Antibody conjugate and use thereof

Abstract

The present invention provides a latex microsphere conjugate or a fluorescent microsphere conjugate, comprising an antibody and a conjugate moiety, wherein 1-27 amino acid residues are chimerically inserted after the C-terminal lysine residue of the heavy chain of the antibody. The conjugate has high activity or high sensitivity.

Description

Antibody conjugates and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202410420186.X, filed on April 08, 2024, entitled “Antibody conjugates and uses thereof”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biotechnology, in particular, the present application relates to an antibody conjugate and uses thereof, more particularly, the present application relates to a latex microsphere conjugate or a fluorescent microsphere conjugate, an antibody, a nucleic acid molecule, a vector, a cell or a host, a test paper or a kit and uses thereof, a method for preparing a conjugate and a method for immunodetection. BACKGROUND

[0004] Conjugates formed by antibodies (or antigens) and solid phases have been widely used in the field of immunodetection, latex microsphere conjugates and fluorescent microsphere conjugates are two commonly used conjugates.

[0005] Latex microspheres are a commonly used solid phase in biochemical detection, which are often used in latex agglutination test and latex immunoturbidimetry to detect target substances. Latex agglutination test (LAT) is an indirect agglutination test using latex microspheres as carriers. The soluble antibody (or antigen) is adsorbed on the surface of the carrier, and then reacts with the corresponding antigen (or antibody). In the presence of electrolytes under suitable conditions, an agglutination reaction can occur. This method has the advantages of being fast and simple, easy to store, and relatively accurate. Latex immunoturbidimetry is a method in which antibodies are combined with latex microspheres. When antigen-antibody complexes are formed, the absorbance of the reaction is enhanced. By comparing the turbidity of the reaction solution with that of a standard solution treated in the same way, the content of antigen in the sample can be calculated. The entire analysis process can be completed in a few minutes using a biochemical analyzer.

[0006] Immunochromatography technology is a solid phase technology that uses a strip-shaped fibrous layer material. The sample solution moves on the chromatography material through capillary action, and at the same time, the labeled analyte in the sample reacts specifically with the antigen / antibody coated on the chromatography material, enriching on the detection line. Through the color or photoelectric signal amplification effect of the label, the detection purpose is achieved. The newly developed immunofluorescence technology is a fluorescent microsphere immunochromatography quantitative detection method. This method fully utilizes the excellent properties of fluorescent microspheres and combines immunochromatography technology to realize fluorescent quantitative detection based on the optimization of test strip structure and composition materials.

[0007] The activity of the conjugate affects the sensitivity and accuracy of the immunoassay, and the optimization of the antibody material is a key factor in preparing a high-activity conjugate. Therefore, there is an urgent need to provide an antibody material suitable for a latex microsphere or fluorescent microsphere platform. SUMMARY

[0008] The present application aims to provide a latex microsphere conjugate or a fluorescent microsphere conjugate, an antibody, a nucleic acid molecule, a vector, a cell or a host, a test paper or a kit, and the use thereof, a method for preparing a conjugate, and a method for immunoassay.

[0009] In a first aspect of the present application, the present application provides a latex microsphere conjugate. According to an embodiment of the present application, the conjugate comprises an antibody and a latex microsphere, wherein 1 to 27 amino acid residues are chimerized after a C-terminal lysine residue of a heavy chain of the antibody,

[0010] i) wherein, when 1 amino acid residue is chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine;

[0011] ii) wherein, when 2 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 2 amino acid residues are two histidines;

[0012] iii) wherein, when 4 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 4 amino acid residues are F-C-P-F or K-L-L-C; and

[0013] iiii) wherein, when 27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 27 amino acid residues are a sequence as shown in SEQ ID NO: 7.

[0014] In a second aspect of the present application, the present application provides a fluorescent microsphere conjugate. According to an embodiment of the present application, the conjugate comprises an antibody and a fluorescent microsphere, wherein 1 to 27 amino acid residues are chimerized after a C-terminal lysine residue of a heavy chain of the antibody,

[0015] i) wherein, when 1 amino acid residue is chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine;

[0016] ii) wherein, when 2 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 2 amino acid residues are two histidines;

[0017] iii) wherein, when 4 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 4 amino acid residues are F-C-P-F or K-L-L-C; and

[0018] iiii) wherein, when 27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is as set forth in SEQ ID NO: 7.

[0019] In a third aspect of the present application, the present application provides an antibody. According to embodiments of the present application, 1-27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody,

[0020] i) wherein, when 1 amino acid residue is chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine;

[0021] ii) wherein, when 2 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 2 amino acid residues are two histidines;

[0022] iii) wherein, when 4 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 4 amino acid residues are F-C-P-F or K-L-L-C; and

[0023] iiii) wherein, when 27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is as set forth in SEQ ID NO: 7.

[0024] In a fourth aspect of the present application, the present application provides a method for preparing the latex microsphere conjugate or the fluorescent microsphere conjugate described above. According to embodiments of the present application, the method comprises: incubating a latex microsphere or a fluorescent microsphere, a crosslinking agent and the antibody described in the third aspect.

[0025] In a fifth aspect of the present application, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the antibody in the conjugate described in the first aspect or the second aspect or the antibody described in the third aspect.

[0026] In a sixth aspect of the present application, the present application provides a vector. According to embodiments of the present application, the vector comprises the nucleic acid molecule described in the fifth aspect.

[0027] In a seventh aspect of the present application, the present application provides a cell or a host. According to embodiments of the present application, the cell or the host comprises: the nucleic acid molecule described in the fifth aspect or the vector described in the sixth aspect.

[0028] In an eighth aspect of the present application, the present application provides use of the latex microsphere conjugate of the first aspect or the antibody of the third aspect in the preparation of a latex agglutination or latex immunoturbidimetric assay kit.

[0029] In a ninth aspect of the present application, the present application provides use of the fluorescent microsphere conjugate of the second aspect or the antibody of the third aspect in the preparation of an immunochromatographic test paper.

[0030] In a tenth aspect of the present application, the present application provides a latex immunoturbidimetric or latex agglutination assay kit, which comprises the latex microsphere conjugate of the first aspect or the antibody of the third aspect according to an embodiment of the present application.

[0031] In an eleventh aspect of the present application, the present application provides an immunochromatographic test paper, which comprises the fluorescent microsphere conjugate of the second aspect or the antibody of the third aspect according to an embodiment of the present application.

[0032] In a twelfth aspect of the present application, the present application provides a method for immunodetection. According to an embodiment of the present application, the method comprises: contacting the latex microsphere conjugate of the first aspect, the fluorescent microsphere conjugate of the second aspect, the antibody of the third aspect, the assay kit of the tenth aspect or the immunochromatographic test paper of the eleventh aspect with a sample to be detected to form an immune complex.

[0033] The amino acid sequence of the present application is shown as follows:

[0034] Additional aspects and advantages of the present application will be in part apparent and in part pointed out below in the description of embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0035] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments, taken in conjunction with the accompanying drawings, in which:

[0036] FIG. 1 is a mass spectrum result chart of each mutation group according to an embodiment of the present application. DETAILED DESCRIPTION

[0037] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0038] It should be noted that the terms "first", "second", and the like, do not denote any order, quantity, combination or important / primary use, but are used in the description for the purpose of clear and distinct identification only. Thus, a feature defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality" is two or more, unless otherwise specified.

[0039] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. For ranges, the endpoints or any other point within the range can be combined with other endpoints or points within the range to form another range. For numerical values, any numerical value can be combined with any other numerical value to form another numerical value.

[0040] In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the general use of their respective fields. Abbreviations for amino acid residues are in accord with the standard three letter and / or one letter codes as set forth in the art.

[0041] In this document, the terms "comprising" or "including" or "containing" means "including but not limited to", and thus specifies the presence of stated features, integers, steps, or components, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0042] In this document, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0043] In this document, the term "at least 80% identity" means at least 80% identity to a reference sequence, which can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.

[0044] In the present context, the terms "identity", "homology" or "similarity" are used when describing an amino acid sequence relative to a reference sequence, are determined by the percent of identical amino acids between two amino acid sequences by conventional methods, see, e.g., Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that can be used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to compare sequences, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or the WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program from Intelligenetics, Mountain View, California.

[0045] In the present context, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable region, e.g. an amino acid sequence providing an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH), and a light chain (L) variable region (abbreviated herein as VL). In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments) as well as whole antibodies, e.g. intact immunoglobulins of the types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chain of an immunoglobulin can be of the kappa type or the lambda type.

[0046] In the present context, the term "vector" generally refers to a nucleic acid molecule that is capable of inserting itself into a suitable host and self-replicating, which transfers the inserted nucleic acid molecule to and / or between cells or hosts. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, the vector can produce a desired expression product by culturing a suitable cell or host containing the vector.

[0047] In the present context, the term "cell" generally refers to a cell having a unique trait stably inherited by modification or recombination of the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant vector can be introduced. The term "transformed" or "transfected" used herein means introducing a nucleic acid (e.g. a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present application, and can be used for expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0048] The present application proposes a latex microsphere conjugate or a fluorescent microsphere conjugate, an antibody, a nucleic acid molecule, a vector, a cell or a host, a test paper or a kit, uses thereof, a method of preparing a conjugate, and a method of immunoassay, which will be described in detail below, respectively.

[0049] Latex microsphere conjugate

[0050] In a first aspect of the present application, the present application provides a latex microsphere conjugate. According to an embodiment of the present application, the conjugate comprises an antibody and a latex microsphere, wherein 1 to 27 amino acid residues are chimerized after a C-terminal lysine residue of a heavy chain of the antibody,

[0051] i) wherein, when 1 amino acid residue is chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine;

[0052] ii) wherein, when 2 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 2 amino acid residues are two histidines;

[0053] iii) wherein, when 4 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 4 amino acid residues are F-C-P-F or K-L-L-C; and

[0054] iiii) wherein, when 27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 27 amino acid residues are a sequence as set forth in SEQ ID NO: 7.

[0055] The conjugate of the present application has improved activity or detection sensitivity.

[0056] Herein, the "C-terminal lysine residue of a heavy chain" refers to a lysine residue at the carboxyl terminus of a heavy chain of an antibody before chimerization, and the antibody before chimerization can be a wild-type antibody or an engineered antibody.

[0057] The C-terminal lysine residue of a heavy chain of a wild-type antibody is well known in the art, for example, see the IMGT website (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). Exemplary C-terminal lysine residues of heavy chains of human, murine, ovine or rabbit wild-type antibodies are as follows (bold subscript):

[0058] Human IgG H:

[0059] Murine IgG H G1:

[0060] Murine IgG H G2a:

[0061] Murine IgG H G2b:

[0062] Murine IgG H G2c:

[0063] Murine IgG H G3:

[0064] Sheep IgHG:

[0065] Rabbit IgHG:

[0066] In the present text, the term "IgHG" refers to the heavy chain of an IgG antibody; the term "IgHG1" refers to the heavy chain of an IgG1 antibody; the term "IgHG2a" refers to the heavy chain of an IgG2a antibody; the term "IgHG2b" refers to the heavy chain of an IgG2b antibody; and other analogies.

[0067] In the present text, the term "wild type" refers to an antibody type that naturally occurs and has not been modified by mutation.

[0068] In the present text, "F-C-P-F" or "K-L-L-C" is a short peptide consisting of four amino acids, the amino acids represented by the one-letter abbreviations are well known to those skilled in the art.

[0069] In some alternative embodiments of the present application, the latex microspheres described above comprise polystyrene latex microspheres.

[0070] Fluorescent microsphere conjugate

[0071] In a second aspect of the present application, the present application proposes a fluorescent microsphere conjugate. According to embodiments of the present application, the conjugate comprises an antibody and a fluorescent microsphere, wherein 1-27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody,

[0072] i) wherein, when 1 amino acid residue is chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine;

[0073] ii) wherein, when 2 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 2 amino acid residues are two histidines;

[0074] iii) wherein, when 4 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 4 amino acid residues are F-C-P-F or K-L-L-C; and

[0075] iiii) wherein, when 27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is shown in SEQ ID NO: 7.

[0076] The conjugate of the present application has improved activity or sensitivity.

[0077] In some alternative embodiments of the present application, the fluorescent microspheres described above comprise fluorescently labeled polystyrene latex microspheres.

[0078] antibody

[0079] In a third aspect of the present application, the present application provides an antibody. According to embodiments of the present application, the antibody has 1-27 amino acid residues chimerized after the C-terminal lysine residue of the heavy chain of the antibody,

[0080] i) wherein, when 1 amino acid residue is chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine;

[0081] ii) wherein, when 2 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 2 amino acid residues are two histidines;

[0082] iii) wherein, when 4 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the 4 amino acid residues are F-C-P-F or K-L-L-C; and

[0083] iiii) wherein, when 27 amino acid residues are chimerized after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is shown as SEQ ID NO: 7.

[0084] The conjugate of the present application has improved activity or sensitivity.

[0085] In some alternative embodiments of the present application, the antibody in the conjugate of the first aspect or the second aspect described above, or the antibody of the third aspect, can further comprise at least one of the following technical features:

[0086] In some alternative embodiments of the present application, the antibody has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 amino acid residues chimerized after the C-terminal lysine residue of the heavy chain.

[0087] In some alternative embodiments of the present application, the antibody is selected from IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA, IgM, IgE, IgD or a combination thereof.

[0088] In some alternative embodiments of the present application, the antibody is a murine antibody, a human antibody, a bovine antibody, a horse antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, or a goose antibody.

[0089] In some alternative embodiments of the present application, the antibody chimeric before contains a heavy chain constant region of SEQ ID NO: 5 or an amino acid sequence having at least 80% identity with SEQ ID NO: 5.

[0090] In some alternative embodiments of the present application, the antibody includes a heavy chain constant region including an amino acid sequence as shown in any one of SEQ ID NO: 9-18 or an amino acid sequence having at least 80% identity with the amino acid sequence as shown in any one of SEQ ID NO: 9-18.

[0091] In some alternative embodiments of the present application, the antibody includes a heavy chain constant region including an amino acid sequence as shown in any one of SEQ ID NO: 9-18 or an amino acid sequence having at least 80% identity with the amino acid sequence as shown in any one of SEQ ID NO: 9-18.

[0092] In some alternative embodiments of the present application, the antibody includes two heavy chains and two light chains.

[0093] In some alternative embodiments of the present application, the antibody includes a light chain constant region including an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% identity with the amino acid sequence as shown in SEQ ID NO: 6.

[0094] In this application, the antibody of the present application also includes a heavy chain variable region and a light chain variable region, which can be determined according to the target antigen, as long as the target antigen can be combined, and the specific sequence is not limited, all within the protection scope of the present application.

[0095] In some alternative embodiments of the present application, the target antigen is an antigen related to myocardial injury, infectious disease, endocrine, tumor or drug.

[0096] In some alternative embodiments of the present application, the antigen is an antigen related to myocardial infarction, viral or bacterial infectious disease.

[0097] In some alternative embodiments of the present application, the antigen includes, but is not limited to, CKMB antigen, HIV antigen, hepatitis A virus antigen, hepatitis B virus antigen, hepatitis C virus antigen, hepatitis D virus antigen, hepatitis E virus antigen, hepatitis G virus antigen, rubella virus antigen, human cytomegalovirus antigen, herpes simplex virus type 1 antigen, herpes simplex virus type 2 antigen, rabies virus antigen, human T lymphotropic leukemia virus antigen, dengue virus antigen, coronavirus antigen, human papillomavirus antigen, West Nile virus antigen, forest encephalitis virus antigen, measles virus antigen, influenza virus antigen, parainfluenza virus antigen, varicella virus antigen, echovirus type antigen, coxsackievirus antigen, Japanese encephalitis virus antigen, coxsackievirus antigen, Epstein-Barr virus antigen, mumps virus antigen, Treponema pallidum antigen, Borrelia burgdorferi antigen, Chlamydia trachomatis antigen, Chlamydia pneumoniae antigen, Chlamydia psittaci antigen, Ureaplasma urealyticum antigen, Mycoplasma pneumoniae antigen, Mycobacterium tuberculosis antigen, Helicobacter pylori antigen, gonococcus antigen, malaria antigen, Trypanosoma cruzi antigen, Toxoplasma gondii antigen.

[0098] In some alternative embodiments of the present application, the antibody has a light chain and a heavy chain selected from any of the following groups or having at least 80% identity thereto:

[0099] It should be noted that based on the amino acid sequences of the antibodies of the present application, it is easy for those skilled in the art to conceive of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibodies, for example, to isolate and purify the antibodies from the culture products of recombinant cells capable of recombinantly expressing the antibodies as described in any of the above, which is easy for those skilled in the art to achieve, and based on this, regardless of the technology used to prepare the antibodies of the present disclosure, they all fall within the scope of protection of the present disclosure.

[0100] Method for preparing conjugate

[0101] In a fourth aspect of the present application, a method for preparing the latex microsphere conjugate or the fluorescent microsphere conjugate described above is provided. According to an embodiment of the present application, the method comprises incubating the latex microspheres or the fluorescent microspheres, a crosslinking agent, and the antibody of the third aspect. In this way, the conjugate can be effectively prepared, and the preparation method is simple.

[0102] Nucleic acid molecule, vector, cell or host

[0103] In the process of preparing or obtaining the antibodies in the conjugate of the first aspect or the second aspect or the antibody of the third aspect, nucleic acid molecules expressing these antibodies can be used, which are linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0104] In a fifth aspect, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the antibody in the conjugate of the first aspect or the second aspect, or the antibody of the third aspect.

[0105] According to embodiments of the present application, the nucleic acid molecule comprises DNA or RNA.

[0106] It should be noted that, for the nucleic acid molecule mentioned herein, it is understood by those skilled in the art that either one of the complementary double strands, or both, are actually included. For convenience, in this document, although only one strand is given in most cases, the other complementary strand is actually disclosed. In addition, the sequence of the molecule in the present application includes DNA or RNA form, and the disclosure of one means the disclosure of the other.

[0107] In a sixth aspect, the present application provides a vector. According to embodiments of the present application, the vector comprises the nucleic acid molecule of the fifth aspect.

[0108] In the process of connecting the above-mentioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid molecule can be operably linked to the control elements. "Operably linked" herein means that the exogenous gene is connected to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be plasmids, bacteriophages, etc. After the vector according to some specific embodiments of the present application is introduced into a suitable recipient cell, the expression of the aforementioned antibody can be effectively realized under the mediation of the regulation system, and then the antibody can be obtained in large quantities in vitro.

[0109] In some specific embodiments of the present application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0110] In an optional embodiment of the present application, the expression vector is a plasmid expression vector or a lentivirus expression vector.

[0111] In a seventh aspect, the present application provides a cell or host. According to embodiments of the present application, the cell or host comprises: the nucleic acid molecule of the fifth aspect or the vector of the sixth aspect.

[0112] Under suitable conditions, the cell can effectively express the aforementioned antibody in the cell.

[0113] According to an embodiment of the present application, the cell is obtained by introducing the vector of the sixth aspect into the cell.

[0114] It should be noted that the cell of the present application is not particularly limited, and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces, or Giardia lamblia, etc. The eukaryotic cell includes Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc. fungal cells, grasshopper cells, tobacco cells, BHK cells, CHO cells, COS cells, myeloma cells, and other mammalian cells.

[0115] In an alternative embodiment of the present application, the cell is a mammalian cell, including BHK cells, CHO cells, NSO cells, or COS cells, and does not include animal reproductive cells, fertilized eggs, or embryonic stem cells.

[0116] It should be noted that the "suitable conditions" in the present application refer to conditions suitable for the expression of the antibody of the present application. Those skilled in the art will readily understand that the conditions suitable for the expression of the antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody according to the specific environment of the laboratory.

[0117] Use, kit, or immunodetection method

[0118] In an eighth aspect of the present application, the present application provides a use of the latex microsphere conjugate of the first aspect or the antibody of the third aspect in the preparation of a latex agglutination or latex immunoturbidimetric detection kit.

[0119] In a ninth aspect of the present application, the present application provides a use of the fluorescent microsphere conjugate of the second aspect or the antibody of the third aspect in the preparation of an immunochromatography test paper.

[0120] In a tenth aspect of the present application, the present application provides a latex immunoturbidimetric or latex agglutination detection kit, according to an embodiment of the present application, the detection kit comprises the latex microsphere conjugate of the first aspect or the antibody of the third aspect.

[0121] In an alternative embodiment of the present application, the latex immunoturbidimetric kit comprises a first reagent, a second reagent, and a calibrator, and the first reagent or the second reagent comprises the latex microsphere conjugate of the first aspect or the antibody of the third aspect.

[0122] In an eleventh aspect, the present application provides an immunochromatographic test strip, according to embodiments of the present application, the immunochromatographic test strip comprises the fluorescent microsphere conjugate of the second aspect or the antibody of the third aspect.

[0123] In an optional embodiment of the present application, the immunochromatographic test strip comprises a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad, and the conjugate pad is provided with the fluorescent microsphere conjugate of the second aspect or the antibody of the third aspect.

[0124] In an optional embodiment of the present application, the conjugate pad is selected from a glass fiber membrane.

[0125] In an optional embodiment of the present application, the nitrocellulose membrane is provided with a detection line.

[0126] In an optional embodiment of the present application, the nitrocellulose membrane is further provided with a quality control line.

[0127] In a twelfth aspect, the present application provides a method for immunodetection, according to embodiments of the present application, the method comprises: contacting the latex microsphere conjugate of the first aspect, the fluorescent microsphere conjugate of the second aspect, the antibody of the third aspect, the detection kit of the tenth aspect or the immunochromatographic test strip of the eleventh aspect with a sample to be detected to form an immune complex.

[0128] In an optional embodiment of the present application, based on the signal of the immune complex, it is determined whether the sample to be detected contains a target substance or the content of the target substance.

[0129] It should be noted that the "sample to be detected" described above can be a sample to be detected of a patient, such as a serum sample; or can be a sample that is not necessarily a patient sample, for example, in scientific research, the above method is only used to detect the presence of an antigen in a sample or its content, and does not involve disease diagnosis.

[0130] The schemes of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0131] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or contemplated herein are standard methodologies. The materials, methods, and examples are illustrative only and not limiting.

[0132] Practicing the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ordinary skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0133] Example 1: Materials and Methods

[0134] 1. Heavy chain C-terminal design

[0135] Five mutations were designed for antibody 1 (heavy chain and light chain sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), see Table 1; eight mutations were designed for antibody 2 (heavy chain and light chain sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively), see Table 2.

[0136] Table 1: Antibody 1 heavy chain end design

[0137] Table 2: Antibody 2 heavy chain end design

[0138] 2, plasmid construction expression

[0139] 2.1 Construction of recombinant antibody expression plasmid

[0140] In this embodiment, the restriction enzymes and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Gene Sequencing Company. pcDNA TM 3.4 vector is a constructed recombinant antibody eukaryotic expression vector, which is modified to introduce a multiple cloning enzyme digestion site, and is hereinafter referred to as 3.4A expression vector.

[0141] Design primers specific for the VL and VH genes of the wild-type antibody, with restriction enzyme digestion sites and protective bases at both ends, and amplify the VL gene fragment and the VH gene fragment by PCR. The VL gene fragment and the VH gene fragment of the corresponding mutation group shown in Table 1 or Table 2 are obtained by primer design.

[0142] The VL gene fragment and the VH gene fragment are double-digested with restriction enzymes, and the 3.4A vector is double-digested with restriction enzymes. After purification and recovery of the fragments and the vector, the VL gene fragment and the VH gene are respectively connected to the 3.4A expression vector, and the recombinant expression plasmids of Heavy Chain and Light Chain are obtained, respectively.

[0143] 2.2, expression of recombinant antibody samples

[0144] The HEK293 cells were recovered in advance and subcultured in a 200ml system to make the cell density reach 3-5x10 6cells / ml, cell viability >95%; centrifugal washing of cells, re-dissolution with culture medium, and adjustment of cell density to 2.9 x 10 6 cells / ml, as a cell dilution. Prepare a plasmid DNA dilution and a transfection reagent dilution containing the recombinant expression plasmid obtained in step 2.3 of the present application, respectively, using culture medium. Add the transfection reagent dilution to the plasmid DNA dilution, mix, and stand at room temperature for 15 min; slowly add the mixture to the cell dilution within 1 min, mix, take a sample for counting, record and observe the cell viability after transfection, and place in a 35°C constant temperature incubator for culture, with a rotation speed of 120 rpm and a CO2 content of 8%; after 13 days, centrifugal sampling. The results show that the expression amounts of the above-mentioned mutant antibodies and the wild-type antibody are equivalent, both reaching about 300 mg / L.

[0145] 3. HPLC-SEC (high performance liquid chromatography - size exclusion chromatography)

[0146] Take 20 μg of the purified multi-group recombinant antibody sample obtained in step 2.2 of the present example, and determine the SEC purity of the recombinant antibody sample by high performance liquid chromatography - size exclusion chromatography (HPLC-SEC). The detection results show that the wild-type antibody and the antibodies of each of the above-mentioned mutant groups are all purified to obtain samples with excellent purity.

[0147] 4. Mass spectrometry detection

[0148] Intact molecular weight of mAb

[0149] Experimental procedure

[0150] Direct injection: mAb sample is dissolved in ddH2O to 1 ug / ul, and directly injected.

[0151] Deglycosylated molecular weight of mAb

[0152] Experimental procedure: 1. Removal of salt from glycoprotein buffer; 2. Release of N-glycan chain; 3. Removal of free N-glycan; 4. Recovery of deglycosylated mAb for standby use.

[0153] Molecular weight of mAb light and heavy chains

[0154] Experimental procedure:

[0155] Experiment 1 (non-denaturing reduction): 1. Reduction: add 95 ul 100 ug mAb to a 0.6 ml EP tube, and then add 5 ul 1M DTT to make the final concentration 50 mM. Metal bath at 57°C, shake, and incubate for 45 min. After incubation, aspirate the sample into a bottle for standby use.

[0156] Experiment 2 (reduction after denaturation): 1. Denaturation: Add 50ul 100ug mAb to a 1.5ml EP tube, then add 50ul 8M guanidine hydrochloride, final concentration of guanidine hydrochloride is 4M. Vigorously shake on a shaker for 15s to make the mAb completely denatured. 2. Reduction: Add 5ul 1M DTT to make the final concentration 50mM. Metal bath 57℃, shake, incubate for 45min. After incubation, suck the sample into a sample bottle and keep for later use.

[0157] 5. Biochemical activity identification

[0158] 5.1. Take 160ul of 10% polystyrene carboxyl latex with a particle size of 188nm, and dilute to 2ml with 10mmol / L HEPES (PH 7.5) buffer solution;

[0159] 5.2. Slowly add 50ul (10mg / ml) EDC solution, 50ul (10mg / ml) NHS solution to step 5.1, after stirring at room temperature for 20min, dilute to 10ml with 10mmol / L HEPES (PH 7.5) buffer solution;

[0160] 5.3. Take 70ul of antibody solution respectively, add 50ul of BSA (10%);

[0161] 5.4. Slowly add the diluted antibody solution of step 5.3 to step 5.2, stir at room temperature for 3h;

[0162] 5.5. Centrifuge at 15000rpm for 40min, resuspend with 10mmol / L PBS (containing 0.2% Tween 20, 0.1% BSA, 10% sucrose) by ultrasonic, which is test reagent 2.

[0163] 5.6. Use 50mmol / L glycine buffer as test reagent 1 to form the experimental test reagent with the above test reagent 2;

[0164] 5.7. Calibration: use positive patient serum for dilution preparation, detection instrument: Mindray BS-480. Activity difference = (absorbance of mutant group-absorbance of wild type) / absorbance of wild type; Average activity difference = (total activity difference-activity difference under zero concentration calibration) / (difference group number-1).

[0165] 6. Fluorescence activity identification

[0166] 6.1. Antibody labeling: Take 100ul 1% fluorescent microspheres (Merck, Eu-030) into 900ul activation buffer, mix well, centrifuge to remove supernatant, add 1mL activation buffer, ultrasonic mixing, then add activator (EDC NHS), avoid light, shake mixing for 20min, centrifuge to remove supernatant, add the same volume of coupling buffer as the microspheres, ultrasonic mixing, then add labeled antibody 0.2-0.4mg, avoid light, shake mixing for 3h, finally add blocking buffer for blocking, avoid light, shake mixing for 45min, then terminate labeling, centrifuge to remove supernatant, re-dissolve the microspheres with microsphere storage solution, ultrasonic mixing, and store at 4°C for use.

[0167] 6.2. Preparation of microsphere working solution: dilute the antibody label to a final concentration of 10-20% with microsphere diluent, and use a spray pad instrument to spray the label on glass fiber.

[0168] 6.3. Preparation of dried microsphere pad: place the sprayed fluorescent pad in a 50°C oven and dry for more than 2h.

[0169] 6.4. Sample pad treatment: dilute the blocking agent to 0.4mg / ml with sample pad diluent, spread on glass fiber, and place in a 50°C oven to dry overnight.

[0170] 6.5. NC membrane coating: dilute the coating antibody to 1.0mg / ml with coating diluent, then coat; place in a 50°C oven to dry overnight.

[0171] 6.6. Preparation of fluorescent chromatography strip: cut the fluorescent chromatography strip with a strip cutter, assemble, and then add sample for detection.

[0172] 6.7. Detection (1) Quality control: recombinant antigen, dilute with sample diluent to correspond to the detection of paired antibodies (2) Detection method: after adding sample for 15min, perform instrument judgment. Activity difference = (mutant group activity - wild type activity) / wild type activity; Average activity difference = total activity difference / number of difference groups

[0173] Example 2 detection results

[0174] 1. Complete molecular weight analysis

[0175] Table 3: Table 1 mutant group mass spectrometry analysis results

[0176] Table 4: Table 2 mutant group mass spectrometry analysis results

[0177] 2. Biochemical detection activity data as follows:

[0178] Table 5: Table 1 mutant group biochemical activity results

[0179] Table 6-1: Biochemical activity results of mutation groups of Table 2

[0180] Table 6-2: Biochemical activity results of mutation groups of Table 2

[0181] As can be seen from Table 5, Table 6-1 and 6-2, the above mutation groups have improved biochemical detection activity compared with the wild type, and the same designed mutation groups have improved detection activity in different projects.

[0182] Meanwhile, Table 6-1 and 6-2 show that the activity of mutation group 2-1, mutation group 2-2, mutation group 2-9, and mutation group 2-10 has been saturated, therefore, the coating concentration of the antibody is reduced for re-determination, and the results show that they have higher biochemical detection activity than the wild type, and the exemplary results are shown in the following Table 6-3.

[0183] Table 6-3: Biochemical activity results of mutation group 2-9 and mutation group 2-10

[0184] 3, the fluorescence POCT detection activity data are as follows:

[0185] Table 7: Fluorescence POCT activity results of mutation groups of Table 1

[0186] Table 8-1: Fluorescence POCT activity results of mutation groups of Table 2

[0187] Table 8-2: Fluorescence POCT activity results of mutation groups of Table 2

[0188] Table 7 and Table 8 show that the mutation groups have significantly improved detection activity compared with the wild type in the fluorescence POCT platform. The improvement effect of mutation group 1-1 and mutation group 1-5 in the fluorescence POCT platform is not as obvious as that in the biochemical platform.

[0189] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction. Industrial applicability

[0190] The latex microsphere conjugate or fluorescent microsphere conjugate provided by the present disclosure has higher sensitivity and accuracy, and the activity of the conjugate formed after the antibody is conjugated with the latex microsphere or fluorescent microsphere is not affected. Therefore, the latex microsphere conjugate or fluorescent microsphere conjugate provided by the present disclosure has excellent practical performance and broad market application prospects.

[0191] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A latex microsphere conjugate comprising an antibody and latex microspheres, wherein: The antibody has 1 to 27 amino acid residues inserted after the C-terminal lysine residue of the heavy chain. i) wherein, when an amino acid residue is chimeric after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine; ii) wherein, when two amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the two amino acid residues are two histidines; iii) wherein, when four amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the four amino acid residues are FCPF or KLLC; and iiii) wherein, when 27 amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is as shown in SEQ ID NO:

7.

2. A fluorescent microsphere conjugate comprising an antibody and fluorescent microspheres, wherein: The antibody has 1 to 27 amino acid residues inserted after the C-terminal lysine residue of the heavy chain. i) wherein, when an amino acid residue is chimeric after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine; ii) wherein, when two amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the two amino acid residues are two histidines; iii) wherein, when four amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the four amino acid residues are FCPF or KLLC; and iiii) wherein, when 27 amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is as shown in SEQ ID NO:

7.

3. An antibody, characterized in that The antibody has 1 to 27 amino acid residues inserted after the C-terminal lysine residue of the heavy chain. i) wherein, when an amino acid residue is chimeric after the C-terminal lysine residue of the heavy chain of the antibody, the amino acid residue is cysteine, histidine, alanine, valine, tyrosine or serine; ii) wherein, when two amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the two amino acid residues are two histidines; iii) wherein, when four amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the four amino acid residues are FCPF or KLLC; and iiii) wherein, when 27 amino acid residues are embedded after the C-terminal lysine residue of the heavy chain of the antibody, the sequence of the 27 amino acid residues is as shown in SEQ ID NO:

7.

4. The latex microsphere conjugate according to claim 1, the fluorescent microsphere conjugate according to claim 2, or the antibody according to claim 3, characterized in that: The antibody is selected from IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA, IgM, IgE or IgD or a combination thereof; Optionally, the antibody is a mouse antibody, a human antibody, a bovine antibody, a horse antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody or a goose antibody; Optionally, the antibody contains a heavy chain constant region of SEQ ID NO: 5 or an amino acid sequence having at least 80% identity with SEQ ID NO: 5 before chimerization; Optionally, the antibody comprises a heavy chain constant region comprising an amino acid sequence selected from any one of SEQ ID NOs: 9 to 18, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 9 to 18; Optionally, the antibody comprises two heavy chains and two light chains; Optionally, the antibody comprises a light chain constant region comprising the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

6.

5. A method for preparing the latex microsphere conjugate according to claim 1 or the fluorescent microsphere conjugate according to claim 2, characterized in that: The method comprises incubating latex microspheres or fluorescent microspheres, a cross-linking agent and the antibody according to claim 3.

6. A nucleic acid molecule, vector, cell or host, characterized in that: The nucleic acid molecule encodes the antibody in the conjugate of claim 1 or 2 or the antibody of claim 3; The vector includes the above-mentioned nucleic acid molecule; the cell or host includes: the above-mentioned nucleic acid molecule or vector.

7. Use of the latex microsphere conjugate according to claim 1 or the antibody according to claim 3 in preparing a latex agglutination or latex immunoturbidimetric detection kit.

8. Use of the fluorescent microsphere conjugate according to claim 2 or the antibody according to claim 3 in preparing immunochromatographic test paper.

9. A latex immunoturbidimetric or latex agglutination detection kit, characterized in that: comprising the latex microsphere conjugate of claim 1 or the antibody of claim 3; Optionally, the latex immunoturbidimetric kit comprises a first reagent, a second reagent and a calibrator, and the first reagent or the second reagent comprises the latex microsphere conjugate according to claim 1 or the antibody according to claim 3.

10. An immunochromatographic test paper, characterized in that: comprising the fluorescent microsphere conjugate according to claim 2 or the antibody according to claim 3; Optionally, the immunochromatographic test paper comprises a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad, and the conjugate pad is provided with the fluorescent microsphere conjugate according to claim 2 or the antibody according to claim 3; Optionally, a detection line is provided on the nitrocellulose membrane; Optionally, a quality control line is also provided on the nitrocellulose membrane.

11. A method of immunoassay, characterized in that: include: The latex microsphere conjugate according to claim 1, the fluorescent microsphere conjugate according to claim 2, the antibody according to claim 3, the detection kit according to claim 9, or the immunochromatographic test paper according to claim 10 is contacted with a sample to be detected to form an immune complex; Optionally, based on the signal of the immune complex, it is determined whether the sample to be tested contains the target substance or the content of the target substance.

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