught, acridinium ester, horseradish peroxidase), electrochemiluminescence markers (such as terpyridine ruthenium), quantum dots (such as gold quantum dots, CdSe quantum dots, ZnCdSe quantum dots, etc.), fluorescent microspheres, etc., or Its combination.
  • chemiluminescent marker such as alkaline phosphatase, luminol, isoluminol. Connaught, acridinium ester, horseradish peroxidase), electrochemiluminescence markers (such as terpyridine ruthenium), quantum dots (such as gold quantum dots, CdSe quantum dots, ZnCdSe quantum dots, etc.), fluorescent microspheres, etc
  • the capture antibody mixture may also include specific capture antibodies for other markers.
  • the detection antibody mixture includes specific detection antibodies for the other markers, and the other markers can be selected.
  • CRP C-reactive protein
  • IL-6 interleukin 6
  • TNF- ⁇ tumor necrosis factor
  • SAA serum atherosin A
  • HBP heparin binding protein
  • suPAR soluble urokinase-type fibrinolysis Proenzyme activator receptor
  • IL-1 ⁇ interleukin-1 ⁇
  • interleukins such as IL-2, IL-4, IL-8, IL-10, IL-12, IL-13, IL -18
  • TIMP-1 Metalloproteinase tissue mimicking factor-1
  • sTREM-1 serum soluble myeloid cell trigger receptor-1
  • MMP-9 metalloproteinase 9
  • TLR2 Toll-like receptor
  • the other markers can be selected from C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor (TNF- ⁇ ), serum atherosin A (SAA), heparin binding protein ( At least one of HBP).
  • CRP C-reactive protein
  • IL-6 interleukin 6
  • TNF- ⁇ tumor necrosis factor
  • SAA serum atherosin A
  • HBP heparin binding protein
  • the PCT capture antibody can also be replaced by the specific capture antibody of other markers.
  • the detection antibody mixture contains the specific detection antibody of the other marker instead of the PCT detection antibody.
  • the other marker may be one of the markers listed above, for example.
  • the second aspect of the application relates to a method for mixed detection of PCT and Presepsin in a sample to be tested, including the following steps:
  • the procalcitonin detection antibody binds to the procalcitonin bound on the procalcitonin capture antibody and the labeled soluble CD14 subtype detection antibody binds to the soluble CD14 subtype bound to the soluble CD14 subtype capture antibody Combine to form a sandwich compound;
  • a detection substrate is added to the aforementioned sandwich complex to detect the mixed concentration level of procalcitonin and soluble CD14 subtypes in the sample to be tested.
  • the method specifically includes:
  • a chemiluminescent substrate is added to the above-mentioned cleaned sandwich complex, and the number of photons produced by the reaction is detected to obtain the mixed signal value of procalcitonin and soluble CD14 subtype in the sample to be tested.
  • the mixed concentration level of procalcitonin and soluble CD14 subtypes in the test sample is directly proportional to the mixed concentration of procalcitonin and soluble CD14 subtypes in the test sample.
  • the procalcitonin capture antibody and the soluble CD14 subtype capture antibody are respectively coated on different solid phases.
  • the procalcitonin capture antibody and the soluble CD14 subtype capture antibody can also be co-coated on the same solid phase.
  • the PCT capture antibody and the Presepsin capture antibody are added in a mixed form, or added separately.
  • the PCT detection antibody and the Presepsin detection antibody are added in a mixed form, or added separately.
  • the addition ratio of the PCT capture antibody and the Presepsin capture antibody is determined based on the principle of maximizing the diagnostic efficacy of diagnosing sepsis, that is, the selection is based on the principle of maximizing the AUC of the ROC curve.
  • the addition ratio of the PCT capture antibody and the Presepsin capture antibody may be in the range of about 5:1 to about 1:5, preferably in the range of about 3:1 to about 1:5, and more preferably in the range of about 3:1 to about 1. In the range of :3, it may be about 3:1 or 1:1 or 1:3, and more preferably it may be about 1:3.
  • the addition ratio of the PCT detection antibody and the Presepsin detection antibody is determined according to the principle of maximizing the diagnostic efficacy of diagnosing sepsis, that is, the selection is based on the principle of maximizing the AUC of the ROC curve.
  • the addition ratio of the PCT detection antibody to the Presepsin detection antibody may be in the range of about 5:1 to about 1:5, preferably in the range of about 3:1 to about 1:5, and more preferably in the range of about 3:1 to about 1. In the range of :3, it may be about 3:1 or 1:1 or 1:3, and more preferably it may be about 1:3.
  • the addition ratio of the PCT capture antibody and the Presepsin capture antibody may be equal to the addition ratio of the PCT detection antibody and the Presepsin detection antibody, preferably about 1:3.
  • the sample to be tested may be blood, blood components such as serum or plasma, and preferably a plasma sample.
  • the third aspect of this application relates to the application of soluble CD14 subtype antibody and procalcitonin antibody in the preparation of analytical reagents for assessing whether a patient suffers from sepsis, wherein the patient’s sample to be tested is coated in a solid
  • the procalcitonin capture antibody on the phase and the soluble CD14 subtype capture antibody coated on the solid phase are mixed, and then the labeled procalcitonin detection antibody and the labeled soluble CD14 subtype detection antibody are added to detect the
  • the mixed concentration level of procalcitonin and soluble CD14 subtype in the sample to be tested, and the increase of the mixed concentration level relative to the reference value is related to the increased possibility of the patient suffering from sepsis.
  • a chemiluminescent substrate is used to detect the patient’s test sample, capture antibody, and the number of photons generated by the antibody reaction to obtain procalcitonin and soluble CD14 in the test sample.
  • the mixed signal value of the subtype which represents the mixed concentration value of procalcitonin and soluble CD14 subtype in the sample to be tested.
  • the increase in the level of the mixed signal value relative to the reference value is positively correlated with an increase in the possibility of the patient suffering from sepsis.
  • the mixed detection kit according to the present application is used to test the sample to be tested, an ROC curve is established, the threshold value is determined according to the ROC curve, and the mixed signal value is compared with the threshold value.
  • the combined detection factor is ⁇ 1
  • the patient is at high risk of sepsis
  • the combined detection factor is ⁇ 1
  • the patient is at low risk of sepsis.
  • the mixed signal value of procalcitonin and soluble CD14 subtype in the test sample of the patient is detected within 72 hours after the patient is suspected of having sepsis. .
  • the fourth aspect of the application relates to the application of soluble CD14 subtype antibody and procalcitonin antibody in the preparation of analytical reagents for assessing the prognosis of patients with suspected sepsis, wherein the patient’s sample to be tested is coated in a solid
  • the procalcitonin capture antibody on the phase and the soluble CD14 subtype capture antibody coated on the solid phase are mixed, and then the labeled procalcitonin detection antibody and the labeled soluble CD14 subtype detection antibody are added to detect the
  • the mixed concentration level of procalcitonin and soluble CD14 subtype in the test sample, and the increase of the mixed concentration level relative to the reference value is related to the increased risk of death in patients with suspected sepsis.
  • a chemiluminescent substrate is used to detect the patient’s test sample, capture antibody, and the number of photons generated by the antibody reaction to obtain procalcitonin and soluble CD14 in the test sample.
  • the mixed signal value of the subtype which represents the mixed concentration value of procalcitonin and soluble CD14 subtype in the sample to be tested.
  • the mixed detection factor is then compared with the reference value to assess the risk of death in patients with suspected sepsis.
  • an increase in the level of the mixed signal value relative to a reference value is positively correlated with an increase in the risk of death of patients with suspected sepsis.
  • the mixed signal value of procalcitonin and soluble CD14 subtype in the test sample of the patient is detected within 72 hours after the patient is suspected of having sepsis .
  • Example 1 Preparation of a kit (mixed detection kit) for the mixed detection of PCT and Presepsin in the sample to be tested
  • the mixed detection kit of the present application is prepared by mixing the PCT capture antibody and Presepsin capture antibody in a certain ratio, then coat it on the surface of a solid carrier, and then mix the PCT detection antibody and Presepsin detection antibody in a certain ratio, Then label the signal marker.
  • the PCT capture antibody and the Presepsin capture antibody are mixed at a concentration ratio of about 5:1 to about 1:5, respectively, and coated on the surface of a solid-phase carrier, wherein the solid-phase carrier is preferably a superparamagnetic magnetic bead.
  • the PCT detection antibody and the Presepsin detection antibody are also mixed in a ratio of about 5:1 to about 1:5 and labeled with a signal marker, wherein the signal marker is preferably alkaline phosphatase.
  • the ratio of PCT capture antibody and Presepsin capture antibody is the same as the ratio of PCT detection antibody and Presepsin detection antibody.
  • the concentration of the captured antibody mixture is in the range of about 1 ⁇ g/mL to about 10 ⁇ g/mL, preferably about 2 ⁇ g/mL, and the concentration of the labeled detection antibody mixture after dilution is about 0.5 ⁇ g/mL to about 5 ⁇ g/mL In the range, it is preferably about 1 ⁇ g/mL.
  • the mixing ratio of the PCT capture antibody and the Presepsin capture antibody and the mixing ratio of the PCT detection antibody and the Presepsin detection antibody are both about 1:3, the diagnostic efficacy of the diagnosis of sepsis is relatively maximum.
  • the mixing ratio of the PCT capture antibody and the Presepsin capture antibody was selected to be about 1:3.
  • the mixing ratio of the PCT detection antibody and the Presepsin detection antibody was selected to be about 1:3.
  • Example 2 Method for mixed detection of PCT and Presepsin in the sample to be tested
  • the mixed detection kit according to the embodiment of the present application for example, the mixed detection kit prepared according to Example 1, the capture antibody mixture coated on a solid phase carrier, the labeled detection antibody mixture, and the sample to be tested are mixed, wherein ,
  • the PCT marker in the sample to be tested combines with the PCT capture antibody and the PCT detection antibody to form a sandwich structure.
  • the Presepsin marker in the sample to be tested combines with the Presepsin capture antibody and the Presepsin detection antibody to form a sandwich structure; wash and remove Unbound detection antibody; by Shenzhen Mindray Bio-Medical Electronics Co., Ltd., such as CL-2000i, CL-900i, CL-6000i, or CL-1000i, the signal generated by the signal marker labeled by the detection antibody is detected,
  • the size of the mixed signal value is proportional to the concentration of Presepsin and PCT in the sample to be tested.
  • the method of this implementation is used to test the above 215 samples to establish an ROC curve, as shown in Figure 2.
  • the threshold is determined to be 200,000, and the sensitivity and specificity corresponding to the threshold are 0.86 and 0.87, respectively.
  • Example 3 Application of Presepsin antibody and PCT antibody in the preparation of analytical reagents for assessing whether a patient suffers from sepsis
  • the mixed detection kit according to the embodiment of the application was used to detect the plasma samples of 141 suspected sepsis patients within 72 hours of admission to obtain the mixed signal value, and simultaneously detect the 141 suspects.
  • PCT concentration and Presepsin concentration in plasma samples of patients with sepsis are established respectively, as shown in Figure 3.
  • the AUC areas of the ROC curves for PCT alone, Presepsin alone, and PCT and Presepsin mixed detection are 0.84, 0.91, and 0.96, respectively. It can be seen that the combined detection of PCT and Presepsin has higher diagnostic efficacy.
  • the mixed detection kit according to the embodiment of the application was used to test 200 samples, including 60 healthy people, 52 locally infected people, 51 septic patients, and 37 septic shock people.
  • the diagnostic criteria are based on Sepsis 3.0 (Singer M, Deutschman CS, Seymour CW, et al.
  • Example 4 Application of Presepsin antibody and PCT antibody in the preparation of analytical reagents for assessing the prognosis of patients with suspected sepsis
  • the mixed detection kit according to the embodiment of the application was used to detect the plasma samples of 88 suspected sepsis patients and track the prognosis of the patients within 30, using the Kaplan-Meier statistical analysis method, The relationship curve chart of the survival rate of patients with suspected sepsis within 30 days of admission to the hospital is obtained, as shown in Figure 4, where N represents the number of patients. For different groups, the survival rate of patients is significantly different. For group III, when the mixed detection factor ⁇ 12.21, the survival rate of patients is lower than 40% (p ⁇ 0.01).
  • the prognostic effect of the mixed detection concentration value, the single PCT detection concentration value, the single Presepsin detection concentration value within 72 hours of admission and the patient's survival rate within 30 days after admission were compared with the prognostic effect of the ROC curve.
  • the AUCs of PCT alone, Presepsin alone, and PCT and Presepsin combined were 0.72, 0.83, and 0.86, respectively. It can be seen that the embodiments of the present application can more accurately predict the survival status of patients with suspected 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 to be 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: cultured in a serum-free FreeStyle TM 293 Expression Medium (Thermo Fisher Scientific) matrix at 37° C. and 5% CO2.
  • Transfect the constructed plasmid in 293-6E expression system by transient transfection method. After culturing for 6 days, the cell culture supernatant was collected. The supernatant was filtered and subjected to affinity purification. The purified protein was digested with optimized experimental conditions, and the target protein was obtained after purification. As shown in Figure 7, the Bradford method was used to determine the concentration of the target protein as 1.55 mg/mL, and SDS-PAGE and Western Blot were used to determine the purity and molecular weight of the target protein.
  • the prokaryotic system and 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.
  • 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 8 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, the preservation number of which is 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.
  • Tables 5-7 summarize the anti-soluble CD14 subtype monoclonal antibody affinity ranking, epitope classification, and the results of paired antibody screening. 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.
  • 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.
  • the embodiments of the present application can detect the mixed signal value of PCT and Presepsin in a sample in a reaction system, and the comparison of the mixed signal value with the reference value can be used to evaluate the patient suffering from sepsis.
  • the possibility of hyperemia and the evaluation of the prognosis of patients with suspected sepsis have a higher diagnostic and prognostic power than PCT and Presepsin in a separate test sample.
  • due to the mixed detection of PCT and Presepsin in the sample it only needs to be tested once, which can reduce the economic cost and time cost of the test, reduce the amount of blood used, and reduce the impact of different reagent production batches on the test results. Conducive to the production of reagents and automated instrument testing.

Abstract

一种混合检测待测样本中的PCT和Presepsin的试剂盒、方法以及应用，该试剂盒包含：捕获抗体混合物，该捕获抗体混合物含有包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体；以及检测抗体混合物，该检测抗体混合物含有经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体。该试剂盒、方法及应用能够实现在一个反应体系中检测样本中的PCT和Presepsin的混合信号值，该混合信号值与参考值的比较能够用于评估患者患有脓毒血症的可能性以及评估疑似脓毒血症患者的预后，相比于单独检测样本中的PCT和Presepsin，具有更高的诊断和预后效力。

Description

混合检测PCT和Presepsin的试剂盒、方法以及应用 技术领域
本申请涉及混合检测待测样本中的降钙素原(procalcitonin，简称PCT)和可溶性CD14亚型(soluble CDl4 subtype，简称sCDl4-ST或Presepsin)的试剂盒、方法以及应用。
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。
发明内容
因此，本申请提供了一种用于混合检测待测样本中的PCT和Presepsin的试剂盒以及一种用于混合检测待测样本中的PCT和Presepsin的方法。此外，本申请还提供了混合检测待测样本中的PCT和Presepsin在患者的脓毒血症诊断和预后中的应用。
本申请第一方面涉及一种用于混合检测待测样本中的PCT和Presepsin的试剂盒，所述试剂盒包含：捕获抗体混合物，所述捕获抗体混合物含有包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体；以及检测抗体混合物，所述检测抗体混合物含有经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体。
本申请第二方面涉及一种用于混合检测待测样本中的PCT和Presepsin的方法，所述方法包括：
将待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶 性CD14亚型捕获抗体混合，使得包被在固相上的降钙素原捕获抗体与待测样本中的降钙素原结合以及包被在固相上的可溶性CD14亚型捕获抗体与待测样本中的可溶性CD14亚型结合；
将上述待测样本、降钙素原捕获抗体和可溶性CD14亚型捕获抗体的混合物进行清洗，除去未结合的物质；
在上述经清洗的待测样本和捕获抗体混合物的混合物中加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，使得所述经标记的降钙素原检测抗体与所述降钙素原捕获抗体上结合的降钙素原结合以及所述经标记的可溶性CD14亚型检测抗体与所述可溶性CD14亚型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，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平。
本申请第三方面涉及可溶性CD14亚型抗体和降钙素原抗体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，将患者的待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，然后加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度值，所述混合浓度水平相对于参考值的升高与患者患有脓毒血症的可能性增加相关。
本申请第四方面涉及可溶性CD14亚型抗体和降钙素原抗体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，将患者的待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，然后加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度值，所述混合浓度水平相对于参考值的升高与疑似脓毒血症患者的死亡风险增加相关。
通过本申请实施例的试剂盒、方法以及应用能够实现在一个反应体系中检测样本中的PCT和Presepsin的混合信号值，该混合信号值与参考值的比较能够用于评估患者患有脓毒血症的可能性以及评估疑似脓毒血症患者的预后，相比于单独检测样本中的PCT和Presepsin具有更高的诊断和预后效力。此外， 由于对样本中的PCT和Presepsin进行混合检测而仅需要检测一次，从而能够降低检测的经济成本和时间成本以及减少用血量，还能够减少不同试剂生产批次对检测结果的影响，有利于试剂生产和仪器自动化检测。
附图说明
在下文中，参考附图描述本申请的优选的实施方式。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为按照不同比例混合PCT捕获抗体和Presepsin捕获抗体以及按照不同比例混合PCT检测抗体和Presepsin检测抗体用于评估患者是否患有脓毒血症的ROC曲线图。
图2为本申请实施例用于评估患者是否患有脓毒血症的ROC曲线图。
图3为单独检测PCT、单独检测Presepsin以及混合检测PCT和Presepsin分别用于评估患者是否患有脓毒血症的ROC曲线图。
图4为本申请实施例用于评估脓毒血症预后的生存率曲线图。
图5为单独检测PCT、单独检测Presepsin以及混合检测PCT和Presepsin分别用于评估脓毒血症预后的ROC曲线图。
图6为原核表达蛋白SDS-PAGE和Western Blot。
图7为真核表达蛋白SDS-PAGE和Western Blot
图8为抗可溶性CD14亚型多克隆抗体SDS-PAGE和Western Blot。
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。
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细胞介素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。
可溶性CD14亚型(soluble CDl4 subtype，简称sCDl4-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。Presepsin是sCDl4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。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。目前Presepsin被认为在评估脓毒症严重程度及预后方面具有高特异性，并能准确的指导脓毒症抗菌药的应用。尽管如此，Presepsin作为诊断标记物在某些感染场景下仍然存在缺陷。
因此，本申请实施例提出一种具有高诊断和预后效力的混合检测待测样本中的PCT和Presepsin的试剂盒、方法以及应用。
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，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。
在本申请的范围中，用于混合检测待测样本中的PCT和Presepsin的试剂 盒也被称作“混合检测试剂盒”。
本申请第一方面提供一种用于混合检测待测样本中的PCT和Presepsin的试剂盒(混合检测试剂盒)，包含：固相组分，所述固相组分包括包被在固相上的降钙素原捕获抗体(PCT捕获抗体)和包被在固相上的可溶性CD14亚型捕获抗体(Presepsin捕获抗体)；和标记组分，所述标记组分包括经标记的降钙素原检测抗体(PCT检测抗体)和经标记的可溶性CD14亚型检测抗体(Presepsin检测抗体)。
在本申请第一方面的一些实施方式中，所述固相组分以捕获抗体混合物的形式存在于所述试剂盒中，所述捕获抗体混合物混合有包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体。在其他实施例中，所述固相组分也可以包括相互独立分装的包被在固相上的降钙素原捕获抗体组分和包被在固相上的可溶性CD14亚型捕获抗体组分。
同样地，在本申请第一方面的一些实施方式中，所述标记组分以检测抗体混合物的形式存在于所述试剂盒中，所述检测抗体混合物混合有经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体。在其他实施例中，所述标记组分也可以包括相互独立分装的经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体。
在本申请第一方面的一些实施方式中，所述降钙素原捕获抗体和所述可溶性CD14亚型捕获抗体分别包被在不同的固相上。当然，所述降钙素原捕获抗体和所述可溶性CD14亚型捕获抗体也可以共同包被在同一固相上。
在本申请第一方面的一些实施方式中，PCT捕获抗体和Presepsin捕获抗体混合比例是依据诊断脓毒血症的诊断效力最大化的原则确定，即ROC曲线的AUC最大化的原则进行选取。固相组分中PCT捕获抗体与Presepsin捕获抗体的混合比例可以在约5:1至约1:5的范围中，优选在约3:1至约1:5的范围中，更优选在约3:1至1:3的范围中，尤其是可以为约3:1或1:1或1:3，更尤其优选可以为约1:3。
在本申请第一方面的一些实施方式中，PCT检测抗体和Presepsin检测抗体混合比例是依据诊断脓毒血症的诊断效力最大化的原则确定，即ROC曲线的AUC最大化的原则进行选取。标记组分中PCT检测抗体与Presepsin检测抗体的混合比例可以在约5:1至约1:5的范围中，优选在约3:1至约1:5的范围中，更优选在约3:1至1:3的范围中，尤其是可以为约3:1或1:1或1:3，更 尤其优选可以为约1:3。
在本申请第一方面的一些实施方式中，固相组分中PCT捕获抗体与Presepsin捕获抗体的混合比例可以等于标记组分中PCT检测抗体和Presepsin检测抗体的混合比例，优选均为约1:3。
在本申请第一方面的一些实施方式中，PCT捕获抗体或PCT检测抗体均可商业购得，其中PCT捕获抗体例如是Anti-hPCT 4003 SPTN-5(Medix Biochemica)，PCT检测抗体例如是Anti-hPCT 4051 SPTN-5(Medix Biochemica)。
在本申请第一方面的一些实施方式中，所述可溶性CD14亚型捕获抗体特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，所述可溶性CD14亚型捕获抗体，所述可溶性CD14亚型捕获抗体包括：
(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。
在本申请第一方面的一些实施方式中，所述可溶性CD14亚型捕获抗体包括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1中记载的氨基酸序列(见表1)。
表1
Figure PCTCN2019119537-appb-000001
Figure PCTCN2019119537-appb-000002
在本申请第一方面的一些实施方式中，所述可溶性CD14亚型捕获抗体包括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2中记载的氨基酸序列(见表2)。
表2
Figure PCTCN2019119537-appb-000003
Figure PCTCN2019119537-appb-000004
在本申请第一方面的一些实施方式中，所述可溶性CD14亚型捕获抗体包括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，所述可溶性CD14亚型捕获抗体包括：
(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，Presepsin捕获抗体可以来自由保藏号为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，而Presepsin检测抗体可以来自由保藏号为CGMCC NO.18536的杂交瘤细胞株分泌产生的抗可溶性CD14亚型多克隆抗体。
在本申请第一方面的一些实施方式中，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。
在本申请第一方面的一些实施方式中，PCT检测抗体和Presepsin检测抗体标记有信号标记物，该信号标记物例如可以是化学发光标记物(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。
在本申请第一方面的一些实施方式中，捕获抗体混合物还可以包括其他标志物的特异性捕获抗体，相应地，检测抗体混合物包含该其他标志物的特异性检测抗体，该其他标志物可以选自C-反应蛋白(CRP)、白细胞介素6(IL-6)、肿瘤坏死因子(TNF-α)、血清粥样蛋白A(SAA)、肝素结合蛋白(HBP)、可溶性尿激酶型纤溶酶原激活物受体(suPAR)、白细胞介素-1β(IL-1β)、白细胞介素例如IL-2、IL-4、IL-8、IL-10、IL-12、IL-13、IL-18、金属蛋白酶组织拟制因子-1(TIMP-1)、血清可溶性髓样细胞触发受体-1(sTREM-1)、金属基质蛋白酶9(MMP-9)、肾上腺髓质素前体(Pro-ADM)、Toll样受体(TLR2)、α2-巨球蛋白(A2M)、肌钙蛋白I(TnI)、趋化因子2(CCL2)、CD163、骨桥蛋白(OPN)、D-二聚体(D-Dimer)、脂多糖结合蛋白(LBP)、(1,3)-β-D-葡聚糖(BG)、凝血因子、促血管生成素1(Ang-1)、血管生成素-2(Ang-2)、血管内皮生长因子、γ-干扰素人、免疫球蛋白E、人免疫球蛋白A、人免疫球蛋白M、人免疫球蛋白G、人尿微量白蛋白中的至少一种。优选地，该其他标志物可以选自C-反应蛋白(CRP)、白细胞介素6(IL-6)、肿瘤坏死因子(TNF-α)、血清粥样蛋白A(SAA)、肝素结合蛋白(HBP)中的至少一种。
在本申请第一方面的一些实施方式中，还可以用其他标志物的特异性捕获抗体替代PCT捕获抗体，相应地，检测抗体混合物包含该其他标志物的特异性检测抗体替代PCT检测抗体，该其他标志物例如可以是上述列举的标志物之一。
本申请第二方面涉及一种用于混合检测待测样本中的PCT和Presepsin的方法，包括以下步骤：
将待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，使得包被在固相上的降钙素原捕获抗体与待测样本中的降钙素原结合以及包被在固相上的可溶性CD14亚型捕获抗体与待测 样本中的可溶性CD14亚型结合；
在上述待测样本、降钙素原捕获抗体和可溶性CD14亚型捕获抗体的混合物中加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，使得所述经标记的降钙素原检测抗体与所述降钙素原捕获抗体上结合的降钙素原结合以及所述经标记的可溶性CD14亚型检测抗体与所述可溶性CD14亚型捕获抗体上结合的可溶性CD14亚型结合，形成夹心复合物；
在上述夹心复合物中加入检测底物，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平。
在本申请第二方面的一些实施方式中，所述方法具体包括：
将待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，使得包被在固相上的降钙素原捕获抗体与待测样本中的降钙素原结合以及包被在固相上的可溶性CD14亚型捕获抗体与待测样本中的可溶性CD14亚型结合；
将上述待测样本、降钙素原捕获抗体和可溶性CD14亚型捕获抗体的混合物进行清洗，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降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，使得所述经标记的降钙素原检测抗体与所述降钙素原捕获抗体上结合的降钙素原结合以及所述经标记的可溶性CD14亚型检测抗体与所述可溶性CD14亚型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化学发光底物，检测反应所产生的光子数，以得到待测样本中的降钙素原和可溶性CD14亚型的混合信号值，该混合信号值表征待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平，与待测样本中的降钙素原和可溶性CD14亚型的混合浓度值成正比。
在本申请第二方面的一些实施方式中，所述降钙素原捕获抗体和所述可溶性CD14亚型捕获抗体分别包被在不同的固相上。当然，所述降钙素原捕获抗体和所述可溶性CD14亚型捕获抗体也可以共同包被在同一固相上。
在本申请第二方面的一些实施方式中，PCT捕获抗体和Presepsin捕获抗体以混合的形式加入，或者分开加入。
在本申请第二方面的一些实施方式中，PCT检测抗体和Presepsin检测抗 体以混合的形式加入，或者分开加入。
在本申请第二方面的一些实施方式中，PCT捕获抗体和Presepsin捕获抗体的加入比例是依据诊断脓毒血症的诊断效力最大化的原则确定，即ROC曲线的AUC最大化的原则进行选取。PCT捕获抗体与Presepsin捕获抗体的加入比例可以在约5:1至约1:5的范围中，优选在约3:1至约1:5的范围中，更优选在约3:1至约1:3的范围中，尤其是可以为约3:1或1:1或1:3，更尤其优选可以为约1:3。
在本申请第二方面的一些实施方式中，PCT检测抗体和Presepsin检测抗体的加入比例是依据诊断脓毒血症的诊断效力最大化的原则确定，即ROC曲线的AUC最大化的原则进行选取。PCT检测抗体与Presepsin检测抗体的加入比例可以在约5:1至约1:5的范围中，优选在约3:1至约1:5的范围中，更优选在约3:1至约1:3的范围中，尤其是可以为约3:1或1:1或1:3，更尤其优选可以为约1:3。
在本申请第二方面的一些实施方式中，PCT捕获抗体与Presepsin捕获抗体的加入比例可以等于PCT检测抗体和Presepsin检测抗体的加入比例，优选均为约1:3。
在本申请第二方面的一些实施方式中，待测样本可以是血液、血液组分例如血清或血浆，优选为血浆样本。
本申请第二方面的其他特征，例如PCT捕获抗体/检测抗体、Presepsin捕获抗体/检测抗体、标记物、固相等，可参照上述对本申请第一方面的相关描述，在此不再赘述。
本申请第三方面涉及可溶性CD14亚型抗体和降钙素原抗体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，将患者的待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，然后加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平，所述混合浓度水平相对于参考值的升高与患者患有脓毒血症的可能性增加相关。
在本申请第三方面的一些实施方式中，利用化学发光底物检测患者的待测样本、捕获抗体以及检测抗体反应所产生的光子数，以得到待测样本中的降钙素原和可溶性CD14亚型的混合信号值，该混合信号值表征待测样本中的 降钙素原和可溶性CD14亚型的混合浓度值。可以将待测样本中的PCT和Presepsin的混合信号值与阈值进行比较，得出混合检测因子，例如混合检测因子＝待测样本的混合信号值/阈值。然后在将所述混合检测因子与参考值进行比较，以评估患者是否患有脓毒血症。
在本申请第三方面的一些实施方式中，所述混合信号值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。
在本申请第三方面的一些实施方式中，用根据本申请的混合检测试剂盒对待测样本进行测试，建立ROC曲线，根据ROC曲线，以确定阈值，将混合信号值与阈值进行比较。当混合检测因子≥1时，患者高风险患有脓毒血症；当混合检测因子<1时，患者低风险患有脓毒血症。
在本申请第三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小时以内检测该患者的待测样本中的降钙素原和可溶性CD14亚型的混合信号值。
本申请第四方面涉及可溶性CD14亚型抗体和降钙素原抗体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，将患者的待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，然后加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平，所述混合浓度水平相对于参考值的升高与疑似脓毒血症患者的死亡风险增加相关。
在本申请第四方面的一些实施方式中，利用化学发光底物检测患者的待测样本、捕获抗体以及检测抗体反应所产生的光子数，以得到待测样本中的降钙素原和可溶性CD14亚型的混合信号值，该混合信号值表征待测样本中的降钙素原和可溶性CD14亚型的混合浓度值。可以将待测样本中的PCT和Presepsin的混合信号值与阈值进行比较，得出混合检测因子，例如混合检测因子＝待测样本的混合信号值/阈值。然后在将所述混合检测因子与参考值进行比较，以评估疑似脓毒血症患者的死亡风险。
在本申请第四方面的一些实施方式中，所述混合信号值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小时以内检测该患者的待测样本中的降钙素原和可溶性CD14亚型 的混合信号值。
实施例1：用于混合检测待测样本中的PCT和Presepsin的试剂盒(混合检测试剂盒)的制备
通过如下方式制备本申请的混合检测试剂盒：将PCT捕获抗体和Presepsin捕获抗体按一定的比例混合，然后包被于固相载体表面，接着将PCT检测抗体和Presepsin检测抗体按一定的比例混合，然后标记信号标记物。
首先，将PCT捕获抗体和Presepsin捕获抗体按照浓度比分别为约5:1至约1:5的比例进行混合，并包被于固相载体表面，其中固相载体优选为超顺磁性磁珠。将PCT检测抗体和Presepsin检测抗体同样按照约5:1至约1:5的比例进行混合并且标记有信号标记物，其中信号标记物优选为碱性磷酸酶。在该实施例中，PCT捕获抗体和Presepsin捕获抗体的比例与PCT检测抗体和Presepsin检测抗体的比例相同。然后，选择约50mM MES(2-吗啉乙磺酸)，约0.5M NaCl，pH约为6.0的缓冲液对经包被的捕获抗体混合物和经标记的检测抗体混合物进行稀释，稀释后经包被的捕获抗体混合物的浓度在约1μg/mL至约10μg/mL的范围中，优选为约2μg/mL，稀释后经标记的检测抗体混合物的浓度在约0.5μg/mL至约5μg/mL的范围中，优选为约1μg/mL。
选择具有明确临床诊断信息的样本，其中脓毒血阴性样本>50例，脓毒血阳性样本>50例。选用以上描述方法制备的试剂盒对待测样本进行测试，建立ROC曲线并计算AUC面积，如表3和图1所示。
表3
Figure PCTCN2019119537-appb-000005
由表3和图1可知，在一定范围内，随着PCT捕获抗体与Presepsin捕获抗体的浓度比或PCT检测抗体与Presepsin检测抗体的浓度比越小，相应的AUC面积越大，诊断脓毒血症的诊断效力越大。当PCT捕获抗体与Presepsin捕获抗体的浓度比或PCT检测抗体与Presepsin检测抗体的浓度比达到一定值时，AUC面积不再增大。在本申请实施例中，PCT捕获抗体与Presepsin捕获抗体的混合比例与PCT检测抗体和Presepsin检测抗体的混合比例均为约1:3时，诊断脓毒血症的诊断效力相对最大。
在后续实验中，选择PCT捕获抗体和Presepsin捕获抗体的混合比例为约1:3，同样地，选择PCT检测抗体和Presepsin检测抗体的混合比例为约1:3。
实施例2：用于混合检测待测样本中的PCT和Presepsin的方法
将根据本申请实施例的混合检测试剂盒、例如根据实施例1所制备的混合检测试剂盒中包被在固相载体上的捕获抗体混合物、经标记的检测抗体混合物与待测样本混合，其中，待测样本中的PCT标志物与PCT捕获抗体和PCT检测抗体结合，形成三明治结构，同样地，待测样本中的Presepsin标志物与Presepsin捕获抗体和Presepsin检测抗体结合，形成三明治结构；清洗去除未结合的检测抗体；通过深圳迈瑞生物医疗电子股份有限公司例如型号为CL-2000i、CL-900i、CL-6000i或CL-1000i的仪器对检测抗体所标记的信号标记物产生的信号进行检测，以得到待测样本中的PCT和Presepsin的混合信号值，其中，混合信号值的大小与待测样本内的Presepsin和PCT的浓度成正比。选择215例具有明确临床诊断信息的样本，其中脓毒血阴性样本95例，脓毒血阳性样本120例。借助检测设备、例如深圳迈瑞生物医疗电子股份有限公司的CL-2000i仪器，采用本实施的方法对上述215例样本进行测试，建立ROC曲线，如图2所示。在该实施例中，根据图2所示的ROC曲线，确定阈值为200000，阈值对应的灵敏度和特异性分别为：0.86和0.87。
实施例3：Presepsin抗体和PCT抗体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
采用本申请实施例的方法，利用根据本申请实施例的混合检测试剂盒对141名疑似脓毒血症患者入院72小时以内的血浆样本检测以得到混合信号值，并同时分别检测这141名疑似脓毒血症患者的血浆样本中的PCT浓度值和 Presepsin浓度值。根据检测结果，分别建立单独检测PCT、单独检测Presepsin以及混合检测PCT和Presepsin对应的ROC曲线，如图3所示。由ROC曲线的AUC面积可知，单独检测PCT、单独检测Presepsin以及混合检测PCT和Presepsin的ROC曲线的AUC面积分别为0.84、0.91、0.96。由此可知，混合检测PCT和Presepsin的诊断效力更高。
用根据本申请实施例的混合检测试剂盒对200例样本进行测试，其中健康人群60例、局部感染人群52例、脓毒血症患者51例、脓毒性休克人群37例。诊断标准依据Sepsis 3.0(Singer M,Deutschman CS,Seymour CW,et al.The Third international consensus definitions for sepsis and septic shock(Sepsis-3)[J])，其中对不同分组人群的测试结果(见表4)显示出：随着感染的发展，混合检测因子的数值不断升高。
表4
Figure PCTCN2019119537-appb-000006
实施例4：Presepsin抗体和PCT抗体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
采用本申请实施例的方法，利用根据本申请实施例的混合检测试剂盒对88名疑似脓毒血症患者的血浆样本进行检测并追踪患者30内的预后情况，采用Kaplan-Meier统计分析方法，得到疑似脓毒血症患者入院医院30天内的生存率的关系曲线图，如图4所示，其中N表示患者人数。对于不同分组，患者的生存率有显著不同，其中对于组Ⅲ，当混合检测因子≥12.21时，患者的生存率低于40％(p<0.01)。
此外，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小时内混合检测浓度值、单独PCT检测浓度值、单独Presepsin检测浓度值与患者入院30天内的生存率预后效力。如图5所示，单独检测PCT、 单独检测Presepsin以及混合检测PCT和Presepsin的AUC分别为0.72、0.83和0.86。由此可见，本申请实施例能够更准确地预测疑似脓毒血症患者的生存状况。
实施例5：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系统和真核表达系统进行表达，下面详细描述相应的过程：
(1)采用原核表达系统来表达可溶性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6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)采用真核表达系统来表达可溶性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％CO2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7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所获得的原核系统和真核系统表达纯化的蛋白、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8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8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所获得的原核系统和真核系统表达纯化的蛋白、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，其保藏号为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5-7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5
Figure PCTCN2019119537-appb-000007
Figure PCTCN2019119537-appb-000008
表6
Figure PCTCN2019119537-appb-000009
表7
Figure PCTCN2019119537-appb-000010
Figure PCTCN2019119537-appb-000011
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 PCTCN2019119537-appb-000012
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6：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。
从上面描述的各个实施例可知，本申请实施例能够实现在一个反应体系中检测样本中的PCT和Presepsin的混合信号值，该混合信号值与参考值的比较能够用于评估患者患有脓毒血症的可能性以及评估疑似脓毒血症患者的预后，相比于单独检测样本中的PCT和Presepsin具有更高的诊断和预后效力。此外，由于对样本中的PCT和Presepsin进行混合检测而仅需要检测一次，从而能够降低检测的经济成本和时间成本以及减少用血量，还能够减少不同试 剂生产批次对检测结果的影响，有利于试剂生产和仪器自动化检测。
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. 一种用于混合检测待测样本中的降钙素原和可溶性CD14亚型的试剂盒，所述试剂盒包含：
    捕获抗体混合物，所述捕获抗体混合物含有包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体；以及
    检测抗体混合物，所述检测抗体混合物含有经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体。
  2. 根据权利要求1所述的试剂盒，其中，所述降钙素原捕获抗体和所述可溶性CD14亚型捕获抗体分别包被在不同的固相上。
  3. 根据权利要求1或2所述的试剂盒，其中，所述捕获抗体混合物中所述降钙素原捕获抗体与所述可溶性CD14亚型捕获抗体的混合比例在5:1至1:5的范围中，优选在3:1至1:5的范围中，更优选在3:1至1:3的范围中。
  4. 根据权利要求3所述的试剂盒，其中，在所述捕获抗体混合物中所述降钙素原捕获抗体与所述可溶性CD14亚型捕获抗体的混合比例为3:1、1:1或1:3，优选为1:3。
  5. 根据权利要求1至4中任一项所述的试剂盒，其中，在所述检测抗体混合物中所述降钙素原检测抗体和所述可溶性CD14亚型检测抗体的混合比例在5:1至1:5的范围中，优选在3:1至1:5的范围中，更优选在3:1至1:3的范围中。
  6. 根据权利要求5所述的试剂盒，其中，在所述检测抗体混合物中所述降钙素原检测抗体和所述可溶性CD14亚型检测抗体的混合比例为3:1、1:1或1:3，优选为1:3。
  7. 根据权利要求1至6中任一项所述的试剂盒，其中，所述捕获抗体混合物中所述降钙素原捕获抗体与所述可溶性CD14亚型捕获抗体的混合比例等于在所述检测抗体混合物中所述降钙素原检测抗体和所述可溶性CD14亚型检测抗体的混合比例，所述混合比例优选均为1:3。
  8. 根据权利要求1至7中任一项所述的试剂盒，其中，所述可溶性CD14亚型捕获抗体特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，所述可溶性CD14亚型捕获抗体包括：
    (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。
  9. 根据权利要求8所述的试剂盒，其中，所述可溶性CD14亚型捕获抗体包括：
    (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。
  10. 根据权利要求1至9中任一项所述的试剂盒，
    其中，所述可溶性CD14亚型捕获抗体特异性地识别由SEQ ID No.42的氨基酸序列构成的表位，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。
  11. 一种用于混合检测待测样本中的降钙素原和可溶性CD14亚型的方法，所述方法包括：
    将待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，使得包被在固相上的降钙素原捕获抗体与待测样本中的降钙素原结合以及包被在固相上的可溶性CD14亚型捕获抗体与待测样本中的可溶性CD14亚型结合；
    将上述待测样本、降钙素原捕获抗体和可溶性CD14亚型捕获抗体的混合 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降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，使得所述经标记的降钙素原检测抗体与所述降钙素原捕获抗体上结合的降钙素原结合以及所述经标记的可溶性CD14亚型检测抗体与所述可溶性CD14亚型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，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平。
  12. 根据权利要求11所述的方法，其中，所述降钙素原捕获抗体和所述可溶性CD14亚型捕获抗体分别包被在不同的固相上。
  13. 根据权利要求11或12所述的方法，其中，所述降钙素原捕获抗体与所述可溶性CD14亚型捕获抗体的加入比例在5:1至1:5的范围中，优选在3:1至1:5的范围中，更优选在3:1至1:3的范围中。
  14. 根据权利要求16所述的方法，其中，所述降钙素原捕获抗体与所述可溶性CD14亚型捕获抗体的加入比例为3:1、1:1或1:3，优选为1:3。
  15. 根据权利要求11至14中任一项所述的方法，其中，所述降钙素原检测抗体和所述可溶性CD14亚型检测抗体的加入比例在5:1至1:5的范围中，优选在3:1至1:5的范围中，更优选在3:1至1:3的范围中。
  16. 根据权利要求11至15中任一项所述的方法，其中，所述降钙素原捕获抗体与所述可溶性CD14亚型捕获抗体的加入比例等于所述降钙素原检测抗体和所述可溶性CD14亚型检测抗体的加入比例，优选均为1:3。
  17. 根据权利要求11至16中任一项所述的方法，其中，所述待测样本为血浆样本。
  18. 可溶性CD14亚型抗体和降钙素原抗体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，将患者的待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，然后加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平，所述混合浓度水平相对于参考值的升高与患者患有脓毒血症的可能性增加相关。
  19. 根据权利要求18所述的应用，其中，在对患者产生患有脓毒血症的 怀疑后72小时以内检测该患者的待测样本中的降钙素原和可溶性CD14亚型的混合浓度值。
  20. 可溶性CD14亚型抗体和降钙素原抗体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，将患者的待测样本、包被在固相上的降钙素原捕获抗体和包被在固相上的可溶性CD14亚型捕获抗体混合，然后加入经标记的降钙素原检测抗体和经标记的可溶性CD14亚型检测抗体，以检测所述待测样本中的降钙素原和可溶性CD14亚型的混合浓度水平，所述混合浓度水平相对于参考值的升高与疑似脓毒血症患者的死亡风险增加相关。
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