Anti-cortisol antibody, reagent and kit for detecting cortisol

Abstract

The present invention discloses an antibody against cortisol or a reagent and kit for detecting cortisol, which relates to the field of antibodies. The anti-cortisol antibody disclosed by the present invention includes a heavy chain complementary determining region and a light chain complementary determining region. This antibody provides an important source of raw materials for the detection of cortisol and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an anti-cortisol antibody, a reagent for detecting cortisol, and a kit. Background Art

[0002] Cortisol, also known as hydrocortisone, hydrocortin or compound F. The regulation pathway of human cortisol secretion is the hypothalamic-pituitary-adrenal axis. When the human brain senses stress, the pituitary gland secretes adrenocorticotropic hormone (ACTH), which promotes the hyperplasia of adrenal cortical tissue and the production and secretion of corticosteroids. The secretion of cortisol in the human body is regular. The level of cortisol in a day rises sharply within 30 minutes after waking up in the morning, reaches the highest value about 30-45 minutes after waking up, and then gradually decreases throughout the day until it reaches the lowest value before going to bed.

[0003] Under normal circumstances, cortisol can reduce sodium loss, briefly improve memory, and help remove toxins from the liver. In a state of stress, cortisol helps maintain blood pressure stability and control excessive inflammation. However, excessive cortisol secretion has negative effects on the human body. Especially when a person repeatedly bears heavy pressure, has a tense life rhythm, diets for a long time, or lacks sleep for a long time, resulting in long-term excessive cortisol secretion. At this time, cortisol can cause elevated blood sugar, increased appetite, weight gain, decreased libido, and extreme fatigue. In addition, abnormal cortisol secretion can also cause diseases in the human body. Cushing's syndrome is a typical disease caused by excessive cortisol secretion, and its manifestations include rapid weight gain, excessive sweating, easy bruising, and psychological disorders. In addition, generalized anxiety disorder (GAD) is also closely related to abnormal cortisol secretion in the human body. The lifetime prevalence of this disease in adults is estimated to be 4.1% - 6.6%, and female patients are twice as many as male patients. It not only seriously affects the quality of life, mental health, and social function of patients, but also coexists with other diseases, increasing the risk of suicide. By detecting the content of cortisol, it is helpful to analyze and judge the health status of the human body, including: diagnosing the functional status of the adrenal gland, indirectly observing the functional status of the pituitary gland, Cushing's syndrome, cardiovascular diseases, stress, and mental diseases, etc. Thus, timely and effective detection of cortisol in the human body is of great significance for us to understand our own physical condition at that time.

[0004] Cortisol determination mostly uses immunoassay or LC-MS method. Although the LC-MS method is accurate and sensitive, it requires expensive instruments, so it is difficult to popularize in general laboratories. The immunoassay has the characteristics of high sensitivity, low detection cost, high feasibility, etc., and is suitable for wide promotion and application. Immunological detection methods all require antibodies against cortisol. Therefore, there is a strong demand in this field for antibodies that can effectively bind to cortisol and detect it. Summary of the Invention

[0005] The present application provides an antibody or an antigen-binding fragment thereof, which provides an important raw material source for the detection of cortisol and has good activity or affinity.

[0006] To achieve the above object, according to one aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises three complementary determining regions of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and three complementary determining regions of any one of the light chain variable regions having the amino acid sequences shown in SEQ ID NO: 18, 19, and 20.

[0007] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment comprises the following complementary determining regions:

[0008] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0009] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0010] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0011] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0012] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same;

[0013] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0014] To achieve the above object, according to a third aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, 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; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 18, 19, and 20.

[0015] To achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 21; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 22, 23, and 24.

[0016] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, which comprises the above-mentioned antibody or its antigen-binding fragment.

[0017] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which comprises the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0018] To achieve the above object, according to the seventh aspect of the present invention, there is provided a method for detecting cortisol, comprising: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, or reagent or kit with cortisol in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0019] To achieve the above object, according to the eighth aspect of the present invention, there is provided a nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment.

[0020] To achieve the above object, according to the ninth aspect of the present invention, there is provided a vector comprising the above-mentioned nucleic acid.

[0021] To achieve the above object, according to the tenth aspect of the present invention, there are provided cells comprising the above-mentioned nucleic acid, vector, or expressing the above-mentioned antibody or its antigen-binding fragment.

[0022] To achieve the above object, according to the eleventh aspect of the present invention, there is provided a method for preparing the above-mentioned antibody or its antigen-binding fragment, which method comprises culturing the above-mentioned cells.

[0023] To achieve the above object, according to the twelfth aspect of the present invention, there is provided the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in detecting or preparing a product for detecting cortisol. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 Results of reducing SDS-PAGE for Anti-CORT 20D9 Rmb1 to Rmb3. DETAILED DESCRIPTION OF THE INVENTION

[0026] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, 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 any one of the amino acid sequences shown in SEQ ID NO: 18, 19, and 20.

[0027] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the same heavy-chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the same light-chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect.

[0028] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region shown in SEQ ID NO: 17; LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the light-chain variable region shown in SEQ ID NO: 18.

[0029] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0030] In the present invention, the terms "complementarity-determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0031] In the present invention, the heavy-chain complementarity-determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light-chain complementarity-determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3.

[0032] The methods for defining CDRs are well-known in the art, and the methods for defining CDRs include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "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). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other methods for defining CDRs that may not strictly follow one of the above-mentioned schemes, but will still overlap with at least a part of the CDR region defined by Kabat, although they may be shortened or extended according to the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of a given antibody, those 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 scope of protection of the present disclosure.

[0033] Table 1: CDR Definitions 1

[0034]

[0035]

[0036] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below), and the amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly correspond this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0037] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0038] 3If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0039] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0040] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0041] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0042] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0043] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0044] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0045] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0046] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0047] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0048] In some alternative embodiments of the present invention, the Kabat, Chothia, AbM, or IMGT system-defined amino acid sequence positions of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 are as follows:

[0049] CDR Kabat AbM IMGT Chothia HCDR1 H31 - H35 H26 - H35 H26 - H33 H26 - H32 HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0050] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising the following complementarity-determining regions:

[0051] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same;

[0052] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same;

[0053] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same;

[0054] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same;

[0055] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same;

[0056] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or consists of the same.

[0057] In an alternative embodiment, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0058] In the present invention, the "framework region" or "FR" region includes the heavy-chain framework region and the light-chain framework region, and refers to the regions other than the CDRs in the heavy-chain variable region and the light-chain variable region of the antibody; wherein, the heavy-chain framework region can be further divided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light-chain framework region can be further divided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0059] In the present invention, the heavy chain variable region is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0060] In some alternative embodiments of the present invention, the antibody or its antigen-binding fragment described in the first aspect or the second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0061] The HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity therewith;

[0062] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity therewith;

[0063] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity therewith;

[0064] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity therewith;

[0065] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity therewith;

[0066] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity therewith;

[0067] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity therewith;

[0068] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity therewith.

[0069] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the antibody or its antigen-binding fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12, 13, or 14) above.

[0070] In some alternative embodiments of the present invention, the LFR2 comprises / is as shown in SEQ ID NO:25.

[0071] In some alternative embodiments of the present invention, the LFR3 comprises / is as shown in SEQ ID NO:26.

[0072] In some alternative embodiments of the present invention, the antibody or its antigen-binding fragment binds cortisol with an affinity of KD < 4.31×10 -9 M.

[0073] In some alternative embodiments of the present invention, the antibody or its antigen-binding fragment binds cortisol with an affinity of KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M, KD ≤ 10 -12 M, KD ≤ 10 -13 M.

[0074] In some alternative embodiments of the present invention, the antibody or its antigen-binding fragment binds cortisol with an affinity of KD ≤ 9.90×10 -13 M.

[0075] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0076] In some alternative embodiments of the present invention, the determination of KD is carried out by referring to the method in the following examples.

[0077] In a third aspect, an embodiment of the present invention provides an antibody or its antigen-binding fragment, 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; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO:18, 19, and 20.

[0078] In some alternative embodiments of the present invention, the heavy chain variable region and the light chain variable region described in the first aspect or the third aspect above are selected from any one of the following combinations:

[0079] Combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:17 SEQ ID NO:18 2 SEQ ID NO:17 SEQ ID NO:19 3 SEQ ID NO:17 SEQ ID NO:20 .

[0080] In some alternative embodiments of the present invention, the antibody or its antigen-binding fragment described in the above first aspect, second aspect or third aspect further comprises a constant region.

[0081] In some alternative embodiments of the present invention, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0082] In some alternative embodiments of the present invention, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE or IgD or a combination of segments of multiple heavy chain constant regions.

[0083] In some alternative embodiments of the present invention, the heavy chain constant region comprises the CH1 region of IgG, the hinge region of IgG, the CH2 region of IgM, the CH3 region of IgM and / or the CH4 region of IgM.

[0084] In some alternative embodiments of the present invention, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0085] In some alternative embodiments of the present invention, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0086] In some alternative embodiments of the present invention, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, murine, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, gamecock or human.

[0087] In some alternative embodiments of the present invention, the species origin of the constant region is murine.

[0088] In some alternative embodiments of the present invention, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0089] It should be noted that in other embodiments, the constant region sequence may 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 constant region (SEQ ID NO:15 or 16).

[0090] In some alternative embodiments of the present invention, the antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0091] The antigen-binding fragments of the above-mentioned antibodies generally have the same binding specificity as the antibodies from which they are derived. It is readily understood by those skilled in the art from the content described in the present invention that the antigen-binding fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the intact antibody in the present invention, those skilled in the art can easily obtain the above-mentioned antigen-binding fragments.

[0092] The antigen-binding fragments of the above-mentioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0093] In a fourth aspect, the present invention provides an antibody or its antigen-binding fragment, including a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 21; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 22, 23, 24.

[0094] In some alternative embodiments of the present invention, the antibody or its antigen-binding fragment described in the first, second, third, or fourth aspect above includes a heavy chain and a light chain in any of the following combinations:

[0095] Combination Heavy chain Light chain 1 SEQ ID NO:21 SEQ ID NO:22 2 SEQ ID NO:21 SEQ ID NO:23 3 SEQ ID NO:21 SEQ ID NO:24

[0096] In a fifth aspect, the present invention provides an antibody conjugate, which includes the above-mentioned antibody or its antigen-binding fragment.

[0097] In some alternative embodiments of the present invention, the above-mentioned antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

[0098] In some alternative embodiments of the present invention, the antibody conjugate further includes a label conjugated to the antibody or its antigen-binding fragment.

[0099] In some alternative embodiments of the present invention, the above-mentioned label refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and through which qualitative or quantitative detection of the corresponding target can be achieved.

[0100] In some alternative embodiments of the present invention, the label includes, but is not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0101] In the actual use process, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. No matter which marker is used, it falls within the protection scope of the present invention.

[0102] In some alternative embodiments of the present invention, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0103] In some alternative embodiments of the present invention, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0104] In some alternative embodiments of the present invention, the radioisotopes 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.

[0105] In some alternative embodiments of the present invention, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosalic acid and its derivatives, and peroxyoxalate and its derivatives.

[0106] In some alternative embodiments of the present invention, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0107] In some alternative embodiments of the present invention, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0108] In some alternative embodiments of the present invention, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0109] In some alternative embodiments of the present invention, the colloidal metal is colloidal gold.

[0110] In some alternative embodiments of the present invention, the above-mentioned antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0111] In some alternative embodiments of the present invention, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0112] In some alternative embodiments of the present invention, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0113] In a sixth aspect, the present invention provides a reagent or kit, which includes the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0114] As described above, the antibody or its antigen-binding fragment in some embodiments or examples of the present invention can effectively bind to cortisol. Therefore, a reagent or kit containing the cortisol antibody or its antigen-binding fragment can effectively perform qualitative or quantitative detection of cortisol. By applying the reagent or kit provided by the present invention, for example, it can be used in detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of cortisol and its antibody. As described above, the antibody or its antigen-binding fragment in some embodiments or examples of the present invention has a higher binding activity or affinity with cortisol. Therefore, a reagent or kit containing the antibody or its antigen-binding fragment has higher detection sensitivity or specificity.

[0115] In a seventh aspect, the present invention provides a method for detecting cortisol, including: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with cortisol in a test sample under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0116] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0117] In a ninth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0118] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

[0119] In an eleventh aspect, the present invention provides a method for preparing an antibody or its antigen-binding fragment, which includes: culturing the cell as described above.

[0120] In a twelfth aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, or the above-mentioned reagent or kit in detecting cortisol or preparing a product for detecting cortisol.

[0121] Based on the disclosure of the amino acid sequence of the antibody or its antigen-binding fragment in the present invention, those skilled in the art can easily think of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibody or its antigen-binding fragment. For example, the antibody or its antigen-binding fragment can be isolated and purified from the culture product of a recombinant cell capable of recombinantly expressing the antibody or its antigen-binding fragment described in any one of the above. This is easily achievable for those skilled in the art. Based on this, no matter which technique is used to prepare the antibody or its antigen-binding fragment of the present invention, it falls within the protection scope of the present invention.

[0122] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicated by the manufacturer can all be obtained as conventional products through commercial purchase.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0124] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual" (Second Edition, Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, Ed., 1984); "Animal Cell Culture" (R.I. Freshney, Ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, Eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P. Calos, Eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., Eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., Eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., Eds., 1991), each of which is hereby expressly incorporated by reference.

[0125] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0126] Example 1 Preparation of Anti-CORT 20D9 Monoclonal Antibody

[0127] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Corporation. The MagExtractor-RNA extraction kit was purchased from TOYOBO Corporation. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-CORT 20D9 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0128] (1) Preparation of antibody gene

[0129] mRNA was extracted from the hybridoma cell line secreting the Anti-CORT 20D9 monoclonal antibody, and a DNA product was obtained by RT-PCR. After adding A to this product with rTaq DNA polymerase, it was inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, 4 clones each of the Heavy Chain and Light Chain genes were cloned and sent to a gene sequencing company for sequencing.

[0130] (2) Sequence analysis of the variable region genes of the Anti-CORT 20D9 antibody

[0131] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the VNTI 11.5 software was used for analysis to confirm that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 321 bp, and there was a 57 bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 357 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

[0132] (3) Construction of the recombinant antibody expression plasmid

[0133] pcDNA TM 3.4 The pcDNA 3.4 vector was the recombinant antibody eukaryotic expression vector constructed. This expression vector had introduced multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI, and was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protection bases at both ends, and a 0.70 kb Light Chain gene fragment and a 1.40 kb Heavy Chain gene fragment were amplified by PCR.

[0134] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0135] 2. Recombinant antibody production

[0136] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select the antibody concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it as the cell diluent. Prepare the plasmid DNA and transfection reagent diluents with the medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in an incubator at 35°C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0137] The obtained antibody was named Anti-CORT 20D9Rmb1, and the Anti-CORT 20D9Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0138] Table 2 Antibody sequences

[0139] Antibody name Heavy chain Light chain Anti - CORT 20D9Rmb1 SEQ ID NO:21 SEQ ID NO:22 Anti - CORT 20D9Rmb2 SEQ ID NO:21 SEQ ID NO:23 Anti - CORT 20D9Rmb3 SEQ ID NO:21 SEQ ID NO:24

[0140] Example 2 Performance detection of the antibody

[0141] 1. Affinity analysis

[0142] Using an AMC sensor, the purified antibody was diluted to 10 μg / ml with PBST, and cortisol-BSA (obtained from Fapon Biotech) was serially diluted with PBST.

[0143] Operation process: Equilibrate in Buffer 1 (PBST) for 60 s, immobilize the antibody in the antibody solution for 300 s, incubate in Buffer 2 (PBST) for 180 s, bind in the antigen solution for 420 s, dissociate in Buffer 2 for 1200 s, regenerate the sensor with 10 mM pH 1.69 GLY solution and Buffer 3 (PBST), and output data. (KD represents the equilibrium dissociation constant, i.e., affinity; kon represents the binding rate; kdis represents the dissociation rate. The main components of PBST are Na2HPO4 + NaCl + TW-20).

[0144] Table 3 Affinity Data

[0145] Sample name KD Kon kdis Control 4.31E-9 1.72E+04 7.42E-05 Anti - CORT 20D9Rmb1 3.03E-14 3.30E+06 1.00E-07 Anti - CORT 20D9Rmb2 9.30E-13 3.04E+06 2.83E-06 Anti - CORT 20D9Rmb3 9.90E-13 3.20E+06 3.17E-06

[0146] 2. Activity Identification

[0147] Dilute cortisol-BSA (from Fapon Biotech) to 3 μg / ml with the coating solution (main component NaHCO3), 100 μL per well, overnight at 4°C; the next day, wash 2 times with the washing solution (main components Na2HPO4 + Nacl), pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0148] Remarks: Solution A (main components citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0149] Table 4 Activity Data

[0150] Concentration (ng / ml) 123.457 41.152 13.717 4.572 1.524 0.000 Control 1.190 0.762 0.391 0.200 0.097 0.016 Anti - CORT 20D9Rmb1 1.896 0.977 0.486 0.250 0.132 0.019 Anti - CORT 20D9Rmb2 1.891 0.979 0.488 0.253 0.136 0.016 Anti - CORT 20D9Rmb3 1.888 0.984 0.479 0.257 0.139 0.023

[0151] 3. Stability Assessment

[0152] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Samples were taken at 7 days, 14 days, and 21 days for status observation, and the 21-day samples were subjected to activity detection. The results showed that there were no obvious protein status changes in the antibody after being placed for 21 days under the three test conditions, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above-mentioned antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-CORT 20D9Rmb3 after 21 days of assessment.

[0153] Table 5 Stability data

[0154] Sample concentration (ng / ml) 123.457 41.152 0.000 4°C, 21 - day sample 1.846 0.972 0.019 - 80°C, 21 - day sample 1.851 0.981 0.017 37°C, 21 - day sample 1.844 0.983 0.021

[0155] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0156] Some of the amino acid sequences involved in this application are shown in Table 6 as follows:

[0157] Table 6 Amino acid sequences

[0158]

[0159]

Claims

1. An anti-cortisol antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment has three complementarity-determining regions of the heavy-chain variable region shown by the amino acid sequence SEQ ID NO: 17 and three complementarity-determining regions of any one of the light-chain variable regions shown by the amino acid sequences SEQ ID NO: 18, 19, or 20, and the complementarity-determining regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

2. An anti-cortisol antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises the following complementarity-determining regions: The amino acid sequence of HCDR1 is as shown by SEQ ID NO: 1; The amino acid sequence of HCDR2 is as shown by SEQ ID NO: 2; The amino acid sequence of HCDR3 is as shown by SEQ ID NO: 3; The amino acid sequence of LCDR1 is as shown by SEQ ID NO: 4; The amino acid sequence of LCDR2 is as shown by SEQ ID NO: 5; The amino acid sequence of LCDR3 is as shown by SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that, The HFR1 comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 80% identity therewith; The HFR2 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 80% identity therewith; The HFR3 comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80% identity therewith; The HFR4 comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% identity therewith; The LFR1 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% identity therewith; The LFR2 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% identity therewith; The LFR3 comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 80% identity therewith; The LFR4 comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80% identity therewith.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and 4, characterized in that, The antibody or its antigen-binding fragment binds cortisol with an affinity of KD < 4.31×10 -9 M.

6. An anti-cortisol antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy-chain variable region is as shown by SEQ ID NO: 17; the amino acid sequence of the light-chain variable region is as shown by any one of SEQ ID NO: 18, 19, or 20.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4 to 6, characterized in that, The antibody or its antigen-binding fragment further comprises a constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that, The constant region comprises a heavy-chain constant region and a light-chain constant region.

9. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The heavy-chain constant region is selected from the heavy-chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD or a combination of segments of multiple heavy-chain constant regions.

10. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The heavy-chain constant region comprises the CH1 region of IgG, the hinge region of IgG, the CH2 region of IgM, the CH3 region of IgM, and / or the CH4 region of IgM.

11. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that, The species origin of the constant region is bovine, equine, porcine, ovine, murine, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

12. The antibody or antigen-binding fragment thereof according to claim 7, wherein The species origin of the constant region is murine.

13. The antibody or antigen-binding fragment thereof according to claim 8, wherein The heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto, and the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4 to 6, wherein The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

15. An anti-cortisol antibody, comprising a heavy chain and a light chain, wherein The amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 21; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 22, 23, and 24.

16. An antibody conjugate, wherein The antibody conjugate comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 14 or the antibody according to claim 15.

17. The antibody conjugate according to claim 16, wherein The antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

18. The antibody conjugate according to claim 16, wherein The antibody conjugate further comprises a label conjugated to the antibody or its antigen-binding fragment.

19. The antibody conjugate according to claim 18, wherein The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

20. The antibody conjugate according to claim 16, wherein The antibody conjugate further comprises a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

21. The antibody conjugate according to claim 20, wherein The solid-phase carrier is selected from microspheres, plates, and membranes.

22. A reagent or kit, wherein The reagent or kit comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 14, the antibody according to claim 15, or the antibody conjugate according to claim 7.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to claims 16 to 21, or the reagent or kit according to claim 22 in the preparation of a product for detecting cortisol, wherein Comprising: a) contacting the antibody or its antigen-binding fragment according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to claims 16 to 21, or the reagent or kit according to claim 22 with cortisol in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

24. A nucleic acid, wherein It encodes the antibody or its antigen-binding fragment according to any one of claims 1 to 14 or the antibody according to claim 15.

25. A vector, wherein It contains the nucleic acid according to claim 24.

26. A cell, wherein It contains the nucleic acid according to claim 24 or the vector according to claim 25.

27. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15, wherein It comprises: culturing the cell according to claim 26.

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