An antibody binding etomidate and uses thereof

Abstract

An antibody or antigen binding fragment that binds etomidate, the antibody or antigen binding fragment comprising: a heavy chain variable region comprising a CDRH1 of the amino acid sequence set forth in SEQ ID NO: 2, a CDRH2 of the amino acid sequence set forth in SEQ ID NO: 3, and a CDRH3 of the amino acid sequence set forth in SEQ ID NO: 4; and a light chain variable region comprising a CDRL1 of the amino acid sequence set forth in SEQ ID NO: 6, a CDRL2 of the amino acid sequence LTS, and a CDRL3 of the amino acid sequence set forth in SEQ ID NO: 7.

Description

TECHNICAL FIELD

[0001] The present specification relates to the technical field of biological medicine, in particular to an antibody binding etomidate and application thereof. BACKGROUND

[0002] Etomidate is a hypnotic intravenous anesthetic drug, belonging to imidazole derivatives, with good safety, and is one of the commonly used drugs for anesthetic induction. Etomidate has good safety and controllability when applied to outpatient and operating room anesthesia, and has a wide application prospect. At present, the detection of etomidate mainly relies on high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), mass spectrometry (MS) and the like, but the instruments are expensive, the detection is time-consuming, and professional technical personnel are required to operate.

[0003] Therefore, it is necessary to provide a technology for rapidly, sensitively and accurately detecting etomidate. SUMMARY

[0004] According to a first aspect of the present specification, an antibody or antigen binding fragment binding etomidate is provided, characterized in that the antibody or antigen binding fragment comprises: a heavy chain variable region comprising CDRH1 with an amino acid sequence as shown in SEQ ID NO: 2, CDRH2 with an amino acid sequence as shown in SEQ ID NO: 3 and CDRH3 with an amino acid sequence as shown in SEQ ID NO: 4; a light chain variable region comprising CDRL1 with an amino acid sequence as shown in SEQ ID NO: 6, CDRL2 with an amino acid sequence of LTS and CDRL3 with an amino acid sequence as shown in SEQ ID NO: 7.

[0005] In some embodiments, the antibody is a murine antibody, a rabbit antibody or a humanized antibody.

[0006] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5.

[0007] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0008] In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, dAb.

[0009] According to a second aspect of the present specification, a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen binding fragment as described above is provided.

[0010] In some embodiments, the nucleic acid molecule comprises: a nucleotide sequence encoding a heavy chain variable region comprising a coding sequence for CDRH1 as set forth in SEQ ID NO: 9, a coding sequence for CDRH2 as set forth in SEQ ID NO: 10, and a coding sequence for CDRH3 as set forth in SEQ ID NO: 11; and a nucleotide sequence encoding a light chain variable region comprising a coding sequence for CDRL1 as set forth in SEQ ID NO: 13, a coding sequence for CDRL2 of the sequence CTCACATCC, and a coding sequence for CDRL3 as set forth in SEQ ID NO: 14.

[0011] In some embodiments, the nucleotide sequence encoding a heavy chain variable region is set forth in SEQ ID NO: 8; and the nucleotide sequence encoding a light chain variable region is set forth in SEQ ID NO: 12.

[0012] According to a third aspect of the present specification, there is provided a recombinant vector comprising the nucleic acid molecule as described hereinbefore.

[0013] According to a fourth aspect of the present specification, there is provided a host cell comprising the nucleic acid molecule as described hereinbefore or the recombinant vector of claim 8.

[0014] According to a fifth aspect of the present specification, there is provided a method of producing an antibody or antigen binding fragment that binds to etomidate, the method comprising: culturing the host cell as described hereinbefore; and isolating the antibody or antigen binding fragment from the cell culture of the host cell.

[0015] According to a sixth aspect of the present specification, there is provided a method of detecting etomidate in a sample, the method comprising: contacting the sample with an antibody or antigen binding fragment that is capable of binding to etomidate to form an antigen-antibody complex, the antibody or antigen binding fragment being an antibody or antigen binding fragment as described hereinbefore; and detecting the antigen-antibody complex to qualitatively or quantitatively detect etomidate in the sample.

[0016] In some embodiments, the antibody or antigen binding fragment is labeled with a detectable label, and the antigen-antibody complex is detected by a signal generated by the detectable label; or the antibody or antigen binding fragment is unlabeled, and the antibody or antigen binding fragment in the antigen-antibody complex as a capture antibody is bound to a detection antibody labeled with a detectable label, and the antigen-antibody complex is detected by a signal generated by the detectable label.

[0017] In some embodiments, the quantitative detection comprises: comparing a first detection signal obtained from the sample and a second detection signal obtained from a control or a calibrator, wherein the first detection signal is generated by an antigen-antibody complex formed by contacting the sample with the antibody or antigen-binding fragment, the second detection signal is generated by an antigen-antibody complex formed by contacting the control or the calibrator with the antibody or antigen-binding fragment, the control or the calibrator contains a predetermined concentration of etomidate; and determining the concentration of etomidate in the sample based on the comparison result and the predetermined concentration.

[0018] In some embodiments, the sample comprises one or more of peripheral blood, urine, saliva, and hair.

[0019] According to a seventh aspect of the present specification, there is provided a kit for detecting etomidate, comprising the antibody or antigen-binding fragment as described hereinbefore.

[0020] In some embodiments, the kit is selected from the group consisting of colloidal gold detection kit, immunochromatographic detection kit, enzyme-linked immunodetection kit, chemiluminescence kit, immunoturbidimetric detection kit.

[0021] According to an eighth aspect of the present specification, there is provided use of the antibody or antigen-binding fragment, nucleic acid molecule, recombinant vector, or host cell as described hereinbefore in detecting etomidate or in preparing a kit for detecting etomidate. DETAILED DESCRIPTION

[0022] It should be understood that, although the terms“first,”“second,”“third,” etc. can be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first detection signal can be termed a second detection signal, and, similarly, a second detection signal can be termed a first detection signal, without departing from the scope of the example embodiments of the present specification.

[0023] As shown in the present specification and claims, unless the context clearly indicates otherwise, the words“a,”“an,”“the,” and / or“this” do not necessarily refer to only one element, but can refer to one or more elements. In general, the terms“including,”“includes,”“containing,”“contains,” and“containing,” are used only to indicate the inclusion of the elements explicitly identified, and do not exclude the presence of other elements.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0025] Etomidate has good hemodynamic stability during general anesthesia induction, fast postoperative recovery, and is currently widely used for anesthesia induction in elderly patients and patients with concurrent cardiovascular diseases and critical conditions. The present specification provides an antibody binding to etomidate and applications thereof. In some embodiments, the therapeutic effect of etomidate needs to be monitored, and the antibody binding to etomidate and applications thereof provided by the present specification can be used for monitoring etomidate therapy, for example, for detecting the concentration of etomidate in a biological sample of a patient, which can assist medical personnel in adjusting / optimizing the treatment regimen. In some embodiments, etomidate has a risk of drug abuse, and the antibody binding to etomidate and applications thereof provided by the present specification can be used for drug management of etomidate, for example, for detecting whether etomidate exists in a biological sample of a subject. Specifically, in some embodiments of the present specification, Balb / c mice are immunized with a chemically synthesized etomidate hapten, and the spleen lymphocytes of the successfully immunized mice are selected. The lymphocytes are fused with mouse myeloma cells SP2 / 0 by cell fusion technology, and after two rounds of subcloning screening, a hybridoma cell strain stably secreting anti-etomidate monoclonal antibodies is obtained. A large amount of mouse ascites is obtained by culture and reimmunization, and a high-specificity anti-etomidate monoclonal antibody is obtained by separation and purification. The antibody or antigen-binding fragment provided by the present specification can provide a stable and reliable raw material source for etomidate colloidal gold immunochromatography technology and other methods based on immunological principles, reduce product costs, and at the same time improve the detection rate and sensitivity of etomidate rapid diagnostic products such as gold standard test strips and ELISA kits, and reduce the false positive rate.

[0026] Some embodiments of the present specification provide an antibody or antigen-binding fragment binding to etomidate, which can bind to an etomidate antigen. The antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three complementarity determining regions (CDRs), i.e., CDRH1, CDRH2, and CDRH3; and the light chain variable region includes three complementarity determining regions, i.e., CDRL1, CDRL2, and CDRL3.

[0027] As used herein, the term “antibody” or “Ab” refers to a molecule comprising at least one immunoglobulin heavy chain (HC) and at least one immunoglobulin light chain (LC). Each heavy chain can include a heavy chain variable region (VH) having 3 complementarity determining regions (CDRs) and 4 framework regions (FRs), and a heavy chain constant region (CH). Each light chain can include a light chain variable region (VL) having 3 complementarity determining regions and 4 framework regions, and a light chain constant region (CL). Antibodies can be classified into IgM, IgG, IgA, IgD, and IgE according to the antigenicity of VH. In some embodiments, antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, dimeric antibodies, trimeric antibodies, and multimeric antibodies, etc.

[0028] The term "antigen-binding fragment" refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. Non-limiting examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, CDR fragments, single-chain antibodies (e.g., scFv or dsFv), diabodies fragments (dAb), single domain antibodies (sdAb), and nanobodies (Nb), etc. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, dAb.

[0029] The term "CDR" or "complementarity determining region" refers to the region of an antibody that specifically recognizes an antigen.

[0030] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising a CDRH1 of the amino acid sequence set forth in SEQ ID NO: 2, a CDRH2 of the amino acid sequence set forth in SEQ ID NO: 3, and a CDRH3 of the amino acid sequence set forth in SEQ ID NO: 4; and a light chain variable region comprising a CDRL1 of the amino acid sequence set forth in SEQ ID NO: 6, a CDRL2 of the amino acid sequence LTS, and a CDRL3 of the amino acid sequence set forth in SEQ ID NO: 7.

[0031] In some embodiments, the antibody or antigen-binding fragment comprises one or more of the following sequences: a variant of the foregoing CDRH1 having no more than 2 or no more than 1 amino acid change; a variant of the foregoing CDRH2 having no more than 2 or no more than 1 amino acid change; a variant of the foregoing CDRH3 having no more than 2 or no more than 1 amino acid change; a variant of the foregoing CDRL1 having no more than 2 or no more than 1 amino acid change; a variant of the foregoing CDRL2 having no more than 2 or no more than 1 amino acid change; a variant of the foregoing CDRL3 having no more than 2 or no more than 1 amino acid change.

[0032] As used herein, the term "variant" refers to a modification of one or more amino acids. A variant of a protein or polypeptide substantially retains the biological characteristics of the protein or polypeptide prior to the modification as compared to the reference protein or polypeptide. For example, an antigen-binding protein (e.g., an antibody or antigen-binding fragment) comprising a heavy chain CDR variant and / or a light chain CDR variant retains 85%, 90%, 95%, or 100% of the biological characteristics of the antigen-binding protein prior to the modification, and has the same or similar function as the antigen-binding protein prior to the modification. In some embodiments, the modification of the amino acid is a substitution, deletion, or addition of an amino acid. In some preferred embodiments, the modification of the amino acid is a substitution of an amino acid.

[0033] The term "substitution of an amino acid" refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / polypeptide comprising the amino acid sequence. For example, a substitution of an amino acid can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Substitutions of an amino acid include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a substitution of a physically or functionally similar residue (e.g., a residue having similar size, shape, charge, chemical properties including ability to form covalent or hydrogen bonds, etc.) for the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), uncharged nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Thus, a corresponding amino acid residue is preferably replaced with another amino acid residue from the same side chain family.

[0034] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment is PWTRELLS LLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYGGSYNYNPSLKNRISITRDTSKN QFFLNLNSVTTEDTATYYCATFYDGYYRAWGLWTLVTVS (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment is QIVLTQSPALMSASPGEK VTMTCSASSSVSYMYWYQQKPRSSPKTWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEA EDAATYYCQEWSGNPPITFGAGTKLE (SEQ ID NO: 5).

[0035] In some embodiments, the amino acid sequence of the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, the amino acid sequence of the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth as SEQ ID NO: 5. As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by alignment and comparison. "Percent identity" refers to the percentage of identical residues between the amino acids or nucleotides in the comparison molecules, and is calculated based on the size of the smallest molecule being compared.

[0036] In some embodiments, the amino acid sequence of the heavy chain variable region having the percent identity described above can have one or more changes (e.g., substitutions, deletions, or additions of amino acids) in the CDR regions and / or non-CDR regions (e.g., FR regions) compared to the amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, the amino acid sequence of the light chain variable region having the percent identity described above can have one or more changes in the CDR regions and / or non-CDR regions (e.g., FR regions) compared to the amino acid sequence set forth as SEQ ID NO: 5. In some preferred embodiments, the changes in amino acids occur in the non-CDR regions.

[0037] The antibodies provided by the embodiments of the present disclosure can be derived from the same species. In some embodiments, the antibodies are murine or rabbit antibodies.

[0038] The antibodies provided by the embodiments of the present disclosure can be derived from different species. In some embodiments, the antibodies are chimeric or humanized antibodies.

[0039] As used herein, a "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of one species with the constant region of an antibody of another species. Chimeric antibodies have the advantage of reducing the immunogenicity of heterologous antibodies. In some embodiments, the variable region of a chimeric antibody can be rabbit or murine, and the constant region of the chimeric antibody can be human. The term "humanized antibody" refers to an antibody form that has been genetically engineered to contain both human antibody sequences and non-human antibody sequences. In some embodiments, the humanized antibody has all or part of the CDR regions derived from a non-human (e.g., murine or rabbit) antibody, and all or part of the non-CDR regions (e.g., constant and variable region frameworks) derived from a human antibody.

[0040] In some embodiments, the antibody can be a monoclonal antibody, a bispecific antibody, or a multispecific antibody. The number and class of the epitope of the antigen that the antibody can recognize can be determined by one of skill in the art according to the needs of the application.

[0041] Methods of making antibodies should be known to one of skill in the art. For example, the hybridoma technique, or using recombinant DNA technology to produce antibodies in vitro, starting with the genes encoding the antibodies. Methods of making antigen-binding fragments should be known to one of skill in the art. For example, antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0042] Some embodiments of the present specification also provide an isolated nucleic acid molecule. The nucleic acid molecule comprises a nucleotide sequence encoding any of the antibodies or antigen-binding fragments as described above.

[0043] In some embodiments, the nucleotide sequence encoding the heavy chain variable region in the nucleic acid molecule comprises: a coding sequence of CDRH1 having the sequence of GGC TACTCCATCACCAGTGGTTATTAC (SEQ ID NO: 9); a coding sequence of CDRH2 having the sequence of ATAAGCTACGGCGGTTCCTAT (SEQ ID NO: 10); and a coding sequence of CDRH3 having the sequence of GCAACGTTCTATGATGGTTACTACAGGGCC (SEQ ID NO: 11). In some embodiments, the nucleotide sequence encoding the light chain variable region in the nucleic acid molecule comprises: a coding sequence of CDRL1 having the sequence of TCAAGTGTAAGTTAC (SEQ ID NO: 13), a coding sequence of CDRL2 having the sequence of CTCACATCC, and a coding sequence of CDRL3 having the sequence of CAGGAGTGGAGTGGTAACCCACCCATCACG (SEQ ID NO: 14).

[0044] In some embodiments, the nucleotide sequence encoding the heavy chain variable region can comprise a nucleotide sequence having codon degeneracy with any of the CDRH coding sequences described above, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding CDRH coding sequence described above.

[0045] As used herein, the term “codon degeneracy” refers to polynucleotides encoding a protein or polypeptide that include sequences that are degenerate due to the genetic code. One amino acid can be encoded by more than one triplet codon, such that different nucleotide sequences having codon degeneracy can encode the same protein or polypeptide.

[0046] In some embodiments, the nucleotide sequence encoding the heavy chain variable region can comprise a nucleotide sequence having codon degeneracy with any of the above-mentioned CDRH3-encoding sequences, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding above-mentioned CDRH3-encoding sequence.

[0047] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is CCGTGGACTCGTGAGCTTCTCAGTCTCTTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGGCGGTTCCTATAACTACAACCCCTCTCTCAAAAATCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTTTTTCCTGAACTTGAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCAACGTTCTATGATGGTTACTACAGGGCCTGGGGCCTCTGGACTCTGGTCACTGTCTCT (SEQ ID NO: 8).

[0048] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is CCGTGGACTCGTGAGCTTCTCAGTCTCTTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGGCGGTTCCTATAACTACAACCCCTCTCTCAAAAATCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTTTTTCCTGAACTTGAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCAACGTTCTATGATGGTTACTACAGGGCCTGGGGCCTCTGGACTCTGGTCACTGTCTCT (SEQ ID NO: 8).

[0049] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is a nucleotide sequence having codon degeneracy with the sequence as set forth in SEQ ID NO: 8, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 8.

[0050] In some embodiments, the nucleotide sequence encoding the light chain variable region is a nucleotide sequence having codon degeneracy with the sequence as set forth in SEQ ID NO: 12, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence as set forth in SEQ ID NO: 12.

[0051] Some embodiments of the present specification also provide a recombinant vector comprising any of the nucleic acid molecules as described previously. Methods of making recombinant vectors should be known to those skilled in the art, for example, the aforementioned nucleic acid molecules can be inserted into one or more expression vectors using recombinant DNA techniques and gene transfection methods, such that the nucleic acid molecules are operably linked to transcriptional and translational regulatory sequences.

[0052] Some embodiments of the present specification also provide a host cell comprising any of the nucleic acid molecules or the recombinant vector as described previously.

[0053] As used herein, the term “host cell” refers to a cell capable of introducing and stably maintaining an exogenous gene.

[0054] In some embodiments, the host cell can be a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic cells that can be used as host cells include, but are not limited to, rodent cells, primate cells, plant cells, insect cells, and yeast cells, etc. In some embodiments, the prokaryotic cells that can be used as host cells include, but are not limited to, Escherichia coli, Bacillus sp., and Salmonella sp., etc.

[0055] Some embodiments of the present specification also provide a method of preparing an antibody or antigen binding fragment that binds to etomidate, which uses any of the host cells as described previously. The method comprises: culturing the host cell; isolating the antibody or antigen binding fragment from the cell culture of the host cell.

[0056] Some embodiments of the present specification also provide a method of detecting etomidate in a sample. The method comprises: contacting the sample with an antibody or antigen binding fragment capable of binding to etomidate to form an antigen-antibody complex, the antibody or antigen binding fragment being an antibody or antigen binding fragment as described previously; detecting the antigen-antibody complex to qualitatively or quantitatively detect etomidate in the sample.

[0057] In some embodiments, the sample can be a biological sample, such as peripheral blood, urine, saliva, hair, interstitial fluid, fecal matter, etc. In some embodiments, the sample can be a non-biological sample, such as a sample taken from an article (e.g., a container) or a surface of an article. In some preferred embodiments, the sample comprises one or more of peripheral blood, urine, saliva, and hair.

[0058] In some embodiments, the antibody or antigen-binding fragment is labeled with a detectable label, and the antigen-antibody complex is detected by a signal generated by the detectable label.

[0059] In some embodiments, the antibody or antigen-binding fragment is unlabeled, and the antibody or antigen-binding fragment in the antigen-antibody complex as a capture antibody is bound with a detection antibody labeled with a detectable label, and the antibody complex is detected by a signal generated by the detectable label.

[0060] In some embodiments, the detectable label can be a radioisotope label or a non-radioisotope label. In some embodiments, the radioisotope label includes, but is not limited to, iodine 123, iodine 125, iodine 126, iodine 131, iodine 133, bromine 77, etc. In some embodiments, the non-radioisotope label can be selected from the group consisting of a dye, a hapten, a luminescent agent (e.g., a radioactive luminescent agent, a chemiluminescent agent, a bioluminescent agent, a fluorescent agent (e.g., fluorescein, GFP, Cy3, or Cy5, etc.), or a phosphorescent agent, etc.), an enzyme (e.g., luciferase, peroxidase, or alkaline phosphatase, etc.), an antibody, a receptor, and a ligand (e.g., biotin, avidin, etc.).

[0061] In some embodiments, the quantitative detection comprises: comparing a first detection signal obtained from the sample and a second detection signal obtained from a control or a calibrator, wherein the first detection signal is generated by an antigen-antibody complex formed by contacting the sample with the antibody or antigen-binding fragment, and the second detection signal is generated by an antigen-antibody complex formed by contacting the control or the calibrator with the antibody or antigen-binding fragment, the control or the calibrator containing a predetermined concentration of etomidate; and determining the concentration of etomidate in the sample based on the comparison result and the predetermined concentration.

[0062] Some embodiments of the present specification also provide a kit for detecting etomidate, comprising any antibody or antigen-binding fragment as described above.

[0063] In some embodiments, the kit is selected from the group consisting of a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme-linked immunodetection kit, a chemiluminescence kit, and an immunoturbidimetric detection kit.

[0064] Some embodiments of the present specification also provide the use of any antibody or antigen binding fragment, nucleic acid molecule, recombinant vector or host cell as described above in the detection of etomidate or in the preparation of a kit for detecting etomidate.

[0065] The following examples are some more specific illustrations of the embodiments related to some of the above embodiments. Some of the contents in these examples can also be replaced or combined with the corresponding contents in other embodiments to form new embodiments. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative experiments in the following examples are all set up with three repeated experiments, and the results are averaged. It should be understood that the following examples are for better explanation of the present application and are not intended to limit the present application.

[0066] Example

[0067] Method

[0068] Method for preparing etomidate-klh conjugate

[0069] The preparation of etomidate-KLH conjugate is as follows:

[0070] 1. Take 240 mg of etomidate and dissolve it in methanol. Add 1 N sodium hydroxide solution to the methanol solution and stir at room temperature overnight.

[0071] 2. After the reaction is completed, spin dry the solvent, add a small amount of water and 1 N hydrochloric acid solution to adjust the pH to neutral or weakly acidic.

[0072] 3. Extract 3 times with 20 mL of dichloromethane, collect the organic phase, add anhydrous sodium sulfate to dry, filter and spin dry the organic phase to obtain a light yellow viscous liquid, which is etomidate acid. Do not purify further, but directly proceed to the next step.

[0073] 4. Take the reaction product from the previous step, etomidate acid, and mix it with glycine hydrochloride and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) in a ratio of 1: (1.2-1.5): (1.5-2) in pyridine and stir at room temperature overnight.

[0074] 5. After the reaction is completed, spin dry the solvent and perform thin layer chromatography (TLC) with a developing solvent of petroleum ether: ethyl acetate = 1:1, and the product Rf = 0.3. After separation, obtain 170 mg of product.

[0075] 6. Dissolve the product from the previous step in methanol, take 2-2.5 eq of sodium hydroxide solution to prepare a 1 mol / L solution and add it to the methanol solution, and stir at room temperature overnight.

[0076] 7. After the reaction is complete, evaporate the solution to dryness, add a small amount of water and 1N hydrochloric acid solution to adjust the pH to neutral or weakly acidic.

[0077] 8. Extract three times with 20 mL of dichloromethane, collect the organic phase, dry with anhydrous sodium sulfate, filter and evaporate the organic phase to obtain etomidate hapten.

[0078] 9. Take 20 mg of etomidate hapten, 10.1 mg of N-hydroxysuccinimide (NHS), and 16.8 mg of EDC and dissolve them in N,N-dimethylformamide (DMF). Place the solution on a turntable and react at room temperature overnight to obtain etomidate hapten solution.

[0079] 10. Weigh 40 mg of hemocyanin (KLH) and dissolve it in 4 mL of PBS buffer. Stir well to obtain a KLH solution with a concentration of 10 mg / mL.

[0080] 11. Under ice bath conditions, slowly add the obtained KLH dropwise to the etomidate hapten solution prepared in step 9. Place the resulting mixture on a turntable at room temperature for 8 hours to obtain the artificial antigen mixture.

[0081] 12. Transfer the artificial antigen mixture into a dialysis bag and dialyze it 7 times with PBS buffer. After dialysis, centrifuge at 10000 r / min for 10 min, discard the precipitate, and take the supernatant to obtain the artificial antigen etomidate-KLH conjugate (hereinafter referred to as etomidate-KLH).

[0082] Method for immunizing animals

[0083] The antigen used for animal immunization is etomidate-KLH prepared using the aforementioned method. The animal immunization method specifically includes the following steps:

[0084] 1. Balb / c mice with the same weight and age were randomly divided into two groups: an aluminum adjuvant group (aluminum hydroxide adjuvant) and a non-aluminum adjuvant group.

[0085] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on the fifth day by blood collection through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice). The collected serum was stored at -80℃.

[0086] 3、Alum adjuvant (aluminum hydroxide adjuvant) group preparation method: before immunization, the antigen is diluted to the required corresponding dose (75 μg per mouse) in 75 μL of PBS to obtain an immunogen diluent, and mixed with aluminum adjuvant (1 mg per mouse), according to the volume immunogen diluent: adjuvant = 3:1 (i.e. 75 μL of immunogen diluent is added with 25 μL of adjuvant). Among them, the aluminum adjuvant is shaken before use, and the aluminum adjuvant (25 μL) is slowly added to the immunogen diluent; the aluminum adjuvant and the immunogen diluent are mixed for 30 min to make the adjuvant effectively adsorb the antigen. Subsequent animal experiment operation is carried out according to the immunization.

[0087] 4、No aluminum adjuvant group preparation method: the antigen is diluted to the required corresponding dose (75 μg per mouse) in 100 μL of PBS to obtain 100 μL of immunogen diluent. Subsequent animal experiment operation is carried out according to the immunization.

[0088] 5, subcutaneous injection at 2-week intervals: the experimental design is 3 times of immunization, but the supernatant of some mice is obtained by centrifugation after 7 days of each immunization injection, and the serum titer is detected first, and after 7 days of the last immunization, the maximum blood volume is taken by heart blood sampling, and the supernatant is obtained by centrifugation and stored at -80℃.

[0089] Method for detecting antigen binding capacity of antibodies

[0090] 1, indirect ELISA detection

[0091] The indirect ELISA detection is to detect the binding activity of etomidate by IgG antibody titer detection method.

[0092] (1) Coating plate: the coating antigen etomidate-KLH is diluted to 1 μg / mL using the coating diluent, and 50 μL of the prepared coating antigen is added to each well of the enzyme-labeled plate, which is placed in a 4℃ refrigerator for 24 h.

[0093] (2) After 24 h, take it out from the refrigerator and place it at 37℃ for 30 min, then discard the liquid in the well; wash the well with washing solution for 3 times, 3 min each time.

[0094] (3) Seal the enzyme-labeled reaction well: add 200 μL of blocking solution (for example, 5% skim milk solution) to each well, and seal at 37℃ for 90 min. After sealing, wash the well with washing solution for 3 times, 3 min each time.

[0095] (4) Add the sample to be detected: dilute the sample to be detected according to the required proportion, add the diluted sample to the enzyme-labeled reaction well, 50 μL per well, and place it in a 37℃ incubator for 30 min; wash the well with washing solution for 3 times, 3 min each time.

[0096] (5) Add enzyme-labeled antibody: add the second antibody (e.g., goat anti-mouse IgG-HRP) at an appropriate concentration according to the instructions, 50 μL per well, and react at 37°C for 30 min; wash as before.

[0097] (6) Add substrate solution: add 100 μL of substrate per well, and place at 37°C in the dark for 15-30 min.

[0098] (7) Stop the reaction: add 50 μL of a stopping solution (e.g., 2 mol / L H2SO4) per well to stop the reaction, and read the A450 value with an enzyme marker within 20 min to determine the antibody titer.

[0099] 2. Indirect competitive ELISA detection

[0100] The process of indirect competitive ELISA detection is different from that of indirect ELISA detection in that 50 μL of diluted small-molecule etomidate (or prototype etomidate) standard solution is first added as a competitive hapten to the reaction well in step (4) of the indirect competitive ELISA detection, and then the sample to be detected is added. The remaining steps of the indirect competitive ELISA detection method are the same as those of the indirect ELISA detection method.

[0101] Establishment of anti-etomidate monoclonal antibody hybridoma cell lines

[0102] Preparation of cell fusion-ready mice

[0103] Select 6-8-week-old healthy female Balb / c mice, and after immunization of the animals according to the above animal immunization method, detect the serum titer in the animals according to the above antibody titer detection method.

[0104] Three mice were immunized, and the mouse numbers were A0, A1, and A2 in order. Among them, A0 and A1 were aluminum adjuvant groups, and A2 was a group without aluminum adjuvant. After three immunizations, the serum titers of the immunized mice were detected by the above-mentioned indirect ELISA method and indirect competitive ELISA method, respectively. The detection data are shown in Table 1.

[0105] Table 1 - Serum titer detection data (A450 value)

[0106]

[0107] As can be seen from Table 1, by analyzing the indirect ELISA detection data, the orbital blood titers of the three mice after three immunizations were all >62500; by comparing the indirect ELISA detection data and the indirect competitive ELISA detection data, the orbital blood dilution concentrations of the three mice after three immunizations were all able to reach a competition rate of more than 50% at a dilution of 1:12500. Among them, the competition rate = (A450 未加竞争半抗原 -A450 加竞争半抗原 ) / A450未加竞争半抗原 *100. In summary, high-titer antibodies were detected in the serum of all three mice, indicating that subsequent cell fusion can be performed.

[0108] Cell fusion and screening of hybridoma cell lines

[0109] Three mice, numbered A0, A1, and A2, were selected for cell fusion with the myeloma cell line SP2 / 0, followed by cell screening and cloning. Specifically:

[0110] Eighteen positive wells were selected from the A0 mouse fusion screening and subcloning was performed. Following fusion screening, 48 positive wells with OD450 values ​​>2.2 were selected. These positive wells were serially diluted to determine their titers, and then subjected to a second subcloning screening. Three hybridoma cell lines were obtained and named A0-1, A0-2, and A0-3, respectively.

[0111] Sixteen positive wells were selected from the A1 mouse fusion screening and subcloning was performed. After fusion screening, 15 positive wells with OD450 values ​​>2.1 were selected. The titers of these positive wells were measured by serial dilution, and then a second and third subcloning screening was performed. Finally, three hybridoma cell lines were obtained and named A1-1, A1-2, and A1-3, respectively.

[0112] A total of 28 positive wells were selected from the A2 mouse fusion screening and subcloning was performed. Then, a second and third subcloning screening was conducted, and finally four hybridoma cell lines were obtained, which were named A2-1, A2-1, A2-3, and A2-4, respectively.

[0113] Immunological characterization of monoclonal antibodies

[0114] (1) Ascites titer detection of monoclonal antibody: Each hybridoma cell line was injected into the peritoneal cavity of F1 generation mice, and a total of 10 ascites samples were prepared. The antibody titer of the ascites was tested by indirect ELISA and indirect competitive ELISA. All ascites titer data are shown in Table 2.

[0115] Table 2 - Ascites antibody titer test data (A450 value)

[0116]

[0117] (2) Antibody titer test after purification: The ascites fluid was purified by 3.3% octanoic acid-thiamine precipitation method, and a total of 10 candidate monoclonal antibodies secreted by hybridoma cell lines were obtained. The titer test data of all candidate monoclonal antibodies are shown in Table 3.

[0118] Table 3 - Candidate Monoclonal Antibody Titer Detection Data (A450 Value)

[0119]

[0120] The data of Table 2 and Table 3 show that the 10 candidate monoclonal antibodies have good specific binding capacity to etomidate hapten. And the candidate monoclonal antibodies have good competition effect on the prototype etomidate, for example, candidate monoclonal antibodies A0-3 and A1-2. The above results show that the candidate monoclonal antibodies of the present embodiment can be applied to the immune detection of etomidate, for example, as the detection antibody of etomidate colloidal gold detection product.

[0121] The above candidate monoclonal antibodies are purified by Protein A / G antibody purification column, and the ELISA titer of the purified antibody is >1:128,000, and the purity is >90%.

[0122] Verification of anti-etomidate monoclonal antibody in etomidate antigen detection

[0123] In this embodiment, the immune colloidal gold detection method is taken as an example, and the anti-etomidate antibodies A0-3 and A1-2 are verified by the immune colloidal gold platform.

[0124] The specific experimental settings are as follows:

[0125] Experimental group: etomidate standard product of Hangzhou Anxu Biological Technology Co., Ltd.

[0126] Negative control group: urine sample.

[0127] The anti-etomidate antibodies A0-3 and A1-2 obtained by the above experiment are used as gold-labeled antibodies, and etomidate-KLH is used as a coated antigen to prepare an etomidate competitive method immune colloidal gold antigen detection kit. The reagents of the above experimental group and negative control group are added to the kit, and then the results are detected by using the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biological Technology Co., Ltd. The experimental data results are shown in Table 4.

[0128] Table 4-detection results

[0129]

[0130] Note: G4-G8 represents the level of strip color depth, the higher the value, the darker the color, + / - represents the color is slightly darker or slightly lighter than the level. The value of the added etomidate standard product is lower, indicating that the small molecule has a competition effect, and the antibody can be combined with the etomidate standard product.

[0131]

[0132]

[0133] ​​As can be seen from Table 4, the gradient of the A0-3 and A1-2 antibodies is good, and can be used in, for example, etomidate detection products, and the cut-off value can be made to be 500 ng / mL.

[0134] The above-mentioned verification experiment was used to evaluate the stability of different batches of A1-2 antibodies. Three batches of antibody samples were prepared, numbered A1-2-(1), A1-2-(2), and A1-2-(3), and the evaluation results are shown in Table 5.

[0135] Table 5 - Stability evaluation results table

[0136]

[0137] The evaluation results of Table 5 show that the stability of the A1-2 antibody is good. The A1-2 antibody is named anti-Etomidate-mab1, and the antibody can be used, for example, to prepare an etomidate antigen detection kit.

[0138] Sequencing analysis of the monoclonal antibody anti-Etomidate-mab1

[0139] The monoclonal antibody anti-HP-urease-mab1 was subjected to sequencing analysis, and the specific steps were as follows:

[0140] 1. Design primers for amplifying the heavy chain variable region (VH) and light chain variable region (VL) genes.

[0141] The primers for amplifying the VH and VL genes are as follows:

[0142] VH forward primer (VH-FOR): GCTCAGGGAAATAGCCCTTGAC (SEQ ID NO: 15);

[0143] VH reverse primer (VH-BACK): GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG (SEQ ID NO: 16);

[0144] VL forward primer (VL-FOR): GATGGTGGGAAGATGGATACAGTT (SEQ ID NO: 17);

[0145] VL reverse primer (VL-BACK): ATTWTCAGCTTCCTGCTAATC (SEQ ID NO: 18).

[0146] 2. Take the hybridoma cell strain of anti-Etomidate-mab1 in the logarithmic growth phase (about 10 7The total RNA of the cells was extracted according to the instruction of Trizol RNA extraction kit, and the first strand of cDNA was synthesized by reverse transcription using the total RNA as template. The VH / VL gene of the antibody was amplified by PCR using the above amplification product as template.

[0147] 3. The VH fragment (about 360 bp) and VL fragment (about 300 bp) of anti-Etomidate-mab 1 were recovered and sequenced. The obtained sequences are as follows:

[0148] VH encoding sequence (anti-Etomidate-mab 1-VH): 324 bp

[0149] TCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGG

[0150] TCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGG

[0151] AATGGATGGGCTACATAAGCTACGGCGGTTCCTATAACTACAACCCCTCTCTCAAAAA

[0152] TCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGG

[0153] TCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGG

[0154] TCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGG

[0155] VH amino acid sequence (anti-Etomidate-mab 1 protein): 108 aa

[0156] PWTRELLSLLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYGGSYNYNPSLK NRISITRDTSKNQFFLNLNSVTTEDTATYYCATFYDGYYRAWGLWTLVTVS.

[0157] VL encoding sequence (anti-Etomidate-mab 1 LVK): 315 bp

[0158] CAAATTGTTCTCACCCAGTCTCCAGCACTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAAGATCCTCCCCCAAAACCTGGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGGAGTGGAGTGGTAACCCACCCATCACGTTCGGTGCTGGGACCAAGCTGGAG.

[0159] VL amino acid sequence (anti-Etomidate-mab1 LV kappa protein): 105 aa

[0160] QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKTWIYLTSNLASGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQEWSGNPPITFGAGTKLE.

[0161] The beneficial effects brought by the antibody or antigen binding fragment binding etomidate and the related application thereof disclosed in the embodiments of the present specification include but are not limited to: (1) the monoclonal antibody binding etomidate provided in the embodiments of the present specification has good specificity and sensitivity; (2) the monoclonal antibody binding etomidate provided in the present application can be used for immunoblotting, immunofluorescence and other immunological detection, for example, it can be used for detecting whether etomidate exists in the sample through a colloidal gold detection platform. It should be noted that different embodiments may have different beneficial effects, and in different embodiments, the beneficial effects that can be produced can be any one or a combination of several of the above, or any other beneficial effects that can be obtained.

[0162] Those skilled in the art should understand that the above embodiments only illustrate the present application, and do not limit the present application. Any modification, equivalent replacement and change made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0163] Also, the specification can use certain terminology to describe embodiments of the specification. "One embodiment," "an embodiment," and / or "some embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the specification. Therefore, it is emphasized and should be appreciated that a described "embodiment" or "one embodiment" or "an alternative embodiment" is not necessarily a separate and different embodiment from the described embodiment or one or more alternative embodiments. Moreover, described features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0164] Some embodiments use numerical terms to describe quantities of ingredients, properties, and the like. It is understood that such numerical terms used in embodiments descriptions are approximations. Unless otherwise specified, "approximately," "about," or "substantially" preceding a numerical depiction means ±20% of the value of the numerical depiction. Accordingly, numerical parameters in the specification and claims are approximations. Although these numerical parameters are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values in some embodiments specifically recited are only approximations because the precise value will vary depending on the desired characteristics of the embodiments described herein.

[0165] Each patent, patent application, publication, and other material cited in this specification is hereby incorporated by reference in its entirety. In the event of inconsistencies between the disclosure of this specification and the materials incorporated by reference, the disclosure of this specification shall prevail. To the extent the description, definitions, and / or terminology used in the materials incorporated by reference is inconsistent or in conflict with the description, definitions, and / or terminology in this specification, the description, definitions, and / or terminology in this specification shall prevail.

[0166] Finally, it should be understood that the embodiments described herein are merely exemplary of the principles of the application. Other variations having essentially the same structure and function are within the scope of the present application. Therefore, this application is not intended to be limited to the embodiments described herein but is to be accorded the full scope consistent with the claims, and their equivalents.

Claims

1. An antibody or antigen binding fragment that binds etomidate, characterized in that, The antibody or antigen-binding fragment comprises: a heavy chain variable region comprising a CDRH1 of an amino acid sequence as set forth in SEQ ID NO: 2, a CDRH2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a CDRH3 of an amino acid sequence as set forth in SEQ ID NO: 4; a light chain variable region comprising a CDRL1 of an amino acid sequence as set forth in SEQ ID NO: 6, a CDRL2 of an amino acid sequence of LTS, and a CDRL3 of an amino acid sequence as set forth in SEQ ID NO:

7.

2. The antibody or antigen-binding fragment of claim 1, wherein The amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:

5.

3. The antibody or antigen-binding fragment of claim 1, wherein The antibody is a monoclonal antibody or a multispecific antibody.

4. The antibody or antigen-binding fragment of claim 1, wherein The antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, and scFv.

5. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment of any one of claims 1-3.

6. The nucleic acid molecule of claim 5, wherein, The nucleic acid molecule comprises: a nucleotide sequence encoding a heavy chain variable region comprising a coding sequence of CDRH1 as set forth in SEQ ID NO: 9, a coding sequence of CDRH2 as set forth in SEQ ID NO: 10, and a coding sequence of CDRH3 as set forth in SEQ ID NO: 11; a nucleotide sequence encoding a light chain variable region comprising a coding sequence of CDRL1 as set forth in SEQ ID NO: 13, a coding sequence of CDRL2 of sequence CTCACATCC, and a coding sequence of CDRL3 as set forth in SEQ ID NO:

14.

7. The nucleic acid molecule of claim 5, wherein The nucleotide sequence encoding the heavy chain variable region is as set forth in SEQ ID NO: 8; the nucleotide sequence encoding the light chain variable region is as set forth in SEQ ID NO:

12.

8. A recombinant vector comprising the nucleic acid molecule of any one of claims 5-7.

9. A host cell comprising the nucleic acid molecule of any one of claims 5-7 or the recombinant vector of claim 8.

10. A method of preparing an antibody or antigen binding fragment that binds to etomidate, characterized in that, The method comprises: culturing the host cell of claim 9; isolating the antibody or antigen-binding fragment from the cell culture of the host cell.

11. A method for detecting etomidate in a sample for non-therapeutic and diagnostic purposes, characterized in that, The method comprises: contacting the sample with an antibody or antigen-binding fragment capable of binding to etomidate to form an antigen-antibody complex, the antibody or antigen-binding fragment being the antibody or antigen-binding fragment of any one of claims 1-4; detecting the antigen-antibody complex to qualitatively or quantitatively detect etomidate in the sample.

12. The method of claim 11, wherein, The sample comprises one or more of peripheral blood, urine, saliva, and hair.

13. A kit for detecting etomidate comprising the antibody or antigen-binding fragment of any one of claims 1-4.

14. Use of the antibody or antigen-binding fragment of any one of claims 1-4 for detecting etomidate for non-therapeutic and diagnostic purposes or for preparing a kit for detecting etomidate.

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