Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.
In this context, the etistin Zhuo Kangyuan binding molecule refers broadly to any molecule capable of specifically binding etistin Zhuo Kangyuan. The etiazepine Zhuo Kangyuan binding molecule may comprise an antibody as defined herein, or an antigen binding fragment thereof, directed against an etiazepine antigen. Etistin Zhuo Kangyuan binding molecules also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
Herein, etiazepine Zhuo Kangyuan includes etiazepine molecules or conjugates of etiazepine molecules with other molecules.
Herein, antibodies generally refer to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). The terms "antibody" and "full length antibody" include polyclonal antibodies and monoclonal antibodies. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the terms "antibodies" and "immunoglobulins" are used interchangeably.
An antigen binding fragment is a fragment comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from, but not limited to, any one of F (ab ')2, fab', fab, fv (consisting of VH and VL), scFv (single chain antibody, with a linker peptide between VH and VL), dsFv (disulfide stabilized Fv antibody (disulfide stabilized FV FRAGMENTS, dsFv)), bispecific antibody, nanobody, and antibody minimal recognition unit. In addition to the functional fragments described above, any fragment whose half-life has been increased is included.
By "variable region" or "variable domain" of an antibody is meant a domain of an antibody that recognizes and binds an antigen at the amino terminus of the heavy or light chain of the antibody, the composition and arrangement of the amino acids of the segment determining the specificity of the antibody for recognizing the antigen. The heavy chain variable domain may be referred to as a "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-DETERMINING REGION (CDRs) (also known as hypervariable regions) linked by 4 Framework Regions (FR). The framework regions and the ranges of the CDRs have been precisely defined, for example in Kabat (see sequence of immunologically important proteins ((Sequences of Proteins of Immunological Interest), E.Kabat et al) and Chothia), any of the CDR determination methods well known in the art, including combinations of methods, can identify the CDRs of the variable domain the CDRs in each chain are held closely together by the FRs to form the variable region, and in general the variable regions VL/VH of the heavy and light chains can be obtained by ligating the CDRs numbered from the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combined arrangement.
The term "constant region" or "constant domain" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination. The heavy chain of an antibody has one variable domain (VH) followed by a number of constant domains or regions, such as one or more of the hinge, CH1, CH2, CH3 and CH4, the CH1 domain being adjacent to the VH domain and at the amino terminus of the hinge region of the heavy chain of the antibody and not forming part of the Fc region of the antibody, the hinge region comprising the portion of the heavy chain molecule linking the CH1 domain to the CH2 domain, the N-terminus of CH2 typically being the CH3 domain, the CH3 domain typically forming the C-terminal portion of the antibody, and in some antibody types, such as IgM and IgE, the constant region also comprising the CH4 domain. The constant regions of antibodies may be derived from IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD, as well as subclasses and mutant forms thereof.
The invention is not limited by the manner in which the etiquette Zhuo Kangyuan binding molecule is obtained. In some alternative embodiments, the corresponding antibody can be obtained upon expression in a cell after ligation to the vector using a polynucleotide encoding the etiquette Zhuo Kangyuan binding molecule. The vector may be introduced into eukaryotic cells, particularly mammalian cells, and constructed to express the etiquette Zhuo Kangyuan binding molecule. In other alternative embodiments, the etiquette Zhuo Kangyuan binding molecule may also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as by an automated peptide synthesizer (e.g., such as those sold by Applied BioSystems, etc.), the antigen binding fragment may also optionally be produced by enzymatic cleavage of the antigen binding molecule (including whole antibodies), such as pepsin or papain cleavage, or by chemical cleavage, such as by chemical reductive cleavage of disulfide bonds to obtain the antigen binding fragment described above.
The terms "specifically recognizes," "selectively binds," and "specifically binds" or the like refer to the binding of an antigen binding molecule to an epitope on a predetermined antigen. Typically, the antigen binding molecules bind with a Kd value of about less than 10-5 M, for example about less than 10-5M、10-6M、10-7M、10-8M、10-9 M or 10-10 M or less. The Kd value of an antibody can be determined using methods well established in the art. Other standard assays for evaluating the binding capacity of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, western blot, RIA, and flow cytometry analysis.
The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide encodes an antibody or antigen binding fragment against the above-described etizepine, optionally encoding the sense or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.
The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.
Such vectors are well known to those skilled in the art and include, but are not limited to, plasmids, episomal plasmids, microcircular DNA, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).
The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. Offspring may differ from primary cells morphologically and/or in genomic DNA by natural, accidental, or deliberate mutations that are not necessarily identical to primary cells. "transformant" and "transformed cell" include primary test cells and cultures derived therefrom. The cells may be prokaryotic cells, such as, but not limited to, E.coli, bacillus or Staphylococcus, or eukaryotic cells. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells including, but not limited to, chinese Hamster Ovary (CHO) cells, NSO, SP2 cells 20 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), a549 cells, 3T3 cells, and HEK-293 cells.
Herein, the term "conjugate" refers to a compound formed by covalent attachment of at least two molecules, which may be either direct covalent attachment or covalent attachment through a linker.
As used herein, the term "signalizing agent" refers to a substance capable of providing a detectable signal that can be directly observed by the naked eye or detected by conventional means acceptable in the art, which may provide a signal directly, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence, or indirectly, such as by catalyzing a reaction with a particular substrate to produce any of the above-described signals, by a subsequent reaction in which the signalizing agent participates.
In this document, unless otherwise indicated, any number is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual such relationship, order, or importance between such entities or actions, e.g., numbers (I), (II).
In this document, unless otherwise indicated, "optionally," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.
In this document, the terms "comprises" or "comprising" are intended to include the stated element, integer or step, but not to exclude any other element, integer or step.
As used herein, the articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.
In a first aspect, there is provided an etiquette Zhuo Kangyuan binding molecule comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region, and complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region;
The HCDR1 comprises an amino acid sequence identical to HCDR1 of the heavy chain variable region shown in SEQ ID NO.1, the HCDR2 comprises an amino acid sequence identical to HCDR2 of the heavy chain variable region shown in SEQ ID NO.1, the HCDR3 comprises an amino acid sequence identical to HCDR3 of the heavy chain variable region shown in SEQ ID NO.1, and the LCDR1 comprises an amino acid sequence identical to LCDR1 of the light chain variable region shown in SEQ ID NO.2, the LCDR2 comprises an amino acid sequence identical to LCDR2 of the light chain variable region shown in SEQ ID NO.2, and the LCDR3 comprises an amino acid sequence identical to LCDR3 of the light chain variable region shown in SEQ ID NO. 2.
It will be appreciated that the other regions of the variable regions shown in SEQ ID NOS.1 and 2, from which CDR regions are removed, are not intended to limit the etiquette Zhuo Kangyuan binding molecules provided by the present invention, e.g. in some embodiments the antigen binding molecules may differ from the framework regions in the variable regions shown in SEQ ID NOS.1 and 2 if they contain framework regions. The CDR regions in the variable regions shown in SEQ ID nos. 1 and 2 may be partitioned according to any means known in the art. Alternatively, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable regions are defined by any one or a combination of multiple definition systems Kabat, chothia, IMGT, ABM or contacts. The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1 are shown in Table 1, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.2 are shown in Table 2, respectively, by way of Kabat, chothia, IMGT, ABM or Contact definition.
TABLE 1
TABLE 2
In alternative embodiments, the etilazide Zhuo Kangyuan binding molecule removes CDR regions, and the remaining sequence source species include one or more of rabbits, cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, chickens, humans, and mutants thereof.
In alternative embodiments, the etiquette Zhuo Kangyuan binding molecule contains at least one framework region of a heavy chain variable region, for example, one, two, three or four framework regions of a heavy chain variable region.
In alternative embodiments, the etiquette Zhuo Kangyuan binding molecule contains at least one framework region of a light chain variable region, for example, one, two, three or four framework regions of a light chain variable region.
In alternative embodiments, the etiquette Zhuo Kangyuan binding molecule comprises a framework region of a heavy chain variable region, wherein framework region FR1 of the heavy chain variable region comprises an amino acid sequence identical to FR1 of the heavy chain variable region shown in SEQ ID NO.1, and/or wherein framework region FR2 comprises an amino acid sequence identical to FR2 of the heavy chain variable region shown in SEQ ID NO.1, and/or wherein framework region FR3 comprises an amino acid sequence identical to FR3 of the heavy chain variable region shown in SEQ ID NO.1, and/or wherein framework region FR4 comprises an amino acid sequence identical to FR4 of the heavy chain variable region shown in SEQ ID NO. 1.
In alternative embodiments, the etiquette Zhuo Kangyuan binding molecule comprises a framework region of a light chain variable region, wherein framework region FR1 of the light chain variable region comprises an amino acid sequence identical to FR1 of the light chain variable region shown in SEQ ID NO.2, and/or wherein framework region FR2 comprises an amino acid sequence identical to FR2 of the light chain variable region shown in SEQ ID NO.2, and/or wherein framework region FR3 comprises an amino acid sequence identical to FR3 of the light chain variable region shown in SEQ ID NO.2, and/or wherein framework region FR4 comprises an amino acid sequence identical to FR4 of the light chain variable region shown in SEQ ID NO. 2.
In an alternative embodiment, the heavy chain variable region of the etigen Zhuo Kangyuan binding molecule is shown as SEQ ID NO.1 and the light chain variable region is shown as SEQ ID NO. 2.
In alternative embodiments, the etiquette Zhuo Kangyuan binding fragment includes one or more of F (ab ')2, fab', fab, fv, scFv, dsFv, and an antibody minimal recognition unit.
In alternative embodiments, the etiquette Zhuo Kangyuan binding molecule comprises part or all of the sequence of a constant region.
In alternative embodiments, the constant region sequence is selected from the group consisting of the sequence of part or all of the constant region of any of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD, including subclasses and mutant forms thereof.
In alternative embodiments, the etiquette Zhuo Kangyuan binding molecule is a full length antibody, including a heavy chain constant region and a light chain constant region, the light chain of the antibody is a kappa chain, and the antibody is an IgG1 antibody.
In alternative embodiments, the full length antibody is a murine antibody.
In a second aspect, there is also provided a biomaterial selected from any one of (i) to (iii):
(i) A polynucleotide comprising a nucleotide sequence encoding the etiquette Zhuo Kangyuan binding molecule of the first aspect.
In an alternative embodiment, the polynucleotide comprises a fragment of nucleotide sequence shown as SEQ ID NO.3 encoding the heavy chain variable region shown as SEQ ID NO. 1.
In an alternative embodiment, the polynucleotide comprises a fragment of nucleotide sequence shown as SEQ ID NO.4 encoding the light chain variable region shown as SEQ ID NO. 2.
(Ii) A vector carrying the heavy polynucleotide of (i) above.
(Iii) A cell carrying the polynucleotide of (i) above, or comprising the vector of (ii) above, or expressing the etiquette Zhuo Kangyuan binding molecule.
In a third aspect, there is also provided an antibody-containing composition comprising the following (I) and (II):
(I) The etiquette Zhuo Kangyuan binding molecule of the first aspect.
(II) a signal and/or a solid support.
The antibody-containing compositions of (I) and (II) may be linked or separated, including physical and/or chemical linkages, such as but not limited to attachment of the etiquette Zhuo Kangyuan binding molecule of the first aspect to a signal species and/or solid support by chemical bonding or adsorption.
In alternative embodiments, the antibody-containing composition is a conjugate.
In alternative embodiments, the signal species include, but are not limited to, one or more of enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, colloidal selenium, colloidal silver, colloidal carbon, quantum dots, biotin, streptavidin, radionuclides, radiocontrast agents, paramagnetic ions, metals, and photosensitizers.
In alternative embodiments, the solid support includes, but is not limited to, microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices, or lateral flow devices, and more specifically, for example, can be, but is not limited to, enzyme-labeled wells, immunochromatographic strips, or magnetic beads.
In a fourth aspect, there is also provided a method of preparing the etigen Zhuo Kangyuan binding molecule of the first aspect, comprising culturing a cell as described in the second aspect, and then isolating and purifying the etigen Zhuo Kangyuan binding molecule.
In an alternative embodiment, the method of making further comprises transforming a polynucleotide encoding a polypeptide comprising the etiquette Zhuo Kangyuan binding molecule into a cell and expressing.
In a fifth aspect there is also provided the use of the etiazepine Zhuo Kangyuan binding molecule of the first aspect, or the biomaterial of the second aspect, or the antibody-containing composition of the third aspect, or the method of preparation of the fourth aspect, for the detection of etiazepine at non-diagnostic and therapeutic destinations, or for the preparation of a product for the detection of etiazepine.
In a sixth aspect, there is also provided a kit for detecting etiazepine, the kit comprising the etiazepine Zhuo Kangyuan-binding molecule of the first aspect, or the antibody-containing composition of the third aspect. The kit comprises one or more reagents, which may be identical to the reagent when the kit comprises one reagent.
In alternative embodiments, the kit comprises an immunochromatographic assay kit, an ELISA assay kit, an immunomagnetic particle assay kit, an immunofluorescent assay kit, or an immunoblotting assay kit.
In alternative embodiments, the kit further comprises reagents and/or consumables for detection including, but not limited to, one or more of primers, probes, buffers, dyes, diluents, wash solutions, chromogenic solutions, lysates, negative controls, positive controls, and blank controls, and consumables including, but not limited to, solid phase carriers such as magnetic beads, enzyme-labeled wells, nitrocellulose membranes, or the like. The reagent composition of the kit can be selected by those skilled in the art according to the specific detection means, and the present invention is not limited thereto.
In an alternative embodiment, the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip. The competitive immunochromatography test paper is coated with the antigen binding molecule in any one of the embodiments, and is used for capturing the etizepine in the sample to be detected.
In an alternative embodiment, the detection line of the immunochromatographic test paper is coated with etiazepine coupled with a protein carrier, and the binding pad is coated with the etilazide Zhuo Kangyuan binding molecule marked by a signal substance. It will be appreciated that the immunochromatographic test strip has a conventional immunochromatographic test strip structure known in the art, and an exemplary immunochromatographic test strip structure is constituted by a sample pad, a binding pad and a detection pad, the detection pad being provided with a detection line and a quality control line along the flow direction of a sample. When the sample contains the etiazepine small molecule, the etiazepine in the sample and the etiazepine Zhuo Kangyuan coated on the detection line simultaneously competitively bind with the etiazepine Zhuo Kangyuan binding molecule on the binding pad, and the etiazepine in the sample can preferentially competitively bind with the etiazepine Zhuo Kangyuan binding molecule on the binding pad. The higher the content of etidiazin in the sample, the less etidiazepine Zhuo Kangyuan binding molecules remain on the detection line capable of binding to the etidiazepine antigen when chromatographed to the detection line, and therefore the less detectable signals on the detection line. The etiquette Zhuo Kangyuan binding molecule marked by the signal substance flows to the quality control line to be captured by the antibody coated by the quality control line, and a measurable signal appears, which indicates that the immunochromatographic test paper can be used.
In an alternative embodiment, the signal substance-labeled etiquette Zhuo Kangyuan binding molecule coated on the immunochromatography test paper is a full-length antibody, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
In an alternative embodiment, the anti-etistin Zhuo Kangyuan binding molecule coated on the binding pad is labeled with colloidal gold.
In alternative embodiments, the protein carrier coupled to the eptifibatide includes, but is not limited to, bovine gamma globulin, bovine serum albumin, ovalbumin, bemycetin, human serum albumin, thyroglobulin, tetanus toxoid, synthetic polylysine, or purified protein derivatives, preferably bovine gamma globulin.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1 preparation of etiazepine-BGG antigen
1. 50Mg of the product I is dissolved in 1ml of DMF to prepare 50mg/ml solution, NHS and EDC are added to activate the room temperature reaction overnight, and the mol ratio of the product I to the NHS and the EDC is 1:1.2-1.5:1.2-1.5.
2. 100 Mg of BGG was weighed and dissolved in 10 ml of PBS buffer to prepare a 10mg/ml solution, and the activated small molecules were slowly added to the protein for 8 hours of reaction.
3. And (3) filling the coupled antigen into a dialysis bag, changing the liquid once every two hours, performing liquid changing for more than six times, collecting samples, and measuring the concentration by Lowry to obtain the synthetic antigen etiazepine-BGG.
The experimental mice were immunized stepwise with the chemically synthesized etiazepine-BGG antigen.
Example 2 preparation of monoclonal antibody anti-Isotonitazene-mab 1.
1. Preparation of monoclonal antibody anti-Isotonitazene-mab 1:
and selecting a 6-8 week old Balb/c healthy female mouse, and performing immune injection according to a pre-designated immunization scheme. As immunogen, immunizing BALB/c mouse, extracting spleen lymphocyte of the immunized mouse, fusing lymphocyte with myeloma cell SP2/0 of the mouse by cell fusion technology, and obtaining hybridoma cell strain for stably secreting anti-eptifibatide monoclonal antibody after two rounds of subcloning and screening, thereby obtaining the anti-eptifibatide monoclonal antibody.
The test mice were immunized stepwise with the etizepine-BGG antigen prepared in example 1.
The animal immunity experiment comprises the following specific steps:
1. Balb/c mice with consistent weight and age average were randomly divided into 2 groups, with aluminum adjuvant (aluminum hydroxide adjuvant) and without aluminum adjuvant.
2. Before the experiment starts, mice are respectively collected with preimmune serum (the preimmune serum is collected on the fifth day, blood is taken through eyeballs, a proper amount of blood is taken, and the normal state of the mice is ensured), and the collected serum is stored at-80 ℃.
3. The aluminum adjuvant (aluminum hydroxide adjuvant) group was prepared by diluting each antigen to the corresponding dose (75. Mu.g/mouse) in 75. Mu.L PBS before immunization, mixing with alum adjuvant (1 mg/mouse) according to the volume of antigen: adjuvant=3:1 (i.e., 25. Mu.L adjuvant was added to 75. Mu.L immunogen dilution), shaking the adjuvant before use, slowly dropping the injected adjuvant (25. Mu.L) into the immunogen solution, and mixing the adjuvant and immunogen dilution thoroughly, and mixing the two thoroughly for 30 minutes. The adjuvant can effectively adsorb antigen, and the subsequent operation is performed according to the experimental operation of immune animals.
4. No aluminum adjuvant group, antigen was diluted in 100. Mu.L PBS at the corresponding dose (75. Mu.g/mouse) in the above table, 100. Mu.L immunogen, and the follow-up was performed according to the experimental procedure of immunized animals.
5. Subcutaneous injections at intervals of 2 weeks, the experiment was designed to be 4 immunization modes, but after 4 days of each immunization, the eyeballs were respectively subjected to blood collection, part of the mouse supernatant was obtained by centrifugation, serum titer detection was performed first, after 7 days of the last immunization, the heart was subjected to blood collection to obtain the maximum blood volume, and the supernatant was obtained by centrifugation and stored at-80 ℃.
6. Serum titers were measured.
(1) 2 Mice were immunized and the mice were numbered A0, A1 in sequence. Serum titers were measured after 3 immunizations were completed. The test data are shown in Table 3 below, and the test method is as follows:
and respectively using the etiazepine-BGG antigen and the etiazepine small molecule as antigen competition detection, and using the etiazepine-BGG antigen as a coating antigen to carry out indirect ELISA and competition ELISA to detect serum titers of immunized mice.
The ELISA plate was coated with 1. Mu.g/ml of coating antigen diluted with coating liquid 50. Mu.l/well and 4 coated overnight, and then the plate was washed 3 times with washing liquid (PBST) (same applies below), 200. Mu.l of blocking liquid (5% nonfat milk powder) was added to each well, and the plate was placed in a 37-incubator for 2 hours, after the washing was removed, 50. Mu.l of diluted serum was added to each well, and reacted in a 37-incubator for 30 minutes, and after the washing, 50. Mu.l of goat anti-mouse IgG-HRP solution was added and reacted in a 37-incubator for 30 minutes. After washing, 100. Mu.l of substrate solution was added, the reaction was developed in a 37℃in a dark place for 10min, and finally, 2mol/L H2SO4 and 50. Mu.l of the reaction were added to terminate the reaction, and the A450 value was read by an ELISA reader. The three post-exemption orbital blood titers of 3 mice were >312500.
The indirect competition ELISA process is mostly the same as the indirect ELISA process, except that after the ELISA plate is blocked by a blocking solution and washed, 50 μl of diluted 200ng/mL small molecule etiapine standard solution is added, and 50 μl of diluted serum antibody is added, so that the final concentration of the small molecule reaches 100ng/mL, and the rest steps are the same. 100ng/ml small molecule etiazepine competition detection can reach more than 80% at the time of 1:62500, and fusion can be arranged.
TABLE 3 serum potency assay data
The immune spleen cells are fused with myeloma cell line SP2/0 cells, the fused cells are screened by HAT selection medium (the HAT selection medium contains hypoxanthine, aminopterin and thymine), ELISA positive screening and subcloning are carried out on the fused cells, the screened positive monoclonal is adopted, ascites is taken, antibodies are purified by Protein A/G antibody purification columns, the ELISA titer of the purified antibodies is more than 1:128,000, and the purity is more than 90%.
2. Enzyme-linked ELISA assay recognizes the binding activity of etiazepine.
IgG antibody titer detection mode
1. Coating the bottom plate, namely diluting the used antigen to 3 mug/ml by using coating diluent, adding 100 mug of prepared coating liquid into each hole, placing the mixture in a 4-refrigerator, and placing the mixture for 24 hours.
2. After 24h, the wells were equilibrated for 30min at 37 after removal from the refrigerator, after which the liquid in the wells was discarded, and washed 3 times with washing liquid full of wells for 3min each time.
3. And (3) sealing the enzyme-labeled reaction holes, namely adding 200 mu.l of 5% calf serum into each hole, sealing for 90min, and washing 3 times by using washing liquid after sealing, wherein each time is 3min.
4. Adding a sample to be detected, namely diluting the sample according to a required proportion, adding 100 mu l of diluted sample into enzyme-labeled reaction holes, placing the diluted sample in 37,90min, and washing the sample with washing liquid for 3 times in full holes for 3min each time.
5. Adding enzyme-labeled antibody, adding secondary antibody with proper concentration according to the instruction, and adding 100 μl of the same washing solution into each well between 37 and 90 min.
6. And adding substrate liquid, namely adding 100 mu l of substrate into each hole, and placing the mixture in a 37-light-shielding place for 15-30 min.
7. Stop reaction 50. Mu.l of stop solution was added to each well to measure the experimental results within 20 min.
3. The monoclonal antibody was tested for binding activity to recognize etiazepine.
1. Cell fusion and clone screening data
The serial numbers of the mice are A0 and A1 in sequence, and two rounds of fusion are completed.
A0 mice were screened for fusion and 34 positive wells were picked for subcloning, and finally 12 cell lines were completed. For the first subclone screening, 25 positive clones with OD450 value more than 3.0 are selected for multiple dilution detection titer. And then carrying out secondary subcloning screening to obtain 7 positive cell strains, and selecting 10 cell strains for third subcloning to obtain 5 positive cell strains.
A1 mice were screened for fusion to pick 44 positive wells, and finally 14 cell lines were completed. For the first subclone selection, 32 positive clones with OD450 values >3.0 were selected in total. And then carrying out secondary subcloning screening to completely obtain 7 cell strains. 18 cell lines were selected for the third subcloning, which completed 5 cell lines. 5 cell lines were selected for the fourth subcloning, which completed 2 cell lines.
A total of 26 complete cell lines were obtained after the secondary cell fusion.
2. Cell supernatant preparation and assay data
30-40 ML of culture supernatant is prepared for each complete cell strain, 26 cell culture supernatants are prepared in total, and the detection titer data of partial supernatants are shown in the following table:
TABLE 4 cell supernatant titer assay data
3. After the conditions for antibody purification and detection data and determination of the supernatant subtype, the antibodies all belong to IGg1 type, and 26 antibodies are obtained in total through ProteinG affinity purification. The potency detection data for a portion of the antibodies are shown in the following table:
TABLE 5 purification of antibody titer assay data
The monoclonal antibodies of 26 cell lines have good specific binding capacity to the etiazepine antigen through detection of the supernatant titer and the antibody titer, and the results of competition experiments performed by using small-molecule etiazepine have good competition, so that 26 antibodies are used for testing etiazepine Zhuo Jiaoti gold products.
Example 3 use of monoclonal antibody anti-Isotonitazene-mab1 in products.
The 26 antibodies to etiazepine were validated by an immune colloidal gold platform.
The immune colloidal gold platform is immune chromatography test paper, and comprises a sample pad, a combination pad and a detection pad according to the chromatography direction, wherein the detection pad is provided with a detection line and a quality control line. The binding pad is coated with the screened monoclonal antibody marked by colloidal gold, and the detection line is coated with the etiazepine-BGG antigen of the embodiment 1.
Specifically, the experimental group is to use 26 antibodies from etiazepine Zhuo Kangti # to 26# obtained in the above experiment to detect etiazepine standard of the biological technology limited company in Hangzhou An Xu. The reagent is loaded, then the result is detected by using POCT detection instrument ACG1000 (ID-A003) of the biological technology limited company of Hangzhou An Xu, the negative and gradient of the 6# antibody are better, and partial experimental data are shown in the following table:
TABLE 6 results of partial antibody initial evaluations
Note that G3-G9 represent a level of the strip color shade of the test strip, with higher values representing darker colors and +/-representing a slightly darker or lighter color than that level. The lower value of the added etiazepine standard substance indicates that the small molecules have competitive action, and the antibody can be combined with the etiazepine standard substance.
The results show that the antibody No. 6 has a better gradient, can be used in the product of etiazepine, and has a cut-off value of 500ng/ml, and then the stability of the antibody No. 6 is evaluated, 3 batches of antibody small samples are prepared, and the evaluation results are shown in the following table:
TABLE 7 batch stability assessment results for 6# antibody 3
750Ng/ml of the eptifibatide standard was tested using antibody # 6 and the test results are shown in Table 8 and FIG. 1.
TABLE 8 detection results of antibody # 6 against 750ng/ml etiazepine standard
The No. 6 antibody is named as anti-Isotonitazene-mab1 according to the evaluation result of the product, and can be used for detecting the etiquette Zhuo Kangyuan detection kit.
Example 4 monoclonal antibody anti-Isotonitazene-mab1 heavy chain V region (VH) and light chain V region (VL) sequence analysis.
1. Primers were designed to amplify the heavy chain V region (VH) and light chain V region (VL) genes.
2. Taking hybridoma cell lines (about 107 cells) in the logarithmic growth phase of anti-Isotonitazene-mab1, extracting total RNA of the cells according to the instruction of a Trizol RNA extraction kit, performing reverse transcription by taking the total RNA as a template to synthesize a cDNA first strand, and performing PCR (polymerase chain reaction) amplification on the VH/VL genes of the antibody by taking the amplified products as templates.
3. The heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-Isotonitazene-mab1 were recovered and sequenced by the company.
4. The VH/VL gene sequences were analyzed and the resulting sequences were as follows:
Variable region sequence of heavy chain:
anti-Isotonitazene-mab1-VH:351bp
GAGGTGCAGCTTCTTGAGTCTGGGGGAGGCCTGGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGTCATGGCTTGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGATGTCTGAGGACACGGCCATGTATTACTGTGTAAGAGGGACTACGGTAGTAGCTGGGGATTACTGGGGCCAAGGAACCGCTCTCACAGTCTCAGCT(SEQ ID NO.3)
anti-Isotonitazene-mab1protein:117aa
EVQLLESGGGLVKPGGSLKLSCAASGFTFSTYVMAWIRQTPEKRLEWVATISSGG STYYPDSVKGRFTISRDNARNILYLQMSSLMSEDTAMYYCVRGTTVVAGDYWGQGT ALTVSA(SEQ ID NO.1)
heavy chain variable region CDR analysis, results are shown in table 1:
TABLE 1
Variable region sequence of light chain:
anti-Isotonitazene-mab1LVκ:321bp
CAAGTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACGTGCACTGGTACCAGCAGAAGTCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTC TGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACACTCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTGGTAACCCACCCATCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO.4)
anti-Isotonitazene-mab1LVκprotein:107aa
QVVLSQSPAILSASPGEKVTMTCRASSSVSYVHWYQQKSGSSPKPWIYATSNLAS GVPARFSGSGSGTSYSLTLSRVEAEDAATYYCQQWSGNPPITFGGGTKLEIK(SEQ ID NO.2)
light chain variable region CDR analysis, results are shown in table 2:
TABLE 2
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.