CN112094352A - anti-IgM monoclonal antibody - Google Patents
anti-IgM monoclonal antibody Download PDFInfo
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Abstract
The invention discloses an anti-IgM monoclonal antibody, which comprises a heavy chain variable region CDR1, a CDR2 and a CDR3 which have amino acid sequences shown in SEQ ID NO.26, 28 and 30 respectively; and light chain variable region CDR1, CDR2, CDR3 having the amino acid sequences shown in SEQ ID Nos. 42, 44, 46, respectively. 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 sensitivity and specificity.
Description
Technical Field
The invention belongs to the fields of cellular immunology and genetic engineering, and relates to an anti-IgM 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.
The IgM antibody with pathogen specificity in human serum is detected by ELISA, which has significance for the clinical early diagnosis of infectious disease infectors and the overall control of the whole infectious disease epidemic situation. The content of IgM antibody in human serum is 0.6-2.0mg/ml, which only accounts for 6-10% of the total amount of serum antibody, and is lower than that of IgG and IgA, and IgM molecules are relatively huge and exist in the form of pentamer, so that IgM detection is more difficult than IgG and IgA detection. In the prior art, indirect ELISA is generally adopted to detect pathogen specific IgM antibody, and the operation is usually to dilute serum to be detected, combine the diluted serum with antigen coated on an ELISA plate, wash the serum, add an enzyme-labeled anti-human IgM second antibody and develop color by using a substrate. However, because the content of IgM in serum is lower than that of IgG and IgA, and the IgM molecule is larger than that of IgG and IgA, IgM is inferior in the process of competitively binding to the antigen on the microplate with IgG and IgA, and sensitivity of the detection result is generally low. In addition, when IgM antibodies are detected by indirect ELISA, the used secondary enzyme-labeled antibodies are mostly polyclonal antibodies (antibodies containing anti-human IgM heavy-light chains at the same time), and the detection result usually has higher false positive or higher background.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide an anti-human IgM monoclonal antibody and a preparation method thereof, which can specifically capture a human IgM antibody, facilitate the combination of enzyme-labeled antigens, increase the specificity of IgM detection and reduce the background of the whole system.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided an anti-IgM monoclonal antibody comprising:
heavy chain variable region CDR1, CDR2, CDR3 having the amino acid sequences shown in SEQ ID Nos. 26, 28, 30, respectively; and light chain variable region CDR1, CDR2, CDR3 having the amino acid sequences shown in SEQ ID Nos. 42, 44, 46, respectively.
Further, the heavy chain variable region further comprises:
heavy chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences set forth in SEQ ID Nos. 32, 34, 36 and 38; and light chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown as SEQ ID NO.48, 50, 52 and 54, respectively.
Further, the heavy chain variable region has an amino acid sequence shown as SEQ ID NO.40, and the light chain variable region has an amino acid sequence 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.
Further, the nucleotide sequences of CDR1, CDR2 and CDR3 encoding the heavy chain variable region 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.
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. The vector of the present invention is not particularly limited, but 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, minichromosome, viral or retroviral vector.
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.
The cells for introducing the vector in the present invention include prokaryotic cells 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 blast, COS, NSO, 293T, Bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc.; 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 "introduction" refers to the delivery of 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 recombinant cells of the invention can then be used for expression as well as culture purposes for antibody expression for large scale drug production. Can also be used as active ingredient of pharmaceutical composition. Any suitable culture technique may be used, including but not limited to static culture, spinner flask culture, ascites fluid, hollow fiber-type bioreactor cartridges, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, and the like.
In a fifth aspect, the present invention provides a method for detecting IgM 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 first 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-IgM-second specific binding agent complex; and
2) detecting the complex formed in 1), thereby detecting IgM in the sample;
further, the microplate is coated with a first specific binding agent.
Further, the second specific binding agent is an antigen.
Further, the second specific binding agent is detectably labeled; the label is a label conventional in the art, including but not limited to fluorescent dyes, such as fluorescein-type labels FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine-type labels, including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; texas Red; and the like; luminescent dyes, such as the chemiluminescent dyes luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, peroxy oxalate based systems and derivatives thereof; electrochemiluminescent dyes such as electrochemiluminescent complexes based on ruthenium and iridium; a radioactive label such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At or 131 Bi; a metal chelate.
In a sixth aspect, the present invention provides a product for detecting IgM, which comprises the monoclonal antibody according to the first aspect of the present invention.
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.
As an alternative embodiment, the product is a chip, which includes 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.
In the present invention, the method for detecting or determining the amount of IgM 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.
As an alternative embodiment, the monoclonal antibodies of the invention may be used for the detection of viruses. The detection method comprises the following steps:
1) contacting a sample to be tested with a first specific binding agent and a second specific binding agent, wherein the first specific binding agent is the monoclonal antibody of the first aspect of the invention; for a time and under conditions sufficient to form a first specific binding agent-IgM-second specific binding agent complex; and
2) detecting the complex formed in 1), thereby detecting IgM in the sample.
Preferably, the microplate is coated with a first specific binding agent and the viral antigen to be detected is used as the second specific binding agent.
More preferably, the second specific binding agent is detectably labeled.
In a specific embodiment of the invention, the detectable label is an HRP label.
In the present invention, 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 comprises 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 IgM.
Specifically, if the functional variants include (but are not limited to): substantially similar in primary structural sequence but comprising chemically and/or biochemically modified derivatives, e.g., in vitro or in vivo, not present in the parent monoclonal antibody of the invention. Such 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, and the like.
Alternatively, the functional variant may be a monoclonal antibody as follows: amino acid sequences comprising substitutions, insertions, deletions or combinations thereof of one or more amino acids 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 IgM. For example, a functional variant according to the invention may have increased or decreased binding affinity for IgM as compared to the parent monoclonal antibody.
It will also be understood by those skilled in the art that the present invention encompasses amino acid sequence modifications of the anti-IgM 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-IgM antibodies are prepared by introducing appropriate nucleotide changes into anti-IgM 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-IgM 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-IgM antibody, such as changing the number or position of glycosylation sites.
Preferably, the amino acid sequence of the variable region including, but not limited to, the framework region, the hypervariable region, and particularly the CDR3 region is modified. Typically, the light or heavy chain region includes three hypervariable regions (including three CDRs) and more conserved regions (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.
In the present invention, the terms "sample" or "test sample" are used interchangeably herein, the sample being an in vitro sample that is to be analyzed in vitro and not transferred back into vivo. 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.
The invention has the advantages and beneficial effects that:
the invention provides an anti-human IgM monoclonal antibody which is used for detecting IgM, can reduce the background of the whole reaction system and amplify a signal of specific binding, and has higher sensitivity and specificity.
Drawings
FIG. 1 is an immunoblot identifying the binding specificity of 7 mouse anti-human IgM.
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 IgM monoclonal antibody
1. Balb/c mouse immunization
Commercial human IgM antibody (sigma-aldrich) was used to immunize 5-week-old female Balb/c mice (100. mu.g/mouse), and 100. mu.g of antigen was mixed with equal volume of Freund's complete adjuvant and injected into the abdominal cavity 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, from each well50 μ l of the supernatant was pipetted and applied to a 96 well ELISA plate coated with human IgM (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 antibody strains were obtained and designated 0035, 0036, 0037, 0038, 0039, 0040 and 0041, 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 mouse anti-human IgM monoclonal antibody
5.1 immunological transfer printing
After denaturation and reduction treatment, the human IgM antibody protein was subjected to SDS-PAGE and NC membrane transfer, and the above 7 monoclonal antibodies were hybridized with the antibody, respectively, to detect the binding.
5.2 feasibility of anti-human IgM monoclonal antibody as a Capture antibody (MAC-ELISA)
The anti-human IgM (heavy chain specificity) monoclonal antibodies total four strains, namely 0035, 0036, 0038 and 0041. The four monoclonal antibody proteins were coated on an enzyme-labeled plate (COSTAR) at 200 ng/well, respectively. Taking as an example the test of specific IgM antibodies in human acute phase serum infected with New bunyavirus (SFTSV): mu.l each of the serum (3 parts) of the acute-phase human serum infected with the new bunyavirus and the normal human serum (1 part) was mixed with 90. mu.l of 5% milk (5g of skim milk powder in 100ml of PBS), and then added to the ELISA plates coated with the monoclonal antibodies 0035, 0036, 0038 and 0041, respectively, and incubated at 37 ℃ for 30 minutes. After 5 washes, 100. mu.l of the new bunyavirus recombinant NP antigen-HRP (1:2000) was added and incubated at 37 ℃ for 30 min. After 5 washes, 100. mu.l/well of substrate was added, and after 5 minutes at room temperature, 50. mu.l/well of stop buffer was added, reading 450 nm.
5.3 identification of heavy and light chain isotypes of monoclonal antibody 0038
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 0038
The 0038 hybridoma cells in the logarithmic growth phase were taken, total RNA was extracted 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 showed that 4 strains bound to human IgM heavy chains (0035, 0036, 0038, 0041) and 3 strains bound to human IgM light chains (0037, 0039, 0040) (fig. 1).
The anti-human IgM monoclonal antibody has a better effect of detecting IgM in SFTSV human acute-phase serum as a capture antibody, wherein the monoclonal antibody No. 0038 is more suitable for being used as the capture antibody in a MAC-ELISA system than other monoclonal antibodies.
TABLE 3 monoclonal antibody as capture antibody for detection of IgM in SFTSV human acute phase serum
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 MHV7(26-mer)/MHCG1(21-mer) combination (SEQ ID NO.7/SEQ ID NO.13) can amplify the heavy chain variable region of the monoclonal antibody No. 0038, and only the primer MKV5(30-mer)/MKC (20-mer) combination (SEQ ID NO.18/SEQ ID NO.25) can amplify the light chain variable region of the monoclonal antibody No. 0038. 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 Niya virus (SFTSV) IgM antibody
1. After 10. mu.l of acute-phase serum (20 parts in total) of a new bunyavirus infected person is mixed with 90. mu.l of sample diluent, the mixture is added into an ELISA plate (coated with 0038 monoclonal antibody protein, 200 ng/hole), positive and negative controls are set at the same time, the ELISA plate is sealed by a sealing plate membrane, and the mixture is incubated 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. Add 100. mu.l/well of recombinant NP antigen-HRP from New bunyavirus into each well, incubate for 30 min at 37 ℃.
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 ﹦ 0.22.22, which indicates that 20 positive sera showed positive IgM on this assay system.
TABLE 5 IgM antibody test results for patients in the acute phase of SFTSV
Second, detection of IgM antibody of human dickinsonia tsutsugamushi (Rickettsia tsutsugamushi)
1. 10 mul of acute-stage serum (25 parts in total) of tsutsugamushi disease infected persons and 90 mul of sample diluent are mixed, added into an enzyme label plate (coated with 0038 monoclonal antibody protein, 200 ng/hole), positive and negative controls are set at the same time, the enzyme label plate is sealed by a sealing plate membrane, and is placed in an incubator at 37 ℃ for incubation 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. Each well was added with 56kDa-HRP 100. mu.l/well (1:2000) of tsutsugamushi disease rickettsia, and incubated at 37 ℃ in an incubator for 30 minutes.
4. Method 2 was repeated.
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 6, Cutoff ﹦ 0.208.208, which indicates that 25 positive sera showed positive IgM on this assay system.
IgM antibody detection result of patient in acute stage of tsutsugamushi disease of TABLE 6
| Serum numbering | OD450nm | Decision (+/-) |
| JSRH001 | 0.450 | + |
| JSRH002 | 1.122 | + |
| JSRH003 | 0.754 | + |
| JSRH004 | 0.231 | + |
| JSRH005 | 0.358 | + |
| JSRH006 | 0.761 | + |
| JSRH007 | 0.301 | + |
| JSRH008 | 1.091 | + |
| JSRH009 | 1.925 | + |
| JSRH010 | 0.584 | + |
| JSRH011 | 0.396 | + |
| JSRH012 | 0.581 | + |
| JSRH013 | 0.752 | + |
| JSRH014 | 1.203 | + |
| JSRH015 | 1.384 | + |
| JSRH016 | 0.749 | + |
| JSRH017 | 0.293 | + |
| JSRH018 | 0.402 | + |
| JSRH019 | 0.791 | + |
| JSRH020 | 0.631 | + |
| JSRH021 | 0.587 | + |
| JSRH022 | 0.247 | + |
| JSRH023 | 0.608 | + |
| JSRH024 | 0.396 | + |
| JSRH025 | 0.903 | + |
| Positive reference | 1.357 | + |
| Negative control 1 | 0.088 | - |
| Negative control 2 | 0.079 | - |
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> an anti-IgM monoclonal antibody
<141> 2020-09-27
<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> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgggcagac ttacattctc attcctg 27
<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 Tyr Thr Phe Ser Ser Tyr Trp
1 5
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggctacacat tcagtagtta ctgg 24
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Ile Leu Pro Gly Ser Gly Ser Ser
1 5
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
attttacctg gaagtggtag tagt 24
<210> 30
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Ala Arg Leu Glu Gly Arg Gly Phe Tyr Tyr Val Met Asp Tyr
1 5 10
<210> 31
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gcaagattgg aaggccgggg tttttattat gttatggact ac 42
<210> 32
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr
20 25
<210> 33
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
caggttcagc tgcagcagtc tggagcagag ctgatgaagc ctggggcctc agtgaagata 60
tcctgcaagg ctact 75
<210> 34
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly
1 5 10 15
Glu
<210> 35
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atagagtggg taaagcagag gcctggacat ggccttgagt ggattggaga g 51
<210> 36
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Asn Tyr Asn Glu Arg Phe Lys Gly Arg Ala Thr Phe Thr Ala Asp Thr
1 5 10 15
Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
20 25 30
Ser Ala Val Tyr Tyr Cys
35
<210> 37
<211> 114
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aactacaatg agaggttcaa gggcagggcc acattcactg cagatacatc ctccaacaca 60
gcctacatgc aactcagtag tctgacatct gaggactctg ccgtctatta ctgt 114
<210> 38
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
1 5 10
<210> 39
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tggggtcaag gaacctcagt caccgtctcc tca 33
<210> 40
<211> 121
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Leu Pro Gly Ser Gly Ser Ser Asn Tyr Asn Glu Arg Phe
50 55 60
Lys Gly Arg Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Glu Gly Arg Gly Phe Tyr Tyr Val Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 41
<211> 363
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
caggttcagc tgcagcagtc tggagcagag ctgatgaagc ctggggcctc agtgaagata 60
tcctgcaagg ctactggcta cacattcagt agttactgga tagagtgggt aaagcagagg 120
cctggacatg gccttgagtg gattggagag attttacctg gaagtggtag tagtaactac 180
aatgagaggt tcaagggcag ggccacattc actgcagata catcctccaa cacagcctac 240
atgcaactca gtagtctgac atctgaggac tctgccgtct attactgtgc aagattggaa 300
ggccggggtt tttattatgt tatggactac tggggtcaag gaacctcagt caccgtctcc 360
tca 363
<210> 42
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Ser Ser Val Arg Tyr
1 5
<210> 43
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tcaagtgtac gttac 15
<210> 44
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Ser Thr Ser
1
<210> 45
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
agcacatcc 9
<210> 46
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gln Gln Arg Ser Ser Ser Pro Pro Thr
1 5
<210> 47
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cagcaaagga gtagttcccc acccacg 27
<210> 48
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser
20 25
<210> 49
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
caaattgttc tcacccagtc tccagcaatc atgtctgcgt ctccagggga gaaggtcacc 60
ataacctgca gtgccagt 78
<210> 50
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile
1 5 10 15
Tyr
<210> 51
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
atgcactggt tccagcagaa gccaggcact tctcccaaac tctggattta t 51
<210> 52
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly
1 5 10 15
Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala
20 25 30
Thr Tyr Tyr Cys
35
<210> 53
<211> 108
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
aacctggctt ctggagtccc tggtcgcttc agtggcagtg gatctgggac ctcttactct 60
ctcacaatca gccgaatgga ggctgaagat gctgccactt attactgc 108
<210> 54
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
1 5 10
<210> 55
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ttcggtgctg ggaccaagct ggagctgaaa 30
<210> 56
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Pro Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 57
<211> 318
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
caaattgttc tcacccagtc tccagcaatc atgtctgcgt ctccagggga gaaggtcacc 60
ataacctgca gtgccagttc aagtgtacgt tacatgcact ggttccagca gaagccaggc 120
acttctccca aactctggat ttatagcaca tccaacctgg cttctggagt ccctggtcgc 180
ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240
gatgctgcca cttattactg ccagcaaagg agtagttccc cacccacgtt cggtgctggg 300
accaagctgg agctgaaa 318
Claims (10)
1. An anti-IgM monoclonal antibody, comprising:
heavy chain variable region CDR1, CDR2, CDR3 having the amino acid sequences shown in SEQ ID Nos. 26, 28, 30, respectively; and light chain variable region CDR1, CDR2, CDR3 having the amino acid sequences shown in SEQ ID Nos. 42, 44, 46, respectively.
2. The method of claim 1, wherein the heavy chain variable region further comprises:
heavy chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences set forth in SEQ ID Nos. 32, 34, 36 and 38; and light chain variable region framework regions FR1, FR2, FR3 and FR4 of the amino acid sequences shown as SEQ ID NO.48, 50, 52 and 54, respectively.
3. The monoclonal antibody according to claim 1 or 2, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID No.40 and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 56.
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 comprising a nucleotide sequence encoding the monoclonal antibody of any one of claims 1-4;
preferably, the nucleotide sequences of the CDRs 1, 2 and 3 encoding the heavy chain variable region are shown in SEQ ID NO.27, 29 and 31; the nucleotide sequences of CDR1, CDR2 and CDR3 for encoding the variable region of the light chain are shown in SEQ ID NO.43, 45 and 47;
preferably, the nucleotide sequences encoding framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region are as shown 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;
preferably, 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.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the nucleic acid molecule of claim 5, or comprising the vector of claim 6.
8. A method for detecting IgM 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 first 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-IgM-second specific binding agent complex; and
2) detecting the complex formed in 1), thereby detecting IgG in the sample;
preferably, the microplate is coated with a first specific binding agent;
preferably, the second specific binding agent is an antigen;
preferably, the second specific binding agent is detectably labeled;
preferably, the detectable label is an HRP label.
9. An IgM detection product comprising the monoclonal antibody according to any one of claims 1 to 4.
10. Use of a monoclonal antibody according to any one of claims 1 to 4 in an immunoassay.
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| CN114835807A (en) * | 2022-06-10 | 2022-08-02 | 青岛汉德森生物科技有限公司 | Monoclonal antibody |
| WO2023088443A1 (en) * | 2021-11-20 | 2023-05-25 | 东莞市朋志生物科技有限公司 | Anti-human igm antibody and preparation method therefor and use thereof |
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