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WO2025092669A1 - Anticorps anti-procalcitonine et son utilisation - Google Patents

Anticorps anti-procalcitonine et son utilisation Download PDF

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Publication number
WO2025092669A1
WO2025092669A1 PCT/CN2024/127819 CN2024127819W WO2025092669A1 WO 2025092669 A1 WO2025092669 A1 WO 2025092669A1 CN 2024127819 W CN2024127819 W CN 2024127819W WO 2025092669 A1 WO2025092669 A1 WO 2025092669A1
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WIPO (PCT)
Prior art keywords
antibody
seq
amino acid
acid sequence
procalcitonin
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PCT/CN2024/127819
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English (en)
Chinese (zh)
Inventor
钟冬梅
唐丽娜
梁碧
孟媛
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Fapon Biotech Inc
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Fapon Biotech Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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

Definitions

  • the present disclosure relates to the field of antibody technology, and in particular, to an anti-procalcitonin antibody and application thereof.
  • PCT procalcitonin
  • the gene encoding procalcitonin (PCT) is located on human chromosome 11.
  • the gene consists of 6 exons and 5 introns, with a total length of 7600bp, of which the coding region is 2800bp.
  • After gene transcription, it is translated into procalcitonin precursor in the rough endoplasmic reticulum of thyroid parafollicular cells, and then modified and processed by endogenous peptidases to generate the final product PCT of 116 amino acids.
  • PCT is mainly produced under the stimulation of bacterial toxins and inflammatory cytokines. In non-infectious inflammatory conditions, serum PCT generally does not increase. In the process of infectious inflammation, PCT is generated very quickly and increases within 2 to 6 hours in response to endotoxin stimulation.
  • PCT can be as high as 1000 times within 24 hours.
  • PCT has been widely recognized as a new infectious inflammation marker. It can not only identify bacterial infection at an early stage, but also is an early warning and diagnostic indicator for sepsis.
  • MODS multiple organ dysfunction syndrome
  • PCT is also produced ectopically and the level is abnormally elevated.
  • the concentration of PCT is usually positively correlated with the degree of infection, and the correlation is very high. Its increase or decrease directly reflects the trend of disease deterioration or improvement, and can provide a good prognostic indication. Therefore, the detection of PCT has important reference value in the differential diagnosis, prognosis judgment, efficacy observation and rational guidance of the use of antibiotics for infectious diseases, sepsis, MODS, etc.
  • PCT detection In the early days, PCT was mainly detected using gel chromatography and high performance liquid chromatography. These two methods are not only time-consuming, but also require high operation, are not easy to automate, and are expensive. In recent years, PCT detection has been mostly carried out using immunological methods with the advantages of strong specificity, high sensitivity, and easy operation, including double antibody sandwich immunochemistry, colloidal gold method, and radioimmunoassay, etc., which are all based on the specific reaction of antibodies and antigens, and use labeled substances to amplify and display the detected signals. An immunological detection method. The above-mentioned immunological detection methods all require antibodies against PCT. Therefore, technicians in this field have a strong demand for anti-PCT antibodies with good performance.
  • the present application provides an anti-procalcitonin antibody, which provides an important source of raw materials for the detection of procalcitonin and has good activity or affinity.
  • an anti-procalcitonin antibody comprising three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19. Three complementarity determining regions in the light chain variable region.
  • an anti-procalcitonin antibody comprising the following complementary determining regions:
  • HCDR1 which comprises or consists of the amino acid sequence shown in SEQ ID NO:1;
  • HCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO:2;
  • HCDR3 which comprises or consists of the amino acid sequence shown in SEQ ID NO:3;
  • LCDR1 which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;
  • LCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO:5;
  • LCDR3 which comprises or consists of the amino acid sequence shown in SEQ ID NO:6.
  • an anti-procalcitonin antibody comprising a heavy chain variable region and/or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:19.
  • an anti-procalcitonin antibody comprising a heavy chain and/or a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO:18; the amino acid sequence of the light chain being as shown in SEQ ID NO:20.
  • an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.
  • a reagent or a kit comprising the above-mentioned antibody or the above-mentioned antibody conjugate.
  • the seventh aspect of the present disclosure there is provided a use of the above-mentioned antibody, antibody conjugate, reagent or kit in detecting procalcitonin or diagnosing procalcitonin-related diseases, and preparing a product for detecting procalcitonin.
  • a method for diagnosing whether a subject suffers from a procalcitonin-related disease comprising: a) contacting the antibody according to any one of claims 1 to 5, or the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with a sample from the subject under conditions sufficient for a binding reaction to occur for a binding reaction; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.
  • a method for detecting procalcitonin comprising: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with procalcitonin in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.
  • the present disclosure also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody.
  • Figure 1 shows the results of reducing SDS-PAGE of Anti-PCT 4H5 Rmb1.
  • Figure 2 is a standard curve diagram of clinical sample correlation.
  • an embodiment of the present disclosure provides an anti-procalcitonin antibody, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:17 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO:19.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity.
  • Antigen-binding fragments include any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv, and generally have the same binding specificity as the antibody from which they are derived.
  • the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present disclosure, those skilled in the art can easily obtain the antigen-binding fragments of the above-mentioned antibodies.
  • Antigen binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.
  • CDR complementarity determining region
  • CDRs refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope.
  • CDRs refers to the highly variable region of the heavy and light chains of the antibody.
  • the heavy chain complementarity determining region is denoted by HCDR, which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is denoted by LCDR, which includes LCDR1, LCDR2 and LCDR3.
  • CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition.
  • Kabat definition refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983).
  • Chothia definition refers to Chothia et al., J Mol Biol 196: 901-917 (1987).
  • CDR definition methods that may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups.
  • Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to those in Table 1 also fall within the protection scope of the present disclosure.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.
  • the HCDR1, HCDR2, HCDR3, The amino acid sequences of LCDR1, LCDR2 or LCDR3 correspond to the Kabat numbering positions as follows:
  • the antibody comprises the following complementary determining regions:
  • HCDR1 which comprises or consists of the amino acid sequence shown in SEQ ID NO:1;
  • HCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO:2;
  • HCDR3 which comprises or consists of the amino acid sequence shown in SEQ ID NO:3;
  • LCDR1 which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;
  • LCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO:5;
  • LCDR3 which comprises or consists of the amino acid sequence shown in SEQ ID NO:6.
  • the present disclosure provides an anti-procalcitonin antibody, wherein the antibody comprises the following complementary determining regions:
  • HCDR1 which comprises or consists of the amino acid sequence shown in SEQ ID NO:1;
  • HCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO:2;
  • HCDR3 which comprises or consists of the amino acid sequence shown in SEQ ID NO:3;
  • LCDR1 which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;
  • LCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO:5;
  • LCDR3 which comprises or consists of the amino acid sequence shown in SEQ ID NO:6.
  • the HCDRs and LCDRs are defined by the Kabat system.
  • framework region or "FR” region includes heavy chain framework region and light chain framework region, and refers to the region other than CDR in the heavy chain variable region and light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.
  • the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.
  • the HFR1 comprises/is such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;
  • the HFR2 comprises/is such as SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;
  • the HFR3 comprises/is such as SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;
  • the HFR4 comprises/is such as SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto;
  • the LFR1 comprises/is such as SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;
  • the LFR2 comprises/is such as SEQ ID NO:12 or an amino acid sequence having at least 80% identity thereto;
  • the LFR3 comprises/is represented by SEQ ID NO:13 or an amino acid sequence having at least 80% identity thereto;
  • the LFR4 includes/is such as SEQ ID NO:14 or an amino acid sequence having at least 80% identity thereto.
  • the antibody binds procalcitonin with an affinity of KD ⁇ 3.32 x 10 -9 M.
  • the antibody binds procalcitonin with an affinity of KD ⁇ 10 " 8M, KD ⁇ 10 “9M , KD ⁇ 10” 10M , KD ⁇ 10 "11M or KD ⁇ 10 "12M .
  • the antibody binds procalcitonin with an affinity of KD ⁇ 8.82 ⁇ 10 ⁇ 11 M.
  • thermodynamic detection methods are commonly used, such as isothermal titration calorimetry (ITC); kinetic detection methods are commonly used, such as surface plasmon resonance (SPR) and biomembrane interferometry (BLI); dynamic equilibrium detection methods are commonly used, such as enzyme-linked immunosorbent assay (ELISA).
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • BLI biomembrane interferometry
  • ELISA enzyme-linked immunosorbent assay
  • KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example by using a kinetic assay such as Biosensor system of the system.
  • the antibody comprises a heavy chain variable region and/or a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19.
  • the embodiments of the present disclosure provide an anti-procalcitonin antibody, comprising a heavy chain variable region and/or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19.
  • the antibody described in the first aspect, the second aspect, and the third aspect further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.
  • the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and/or CH4 of IgM.
  • the IgG is selected from IgG1, IgG2, IgG3 or IgG4.
  • the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.
  • the species of origin of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.
  • the species origin of the constant region is mouse.
  • variable and constant region sequences refers to the IMGT division method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire.
  • variable regions divided by different methods may have some amino acid differences with the variable region C-terminus or constant region N-terminus divided by IMGT.
  • variable regions or constant regions divided by other methods known in the art are also within the protection scope of the present disclosure.
  • the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15
  • the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.
  • the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned constant region (SEQ ID NO: 15 or 16).
  • the antibody comprises any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.
  • the antibody comprises a heavy chain and/or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO:18, and the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • the present disclosure provides an anti-procalcitonin antibody, comprising a heavy chain and/or a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO:18, and the amino acid sequence of the light chain being as shown in SEQ ID NO:20.
  • the present disclosure provides an antibody conjugate, which comprises the above-mentioned antibody.
  • the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.
  • the antibody conjugate further comprises a label coupled to the antibody.
  • the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.
  • the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.
  • the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc.
  • fluorescein dyes and their derivatives for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs
  • rhodamine dyes and their derivatives for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B
  • Cy series dyes and their derivatives for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc.
  • Alexa series dyes and their derivatives for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.
  • the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
  • the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.
  • the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.
  • the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.
  • the colloidal metal is colloidal gold.
  • the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.
  • the solid support is selected from microspheres, plates and membranes.
  • the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.
  • the present disclosure provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.
  • the antibodies in some embodiments or examples of the present disclosure can effectively bind to procalcitonin. Therefore, the reagents or kits containing the procalcitonin antibodies can effectively detect procalcitonin qualitatively or quantitatively.
  • the reagents or kits provided by the present disclosure can be used, for example, for detection involving the use of the specific binding properties of procalcitonin and its antibodies, such as immunoblotting and immunoprecipitation.
  • the antibodies in some embodiments or examples of the present disclosure have higher binding activity or affinity to procalcitonin, and thus the reagents or kits containing the antibodies have higher detection sensitivity or specificity.
  • the present disclosure provides a method for detecting procalcitonin, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with procalcitonin in a sample to be detected to form an immune complex under conditions sufficient for an antibody/antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;
  • the immune complex further comprises a second antibody that binds to the antibody.
  • the immune complex further comprises a second antibody, which binds to procalcitonin.
  • the present disclosure provides use of the above-mentioned anti-procalcitonin antibody, antibody conjugate or the above-mentioned reagent or kit in detecting procalcitonin or diagnosing procalcitonin-related diseases, and preparing products for detecting procalcitonin.
  • the disease includes infectious diseases, sepsis, MODS, etc.
  • the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody.
  • the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.
  • the present disclosure provides a cell containing the above-mentioned vector.
  • the present disclosure provides a method for preparing an anti-procalcitonin antibody, comprising: culturing the cells as described above.
  • the present disclosure provides use of the above-mentioned antibody, antibody conjugate, or the above-mentioned reagent or kit in detecting procalcitonin or indicating a procalcitonin-related disease.
  • the present disclosure provides a method for indicating a procalcitonin-related disease in a subject, comprising:
  • the immune complex further comprises a second antibody that binds to the antibody.
  • the immune complex further comprises a second antibody, which binds to procalcitonin.
  • the procalcitonin-related disease of the thirteenth aspect or the fourteenth aspect is selected from sepsis.
  • the restriction endonucleases and Prime Star DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting Anti-PCT 4H5 monoclonal antibody was prepared in this laboratory and revived for use.
  • mRNA was extracted from the hybridoma cell line secreting Anti-PCT 4H5 monoclonal antibody, and the DNA product was obtained by RT-PCR.
  • the product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5 ⁇ competent cells. After the colonies grew, the heavy chain and light chain gene clones were taken and 4 clones each were sent to a gene sequencing company for sequencing.
  • the gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct.
  • the light chain variable region gene sequence was 324 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the heavy chain primer pair, the heavy chain variable region gene sequence was 348 bp, belonging to the VH1 gene family, preceded by a 57 bp leader peptide sequence.
  • pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector, which has been introduced with HindIII, BamHI, EcoRI and other multiple cloning restriction sites were added, and the expression vector was named pcDNA3.4A, hereinafter referred to as 3.4A expression vector; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and a 0.70kb light chain gene fragment and a 1.48kb heavy chain gene fragment were amplified by PCR amplification method.
  • the heavy chain and light chain gene fragments were double-digested with HindIII/EcoRI, and the 3.4A vector was double-digested with HindIII/EcoRI. After the fragments and vectors were purified and recovered, the heavy chain gene and the light chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the heavy chain and light chain, respectively.
  • HEK293 cells in advance, subculture to 200mL system, make the cell density reach 3-5 ⁇ 10 6 cells/ml, reach the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, adjust the cell density to 2.9 ⁇ 10 6 cells/ml, wash the cells, re-dissolve with culture medium, and use as cell diluent.
  • transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15min; slowly add the mixture to cell diluent within 1min, mix well and count, record and observe the viability of cells after transfection, and place them in a 35°C constant temperature incubator for culture, speed 120rmp, CO 2 content 8%, centrifuge and collect samples after 13 days.
  • the centrifuged supernatant was affinity purified using proteinA affinity chromatography column. Take 6ug layer of purified antibody for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands were shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).
  • the obtained antibody was named Anti-PCT 4H5Rmb1.
  • the heavy chain amino acid sequence of antibody Anti-PCT 4H5Rmb1 is shown in SEQ ID NO:18, and the light chain amino acid sequence is shown in SEQ ID NO:20.
  • the purified antibody was diluted in advance, and the PCT recombinant antigen (from Feipeng Bio) was diluted in a gradient manner; the binding and dissociation curves of the antigen and antibody were tested on the Biacore 8K+ device using the CM5 chip that had been pre-coupled with goat anti-mouse IgG.
  • the instrument automatically fitted the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., affinity constant; ka represents the binding rate; kd represents the dissociation rate)
  • the coating solution (main component NaHCO 3 ) was used to dilute PCT recombinant antigen (from Feipeng Bio) to 1ug/ml, 100uL per well, and incubated at 4°C overnight. The next day, the cells were washed twice with washing solution (main component Na 2 HPO 4 +NaCl), and patted dry.
  • the blocking solution (20% BSA + 80% PBS) was added to each well.
  • Solution A main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide
  • Solution B main ingredients: citric acid + EDTA ⁇ 2Na + TMB + concentrated HCl
  • Stop solution EDTA ⁇ 2Na + concentrated H 2 SO 4
  • the above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested.
  • the results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable.
  • Table 4 below shows the OD results of the enzyme immunoassay activity test of the antibody Anti-PCT 4H5Rmb1 for 21 days.
  • 3mg/ml of the antibody Anti-PCT 4H5Rmb1 was replaced with PBS (100mM PB, 50mM NaCl, pH8.0) using a Zeba desalting column (10K MWCO).
  • Acridinium ester was prepared into a 4mM solution using DMSO. 10eq of acridinium ester was added to the Anti-PCT 4H5Rmb1 antibody solution and reacted at 25°C for 2 hours. Excess reagents were removed by desalting, and the acridinium ester-modified antibody was used as the acridinium ester working solution and placed at 4°C Save for later use.
  • the test was performed on the Yingkai shine i2910 fully automatic chemiluminescence immunoassay analyzer using the double antibody sandwich method, that is, 10 ⁇ L of sample (Roche fixed value clinical sample), 50 ⁇ L of magnetic particle working solution and 50 ⁇ L of acridinium ester working solution were added to the instrument in sequence, mixed and incubated for 10 minutes, and the reaction mixture was rinsed after incubation, and the pre-excitation solution and excitation solution were added to detect the relative luminescence intensity (RLU).
  • RLU relative luminescence intensity
  • the Roche assigned samples were tested on the chemiluminescence platform, and the test results are shown in Table 5.
  • the standard curve of clinical sample correlation is shown in Figure 2. The results showed that on the chemiluminescence platform, the correlation R 2 of the clinical samples assigned by the Roche reagent with the magnetic microparticle chemiluminescence reagent composed of the antibody Anti-PCT 4H5Rmb1 was greater than 0.98.
  • the anti-procalcitonin antibody provided by the present disclosure can specifically recognize and bind to procalcitonin, and has high detection sensitivity and specificity. Therefore, the anti-procalcitonin antibody provided by the present disclosure has excellent practical performance and broad market application prospects.

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Abstract

L'invention concerne un anticorps anti-procalcitonine et son utilisation, se rapportant au domaine des anticorps. L'anticorps anti-procalcitonine comprend une région déterminant la complémentarité de chaîne lourde et une région déterminant la complémentarité de chaîne légère. L'anticorps constitue une source de matière première importante pour la détection de procalcitonine, et présente une affinité ou une activité satisfaisante.
PCT/CN2024/127819 2023-10-31 2024-10-28 Anticorps anti-procalcitonine et son utilisation Pending WO2025092669A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010007022A1 (en) * 1999-12-22 2001-07-05 Dade Behring Marburg Gmbh. Anti-procalcitonin antibodies and the preparation and use thereof
CN102702324A (zh) * 2011-09-15 2012-10-03 重庆业为基生物科技有限公司 人降钙素原b细胞表位肽段及其单克隆抗体的应用
CN104745534A (zh) * 2015-03-02 2015-07-01 南方医科大学 一种降钙素原单克隆抗体杂交瘤2h4及单克隆抗体
US20190031744A1 (en) * 2015-07-09 2019-01-31 Nanjing Norman Biological Technology Co., Ltd. Monoclonal antibody of human-derived procalcitonin, and preparation method and application thereof
CN114181308A (zh) * 2021-12-28 2022-03-15 广州市雷德生物科技有限公司 一种降钙素原抗体及其应用
CN115873112A (zh) * 2022-12-23 2023-03-31 杭州华葵金配生物科技有限公司 一种降钙素原抗体及其应用
CN116284382A (zh) * 2022-12-23 2023-06-23 杭州华葵金配生物科技有限公司 抗降钙素原抗体及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010007022A1 (en) * 1999-12-22 2001-07-05 Dade Behring Marburg Gmbh. Anti-procalcitonin antibodies and the preparation and use thereof
CN102702324A (zh) * 2011-09-15 2012-10-03 重庆业为基生物科技有限公司 人降钙素原b细胞表位肽段及其单克隆抗体的应用
CN104745534A (zh) * 2015-03-02 2015-07-01 南方医科大学 一种降钙素原单克隆抗体杂交瘤2h4及单克隆抗体
US20190031744A1 (en) * 2015-07-09 2019-01-31 Nanjing Norman Biological Technology Co., Ltd. Monoclonal antibody of human-derived procalcitonin, and preparation method and application thereof
CN114181308A (zh) * 2021-12-28 2022-03-15 广州市雷德生物科技有限公司 一种降钙素原抗体及其应用
CN115873112A (zh) * 2022-12-23 2023-03-31 杭州华葵金配生物科技有限公司 一种降钙素原抗体及其应用
CN116284382A (zh) * 2022-12-23 2023-06-23 杭州华葵金配生物科技有限公司 抗降钙素原抗体及其应用

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