Anti-procalcitonin antibody and use thereof

Abstract

An anti-procalcitonin antibody and a use thereof, relating to the field of antibodies. The anti-procalcitonin antibody comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for detection of procalcitonin, and has good affinity or activity.

Description

Anti-procalcitonin antibody and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number 202311439580.X, filed with the Chinese Patent Office on October 31, 2023, entitled “An Anti-Procalcitonin Antibody and Its Application,” the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present disclosure relates to the field of antibody technology, and in particular to an anti-procalcitonin antibody and applications thereof. Background Art

[0004] The gene encoding procalcitonin (PCT) is located on human chromosome 11. It consists of six exons and five introns, with a total length of 7600 base pairs (bp), of which the coding region is 2800 bp. Following gene transcription, it is translated into a procalcitonin precursor within the rough endoplasmic reticulum of thyroid parafollicular cells. This precursor is then modified and processed by endogenous peptidases to produce the 116-amino acid final product, PCT. PCT is primarily produced in response to bacterial toxins and inflammatory cytokines. While serum PCT levels generally remain low in non-infectious inflammatory states, PCT production is very rapid during infectious inflammation, rising within 2 to 6 hours in response to endotoxin stimulation. In patients with severe systemic infection, PCT levels can rise as much as 1000-fold within 24 hours. PCT has become widely recognized as a new marker of infectious inflammation, not only for early identification of bacterial infection but also as a warning and diagnostic indicator for sepsis. Furthermore, numerous studies have found that PCT is also ectopically produced and abnormally elevated in cases of severe bacterial, fungal, and parasitic infections, as well as in multiple organ dysfunction syndrome (MODS). PCT concentration is generally positively correlated with the severity of infection, with a high correlation. Its rise or fall directly reflects the trend of disease worsening or improvement, providing a good prognostic indicator. Therefore, PCT testing plays an important role in the differential diagnosis, prognosis, efficacy observation, and rational guidance of antimicrobial use in infectious diseases, sepsis, and MODS.

[0005] 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 requirements, are difficult to automate, and are expensive. In recent years, PCT detection has mostly been 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, those skilled in the art have a strong demand for anti-PCT antibodies with good performance.

[0006] Summary of the Invention

[0007] 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.

[0008] To achieve the above objectives, according to one aspect of the present disclosure, an anti-procalcitonin antibody is provided, which comprises three complementarity determining regions of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having the amino acid sequence shown in SEQ ID NO: 19.

[0009] In order to achieve the above object, according to a second aspect of the present disclosure, an anti-procalcitonin antibody is provided, wherein the antibody comprises the following complementary determining regions:

[0010] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0012] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0013] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0014] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0015] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0016] To achieve the above objectives, according to the third aspect of the present disclosure, an anti-procalcitonin antibody is provided, 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 shown in SEQ ID NO: 17; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19.

[0017] To achieve the above objectives, according to the fourth aspect of the present disclosure, an anti-procalcitonin antibody is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0018] In order to achieve the above object, according to a fifth aspect of the present disclosure, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0019] In order to achieve the above-mentioned object, according to the sixth aspect of the present disclosure, a reagent or a kit is provided, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0020] To achieve the above objectives, according to 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, or preparing a product for detecting procalcitonin.

[0021] To achieve the above objectives, according to an eighth aspect of the present disclosure, a method for diagnosing whether a subject suffers from a procalcitonin-related disease is provided, 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 to perform 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.

[0022] To achieve the above objectives, according to a ninth aspect of the present disclosure, a method for detecting procalcitonin is provided, comprising: a) contacting the antibody of any one of claims 1 to 5, the antibody conjugate of claim 6, or the reagent or kit of 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.

[0023] To achieve the above objectives, the present disclosure also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without any creative work.

[0025] FIG1 shows the results of reducing SDS-PAGE of Anti-PCT 4H5 Rmb1.

[0026] Figure 2 is a standard curve diagram of the correlation of clinical samples. DETAILED DESCRIPTION

[0027] In a first aspect, the embodiments of the present disclosure provide an anti-procalcitonin antibody, comprising three complementarity determining regions of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having the amino acid sequence shown in SEQ ID NO: 19.

[0028] In the present disclosure, the term "antibody" is used in the broadest sense and 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. It will be readily understood by those skilled in the art based on the contents of the present disclosure that 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, it is easy for those skilled in the art to obtain the antigen-binding fragments of the above-mentioned antibodies.

[0029] Antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0030] In the present disclosure, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more, or even all, of the primary amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibodies.

[0031] In the present disclosure, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0032] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200,255,854,554, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but may still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened 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 literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0033] Table 1: CDR Definition 1 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers in the heavy chain represented by "H+number" and amino acid numbers in the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software. 3If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B. 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34. 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0034] According to an embodiment of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0035] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0036] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0037] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0038] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0039] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0040] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0041] According to an embodiment of the present disclosure, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0042] According to an embodiment of the present disclosure, the antibody comprises the following complementarity determining regions:

[0043] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0044] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0045] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0046] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0047] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0048] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0049] In a second aspect, the present disclosure provides an anti-procalcitonin antibody, wherein the antibody comprises the following complementary determining regions:

[0050] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0051] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0052] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0053] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0054] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0055] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0056] According to an embodiment of the present disclosure, the HCDRs and LCDRs are defined by the Kabat system.

[0057] In the present disclosure, "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; 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.

[0058] In the present disclosure, the heavy chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0059] In an alternative embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0060] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0061] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0062] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0063] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0064] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0065] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0066] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0067] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0068] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-procalcitonin antibodies provided herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0069] In an alternative embodiment, the antibody has a KD < 3.32×10 -9 M binds procalcitonin with high affinity.

[0070] In an alternative embodiment, the antibody has a KD ≤ 10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 M binds procalcitonin with high affinity.

[0071] In an alternative embodiment, the antibody has a KD ≤ 8.82×10 -11 M binds procalcitonin with high affinity.

[0072] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0073] In alternative embodiments, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example, by using a kinetic assay such as System of biosensor systems.

[0074] In an optional embodiment, 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.

[0075] In a third aspect, the presently disclosed embodiments 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.

[0076] In an optional embodiment, the antibody described in the first, second, and third aspects above further comprises a constant region.

[0077] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0078] In an optional embodiment, 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.

[0079] In an alternative embodiment, 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.

[0080] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0081] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0082] In an alternative embodiment, the species origin of the constant region is cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0083] In an alternative embodiment, the species origin of the constant region is mouse.

[0084] In this article, the division of 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. the international ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Variable regions divided by different methods may differ from 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 scope of protection of this disclosure.

[0085] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0086] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0087] In an alternative embodiment, the antibody comprises any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.

[0088] In an optional embodiment, 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.

[0089] In a fourth aspect, the present disclosure provides an anti-procalcitonin antibody, comprising a heavy chain and / or a light chain, wherein 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.

[0090] In a fifth aspect, the present disclosure provides an antibody conjugate comprising the above-mentioned antibody.

[0091] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0092] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody.

[0093] In an optional embodiment, 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.

[0094] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0095] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present disclosure.

[0096] In an optional embodiment, the fluorescent dyes include but are 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. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), 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) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0097] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0098] In an optional embodiment, 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.

[0099] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0100] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0101] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0102] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0103] In an optional embodiment, the colloidal metal is colloidal gold.

[0104] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0105] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0106] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0107] In a sixth aspect, the present disclosure provides a reagent or a kit, which comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0108] As previously mentioned, the antibodies in some embodiments or examples of the present disclosure are capable of effectively binding to procalcitonin. Therefore, reagents or kits comprising the procalcitonin antibodies are capable of effectively performing qualitative or quantitative detection of procalcitonin. The reagents or kits provided herein can be used, for example, in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of procalcitonin and its antibodies. As previously mentioned, the antibodies in some embodiments or examples of the present disclosure have higher binding activity or affinity for procalcitonin. Therefore, reagents or kits comprising the antibodies have higher detection sensitivity or specificity.

[0109] In a seventh aspect, 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 tested 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;

[0110] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0111] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to procalcitonin.

[0112] In an eighth aspect, the present disclosure provides uses of the aforementioned anti-procalcitonin antibodies, antibody conjugates, or the aforementioned reagents or kits in detecting procalcitonin or diagnosing procalcitonin-related diseases, or in preparing products for detecting procalcitonin.

[0113] In an optional embodiment, the disease includes infectious diseases, sepsis, MODS, etc.

[0114] In a ninth aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody.

[0115] In a tenth aspect, the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

[0116] In an eleventh aspect, the present disclosure provides cells containing the above-mentioned vector.

[0117] In a twelfth aspect, the present disclosure provides a method for preparing an anti-procalcitonin antibody, comprising: culturing the cells as described above.

[0118] In a thirteenth aspect, 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.

[0119] In a fourteenth aspect, the present disclosure provides a method for indicating a procalcitonin-related disease in a subject, comprising:

[0120] a) contacting the above-mentioned antibody, antibody conjugate, or the above-mentioned reagent or kit with procalcitonin in a sample from a subject under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and

[0121] b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence or status of a procalcitonin-related disease in the subject.

[0122] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0123] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to procalcitonin.

[0124] In an optional embodiment, the procalcitonin-related disease of the thirteenth aspect or the fourteenth aspect is selected from sepsis.

[0125] Based on the amino acid sequence of the anti-procalcitonin antibody disclosed in the present disclosure, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-procalcitonin antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-procalcitonin antibody of the present disclosure, it falls within the scope of protection of the present disclosure.

[0126] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0128] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0129] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0130] Example 1 Preparation of Anti-PCT 4H5 Monoclonal Antibody

[0131] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the anti-PCT 4H5 monoclonal antibody was generated in our laboratory and revived for future use.

[0132] (1) Antibody gene preparation

[0133] mRNA was extracted from a hybridoma cell line secreting the Anti-PCT 4H5 monoclonal antibody. DNA products were obtained by RT-PCR. This product was PCR-polymerized with rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were then transformed into DH5α competent cells. After colonies grew, the heavy and light chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0134] (2) Sequence analysis of the variable region gene of the Anti-PCT 4H5 antibody

[0135] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the light chain, 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, and preceded by a 57 bp leader peptide sequence.

[0136] (3) Construction of recombinant antibody expression plasmid

[0137] pcDNA TM 3.4 The vector was a recombinant antibody eukaryotic expression vector constructed using pMD-18T. Multiple cloning restriction sites, including HindIII, BamHI, and EcoRI, had been introduced into the vector, which was named pcDNA3.4A expression vector and will be referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.70 kb light chain gene fragment and a 1.48 kb heavy chain gene fragment.

[0138] The heavy chain and light chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the heavy chain gene and light chain gene were connected to the 3.4A expression vector to obtain recombinant expression plasmids for the heavy chain and light chain, respectively.

[0139] 2. Recombinant Antibody Production

[0140] Resuscitate HEK293 cells in advance and subculture them into 200 mL system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 μg of culture medium and re-dissolved. This was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and allowed to stand at room temperature for 15 minutes. The mixture was slowly added to the cell diluent over 1 minute, mixed, and sampled. The viability of the cells after transfection was recorded and observed. The cells were cultured in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electrophoresis pattern is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with an Mr of 50 KD (heavy chain) and the other with an Mr of 28 KD (light chain).

[0141] The obtained antibody was named Anti-PCT 4H5Rmb1. The heavy chain amino acid sequence of the 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.

[0142] Example 2 Performance testing of antibodies

[0143] 1. Affinity Analysis

[0144] Purified antibodies were diluted in advance, and PCT recombinant antigen (from Feipeng Bio) was serially diluted. Binding and dissociation curves were measured on a Biacore 8K+ instrument using a CM5 chip pre-coupled with goat anti-mouse IgG. The instrument automatically fitted the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, also known as the affinity constant; ka represents the association rate; and kd represents the dissociation rate.)

[0145] Table 2 Affinity data

[0146] 2. Activity Identification

[0147] Dilute PCT recombinant antigen (from Feipeng Bio) to 1ug / ml in coating solution (main component NaHCO3), 100uL per well, 4℃ overnight; the next day, wash twice with washing solution (main component Na2HPO4+NaCl), pat dry; add blocking solution (20% BSA+80% PBS), 120uL per well, 37℃, 1h, pat dry; add diluted purified antibody and control antibody, 100uL / well, 37℃, 30min; wash 5 times with washing solution, pat dry; add goat anti-mouse IgG-HRP, 100uL per well, 37℃, 30min; wash 5 times with washing solution, pat dry; add color development solution A (50uL / well), add color development solution B (50uL / well), 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on microplate reader.

[0148] Note: 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 H2SO4)

[0149] Table 3: Activity data

[0150] 3. Stability assessment

[0151] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under all three test conditions for 21 days, and the activity did not decrease with increasing test temperature, indicating that the above-mentioned antibody is stable. Table 4 below shows the OD results of the enzyme immunoassay activity assay of the antibody Anti-PCT 4H5Rmb1 after 21 days of testing.

[0152] Table 4: Stability data

[0153] 4. Antibody Performance in Magnetic Microparticle Chemiluminescence Detection

[0154] 4.1 Antibody Coating Process

[0155] Wash 10mg / mL carboxyl magnetic beads three times with MES buffer. Resuspend the beads in MES buffer and add EDC to a final concentration of 1mg / mL. Mix on a shaker at 25°C and incubate for 30 minutes. Resuspend the beads in MES buffer and add Anti-PCT-A (from Feipeng Bio) to a final concentration of 0.2mg / mL. This solution serves as the magnetic particle working solution. Mix on a shaker at 25°C and incubate for 120 minutes. Store in Tris buffer at 2-8°C.

[0156] 4.2 Antibody labeling process

[0157] 3 mg / ml of anti-PCT 4H5Rmb1 antibody was exchanged into PBS (100 mM PB, 50 mM NaCl, pH 8.0) using a Zeba desalting column (10K MWCO). A 4 mM acridinium ester solution was prepared in DMSO. 10 eq of acridinium ester was added to the anti-PCT 4H5Rmb1 antibody solution and reacted at 25°C for 2 hours. Excess reagent was removed by desalting, and the acridinium ester-modified antibody was used as the acridinium ester working solution and stored at 4°C until use.

[0158] 4.3 Detection process

[0159] The test was performed on the Yingkai shine i2910 fully automatic chemiluminescence immunoassay using the double antibody sandwich method. That is, 10 μL of sample (Roche-certified clinical sample), 50 μL of magnetic particle working solution, and 50 μL of acridinium ester working solution were sequentially added to the instrument, mixed, and incubated for 10 minutes. After incubation, the reaction mixture was rinsed, and pre-excitation solution and excitation solution were added to detect the relative luminescence intensity (RLU).

[0160] 4.4 Test results

[0161] The Roche assigned value samples were tested on the chemiluminescence platform. The test results are shown in Table 5, and the standard curve of clinical sample correlation is shown in Figure 2. The results showed that the magnetic particle chemiluminescence reagent composed of the antibody Anti-PCT 4H5Rmb1 on the chemiluminescence platform was correlated with the clinical sample assigned value R 2 >0.98.

[0162] Table 5: Clinical sample test results

[0163] Some of the amino acid sequences involved in this application are shown in Table 6:

[0164] Table 6: Amino acid sequence listing

[0165] The foregoing description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure. Industrial Applicability

[0166] The anti-procalcitonin antibodies provided herein can specifically recognize and bind to procalcitonin, have high detection sensitivity and specificity, and can be used for the detection of procalcitonin. Therefore, the anti-procalcitonin antibodies provided herein have excellent practical performance and broad market application prospects.

Claims

1. An anti-procalcitonin antibody, comprising 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.

2. The antibody according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-procalcitonin antibody, characterized in that The antibody comprises the following complementarity 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; and LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; Optionally, the antibody further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, the HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody has a KD of less than 3.32×10 -9 M binds procalcitonin with high affinity.

4. An anti-procalcitonin antibody comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, 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; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

5. An anti-procalcitonin antibody comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 18; the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of 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 in detecting procalcitonin or diagnosing procalcitonin-related diseases, or in preparing a product for detecting procalcitonin.

9. The use according to claim 8, characterized in that The procalcitonin-related diseases include infectious diseases, sepsis, and MODS.

10. 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 to perform a binding reaction; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to procalcitonin.

11. A method for detecting procalcitonin, characterized in that: The use comprises: 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 tested under conditions sufficient for an antibody / antigen binding reaction 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; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to procalcitonin.

12. A nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment according to any one of claims 1 to 5.

13. A vector, wherein The vector comprises the nucleic acid of claim 12.

14. A cell, characterized in that The cell comprises the nucleic acid of claim 12 or the vector of claim 13.

15. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 5, characterized in that: The method comprises culturing the cell of claim 14.

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