Anti-methamphetamine antibodies, reagents and kits for detecting methamphetamine

Abstract

The present invention discloses an anti-methamphetamine antibody or a reagent and kit for detecting methamphetamine, relating to the field of antibodies. The anti-methamphetamine antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important source of raw materials for the detection of methamphetamine 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-methamphetamine antibody, a reagent and a kit for detecting methamphetamine. Background Art

[0002] Methamphetamine (Met), also known as methylamphetamine, has the chemical formula C10H15N (N-methyl-1-phenylpropan-2-amine, N-methyl-α-methylphenethylamine). Its crystalline form is commonly known as ice. It can be taken orally, inhaled, directly through the nose, or injected intravenously. It has high bioavailability and is a potent central nervous system stimulant, primarily used as a narcotic. Methamphetamine was discovered in 1893 and has two enantiomers: L-methamphetamine and D-methamphetamine.

[0003] Methamphetamine is an indirect agonist of dopamine and norepinephrine in the central nervous system. Long-term methamphetamine use can cause abnormal brain function, leading to psychotic symptoms such as auditory hallucinations, hyperactivity, delusions of persecution, and cognitive impairment. Furthermore, methamphetamine primarily produces a series of physiological effects by indirectly stimulating neurotransmitters, damaging neurons and making its abuse highly addictive and difficult to quit. Due to its harmful effects on the human body and society, my country has designated methamphetamine as a controlled substance, prohibiting its abuse. The development of a rapid methamphetamine detection method is of great significance for combating crime and drug control, while also addressing the need for safety screenings for individuals in high-risk occupations involving drugs.

[0004] At present, the main methods for detecting methamphetamine include colloidal gold immunochromatography, fluorescent immunochromatography, chemiluminescence immunoassay, etc., which have the advantages of being rapid and easy to operate. The aforementioned methods are based on the specific reaction of antibodies and antigens, and are an immunological detection method that amplifies and displays the detected signal by utilizing colloidal gold, fluorescent substances, and luminescent substances. Obtaining anti-Met antibodies is the key to achieving immunoassay.

[0005] The above immunological detection methods all require antibodies against methamphetamine. Therefore, those skilled in the art have a strong demand for anti-methamphetamine antibodies with good performance. Summary of the Invention

[0006] The present application provides an antibody or an antigen-binding fragment thereof, which provides an important source of raw materials for the detection of methamphetamine and has good activity or affinity.

[0007] To achieve the above-mentioned object, according to one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises three complementarity determining regions of a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 19, 20, and 21 and three complementarity determining regions of a light chain variable region having the amino acid sequence of SEQ ID NO: 25.

[0008] 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, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity determining regions:

[0009] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0010] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0011] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0012] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0013] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5;

[0014] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0015] To achieve the above-mentioned object, according to the third aspect of the present invention, an antibody or an antigen-binding fragment thereof 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 any one of SEQ ID NOs: 19, 20, and 21; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25.

[0016] To achieve the above-mentioned object, according to the fourth aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 22, 23, and 24; and the amino acid sequence of the light chain is shown in SEQ ID NO: 26.

[0017] In order to achieve the above object, according to a fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or antigen-binding fragment thereof.

[0018] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or kit is provided, wherein the reagent or kit comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0019] To achieve the above-mentioned object, according to a seventh aspect of the present invention, a method for detecting methamphetamine is provided, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit with methamphetamine 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.

[0020] In order to achieve the above object, according to the eighth aspect of the present invention, a nucleic acid is provided, which encodes the above-mentioned antibody or antigen-binding fragment thereof.

[0021] In order to achieve the above object, according to the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0022] To achieve the above object, according to the tenth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above antibody or antigen-binding fragment thereof.

[0023] To achieve the above object, according to the eleventh aspect of the present invention, there is provided a method for preparing the above antibody or antigen-binding fragment thereof, the method comprising culturing the above cell.

[0024] In order to achieve the above-mentioned object, according to the twelfth aspect of the present invention, there is provided a use of the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit in detecting or preparing methamphetamine products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, 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 invention 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 paying any creative work.

[0026] Figure 1 These are the results of reducing SDS-PAGE of Anti-MET 20E8 Rmb1 to Anti-MET 20E8 Rmb3. DETAILED DESCRIPTION

[0027] In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, comprising three complementarity determining regions of a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 19, 20, and 21 and three complementarity determining regions of a light chain variable region having the amino acid sequence of SEQ ID NO: 25.

[0028] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect.

[0029] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 19; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 25.

[0030] 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, so long as they exhibit the desired biological activity.

[0031] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0032] In the present invention, 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.

[0033] 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. 200,825,754,554, "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 documents 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.

[0034] Table 1: CDR Definition 1

[0035] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> 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

[0036] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on 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).

[0037] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] According to an embodiment of the present invention, 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.

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

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

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

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

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

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

[0048] According to an embodiment of the present invention, 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:

[0049]

[0050]

[0051] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity determining regions:

[0052] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0053] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0054] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0055] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0056] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0057] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0058] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0059] 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 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.

[0060] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0061] In an optional embodiment, the antibody or antigen-binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0062] The HFR1 comprises / is as SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0063] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0064] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0065] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0066] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0067] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0068] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0069] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto;

[0070] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or antigen-binding fragments thereof provided by the present invention 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).

[0071] In an optional embodiment, the HFR1 comprises / is represented by the amino acid sequence shown in SEQ ID NO:17.

[0072] In an alternative embodiment, the HFR3 comprises / is represented by the amino acid sequence of SEQ ID NO:18.

[0073] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD < 5.28×10 -9 The affinity of M for binding to methamphetamine.

[0074] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD ≤ 10 -10 M, KD≤10 -11 M, KD≤10 -12 M, KD≤10 -13 M or KD≤10 -14 The affinity of M for binding to methamphetamine.

[0075] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD ≤ 4.91×10 -10 The affinity of M for binding to methamphetamine.

[0076] 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.

[0077] 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.

[0078] In a third aspect, an embodiment of the present invention provides 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 shown in any one of SEQ ID NOs: 19, 20, and 21, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25.

[0079] In an optional embodiment, the heavy chain variable region and the light chain variable region of the first aspect or the third aspect are selected from any one of the following combinations:

[0080] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO: 19 SEQ ID NO:25 2 SEQ ID NO:20 SEQ ID NO:25 3 SEQ ID NO:21 SEQ ID NO:25

[0081] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0082] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0083] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the kappa or lambda type light chain constant region.

[0084] 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.

[0085] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0086] In an alternative embodiment, the species origin of the constant region is mouse.

[0087] 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.

[0088] 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).

[0089] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0090] The antigen-binding fragments of the aforementioned antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that the antigen-binding fragments of the aforementioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0091] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0092] In a fourth aspect, the present invention provides 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 shown in any one of SEQ ID NO: 22, 23, and 24, and the amino acid sequence of the light chain is shown in any one of SEQ ID NO: 26.

[0093] In an optional embodiment, the antibody or antigen-binding fragment thereof of the first, second, third or fourth aspect comprises any combination of the following heavy chains and light chains:

[0094] combination heavy chain light chain 1 SEQ ID NO:22 SEQ ID NO:26 2 SEQ ID NO:23 SEQ ID NO:26 3 SEQ ID NO:24 SEQ ID NO:26

[0095] In a fifth aspect, the present invention provides an antibody conjugate comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0096] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

[0097] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

[0098] 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.

[0099] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0100] 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 invention.

[0101] 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.).

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0107] In an optional embodiment, the above-mentioned antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0108] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0109] 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.

[0110] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0111] As previously mentioned, the antibody or its Fab in some embodiments or examples of the present invention can effectively be combined with methamphetamine, therefore, the reagent or test kit comprising the methamphetamine antibody or its Fab can effectively carry out qualitative or quantitative detection to methamphetamine. Application reagent or test kit provided by the present invention, for example, can be used for immunoblotting, immunoprecipitation etc. and relate to the detection utilizing methamphetamine and its antibody specific binding performance. As previously mentioned, the antibody or its Fab in some embodiments or examples of the present invention have higher binding activity or affinity with methamphetamine, therefore the reagent or test kit comprising the antibody or its Fab has higher detection sensitivity or specificity.

[0112] In a seventh aspect, the present invention provides a method for detecting methamphetamine, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with methamphetamine 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;

[0113] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof.

[0114] In an alternative embodiment, the immune complex further comprises a second antibody that binds to methamphetamine.

[0115] In an eighth aspect, the present invention also provides another method for detecting methamphetamine, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with methamphetamine in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form a first immune complex; and b) combining methamphetamine with a marker with the antibody to form a second immune complex, and determining the methamphetamine content in the sample by the content of the second immune complex and the amount of antibody used.

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

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

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

[0119] In a twelfth aspect, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising: culturing the cells as described above.

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

[0121] Based on the amino acid sequence of the antibody or antigen-binding fragment thereof disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or antigen-binding fragment thereof. For example, the antibody or antigen-binding fragment thereof can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody or antigen-binding fragment thereof as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, regardless of the technology used to prepare the antibody or antigen-binding fragment of the present invention, it falls within the scope of protection of the present invention.

[0122] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0123] 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.

[0124] The practice of the present invention 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 person skilled in the art. 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.

[0125] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0126] Example 1 Preparation of Anti-MET 20E8 Monoclonal Antibody

[0127] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM The 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-MET 20E8 monoclonal antibody was generated in our laboratory and revived for future use.

[0128] (1) Antibody gene preparation

[0129] mRNA was extracted from a hybridoma cell line secreting the Anti-MET 20E8 monoclonal antibody. DNA products were obtained by RT-PCR. This product was PCR-amplified 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 Chain and Light Chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0130] (2) Sequence analysis of the variable region gene of Anti-MET 20E8 antibody

[0131] 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 VL gene sequence was 336 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 354 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

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

[0133] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter 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 0.71kb Light Chain gene fragment and a 1.40kb Heavy Chain gene fragment.

[0134] 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 respectively connected to 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 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).

[0137] The obtained antibody was named Anti-MET 20E8Rmb1. Anti-MET 20E8Rmb1 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-MET 20E8Rmb1 SEQ ID NO:22 SEQ ID NO:26 Anti-MET 20E8Rmb2 SEQ ID NO:23 SEQ ID NO:26 Anti-MET 20E8Rmb3 SEQ ID NO:24 SEQ ID NO:26

[0140] Example 2 Performance testing of antibodies

[0141] 1. Affinity Analysis

[0142] Purified antibodies were diluted in advance, and MET antigen (from Qingdao Henderson) 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.)

[0143] Table 3 Affinity data

[0144] Sample name KD ka kd comparison 9.53E-09 7.28E+04 8.84E-04 Anti-MET 20E8Rmb1 4.15E-10 9.97E+05 4.14E-04 Anti-MET 20E8Rmb2 4.91E-10 8.82E+05 4.34E-04 Anti-MET 20E8Rmb3 4.37E-10 9.01E+05 3.94E-04

[0145] 2. Activity Identification

[0146] MET antigen (from Feipeng Bio) was diluted to 3 μg / ml in coating buffer (mainly NaHCO₃), and 100 μL was added to each well at 4°C overnight. The next day, the wells were washed twice with wash buffer (mainly Na₂HPO₄ + NaCl) and patted dry. Blocking buffer (20% BSA + 80% PBS) was added at 120 μL per well and incubated at 37°C for 1 hour, then patted dry. Diluted purified antibody and control antibody were added at 100 μL per well and incubated at 37°C for 30 minutes. The wells were washed five times with wash buffer and patted dry. Goat anti-mouse IgG-HRP was added at 100 μL per well and incubated at 37°C for 30 minutes. The wells were washed five times with wash buffer and patted dry. Chromogenic Buffer A (50 μL / well) and Chromogenic Buffer B (50 μL / well) were added for 10 minutes. Stop buffer (50 μL / well) was added. The OD values were read on a microplate reader at 450 nm (reference 630 nm).

[0147] 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)

[0148] Table 4 Activity data

[0149] Concentration (ng / ml) 31.25 15.63 7.81 3.91 1.95 0.00 comparison 1.458 0.786 0.493 0.199 0.020 0.015 Anti-MET 20E8Rmb1 1.939 1.537 0.898 0.523 0.264 0.027 Anti-MET 20E8Rmb2 1.933 1.533 0.833 0.526 0.218 0.024 Anti-MET 20E8Rmb3 1.954 1.547 0.865 0.553 0.254 0.023

[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 the 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 5 below shows the OD results of the enzyme immunoassay activity assay of the antibody Anti-MET 20E8Rmb1 after 21 days of testing.

[0152] Table 5 Stability data

[0153] Sample concentration (ng / ml) 15.63 7.81 0.00 4℃, 21-day samples 1.536 0.799 0.018 -80℃, 21-day sample 1.558 0.785 0.017 37℃, 21-day samples 1.524 0.739 0.016

[0154] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

[0155] Some of the amino acid sequences involved in this application are shown in Table 6:

[0156] Table 6 Amino acid sequence table

[0157]

[0158]

Claims

1. An anti-methamphetamine antibody or antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions of a heavy chain variable region represented by any one of the amino acid sequences of SEQ ID NOs: 19, 20, and 21, and three light chain complementarity determining regions of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 25, wherein the complementarity determining regions of the variable regions are defined by any one of the systems of Kabat, Chothia, IMGT, AbM, or Contact, and are numbered by the Kabat numbering system.

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

6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4, The HFR1 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:7; the HFR2 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:8; the HFR3 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:9; the HFR4 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:10; the LFR1 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:11; the LFR2 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:12; the LFR3 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:13; and the LFR4 includes an amino acid sequence that is at least 80% identical to SEQ ID NO:

14.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The amino acid sequence of the HFR1 is shown in SEQ ID NO:7; the amino acid sequence of the HFR2 is shown in SEQ ID NO:8; the amino acid sequence of the HFR3 is shown in SEQ ID NO:9; the amino acid sequence of the HFR4 is shown in SEQ ID NO:10; the amino acid sequence of the LFR1 is shown in SEQ ID NO:11; the amino acid sequence of the LFR2 is shown in SEQ ID NO:12; the amino acid sequence of the LFR3 is shown in SEQ ID NO:13; and the amino acid sequence of the LFR4 is shown in SEQ ID NO:

14.

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 antigen-binding fragment thereof has a KD of less than 5.28×10 -9 The affinity of M for binding to methamphetamine.

6. An anti-methamphetamine 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 shown in any one of SEQ ID NOs: 19, 20, and 21; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4, and 6, wherein: The antibody or antigen-binding fragment thereof further comprises a constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The constant region includes 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 any one of IgG, IgA, IgM, IgE, and IgD heavy chain constant regions or a combination of multiple constant regions; the light chain constant region is selected from κ-type or λ-type light chain constant regions.

10. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that: 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.

11. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

12. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The species origin of the constant region is mouse.

13. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that: The heavy chain constant region sequence is an amino acid sequence that is at least 80% identical to SEQ ID NO: 15; the light chain constant region sequence is an amino acid sequence that is at least 80% identical to SEQ ID NO:

16.

14. The antibody or antigen-binding fragment thereof according to claim 13, characterized in that: The heavy chain constant region sequence is shown in SEQ ID NO: 15; the light chain constant region sequence is shown in SEQ ID NO:

16.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4, and 6, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

16. An anti-methamphetamine antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 22, 23, and 24; the amino acid sequence of the light chain is shown in SEQ ID NO:

26.

17. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16 and a label conjugated to the antibody or antigen-binding fragment thereof.

18. The antibody conjugate according to claim 17, characterized in that: The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, nanoparticle labels and biotin.

19. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16 and a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

20. The antibody conjugate according to claim 19, characterized in that: The solid support is selected from microspheres, plates and membranes.

21. A reagent or kit, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the antibody according to claim 16, or the antibody conjugate according to claim 7.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the antibody according to claim 16, the antibody conjugate according to any one of claims 17 to 20, or the reagent or kit according to claim 21 in the preparation of a method for detecting methamphetamine products, characterized in that: This includes one of the following methods: a) contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the antibody according to claim 16, the antibody conjugate according to any one of claims 17 to 20, or the reagent or kit according to claim 21 with methamphetamine in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample; b) contacting the antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with the methamphetamine in the sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form a first immune complex; combining the labeled methamphetamine with the antibody to form a second immune complex, and determining the methamphetamine content in the sample based on the content of the second immune complex and the amount of antibody used.

23. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16.

24. A carrier, characterized in that It contains the nucleic acid according to claim 23.

25. A cell, characterized in that It contains the nucleic acid according to claim 23 or the vector according to claim 24.

26. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16, characterized in that: It includes: Cultivate the cell of claim 25.

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