CN112010981B

CN112010981B - Mouse anti-human IgG monoclonal antibody

Info

Publication number: CN112010981B
Application number: CN202010947844.2A
Authority: CN
Inventors: 姬艺洪
Original assignee: Nanjing Miaodi Biotechnology Co ltd
Current assignee: Nanjing Miaodi Biotechnology Co ltd
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2021-05-18
Anticipated expiration: 2040-09-10
Also published as: CN112010981A

Abstract

The invention discloses a mouse anti-human IgG monoclonal antibody, which has the amino acid sequences of heavy chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO.26, 28 and 30, and the amino acid sequences of light chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO.42, 44 and 46. The invention also provides 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 sensitivity and specificity when being applied to immunoassay.

Description

Mouse anti-human IgG monoclonal antibody

Technical Field

The invention belongs to the fields of cellular immunology and genetic engineering, and relates to a mouse anti-human IgG monoclonal antibody.

Background

Enzyme-Linked Immunosorbent Assay (ELISA) is a commonly used type of immunoenzyme technology. The main method is that known antigen or antibody is adsorbed on the surface of solid phase carrier, then the enzyme-labeled antibody or antigen is used for incubation, color developing agent is added for color development, and the difference between the color of the object to be measured and the color of the standard substance is measured by an enzyme-labeling instrument to obtain qualitative or quantitative result. ELISA can be divided into a plurality of types according to different analytes (detection antigens or detection antibodies) and different measurement principles, such as direct methods, indirect methods, competitive methods, sandwich methods and the like.

Indirect ELISA is an effective method for measuring unknown antibodies with known antigens, and plays an important role in laboratory diagnosis of infectious diseases and evaluation of vaccination effect. The basic operation steps are that antigen is firstly combined on an enzyme label plate, and then detection is carried out in two steps: firstly adding an antibody (primary antibody) to be detected to be specifically combined with the antigen, and then adding an enzyme-labeled secondary antibody to detect and utilize a substrate to develop color. Compared with direct ELISA, indirect ELISA uses enzyme-labeled secondary antibody and has higher sensitivity. Indirect ELISA also provides greater flexibility because primary antibodies directed against different antigen targets are used together.

The traditional indirect ELISA has the defects of the possibility of cross reaction, high background and easy false positive misjudgment. The reason is that the enzyme-labeled secondary antibody is mainly designed in the process of eliminating the problems in the process of antigen coating and blocking. First, conventional secondary antibodies, mostly polyclonal antibodies such as rabbit anti-human IgG, are obtained by immunizing rabbits with purified human IgG. The human IgG antibody is composed of a heavy chain and a light chain (kappa or lambda), the immune system of an animal can be stimulated to produce antibodies aiming at the heavy chain and the light chain of the human IgG antibody after a rabbit is immunized, and the light chain of the antibodies such as IgM, IgA and IgE in human serum is the same as the light chain of the IgG, namely the kappa or lambda, so that when the human IgG with a certain coating antigen specificity is detected by indirect ELISA, false positive results or high background can be caused due to impurity of the secondary antibody (containing the antibodies aiming at the heavy chain and the light chain at the same time). Secondly, since the immune background of animals (such as rabbits) for preparing the secondary antibody is not clear, the rabbit has antibodies (caused by recessive infection) aiming at the coating antigen, and the antibodies aiming at the coating antigen in the secondary antibody can be directly combined with the coating antigen in indirect ELISA detection, so that a false positive result or high background is caused.

In view of the above-mentioned deficiencies, the present patent describes the preparation and identification of monoclonal antibodies specific for the heavy chain of human IgG antibodies. Compared with the antibody prepared in the same batch, 0070 is very suitable for being used as a secondary antibody of indirect ELISA for determining the titer of the human IgG antibody, and can reduce the background of the whole reaction system and amplify the signal of specific binding.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide a human IgG antibody heavy chain specific monoclonal antibody and a preparation method thereof.

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

in a first aspect, the invention provides an anti-IgG monoclonal antibody comprising three CDR heavy chain variable regions and three CDR light chain variable regions; wherein, the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.26, 28 and 30, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.42, 44 and 46.

Further, the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.32, 34, 36 and 38; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.48, 50, 52 and 54.

Furthermore, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.40, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 56.

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. 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 the invention, the nucleotide sequences of CDR1, CDR2 and CDR3 encoding the heavy chain variable region of the antibody are shown in SEQ ID NO.27, 29 and 31; the nucleotide sequences of CDR1, CDR2 and CDR3 encoding the variable region of the light chain are shown in SEQ ID NO.43, 45 and 47.

Further, the nucleotide sequences encoding framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region are represented by SEQ ID Nos. 33, 35, 37 and 39; the nucleotide sequences encoding framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.49, 51, 53 and 55.

Further, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.41, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 57.

Nucleic acid sequences encoding the antibody molecules of the invention may be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the antibody heavy and light chains can be synthesized from the determined DNA sequences or based on the corresponding amino acid sequences, as desired.

In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention. Many suitable vectors are known to those skilled in the art of molecular biology, the choice of which depends on the desired function. Non-limiting examples of vectors include plasmids, cosmids, viruses, bacteriophages and other vectors routinely used in, for example, genetic engineering. Methods well known to those skilled in the art can be used to construct various plasmids and vectors.

In one embodiment, 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 and thus ensuring the expression of the variable chain domain of the anti-IgG antibody of the invention encoded thereby in the selected host. In a further embodiment, the one or more vectors comprise further sequences to ensure that not only said variable chain domain of the invention is expressed, but also a full length IgG antibody comprising said variable chain domain of the invention is expressed.

The expression vector may be, for example, a cloning vector, a binary vector or an integrative vector. Expression includes transcription of the nucleic acid molecule, e.g., into translatable mRNA.

Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, pBBR 1-MCS-series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitroen), pMC1neo (Stratagene), pXT1(Stratagene), pSG5(Stratagene), EBO-V2 neo, pBPV-1, PDBPVMneo, SVgMTpttherein, pXT1(Stratagene), pSSVpGSV 6323-Pro-DNA (pRpCAP9), pRpCAPJpA-7-cDNA (pRpCpIVcDNA (pRpCmS 6326), pAK-7), pEPcDNAs (pRpIVcDNAs), pIcDNAs (pR-7) and pGpCmAGrDNA (L6335). 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 +.

In general, a vector may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in a host (e.g., antibiotic resistance), and one or more expression cassettes. In addition, the coding sequences contained in the vector may be linked to transcriptional regulatory elements and/or to other amino acid coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure initiation of transcription, an Internal Ribosome Entry Site (IRES), and optionally regulatory elements that ensure termination of transcription and stabilization of the transcript. Non-limiting examples of such regulatory elements that ensure initiation of transcription include promoters, translation initiation codons, enhancers, insulators, and/or regulatory elements that ensure termination of transcription, which are included downstream of the nucleic acid molecules of the invention. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences which, depending on the expression system used, are capable of directing the expressed protein to a cell compartment or culture medium. The vector may also contain additional expressible polynucleotides encoding one or more chaperones to facilitate proper protein folding.

Additional examples of suitable origins of replication include, for example, full-length ColE1, truncated ColEI, SV40 virus, and M13 origins of replication, yet additional examples of suitable promoters include, but are not limited to, the Cytomegalovirus (CMV) promoter, SV 40-promoter, RSV-promoter (rous sarcoma virus), lacZ promoter, tetracycline promoter/operator (tetp/o), chicken β -actin promoter, CAG-promoter (a combination of chicken β -actin promoter and cytomegalovirus immediate early enhancer), gai10 promoter, human elongation factor 1 α -promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, lac, trp or tac promoter, T7 or T5 promoter, lacUV5 promoter, Autographa californica (Autographa) polynedron promoter or globin intron in mammalian and other animal cells. An example of an enhancer is, for example, the SV 40-enhancer. Additional non-limiting examples of regulatory elements that ensure transcription termination include the SV 40-polyadenylation site, tk-polyadenylation site, rho factor-independent lpp terminator or AcMNPV polyhedric polyadenylation signal. Further non-limiting examples of selectable markers include dhfr, which confers resistance to methotrexate, npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromomycin (paromycin), and hygro, which confers resistance to hygromycin. An additional selection gene has been described, trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to utilize histidinol instead of histidine; mannose 6-phosphate isomerase, which allows the cell to utilise mannose and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2- (difluoromethyl) -DL-ornithine DFMO or confers resistance to blasticidin S a deaminase from Aspergillus terreus (Aspergillus terreus).

In a further embodiment, the vector is a eukaryotic expression plasmid containing an expression cassette consisting of a5 'CMV promoter including intron a and a 3' BGH polyadenylation sequence.

Suitable bacterial expression hosts include, for example, those derived from JM83, W3110, KS272, TG1, K12, BL21 (e.g.BL 21(DE3), BL21(DE3) PlysS, BL21(DE3) RIL, BL21(DE3) PRARE) or

The strain of (1).

The nucleic acid molecules and/or vectors of the invention can be designed to be introduced into cells by, for example, chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nuclear transfection, non-chemical methods (electroporation, sonoporation, light transfection, gene electrotransfer, fluid delivery, or transformation that occurs naturally when cells are contacted with the nucleic acid molecules of the invention), particle-based methods (gene gun, magnetic transfection, transfections by puncture), phage-based vector methods, and viral methods.

To facilitate purification of the nucleic acid molecules of the invention, a tag (tag) sequence may be inserted into the expression vector. Examples of tags include, but are not limited to, a hexa-histidine tag, a myc tag, or a FLAG tag. Any tag known to those skilled in the art to facilitate purification may be used in the present 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 an expression vector according to the third aspect of the invention.

In the present invention, any suitable host cell/vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. Bacterial (e.g., E.coli) and other microbial systems may be used, or eukaryotic (e.g., mammalian) host cell expression systems may also be used. Such cells include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells, or cells of bacterial origin. As the mammalian cell, one selected from the group consisting of, but not limited to, CHO cell, F2N cell, CSO cell, BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, a549 cell, HEK293 cell, and HEK293T cell can be preferably used as the host cell. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.

A fifth aspect of the invention provides a method of detecting IgG in a sample, the method comprising the steps of:

1) contacting the sample with a first specific binding agent and a second specific binding agent, wherein the second specific binding agent is a monoclonal antibody according to the first aspect of the invention; for a time and under conditions sufficient to form a first specific binding agent-IgG-second specific binding agent complex; and

2) detecting the complex formed in 1), thereby detecting IgG in the sample;

further, the second specific binding agent is detectably labeled.

A number of markers (also referred to as dyes) are available which can be generally classified into the following categories, which together and each of them represent an embodiment according to the present disclosure:

(a) fluorescent dyes

The fluorescent label or fluorophore comprises rare earth chelate (europium chelate), fluorescein type label, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine-type labels, including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; texas Red; and the like. Fluorescent labels can be conjugated to aldehyde groups contained in the target molecule using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen/Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

(b) Luminescent dyes

Luminescent dyes or labels can be further subdivided into chemiluminescent and electrochemiluminescent dyes.

Different classes of chemiluminescent labels include luminol, acridinium compounds, coelenterazine and analogs, dioxetanes, peroxy oxalate based systems and derivatives thereof. For immunodiagnostic procedures, acridine-based labels are mainly used.

The labels used as electrochemiluminescent labels which have a more important relationship are electrochemiluminescent complexes based on ruthenium and iridium, respectively. Electrochemiluminescence (ECL) has proven to be very useful in analytical applications as a highly sensitive and selective method. It combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by application of electrode potentials. Typically, ruthenium complexes, especially [ Ru (Bpy)3]2+ (releasing photons at-620 nm), regenerated by TPA (tripropylamine) in the liquid phase or liquid-solid interface are used as ECL-labels.

(c) Radiolabelling utilizes radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131 Bi.

(d) Metal chelates suitable as labels for imaging and therapeutic purposes.

Further, the first specific binding agent is an antigen that binds to IgG.

The terms "sample" or "test sample" are used interchangeably herein, and a 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 one embodiment, the sample is selected from the group consisting of blood, serum, and plasma. In one embodiment, the sample is serum or plasma.

The term "reference sample" as used herein refers to a sample that is analyzed in substantially the same manner as the test sample and whose information is compared to that of the test sample. Thus, the reference sample provides a criterion that allows the information obtained from the test sample to be evaluated. The reference sample may be from a healthy or normal tissue, organ or individual, thereby providing a standard of health status of the tissue, organ or individual. The difference between the state of the normal reference sample and the state of the test sample may be a sign of the risk of disease development or the presence or further progression of such disease or condition. The reference sample may be from an abnormal or diseased tissue, organ or individual, thereby providing a criterion for the diseased state of the tissue, organ or individual. The difference between the status of the abnormal reference sample and the status of the test sample may be a sign of a reduced risk of disease development or absence or amelioration of such disease or disorder.

In a sixth aspect, the invention provides a product for the detection of IgG, the product comprising a monoclonal antibody according to the first aspect of the invention.

The product further comprises a kit, test paper, a chip and the like. Wherein the chip 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; the 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 IgG 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, and the like.

In a seventh aspect, the invention provides the use of a monoclonal antibody according to the first aspect of the invention in an immunoassay. 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, and the like.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In the present invention, a monoclonal antibody encompasses a sequence having a certain degree of sequence identity or sequence homology with the amino acid sequence of the antibody or any nucleotide sequence encoding the antibody, and in the present invention, "homology" may be equivalent to "identity".

One skilled in the art will also appreciate that antibodies may be subjected to various post-translational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Common modifications are the deletion of a basic residue at the carboxy terminus (such as lysine or arginine) due to the action of carboxypeptidase.

The invention also includes all antibodies which are obtained by adding, deleting and modifying the amino acid residues of the amino acid sequence of the antibody, comprise human antibodies and non-human antibodies and have the same functions as the FHA3 antibody or are modified and optimized. The deletion, substitution, insertion or addition may occur simultaneously, and the amino acid to be substituted, inserted or added may be of a natural type or a non-natural type.

The CDRs of the invention can include variants, for example, when the CDRs disclosed herein are back mutated to different framework regions. Typically, individual variant CDRs are at least 70% or 80% amino acid identity to the sequences described herein, more typically with increasing identity of preferably at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%.

As used herein, "identity" indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are identical. As used herein, "similarity" indicates that at any particular position of the aligned sequences, the amino acid residues between the sequences are of a similar type. For example, leucine may be substituted with isoleucine or valine. Other amino acids that may be substituted for one another in general include (but are not limited to): phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains), lysine, arginine and histidine (amino acids having basic side chains), aspartic acid and glutamic acid (amino acids having acidic side chains), asparagine and glutamine (amino acids having amide side chains), and cysteine and methionine (amino acids having sulfur-containing side chains).

Generally, modification of one or more amino acids in a protein does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein results in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative or variant. Typically, these changes are made over several amino acids to minimize changes in the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, greater variations may be tolerated in some cases. Amino acid substitutions are typically of a single base; insertions will typically be on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases, deletions can be much larger.

The antibodies and fragments disclosed herein are expressed at good levels from host cells. Thus, the properties of the antibody and/or binding fragment are suitable for expression on a commercial scale.

Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. "Fab" refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded; "Fab'" refers to a Fab fragment comprising part of the hinge region; "F (ab ') 2" refers to a dimer of Fab'; "Fv" refers to the smallest antibody fragment containing the variable regions of the heavy and light chains of an antibody and having all antigen binding sites.

The invention has the advantages and beneficial effects that:

the invention provides an anti-human IgG monoclonal antibody, which can reduce the background of the whole reaction system and amplify a signal of specific binding, and has higher sensitivity and specificity when used for immunodetection.

Drawings

FIG. 1 is an immunoblot identifying the binding specificity of 7 strains of mouse anti-human IgG.

Detailed Description

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.

EXAMPLE 1 preparation of mouse anti-human IgG monoclonal antibody

1. Balb/c mouse immunization

Human serum was purified by Protein G affinity column chromatography to obtain human IgG antibody, dialyzed into PBS, and the concentration was measured. Immunizing female Balb/c mice (100 mu g/mouse) with 5 weeks of age by using human IgG, and mixing 100 mu g of antigen and an equal volume of Freund complete adjuvant for intraperitoneal injection during the first immunization; after 3 weeks, the same amount of antigen is mixed with Freund's incomplete adjuvant and then the abdominal cavity is immunized; week 5, 3 rd immunization without adjuvant.

2. Fusion of splenocytes with myeloma cells

One week before fusion, the mouse myeloma cells sp2/0 were recovered to OPTI-MEM medium (containing 10% fetal calf serum) and placed at 37 ℃ in 5% CO₂The cells were passaged once 3 days before fusion. On the day of fusion, myeloma cells were harvested, counted, and 5.0X 10 cells were added⁷Myeloma cells were washed 2 times with serum-free medium for use. 3-5 days after 3 rd immunization, the mice are removed of eyeballs, bled and killed. Taking out the spleen of the mouse by aseptic operation, placing the spleen in a sterilized plate, separating splenocytes, and counting for later use.

Spleen cells equivalent to mouse 1/2 spleen were mixed with myeloma cells, centrifuged at 1300rpm for 5min, and the supernatant was removed as much as possible. Adding 1.5ml of 50% PEG within 1.5 min, and shaking up while adding; then 20ml of serum-free medium was added over 8.5 minutes, and the mixture was shaken while adding.

PEG-fused cells were centrifuged at 1000rpm for 5 minutes, the supernatant was removed, 150ml HAT selection medium was added for resuspension, the fused cells were inoculated into a sterile 96-well plate at 150. mu.l/well and placed at 37 ℃ in 5% CO₂The cells were incubated in an incubator for 4 days and 100. mu.l of selection medium was added to each well.

3. Screening and cloning of hybridoma cells

10 days after fusion, 50. mu.l of supernatant was aspirated from each well, added to a 96-well ELISA plate coated with human IgG (blocked with 1% BSA), incubated for 1.0 hour at room temperature, and washed 5 times. Diluting goat anti-mouse labeled with Horseradish Peroxidase (HRP) 1:4000, adding 50 μ l per well, and incubating at room temperature for 0.5 hr; washing is carried out for 5 times. Add 100. mu.l HRP substrate (H) per well₂O₂+ TMB), incubated for 10 minutes at room temperature, 50. mu.l of 0.5M H was added per well₂SO₄And measuring the A450nm value.

The positive well cells were collected, resuspended in HT selection medium, diluted by limiting dilution, and plated in 96 well cell culture plates, observed after 5 days, and wells where only one cell clone was determined to grow were identified by ELISA. And (3) carrying out limiting dilution on the positive hole cells, and carrying out subclone culture for 3-4 times until stable hybridoma cell clones are obtained. A total of 7 anti-human IgG monoclonal antibodies were obtained and designated 0023, 0024, 0066, 0067, 0068, 0069, and 0070, respectively.

4. Preparation of monoclonal antibodies

Culturing hybridoma cells in large scale, injecting BALB/c mouse abdominal cavity, collecting mouse ascites after 2 weeks, purifying with Protein G affinity chromatography column, dialyzing into PBS, measuring concentration, and freezing at-20 deg.C.

5. Identification of monoclonal antibodies

5.1 immunological transfer printing

After the human IgG antibody protein was denatured and reduced, SDS-PAGE was performed, NC membrane was applied, and the above 7 monoclonal antibodies were hybridized with each other to detect the binding.

5.2 detection Performance of anti-human IgG monoclonal antibody as Secondary antibody

Three monoclonal antibodies against human IgG (heavy chain specificity) were 0066, 0068 and 0070. The three monoclonal antibody proteins were labeled with horseradish peroxidase (HRP), respectively. For example, the human serum IgG antibodies infected with the new bunyavirus (SFTSV) were tested: mu.l of each of convalescent serum of a human infected with the new bunyavirus and normal serum of a human is mixed with 100 mu.l of 5% milk (5g of skimmed milk powder dissolved in 100ml of PBS), added into an ELISA plate coated with a recombinant NP antigen of the new bunyavirus, and incubated at 37 ℃ for 30 minutes. After washing 5 times, 100. mu.l (1:10000) of 0066-HRP, 0068-HRP and 0070-HRP enzyme-labeled secondary antibody was added, and the mixture was incubated at 37 ℃ for 30 minutes. After 5 times of washing, 100 ul/well of substrate is added, after 5 minutes of action at room temperature, 50 ul/well of stop solution is added, and the reading value is 450 nm.

5.3 identification of heavy and light chain isoforms of monoclonal antibody 0070

The procedure was carried out using SBA cloning system-HRP product from Southern Biotech according to the instructions provided by the manufacturer.

5.4 determination of nucleic acid sequence of heavy and light chain variable region of monoclonal antibody 0070

Total RNA was extracted from 0070 hybridoma cells in the logarithmic growth phase using Trizol from Invitrogen, and cDNA was generated by reverse transcription using oligo (dT)20 as a primer. Then, specific primers are used for PCR amplification of the heavy chain variable region gene and the light chain variable region gene respectively. And after the PCR product is purified by electrophoresis, inserting the PCR product into a pMD-18T vector by TA cloning, sequencing and carrying out sequence analysis. Table 1 and Table 2 show primers for amplifying the heavy and light chain variable region genes, respectively.

TABLE 1 primers for amplification of hybridoma heavy chain variable region genes

Note: MHV indicates the hybridization primer of the leader sequence of the mouse heavy chain variable region gene

MHCG shows a primer that hybridizes to a mouse gamma constant region gene

TABLE 2 primers for the amplification of the light chain variable region genes of hybridomas

Note: MKV is the primer for hybridizing the leader sequence of mouse kappa chain variable region gene

MKC denotes primers hybridizing with mouse kappa constant region gene

6. Results

The results of the immunoblots (FIG. 1) showed that 3 were bound to the human IgG heavy chain (0066, 0068, 0070) and 4 were bound to the human IgG light chain.

The results of the detection performance of the anti-human IgG monoclonal antibody as a secondary antibody are shown in table 3, and compared with 0066 and 0068, 0070 as a secondary antibody has a specific amplification effect on an antigen/antibody binding signal, has a high signal-to-noise ratio, and is suitable for use as a secondary antibody.

TABLE 3 monoclonal antibody as secondary antibody for detecting IgG in serum of SFTSV human in convalescent period

	0066-HRP	0068-HRP	0070-HRP
				Positive serum 1	0.346	0.840	1.437
Positive serum 2	0.534	0.938	1.494
				Positive serum 3	0.214	0.872	1.543
Positive serum 4	0.149	1.107	1.426
				Negative serum 1	0.077	0.091	0.075
Negative serum 2	0.056	0.102	0.085
				Negative serum 3	0.069	0.087	0.073
Negative serum 4	0.057	0.079	0.074

The identification of heavy and light chain isotypes of the monoclonal antibody 0070 shows that the heavy and light chain isotypes of the 0070 monoclonal antibody are gamma 1 and kappa respectively.

Sequencing results show that only the primer MHV6(27-mer)/MHCG1(21-mer) combination (SEQ ID NO.6/SEQ ID NO.13) can amplify the 0070 heavy chain variable region, and only the primer MKV7(31-mer)/MKC (20-mer) combination (SEQ ID NO.20/SEQ ID NO.25) can amplify the 0070 light chain variable region. The amino acid sequence of the monoclonal antibody is shown in table 4.

TABLE 4 sequences of monoclonal antibodies

EXAMPLE 2 use of monoclonal antibodies

Detection of human dengue virus (DENV) IgG antibody

1. After mixing 1. mu.l of recovery-period serum (total 14 parts) of a dengue virus II infected person with 100. mu.l of sample diluent, adding the mixture into an ELISA plate (coated with recombinant dengue virus serum II NS1 antigen, 100 ng/hole), setting positive and negative controls, sealing the ELISA plate by using a sealing plate membrane, and incubating the ELISA plate in an incubator at 37 ℃ for 30 minutes.

2. And (3) taking out the enzyme-labeled plate, placing the enzyme-labeled plate on a plate washing machine, washing the enzyme-labeled plate for 5 times by using washing liquor, and then spin-drying the residual liquid in the hole or patting the enzyme-labeled reaction plate on absorbent paper to remove the residual liquid in the hole.

3. 100. mu.l/well of 0070-HRP working solution was added to each well, and the mixture was incubated in an incubator at 37 ℃ for 30 minutes.

4. And (5) repeating the step (2).

5. Mixing the color development solution A, B solution at a ratio of 1:1, adding 100 μ l of mixed color development solution into each well, tapping the enzyme-labeled reaction plate, mixing, and standing at room temperature for 5 minutes.

6. Add stop solution 50. mu.l/well into each well, tap and mix well.

7. And (3) placing the ELISA plate under the wavelength of 450nm of an ELISA reader, and determining the light absorption OD value of each hole.

8. Critical value (Cutoff) calculation: cutoff ﹦ 0.748 times negative control duplicate OD mean +0.146, results: the sample to be detected is positive when the OD value is larger than Cutoff, and the sample to be detected is negative when the OD value is smaller than Cutoff.

9. Results

The results are shown in Table 5, Cutoff ﹦ 0.201.201, which indicates that 14 positive sera were IgG positive in this assay system.

TABLE 5 serum II type dengue positive patient IgG antibody test results

Serum numbering	OD450nm	Decision (+/-)
			D191002	0.833	+
D191016	2.112	+
			D191205	0.302	+
D191207	0.250	+
			D191211	1.135	+
D191212	0.688	+
			D191225	0.347	+
D191227	0.817	+
			D191229	0.232	+
D191236	0.225	+
			D191243	1.641	+
D191244	1.665	+
			D191246	0.682	+
D191247	2.591	+
			Positive reference	1.701	+
Negative control 1	0.079	-
			Negative control 2	0.069	-

Second, human infection with novel coronavirus (SARS-CoV-2) IgG antibody detection

1. After mixing 1 mul of novel coronavirus infected patient convalescent period serum (26 parts in total) and 100 mul of sample diluent, adding the mixture into an enzyme label plate (coated with recombinant new coronavirus NP antigen, 100 ng/hole), setting positive and negative controls, sealing the enzyme label plate by using a sealing plate membrane, and incubating for 30 minutes in an incubator at 37 ℃.

4. Method 2 was repeated.

6. Add stop solution 50. mu.l/well into each well, tap and mix well.

9. Results

The results are shown in Table 6, Cutoff ﹦ 0.216.216, which indicates that 26 positive sera were IgG positive in this assay system.

TABLE 6 IgG antibody test results for patients with positive coronavirus

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

<110> Nanjing Miaodi Biotech Co., Ltd

<120> a mouse anti-human IgG monoclonal antibody

<141> 2020-09-10

<160> 57

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaaatgca gctggggcat sttcttc 27

<210> 2

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggatgga gctrtatcat sytctt 26

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaagwtgt ggttaaactg ggttttt 27

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgractttg ggytcagctt grttt 25

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggactcca ggctcaattt agttttcctt 30

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggcttgtc ytrgsgctrc tcttctgc 28

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggratgga gckggrtctt tmtctt 26

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgagagtgc tgattctttt gtg 23

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggmttggg tgtggamctt gctattcctg 30

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggmttggg tgtggamctt gctattcctg 30

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggattttg ggctgatttt ttttattg 28

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgatggtgt taagtcttct gtacctg 27

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cagtggatag acagatgggg g 21

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgaagttgc ctgttaggct gttggtgctg 30

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggagwcag acacactcct gytatgggtg 30

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgagtgtgc tcactcaggt cctggsgttg 30

<210> 17

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgaggrccc ctgctcagwt tyttggmwtc ttg 33

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggatttwc aggtgcagat twtcagcttc 30

<210> 19

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgaggtkcy ytgytsagyt yctgrgg 27

<210> 20

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgggcwtca agatggagtc acakwyycwg g 31

<210> 21

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atgtggggay ctktttycmm tttttcaatt g 31

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggtrtccw casctcagtt ccttg 25

<210> 23

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atgtatatat gtttgttgtc tatttct 27

<210> 24

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atggaagccc cagctcagct tctcttcc 28

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

actggatggt gggaagatgg 20

<210> 26

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gly Phe Ser Leu Thr Ser Tyr Gly

1 5

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gggttttcat tgaccagcta tgga 24

<210> 28

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Ile Trp Ala Gly Gly Ser Thr

1 5

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atatgggctg gtggaagtac a 21

<210> 30

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Ala Arg Gly Gly Val Thr Ala Trp Phe Gly Tyr

1 5 10

<210> 31

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gccagaggtg gtgttacggc ctggtttggt tac 33

<210> 32

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Gln Val His Leu Lys Glu Ser Gly Pro Gly Gln Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser

20 25

<210> 33

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

caggtgcacc tgaaggagtc aggacctggc caggtggcgc cctcacagag cctgtccatc 60

acttgcactg tctct 75

<210> 34

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly

1 5 10 15

Val

<210> 35

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gtacactggg ttcgccagtc tccaggaaag ggtctggagt ggctgggagt a 51

<210> 36

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Asn Tyr Lys Ser Thr His Met Ser Arg Leu Ser Ile Ser Lys Asp Asn

1 5 10 15

Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 37

<211> 114

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

aattacaaat cgactcacat gtccagactg agcatcagca aagacaactc caagagccaa 60

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatgtacta ctgt 114

<210> 38

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210> 39

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

tggggccagg ggactctggt cactgtctct gca 33

<210> 40

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Gln Val His Leu Lys Glu Ser Gly Pro Gly Gln Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Lys Ser Thr His Met

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Val Thr Ala Trp Phe Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 41

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

caggtgcacc tgaaggagtc aggacctggc caggtggcgc cctcacagag cctgtccatc 60

acttgcactg tctctgggtt ttcattgacc agctatggag tacactgggt tcgccagtct 120

ccaggaaagg gtctggagtg gctgggagta atatgggctg gtggaagtac aaattacaaa 180

tcgactcaca tgtccagact gagcatcagc aaagacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccatgtact actgtgccag aggtggtgtt 300

acggcctggt ttggttactg gggccagggg actctggtca ctgtctctgc a 351

<210> 42

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Gln Asp Val Gly Ser Ala

1 5

<210> 43

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

caggatgtgg gttctgct 18

<210> 44

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Trp Ala Ser

1

<210> 45

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tgggcatcc 9

<210> 46

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

Gln Gln Tyr Thr Tyr Phe Gly Gly Thr

1 5

<210> 47

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

cagcaatata cctactttgg tggtacg 27

<210> 48

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Phe Ile Thr Cys Lys Ala Ser

20 25

<210> 49

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtgggaga cagggtcttc 60

atcacctgca aggccagt 78

<210> 50

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Val Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Ser Leu Ile

1 5 10 15

Tyr

<210> 51

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

gtagcctggt atcaacagac accaggacaa tctcctaaat cactgattta c 51

<210> 52

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Glu Ser Glu Asp Leu Ala

20 25 30

Asp Tyr Phe Cys

35

<210> 53

<211> 108

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

acccggcaca ctggagtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 60

ctcaccatta caaatgtgga gtctgaagac ttggcagatt atttctgt 108

<210> 54

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 55

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

ttcggagggg ggaccaaact tgaaataaaa 30

<210> 56

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Phe Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ser Ala

20 25 30

Val Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Ser Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Glu Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Thr Tyr Phe Gly Gly

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 57

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtgggaga cagggtcttc 60

atcacctgca aggccagtca ggatgtgggt tctgctgtag cctggtatca acagacacca 120

ggacaatctc ctaaatcact gatttactgg gcatccaccc ggcacactgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccattacaaa tgtggagtct 240

gaagacttgg cagattattt ctgtcagcaa tatacctact ttggtggtac gttcggaggg 300

gggaccaaac ttgaaataaa a 321

Claims

1. An anti-IgG monoclonal antibody comprising a three CDR heavy chain variable region and a three CDR light chain variable region; wherein, the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.26, 28 and 30, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.42, 44 and 46.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.32, 34, 36 and 38; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.48, 50, 52 and 54.

3. The monoclonal antibody according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No.40 and the amino acid sequence of the light chain variable region is represented by SEQ ID No. 56.

4. The monoclonal antibody of claim 1 or 2, 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 comprising a nucleotide sequence encoding the monoclonal antibody of any one of claims 1-4.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequences of the CDRs 1, 2, 3 encoding the heavy chain variable region of the antibody are set forth in SEQ ID nos. 27, 29, 31; the nucleotide sequences of CDR1, CDR2 and CDR3 encoding the variable region of the light chain are shown in SEQ ID NO.43, 45 and 47.

7. The nucleic acid molecule of claim 5, wherein the nucleotide sequences encoding framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region are as set forth in SEQ ID nos. 33, 35, 37 and 39; the nucleotide sequences encoding framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.49, 51, 53 and 55.

8. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID No.41 and the nucleotide sequence encoding the light chain variable region is set forth in SEQ ID No. 57.

9. A vector comprising the nucleic acid molecule of any one of claims 5 to 8.

10. A host cell comprising the nucleic acid molecule of any one of claims 5 to 8, or comprising the vector of claim 9.

11. A method for detecting IgG in a sample for non-diagnostic purposes, said method comprising the steps of:

1) contacting the sample with a first specific binding agent and a second specific binding agent, wherein the second specific binding agent is a monoclonal antibody according to any one of claims 1-4; for a time and under conditions sufficient to form a first specific binding agent-IgG-second specific binding agent complex; and

2) detecting the complex formed in 1), thereby detecting IgG in the sample;

wherein the first specific binding agent is an antigen.

12. The method of claim 11, wherein the second specific binding agent comprises a detectable label.

13. A kit for detecting IgG, said kit comprising the monoclonal antibody of any one of claims 1-4.

14. Use of a monoclonal antibody according to any one of claims 1 to 4 in the preparation of a reagent for the detection of IgG.