CN114835807B - Monoclonal antibody - Google Patents

Abstract

The invention discloses a monoclonal antibody, which comprises a heavy chain variable region CDR1, a CDR2 and a CDR3 of amino acid sequences respectively shown as SEQ ID No.1, 2 and 3; and light chain variable region CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID No.9, 10 and 11. The invention also discloses a nucleic acid molecule for encoding the monoclonal antibody, an expression vector containing the nucleic acid molecule and a host cell. The monoclonal antibody of the invention has higher affinity activity.

Description

Monoclonal antibody

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a monoclonal antibody.

Background

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone, directed against only a particular epitope. Usually, hybridoma (hybridoma) antibody technology is used to prepare hybridoma, which is a method of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on cell fusion technology. Monoclonal antibodies have three unique mechanisms of action, namely a targeting effect, a blocking effect and a signaling effect.

Mycoplasma pneumoniae belongs to Mycoplasma of Mycoplasmataceae of mollicutes, is a non-staining cell wall-free prokaryotic microorganism that can grow and reproduce on a non-living medium, and is minimally between viruses and bacteria. The mycoplasma pneumoniae is infected by droplets, the incubation period and the disease course are long, and people are generally susceptible to infection. The main method for detecting human mycoplasma pneumoniae in the market at present is a detection kit for detecting anti-mycoplasma pneumoniae IgM antibody by serum, and the detection kit has lower accuracy and can not carry out effective detection in the initial infection stage.

In order to improve the sensitivity and specificity of detecting mycoplasma pneumoniae, it is necessary to establish a rapid, simple and easy diagnostic method with good specificity, and the premise is to prepare a monoclonal antibody with strong specificity and high affinity.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides the monoclonal antibody with strong specificity and high sensitivity, which can be used for detecting at the initial stage of infection, and is convenient, quick and high in accuracy.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a monoclonal antibody comprising:

heavy chain variable region CDR1, CDR2 and CDR3 of amino acid sequences shown in SEQ ID NO.1, 2 and 3 respectively, and light chain variable region CDR1, CDR2 and CDR3 of amino acid sequences shown in SEQ ID NO.9, 10 and 11 respectively.

Further, the heavy chain variable region further comprises:

the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown in SEQ ID NO.4, 5, 6 and 7, and the light chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown in SEQ ID NO.12, 13, 14 and 15, respectively.

Further, the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.8, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 16.

Further, the monoclonal antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody of the first aspect of the invention.

In a third aspect, the invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention.

In a fifth aspect, the invention provides a method of detecting mycoplasma pneumoniae, said method comprising contacting a sample with a monoclonal antibody according to the first aspect of the invention, thereby detecting mycoplasma pneumoniae in said sample.

In a sixth aspect, the invention provides a product comprising a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention.

The seventh aspect of the present invention provides the use of a monoclonal antibody according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention or a product according to the sixth aspect of the present invention for the detection of mycoplasma pneumoniae or for the manufacture of a product for the diagnosis of a disease associated with mycoplasma pneumoniae.

Further, the mycoplasma pneumoniae related diseases include pneumonia, bronchitis, cold or pharyngitis.

Further, the mycoplasma pneumoniae-associated disease is pneumonia.

In an eighth aspect, the invention provides the use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a product according to the sixth aspect of the invention for inhibiting infection by mycoplasma pneumoniae or for the manufacture of a medicament for the treatment of a disease associated with mycoplasma pneumoniae.

Further, the mycoplasma pneumoniae related diseases include pneumonia, bronchitis, cold or pharyngitis.

Further, the mycoplasma pneumoniae related disease is pneumonia.

The ninth aspect of the present invention provides a colloidal gold product, which is characterized in that the colloidal gold product comprises an analysis membrane, wherein a detection line T is arranged on the analysis membrane, and the detection line T coats the monoclonal antibody of the first aspect of the present invention.

Further, the colloidal gold product includes a gold label pad.

Further, the gold-labeled pad is attached with mycoplasma pneumoniae antibodies.

Further, the gold-labeled pad is a nitrocellulose membrane.

Further, the colloidal gold product further comprises a sample pad, absorbent paper and a bottom plate.

Further, the analysis membrane includes a quality control line C.

Further, the quality control line C is coated with a goat anti-mouse antibody.

Further, the coating concentration of the quality control line C is 1.0mg/ml.

Further, the coating solution of the quality control line C is PBS.

Further, the pH of the PBS was 7.2.

Further, the coating concentration of the detection line T is 1.5mg/ml.

Further, the coating solution of the detection line T is PBS.

Further, the pH of the PBS was 7.2.

The invention has the advantages and beneficial effects that: the monoclonal antibody provided by the invention has higher affinity activity, can be directly used for detecting corresponding antigens of pathogens, and is convenient and quick, and high in accuracy.

Drawings

FIG. 1 is a graph of antibody affinity activity detection.

Detailed Description

The invention provides a monoclonal antibody, which comprises the following components:

a heavy chain variable region CDR1, CDR2 and CDR3 of amino acid sequences shown in SEQ ID NO.1, 2 and 3 respectively; and light chain variable region CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO.9, 10 and 11.

Preferably, the heavy chain variable region further comprises:

heavy chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown as SEQ ID No.4, 5, 6, 7; and light chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown as SEQ ID NO.12, 13, 14 and 15, respectively.

Preferably, the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.8, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 16.

The term "monoclonal antibody" refers to an antibody molecule having a single molecular composition, obtained from a population of substantially identical antibodies. The monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. Typically, an immunoglobulin has a heavy chain and a light chain. Each heavy and light chain includes a constant region and a variable region (regions are also referred to as "domains"). The light and heavy chain variable regions comprise four framework regions, interrupted by three hypervariable regions, also known as "complementarity determining regions" (CDRs). The CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs for each chain are typically CDR1, CDR2 and CDR3, numbered consecutively from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.

The monoclonal antibodies of the invention also include functional variants of the antibodies that bind to mycoplasma pneumoniae.

Specifically, the functional variants include, but are not limited to: chemically and/or biochemically modified derivatives that are substantially similar in primary structural sequence but that are not included in the parent monoclonal antibody of the invention, e.g., in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation.

Alternatively, the functional variant may be a monoclonal antibody as follows: amino acid sequences comprising substitutions, insertions, deletions of one or more amino acids, or combinations thereof, as compared to the amino acid sequence of a parent monoclonal antibody. Further, the functional variant may comprise a truncation of the amino acid sequence at one or both of the amino terminus or carboxy terminus. Functional variants according to the invention may have the same or different, higher or lower binding affinity compared to the parent monoclonal antibody, but are still capable of binding to mycoplasma pneumoniae. For example, a functional variant according to the invention may have an increased or decreased binding affinity for mycoplasma pneumoniae compared to the parent monoclonal antibody.

Preferably, the amino acid sequence of the variable region including, but not limited to, the framework region, the hypervariable region, especially the CDR3 region, is modified. Typically, the light or heavy chain region includes three hypervariable regions (including three CDRs) and more conserved regions, the so-called Framework Regions (FRs). The hypervariable region comprises amino acid residues from the CDRs and amino acid residues from the hypervariable loops. Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally align the amino acid sequences for comparison, and to define similar or identical amino acid residues. The parental monoclonal antibody or a portion thereof can be altered by general molecular biology methods known in the art, including PCR, oligonucleotide-directed mutagenesis, and site-directed mutagenesis, or functional variants can be obtained by organic synthetic methods.

It will also be understood by those skilled in the art that the invention encompasses amino acid sequence modifications of the anti-mycoplasma pneumoniae antibodies. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of anti-mycoplasma pneumoniae antibodies are prepared by introducing appropriate nucleotide changes into anti-mycoplasma pneumoniae antibody nucleic acids or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the anti-mycoplasma pneumoniae antibody. Any combination of deletions, insertions and substitutions are made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processes of the anti-mycoplasma pneumoniae antibody, such as changing the number or position of glycosylation sites.

The invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody described above. The nucleic acid molecules of the invention can be synthesized, for example, by standard chemical synthesis methods and/or recombinant methods, or semi-synthetically produced, for example, by combined chemical synthesis and recombinant methods. Ligation of the coding sequence to transcriptional regulatory elements and/or to other amino acid coding sequences can be performed using established methods, such as restriction digest, ligation, and molecular cloning.

In some embodiments of the invention, a nucleic acid molecule generally refers to any ribonucleic acid molecule or deoxyribonucleic acid molecule, which may be unmodified RNA or DNA or modified RNA or DNA. As used herein, a nucleic acid molecule refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The strands in such a region may be from the same molecule or from different molecules. These regions may include all of one or more molecules, but more typically involve only one region of certain molecules. One of the molecules of the triple-helical region is often an oligonucleotide. As used herein, the term "nucleic acid molecule" includes DNA or RNA as described above which contains one or more modified bases. Thus, a DNA or RNA whose backbone is modified for stability or other reasons is a "nucleic acid molecule" as referred to herein when referring to the term. Furthermore, DNAs or RNAs containing unusual bases such as inosine or modified bases such as tritylated bases are nucleic acid molecules to which this term is applied as used in the present invention, and these are just two examples. It will be appreciated that various types of modifications have been made to DNA and RNA for many beneficial purposes known to those skilled in the art. The term nucleic acid molecule as used herein includes chemically, enzymatically or metabolically modified forms of nucleic acid molecules as well as chemical forms of the DNA and RNA properties of viruses and cells, including simple and complex cells. Nucleic acid molecules also include short nucleic acid molecules often referred to as oligonucleotides.

In addition, modifications may be made to the nucleic acid molecules and polypeptides encompassed by the present invention. For example, nucleotide substitutions may be made that do not affect the polypeptide encoded by the nucleic acid, and thus any nucleic acid molecule encoding a hyperimmune serum reactive antigen or fragment thereof is included within the scope of the present invention.

The nucleic acid molecules of the invention may be in the form of RNA such as mRNA or cRNA or DNA, including for example cDNA and genomic DNA, obtained by cloning or produced by chemical synthesis techniques or a combination of both. The DNA may be triplex, double stranded or single stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or may be the non-coding strand, also known as the antisense strand.

The invention also relates to variants of the above-described nucleic acid molecules. The nucleic acid molecule variant may be a naturally occurring variant, such as a naturally occurring allelic variant, or it may be a non-naturally occurring variant. These non-naturally occurring variants of a nucleic acid molecule can be prepared by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms.

In variants in this regard, a variant is one that differs from the nucleic acid molecule described above by nucleotide substitution, deletion, or addition. The substitution, deletion or addition may involve one or more nucleotides. The variants may be altered within the coding region or within the non-coding region or both. Changes in the coding regions may result in conservative or non-conservative amino acid substitutions, deletions or additions.

The invention provides a vector comprising the nucleic acid molecule described above. Many suitable vectors are known to those skilled in the art of molecular biology, the choice of which depends on the desired function. The vector of the present invention is not particularly limited, and may be a vector capable of replicating and/or expressing a polynucleotide in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), plant cells, yeast cells, insect cells, and bacterial cells (e.g., escherichia coli). Preferably, it may be a vector comprising at least one selectable marker operably linked to a suitable promoter such that the polynucleotide may be expressed in a host cell. For example, the vector may comprise a polynucleotide introduced into a phage, plasmid, cosmid, mini-chromosome, viral, or retroviral vector or other vector conventionally used, for example, in genetic engineering.

As an alternative to the present invention, the vector is a virus. Viral vectors are used to introduce non-endogenous nucleic acid sequences encoding target-specific polypeptides. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also include a nucleic acid sequence encoding a transduction marker. Suitable viral vectors include RNA virus-based vectors, such as retroviral-derived vectors, e.g., moloney Murine Leukemia Virus (MLV) -derived vectors, and more complex retroviral-derived vectors, such as lentiviral-derived vectors. HIV-1 derived vectors belong to this class.

Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive strand RNA viruses (e.g., picornaviruses and viruses A), and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2 and Epstein-Barr and cytomegalovirus) and poxviruses (e.g., vaccinia, fowlpox, and canarypox).

As an alternative of the invention, the vector is an expression vector. The expression vector according to the invention is capable of directing the replication and expression of the nucleic acid molecule of the invention in a host.

Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45 a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, clopPV-1, pdBPVMneo, pRSVgpo, pRSVnefro, pGsubpidyVdhyV 2-35, bepcCMV-35, EPCMYO-DNA (pReXpIPsec-DNA), pRpIPcDNAs (LgP-expression vectors (pRepGE) and expression vectors (pRepGE-pIPcDNAs) (pRepGE-10). Non-limiting examples of plasmid vectors suitable for Pichia pastoris (Pichia pastoris) include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus (Xenopus) embryos, zebrafish embryos, and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2+.

The present invention provides a host cell comprising a nucleic acid molecule or vector as described above.

In embodiments of the invention, host cells for introducing the vector include prokaryotic and eukaryotic cells, including but not limited to bacterial cells such as E.coli, streptomyces and Salmonella typhimurium; a yeast cell; fungal cells such as pichia pastoris; insect cells such as Drosophila or Spodoptera Sf9 cells; animal cells, such as Chinese hamster ovary cells, SP2/0, human lymphoid mother cells, COS, NSO,293T, bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells); and plant cells. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.

The term "introducing" refers to delivering a vector comprising a polynucleotide encoding a monoclonal antibody into a host cell. This introduction can be performed by various methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome-mediated transfection, liposome fusion, lipofection, and protoplast fusion. In addition, transfection refers to the use of viral particles through infection to deliver the desired material into cells. In addition, the vector may be introduced into a host cell by gene bombardment. In the present invention, introduction and transfection may be used interchangeably.

The invention provides a method for detecting mycoplasma pneumoniae, which comprises the step of contacting a sample with the monoclonal antibody so as to detect the mycoplasma pneumoniae in the sample.

In the present invention, the terms "sample" or "test sample" are used interchangeably herein, and the sample is an in vitro sample that is to be analyzed in vitro and is not transferred back into the body. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymph fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. In one embodiment, the sample is selected from the group consisting of blood, serum, plasma, synovial fluid and urine. In some embodiments of the invention, the sample is selected from the group consisting of blood, serum, and plasma.

The invention provides a product comprising the monoclonal antibody, the nucleic acid molecule, the vector or the host cell.

In an embodiment of the invention, the product includes, but is not limited to, a pharmaceutically acceptable carrier or pharmaceutical composition.

Pharmaceutically acceptable carriers may comprise inert ingredients that do not unduly inhibit the biological activity of the compound. The pharmaceutically acceptable carrier should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or free of other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical formulation techniques can be used.

Some examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as tween80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropylmethylcellulose, wool fat; sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

Can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. For tablets intended for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral administration, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also have a composition such that they release the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Microencapsulated forms having one or more of the above excipients may also be used in the present invention. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose or starch. As is conventional, such dosage forms may also contain additional substances other than inert diluents, for example, tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also have a composition such that they release the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

In the present invention, the pharmaceutical composition may be prepared using various additives, such as buffers, stabilizers, bacteriostats, isotonizing agents, chelating agents, pH controlling agents, and surfactants.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, pharmaceutically acceptable acids, bases or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form in which it is administered. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracutaneous, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may be administered to a subject by any route as long as the target tissue is reached.

In some embodiments of the invention, the product includes, but is not limited to, a detection reagent, a kit, a chip, or a strip.

As an alternative embodiment, the product is a kit; the kit comprises the antibody prepared by the invention. As another alternative embodiment, the kits of the present invention comprise a diagnostic composition comprising at least one detectable label, such as a detectable moiety/agent. The detectable moiety/agent as a label is preferably one selected from the group consisting of, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials and non-radioactive paramagnetic metal ions. The kit further comprises a conjugate of an antibody linked to a signal producing compound, wherein the antibody of the conjugate is different from the isolated monoclonal antibody of the invention.

As mentioned above, the conjugate (or indicator) should comprise an antibody (perhaps an anti-antibody, depending on the assay) to which a signal producing compound or label is attached. The signal generating compound or "label" is detectable by itself or is reactive with one or more other compounds to produce a detectable product. Examples of signal-generating compounds include chromophores, radioisotopes (e.g., 125I, 131I, 32P, 3H, 35S, and 14C), chemiluminescent compounds (e.g., acridinium), particles (visible or fluorescent), nucleic acids, complexing agents or catalysts such as enzymes (e.g., alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase, and ribonuclease). In the case of enzymes such as alkaline phosphatase or horseradish peroxidase, the addition of a chromogenic, fluorogenic or luminescent substrate can cause the generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal reflection fluorescence, amplification (e.g., polymerase chain reaction), and raman spectroscopy are also useful.

Biological samples that can be tested by the above immunoassays include, but are not limited to, plasma, whole blood, dry whole blood, serum, or aqueous or organic-aqueous extracts of tissues and cells.

As an alternative embodiment, the product is a chip, which comprises a protein chip; the protein chip comprises a solid phase carrier and the monoclonal antibody or the fragment thereof fixed on the solid phase carrier.

As an alternative embodiment, the kit comprises a protein immunoassay kit; the protein immunoassay kit comprises the monoclonal antibody or the fragment thereof.

In the present invention, the method for detecting or determining the amount of Mycoplasma pneumoniae may be any known method. For example, it includes immunodetection or assay methods. The immunoassay or measuring method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method.

In some embodiments of the invention, the enzyme immunoassay includes, but is not limited to, double antibody sandwich, indirect ELISA, double sandwich ELISA, competition ELISA, blocking ELISA, antibody capture ELISA, spot ELISA, cloth ELISA.

In a particular embodiment of the invention, the method used for detection is a double antibody sandwich method.

The invention provides application of the monoclonal antibody, the nucleic acid molecule, the vector, the host cell or the product in detection of mycoplasma pneumoniae or preparation of products for diagnosing diseases related to the mycoplasma pneumoniae.

The term "mycoplasma pneumoniae-associated disease" as used herein refers to a disease caused by infection with mycoplasma pneumoniae. Examples of mycoplasma pneumoniae-associated diseases include, but are not limited to, mycoplasma pneumonia, bronchitis, common cold, pharyngitis, asthma, neurological diseases, and infections of the urogenital system, such as urethritis or pelvic inflammation; severe mycoplasma infections, even with systemic symptoms such as sepsis, joint pain or immune syndrome.

In some embodiments of the invention, the mycoplasma pneumoniae-associated disease comprises pneumonia, bronchitis, cold, or pharyngitis.

In a particular embodiment of the invention, the mycoplasma pneumoniae related disease is pneumonia.

The invention also provides application of the monoclonal antibody, the nucleic acid molecule, the vector, the host cell or the product in inhibiting mycoplasma pneumoniae infection or preparing a medicament for treating diseases related to mycoplasma pneumoniae.

The term "mycoplasma pneumoniae-associated disease" as used herein refers to a disease caused by infection with mycoplasma pneumoniae. Examples of mycoplasma pneumoniae-associated diseases include, but are not limited to, mycoplasma pneumonia, bronchitis, common cold, pharyngitis, asthma, neurological diseases, and infections of the urogenital system, such as urethritis or pelvic inflammation; severe mycoplasma infections may even present with systemic symptoms such as sepsis, joint pain or immune syndrome.

In some embodiments of the invention, the mycoplasma pneumoniae-associated disease comprises pneumonia, bronchitis, cold, or pharyngitis.

In a particular embodiment of the invention, the mycoplasma pneumoniae related disease is pneumonia.

The invention also provides a colloidal gold product, which comprises an analysis membrane, wherein a detection line T is arranged on the analysis membrane, and the detection line T is coated with the monoclonal antibody.

In a specific embodiment of the invention, the colloidal gold product comprises a sample pad, a gold label pad, an analysis membrane, absorbent paper and a bottom plate, wherein the absorbent paper is attached to the bottom plate, the absorbent paper presses the analysis membrane for 2mm, the gold label pad and the sample pad are attached, and the gold label pad presses the analysis membrane for 2mm; and the analysis membrane is provided with a detection line T and a quality control line C, and the detection line T is coated with the monoclonal antibody.

In some embodiments of the present invention, the colloidal gold product refers to a product prepared by using colloidal gold as a developing medium and utilizing the principle of antigen-antibody binding specific binding. In some embodiments of the invention, the colloidal gold product includes, but is not limited to, a colloidal gold test strip, a colloidal gold test card; in a specific embodiment of the present invention, the colloidal gold product is a colloidal gold test strip.

In an embodiment of the invention, the mycoplasma pneumoniae antibody may be any antibody that binds mycoplasma pneumoniae.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.

EXAMPLE 1 preparation of monoclonal antibodies

1.1 immunization of mice

TABLE 1 methods of immunizing mice

1.2 Tail blood titer detection and results

1. Antigen coating: the antigen was diluted to 0.1. Mu.g/ml, 100. Mu.l/well at 25 ℃ for 2h.

2. And (3) sealing: 1% BSA blocking solution, 200. Mu.l/well, 25 ℃ for 1h.

3. A first antibody: collecting blood of mouse tail, centrifuging at high speed to obtain supernatant, and performing gradient dilution of the supernatant at 1000 × by 2 times, at 25 deg.C and 25 deg.C for 30min.

4. Secondary antibody: the secondary antibody was diluted at 10000X, 100. Mu.l, 25 ℃ for 30min.

5. Color development liquid: 100 μ l,25 ℃,20min.

6. Stopping liquid: 50 μ l, reading at double wavelength under microplate reader.

TABLE 2 Tail blood titer test results

1.3 mouse cell fusion

1. Mice were killed by sudden death and soaked in 75% alcohol for 3min.

2. And (3) cutting spleen and leg lymph node cells of the mouse from a super clean bench.

3. Spleen and lymph nodes are fully ground in a super clean bench, so that cells are fully released.

4. Standing and collecting supernatant, removing large impurities as much as possible, centrifuging for 3min at 1000r/min, removing supernatant, and collecting lymphocytes.

5. Adding 5ml of erythrocyte lysate, slightly beating the cells for 5 times to uniformly suspend the cells, and removing the erythrocytes in the cells. Adding an isovolumetric DMEM culture medium, centrifuging for 3min at 1000r/min, removing supernatant, collecting lymphocytes, washing for 3 times by using DMEM, and centrifuging to collect cells.

6. Taking myeloma cells, and enabling the ratio of the myeloma cells to the lymphocytes to be 1:5, mixing well, washing with DMEM for 3 times, and centrifuging to collect cells.

7. Fusing: adding 1ml of PEG, adding 1min completely, then adding 9ml of DMEM medium, and adding 5min completely.

8. And (4) centrifugally collecting hybridoma cells, supplementing HAT culture medium, and dropwise adding the hybridoma cells into the feeder cells prepared in advance. 1.4 cell fusion mother cloning and screening results

1. Antigen coating: the antigen was diluted to 0.1. Mu.g/ml, 100. Mu.l/well at 25 ℃ for 2h.

2. And (3) sealing: 1% BSA blocking solution, 200. Mu.l/well, 25 ℃ for 1h.

3. A first antibody: supernatant, 50. Mu.l/well, 25 ℃ for 30min.

4. Secondary antibody: the secondary antibody was diluted at 10000X, 100. Mu.l, 25 ℃ for 30min.

5. Color development liquid: 100 μ l,25 ℃,20min.

6. Stopping liquid: 50 μ l, reading at double wavelength under microplate reader.

TABLE 3 Master clone screening results

1.5 detection of subclones

1. The parent clone E5 was selected and cultured to obtain the supernatant.

2. Antigen coating: the antigen was diluted to 0.1. Mu.g/ml, 100. Mu.l/well at 25 ℃ for 2h.

3. And (3) sealing: 1% BSA blocking solution, 200. Mu.l/well, 25 ℃ for 1h.

4. A first antibody: cell culture supernatant, 50. Mu.l/well, 25 ℃ for 30min.

5. Secondary antibody: the secondary antibody was diluted at 10000X, 100. Mu.l, 25 ℃ for 30min.

6. Color development liquid: 100 μ l,25 ℃,20min.

7. Stopping liquid: 50 μ l, readings were taken with a microplate reader at dual wavelengths.

TABLE 4 subclone assay results

1.6 purification of antibodies

1. Ascites fluid was filtered through two layers of filter paper, glass fiber and nonwoven fabric, and OD was measured.

2. After filtration, ascites fluid was diluted with A fluid (20mMPB +0.3MNaCl PH = 7.5) 1:1.

3. And mixing the diluted samples uniformly, loading at the loading speed of 1ml/min, collecting effluent, and repeating for 3 times.

4. After loading, the sample is completely balanced by the solution A, and the balancing speed is 1ml/min.

5. After equilibration of solution A, elution and sample inoculation were performed with solution B (0.1M glycine pH = 3), elution rate 2ml/min.

6. During the inoculation, tris (PH = 7.5) was used to adjust the PH to 7.2.

7. Then, the solution is changed over G50 to 10mMPBS, and the pH is =7.2 buffer solution (PBS: 10mMPB + 150mMNaCl).

8. After the solution B is eluted and completely balanced, the solution A is completely balanced.

9. The samples were run for electrophoresis and assayed for activity.

1.7 antibody purification test results

1. Antigen coating: the antigen was diluted to 0.1. Mu.g/ml, 100. Mu.l/well at 25 ℃ for 2h.

2. And (3) sealing: 1% BSA blocking solution, 200. Mu.l/well, 25 ℃ for 1h.

3. A first antibody: the antibody was diluted to the corresponding concentration, 100. Mu.l/well, 25 ℃ for 30min.

4. Secondary antibody: the secondary antibody was diluted at 10000X, 100. Mu.l, 25 ℃ for 30min.

5. Color development liquid: 100 μ l,25 ℃,20min.

6. Stopping liquid: 50 μ l, reading at dual wavelength of microplate reader.

TABLE 5 antibody purification results

1.8 monoclonal antibody sequences

The sequence of the selected monoclonal antibody is shown in Table 6.

TABLE 6 sequences of monoclonal antibodies

Example 2 detection of antibody affinity Activity and results

1. Coating: the C-coated goat mice had a concentration of 1.0mg/ml and the coating solution was 0.01MPBS pH7.2. The T-line is coated with the monoclonal antibody at a concentration of 1.5mg/ml, the coating solution is 0.01MPBS (Methylpolympb) pH7.2, and the mixture is dried at 37 ℃ overnight.

2. Preparing a gold label pad: diluting mycoplasma pneumoniae antibody to 1mg/ml with water, taking 10ml colloidal gold, adding 180 μ l of 1% potassium carbonate, uniformly mixing, adding 100 μ l of mycoplasma pneumoniae antibody, uniformly mixing, reacting at room temperature for 10min, adding 10 μ l of BSA, reacting at room temperature for 5min, centrifuging at room temperature for 5min, removing supernatant, resuspending the precipitate with 150 μ l of heavy suspension (0.01M PBS +1% BSA +2% sucrose), testing light absorption value at 532nm of an ultraviolet spectrophotometer, diluting the concentrated colloidal gold antibody to 10d/ml with the heavy suspension, uniformly coating 600 μ l of the suspension on glass fiber with the specification of 0.5 × 30cm, and drying at 37 ℃ overnight. Drying and storing for later use.

3. Assembling: adsorbing water-absorbing paper, pressing the NC film by the water-absorbing paper for 2mm; attaching a gold label pad and a sample pad, pressing the NC film by the gold label pad for 2mm, and installing a card shell for later use.

4. And (3) testing: the quality control substances were diluted with diluents to 100 ×,1000 ×,10000 × and 100000 ×, respectively, and the diluents were used as negative controls, with a loading of 65 μ l.

5. And (3) testing results: FIG. 1 shows from left to right negative controls, dilutions 100X, 10 ³ ×、10 ⁴ ×、10 ⁵ ×、10 ⁶ X, blank control, as can be seen from FIG. 1, the antibody is diluted to 10 ⁴ And x, the test strip can still detect the affinity activity of the antibody.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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<141> 2022-06-10

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Claims (25)

1. A monoclonal antibody, wherein the monoclonal antibody comprises:

heavy chain variable region CDR1, CDR2 and CDR3 of amino acid sequences shown in SEQ ID NO.1, 2 and 3 respectively, and light chain variable region CDR1, CDR2 and CDR3 of amino acid sequences shown in SEQ ID NO.9, 10 and 11 respectively.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises:

the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown in SEQ ID NO.4, 5, 6 and 7, and the light chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown in SEQ ID NO.12, 13, 14 and 15, respectively.

3. The monoclonal antibody of claim 2, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID No.8 and the light chain variable region has the amino acid sequence shown in SEQ ID No. 16.

4. The monoclonal antibody of any one of claims 1-3, wherein the monoclonal antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

5. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-4.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method for detecting Mycoplasma pneumoniae for non-diagnostic purposes, the method comprising contacting a sample with a monoclonal antibody according to any one of claims 1-4, thereby detecting Mycoplasma pneumoniae in the sample.

9. A product for detecting Mycoplasma pneumoniae, said product comprising a monoclonal antibody according to any one of claims 1-4, a nucleic acid molecule according to claim 5, a vector according to claim 6, or a host cell according to claim 7.

10. Use of the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7, or the product of claim 9 for the detection of mycoplasma pneumoniae for non-diagnostic purposes or for the preparation of a product for the diagnosis of a disease associated with mycoplasma pneumoniae.

11. The use according to claim 10, wherein the mycoplasma pneumoniae-associated disease comprises pneumonia, bronchitis, cold or pharyngitis.

12. The use according to claim 11, wherein the mycoplasma pneumoniae-related disease is pneumonia.

13. A colloidal gold product comprising an analysis membrane having a detection line T coated with the monoclonal antibody of any one of claims 1 to 4.

14. A colloidal gold product as claimed in claim 13, wherein the colloidal gold product comprises a gold-labelled pad.

15. A colloidal gold product according to claim 14, wherein the gold-labeled pad is attached with mycoplasma pneumoniae antibodies.

16. A colloidal gold product according to claim 14, wherein the gold pad is a nitrocellulose membrane.

17. A colloidal gold product according to claim 13, further comprising a sample pad, absorbent paper and a base plate.

18. A colloidal gold product according to claim 13, wherein the analysis membrane comprises a quality control line C.

19. A colloidal gold product according to claim 18, wherein the quality control line C is coated with goat anti-mouse antibody.

20. A colloidal gold product according to claim 19, wherein the coating concentration of the quality control line C is 1.0mg/ml.

21. A colloidal gold product according to claim 18, wherein the coating solution of the quality control line C is PBS.

22. A colloidal gold product according to claim 21 wherein the PBS has a pH of 7.2.

23. A colloidal gold product according to claim 13, wherein the detection line T is coated at a concentration of 1.5mg/ml.

24. A gold colloid product according to claim 13, wherein the coating liquid of the detection line T is PBS.

25. A colloidal gold product as in claim 24 wherein the PBS has a pH of 7.2.

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