CN115677856B - Anti-human IgM antibodies and uses thereof - Google Patents

Anti-human IgM antibodies and uses thereof Download PDF

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CN115677856B
CN115677856B CN202110861408.8A CN202110861408A CN115677856B CN 115677856 B CN115677856 B CN 115677856B CN 202110861408 A CN202110861408 A CN 202110861408A CN 115677856 B CN115677856 B CN 115677856B
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binding fragment
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CN115677856A (en
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孟媛
钟冬梅
唐丽娜
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention belongs to the technical field of antibodies. In particular, it relates to anti-human IgM antibodies and uses thereof. The anti-human IgM antibody provided by the invention has good stability and high binding affinity with human IGM, can obviously reduce or even eliminate endogenous interference, further improves the accuracy of immunodetection, and is efficient and convenient; therefore, the antibody or antigen binding fragment thereof, and the related nucleic acid, vector or cell thereof can be widely applied to the field of immunodetection.

Description

Anti-human IgM antibodies and uses thereof
Technical Field
The invention belongs to the technical field of antibodies. More particularly, to anti-human IgM antibodies and uses thereof.
Background
Immunodetection methods based on antigen-antibody reactions are widely used, and are classified into different detection methods according to antibody markers, such as: enzyme-linked immunity, radioimmunoassay, chemiluminescence, and the like. In clinical applications, the accuracy of the immunoassay results is often affected to varying degrees by interferents in the serum of the patient, resulting in erroneous detection results. The interferents in serum can be classified into endogenous interference and exogenous interference, wherein the exogenous interference comprises hemolysis, sample bacterial contamination, incomplete blood coagulation, stability and preservation condition of a sample and the like; endogenous disturbances include Rheumatoid Factors (RF), xenotropic antibodies (HA), autoantibodies, complement, jaundice, high lipids, etc.; among them, RF and HA are important interference factors. Studies have demonstrated that about 3% -15% of healthy people contain endogenous interference factors, more than 10% (30% -40%) of patients have HA interference, and 10% -40% of people have HAMA interference. Of the endogenous interferences, RF, HA are most common. Therefore, research and development of effective means for reducing or even eliminating RF interference and HA interference are important subjects for guaranteeing the reliability of medical immune test results and ensuring the benefits of doctors and patients. Aiming at eliminating RF interference and HA interference in immunodiagnosis, the simplest and most effective method is to add a blocking agent into a detection system to directly block the binding of an interfering substance and an antibody or antigen in the detection system.
The blocking agent is a biological agent which is added into an immunoassay system and can react with endogenous antibodies, thereby effectively preventing non-analyte-mediated antibody bridging. Blocking agents can be classified as passive blocking agents and active blocking agents. Active blockers are specific to human immunoglobulins and can specifically, actively and efficiently neutralize the components of interfering antibodies, thereby blocking the generation of unintended binding, such as IIR, HBR, etc., in commercial reagents. The preparation can eliminate various heterotrophic interferences, has specific binding force on the interfering heterotrophic antibodies, can be blocked efficiently only by low concentration, and minimizes the influence. IIR is a mixed murine McAb produced from HA and HAAA as immunogens, which HAs a higher affinity (109L/mol) for HAAA. HBR is McAb of murine anti-human IgM. In the active blocking process, the effect of eliminating the interference depends on the affinity of the active blocking agent for the allophilic antibody. Active blockers have a higher blocking capacity in some assays than passive blockers due to their high affinity.
The blocking agent products in the market have certain performance defects although more, and the blocking agent is very large in use amount, and the main flow blocking agent is most imported and is high in price, so that the blocking agent with better performance and lower cost is needed in the market.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of high cost and poor performance of the existing immune blocking agent, and provides an anti-human IgM antibody and application thereof, wherein the anti-human IgM antibody has high affinity with human IgM, good stability and low cost, can be applied to the field of immunodetection as or for preparing the immune blocking agent and for preparing an immunodetection reagent or a kit so as to reduce or even eliminate endogenous interference, and the blocking effect of the anti-human IgM antibody is obviously better than that of a market blocking agent raw material.
It is an object of the present invention to provide anti-human IgM antibodies or antigen binding fragments thereof, said antibodies comprising the following heavy chain complementarity determining regions HCDRs:
HCDR1 with the amino acid sequence shown as SEQ ID NO.1, HCDR2 with the amino acid sequence shown as SEQ ID NO.2, and HCDR3 with the amino acid sequence shown as SEQ ID NO. 3.
It is another object of the present invention to provide nucleic acids, vectors or cells related to said anti-human IgM antibodies or antigen binding fragments thereof.
The invention also provides application of the anti-human IgM antibody or antigen binding fragment thereof and related nucleic acid, vector or cell thereof in immunodetection.
The invention also provides the use of said anti-human IgM antibodies or antigen binding fragments thereof, and related nucleic acids, vectors or cells as/in the preparation of immune blockers.
The invention also provides an immune blocking agent comprising the anti-human IgM antibody or antigen binding fragment thereof, the vector, the nucleic acid or the cell.
The invention also provides a method for reducing/eliminating endogenous interference, and the immune blocking agent is added into an immune detection system.
The invention also provides an immunodiagnostic reagent/kit comprising the immunoblocker.
Drawings
FIG. 1 is a diagram showing the result of reducing SDS-PAGE of the Ig-M-9G1RMb1 antibodies prepared in example 1.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The present invention relates to anti-human IgM antibodies or antigen binding fragments thereof, said antibodies comprising the following heavy chain complementarity determining regions HCDRs:
HCDR1 with the amino acid sequence shown as SEQ ID NO.1, HCDR2 with the amino acid sequence shown as SEQ ID NO.2, and HCDR3 with the amino acid sequence shown as SEQ ID NO. 3.
In some embodiments, the antibody further comprises the following light chain complementarity determining regions LCDRs:
LCDR1 with the amino acid sequence shown as SEQ ID NO.4, LCDR2 with the amino acid sequence shown as SEQ ID NO.5, and LCDR3 with the amino acid sequence shown as SEQ ID NO.6 or SEQ ID NO. 7.
An important advantage of the antibody or antigen binding fragment thereof is that it has a high affinity for human IGM.
An important advantage of the antibody or antigen binding fragment thereof is that the antibody has a remarkable blocking effect on endogenous interferences such as a pseudo-cation sample, an RF sample and the like, and the blocking effect is even better than that of a market blocker raw material, so that the endogenous interferences can be reduced or even eliminated.
In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity. The term "antigen binding fragment" is a substance comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab (consisting of intact light chains and Fd), fv (consisting of VH and VL), scFv (single chain antibody, with a linker peptide between VH and VL) or single domain antibody (consisting of VH only). Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 scFv, fv, fd, single chain antibody, diabody or domain antibody. In a specific embodiment of the invention, the antigen binding fragment of an anti-human IgM antibody has a high affinity for human IgM. In specific embodiments of the invention, the antigen binding fragments of the anti-human IgM antibodies are capable of significantly blocking endogenous interference, such as a false positive sample and an RF sample, and reducing or even eliminating the endogenous interference.
In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues contributing to the binding affinity of an antibody or antigen binding fragment thereof to an antigen or epitope recognized by the antibody or antigen binding fragment thereof. In a specific embodiment of the invention, the CDRs refer to the highly variable regions of the heavy and light chains of an anti-human IgM antibody.
In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR1; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR1. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, chothia and Lesk numbering scheme, and the 1997 Lefranc et al have introduced a new standardized numbering system for all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
In some embodiments, the antibody further comprises at least one of a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 9 or SEQ ID NO. 10.
In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE or IgM, and the light chain constant region is a kappa chain or a lambda chain.
In some embodiments, the species source of the heavy and light chain constant regions is cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock, or human.
In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 11 and the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 12 or SEQ ID NO. 13.
The invention also relates to nucleic acids encoding the antibodies or antigen binding fragments thereof.
Nucleic acids are typically RNA or DNA, and nucleic acid molecules may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are used when they are incorporated into vectors.
The invention also relates to a vector containing said nucleic acid.
The invention also relates to a cell containing said nucleic acid or said vector.
The use of said antibodies or antigen binding fragments thereof, said nucleic acids, said vectors or said cells in immunoassays, and their use as/in the preparation of immune blockers shall all be within the scope of the invention.
The invention also relates to an immune blocking agent comprising said antibody or antigen binding fragment thereof, said nucleic acid, said vector or said cell.
The invention also relates to a method for reducing/eliminating endogenous interference, wherein the immune blocking agent is added in an immune detection system. In a specific embodiment of the invention, the endogenous interference is rheumatoid factor interference or xenotropic antibody interference.
The invention also relates to an immunodiagnostic reagent/kit comprising said immunoblocker.
In some embodiments, the kit is an immunochromatographic assay kit, an enzyme-wash test kit, a chemiluminescent kit, or an immunonephelometric assay kit.
In some embodiments, the kit may include a test strip or a test card onto which the liquid sample from the subject is placed, or an ELISA assay plate with wells in which liquid samples from individual subjects may be placed. In some embodiments, the kit may include a testing device configured for use in a flow cytometer, a biological analyzer, a biosensor.
In some embodiments, the immune blocking agent contained in the kit may be in the form of a liquid solution, attached to a solid support, or as a dry powder. When the immune blocking agent is a liquid solution, the liquid solution may be an aqueous solution. When the immune blocking agent is in the form of an attached solid support, the preferred solid support may be a chromatographic medium such as a film, test strip, plastic bead or plate, or a microscope slide. When the immune blocking agent is a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
The invention has the following beneficial effects:
compared with the raw materials of market blocking agents, the anti-human IgM antibody has the advantages of good stability, low cost, high binding affinity with human IGM, obvious blocking effect on endogenous interference such as a pseudo-cation sample, an RF sample and the like, and can reduce or even eliminate the endogenous interference by directly adding the anti-human IgM antibody into a detection system when in use, thereby improving the accuracy of immunodetection, along with high efficiency and convenience; therefore, the antibody or antigen binding fragment thereof, and the related nucleic acid, vector or cell thereof can be used as or for preparing an immune blocking agent, and for preparing an immune detection reagent or kit, and are widely applied to the field of immune detection.
Embodiments of the present invention will be described in detail below with reference to examples.
In the following examples, restriction enzymes, prime Star DNA polymerase were purchased from Takara corporation. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.
Example 1 preparation of anti-human IgM antibody (Ig-M-9G 1RMb1 antibody)
1. Construction of expression plasmid
(1) Ig-M-9G1RMb1 antibody Gene preparation
mRNA is extracted from hybridoma cell strain secreting Ig-M-9G1RMb1 antibody, DNA product is obtained by RT-PCR method, the product is inserted into pMD-18T vector after A adding reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, after bacterial colony is grown, 4 clones of heavy chain and light chain gene clone are respectively taken, and the heavy chain and light chain gene clone are sent to gene sequencing company for sequencing.
(2) Sequence analysis of Ig-M-9G1RMb1 antibody variable region Gene
The gene sequences obtained by sequencing are placed in an IMGT antibody database for analysis, and are analyzed by using VNTI11.5 software.
Analysis determines that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct; in the gene fragment amplified by the light chain primer pair, the VL gene sequence is 396bp, belongs to the VkII gene family, and a 57bp leader peptide sequence is arranged in front of the VL gene sequence; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 432bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene family.
(3) Construction of recombinant antibody expression plasmids
pcDNA TM 3.4vector is a constructed Ig-M-9G1RMb1 antibody eukaryotic expression vector, which has been introduced into HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and is named pcDNA3.4A expression vector, and is subsequently abbreviated as 3.4A expression vector; according to the result of the variable region gene sequencing of the Ig-M-9G1RMb1 antibody, VL and VH gene specific primers of the Ig-M-9G1RMb1 antibody are designed, and a HindIII, ecoRI restriction site and a protecting base are respectively arranged at two ends, and a light chain gene fragment of 0.73KB and a heavy chain gene fragment of 1.43KB are amplified by a PCR amplification method.
The heavy chain and light chain gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the heavy chain gene and the light chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the heavy chain and the light chain are respectively obtained.
2. Stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
Ig-M-9G1RMb1 antibody-expressing plasmid was diluted with ultrapure water to 40. Mu.g/100. Mu.L, and CHO cells were regulated to 1.43X10 × 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sample counted on days 3, 5, 7, and harvested on day 7.
Coating liquid (main ingredient is NaHCO) 3 ) Diluting human IgM to 1. Mu.g/mL, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing liquid (main component is Na 2 HPO 4 +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; the diluted cell supernatant was added at 100. Mu.L/well for 30min at 37℃to obtain a fraction of the supernatant (1 h). Meanwhile, taking the supernatant of the cells which are not added as a blank control; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding color development solution A (50 μl/hole, wherein the main components of color development solution A are citric acid, sodium acetate, acetanilide and carbamide peroxide), adding color development solution B (50 μl/hole, wherein the main components of color development solution B are citric acid, EDTA.2Na, TMB and concentrated HCl) for 10min; adding stop solution (the main components of the stop solution are EDTA.2Na and concentrated H) 2 SO 4 ) 50. Mu.L/well; OD was read on the microplate reader at 450nm (reference 630 nm).
The results showed that the OD of the reaction was still greater than 1.0 after 1000-fold dilution of the cell supernatant, and less than 0.1 without cell supernatant, indicating that the antibodies produced after transient plasmid transformation were active on human IGM.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, pvuI enzyme 10 mu L, sterile water to 500 mu L, water bath at 37℃overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
The plasmid was diluted to 40. Mu.g/100. Mu.L with ultrapure water, and CHO cells were regulated to 1.43X 10 7 Placing cells/mL in a centrifuge tube, mixing 100 mu L of plasmid with 700 mu L of cells, transferring into an electrorotating cup, electrorotating, and counting the next day; 25 mu mol/L MSX 96 wells were incubated under pressure for approximately 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/mL and 2.2mL, and cell density was 0.3X10 6 Performing seed preservation by using cells/mL and 2 mL; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3. Ig-M-9G1RMb1 antibody preparation
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125mL shake flasks with an inoculation volume of 30mL and a medium of 100% Dynamis, and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/mL, and calculating the expanded culture volume according to the production requirement, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/mL for production.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. And carrying out affinity purification by using a protein A affinity chromatography column to obtain the Ig-M-9G1RMb1 antibody. 6.6. Mu.g of Ig-M-9G1RMb1 antibody was subjected to reducing SDS-PAGE.
The results of the reducing SDS-PAGE of the Ig-M-9G1RMb1 antibody are shown in FIG. 1, and show two bands, 1 Mr 50KD (heavy chain) and the other Mr 28KD (light chain).
The amino acid sequence of HCDR1 of Ig-M-9G1RMb1 antibody is shown as SEQ ID NO.1, the amino acid sequence of HCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 3; the amino acid sequence of LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 6;
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9;
the amino acid sequence of the heavy chain is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain is shown as SEQ ID NO. 12.
Example 2 preparation of anti-human IgM antibody (Ig-M-9G 1RMb2 antibody)
The sequence of the Ig-M-9G1RMb1 antibody prepared in example 1 was analyzed, the LCDR3 st amino acid I (IIe, isoleucine) of the Ig-M-9G1RMb1 antibody was mutated to L (Leu, leucine) by a conventional amino acid-introduced site-directed mutation method, and another antibody (Ig-M-9G 1RMb2 antibody) was constructed by the same method as in step (3), step 2 and step 3 of step 1 of example 1.
The amino acid sequence of HCDR1 of Ig-M-9G1RMb2 antibody is shown as SEQ ID NO.1, the amino acid sequence of HCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 3; the amino acid sequence of LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 7;
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10;
the amino acid sequence of the heavy chain is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain is shown as SEQ ID NO. 13.
Example 3 affinity analysis of anti-human IgM antibodies
The Ig-M-9G1RMb1 antibody prepared in example 1, the Ig-M-9G1RMb2 antibody prepared in example 2, and the market blocker raw material were analyzed for affinity by the following methods:
using an AMC sensor, ig-M-9G1RMb1 antibody and Ig-M-9G1RMb2 antibody were diluted to 10. Mu.g/mL with PBST, and human IGM was gradient diluted with PBST;
the operation flow is as follows: buffer 1 (PBST, main ingredient Na 2 HPO 4 +NaCl+TW-20), antibody solution solidified antibody 300s, buffer 2 (PBST, main ingredient is Na) 2 HPO 4 +NaCl+TW-20), binding 420s in antigen solution, dissociating 1200s in buffer 2, and incubating with 10mM pH 1.69 GLY solution and buffer 3 (PBST, main component Na 2 HPO 4 +NaCl+TW-20) to perform sensor regeneration and output data.
The results of the affinity analysis of the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody are shown in Table 1, and the results show that the affinity of the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody obtained by the invention to human IGM is significantly higher than that of the raw materials of the market blocker.
TABLE 1
Sample name KD(M) kon(1/Ms) kdis(1/s)
Market blocker raw material 2.49E-10 8.15E+05 2.03E-04
Ig-M-9G1RMb1 antibodies 3.02E-12 9.41E+06 2.84E-05
Ig-M-9G1RMb2 antibodies 4.27E-12 1.42E+07 6.07E-05
Note that: in table 1, KD represents equilibrium dissociation constant, i.e. affinity; kon represents the binding rate; kdis represents the dissociation rate. The lower the KD value, the higher the affinity.
Example 4 determination of blocking Properties of anti-human IgM antibodies
1. Verification of blocking Performance on CTNI fluorescent platform
In CTNI fluorescent platform pairing detection, an experimental group respectively treats sample pads with the Ig-M-9G1RMb1 antibody prepared in example 1, the Ig-M-9G1RMb2 antibody prepared in example 2 and a market blocker raw material, and a blank control group sample pad is not treated; samples (L1-L10) were separately examined.
The blocking effects of the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody on the CTNI fluorescent platform are shown in Table 2, and the results show that the experimental group has remarkable elimination effect on the pseudo-cation sample; the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody have obvious blocking effects on the false positive samples, and the blocking effects are obviously better than those of the market blocker raw materials.
TABLE 2
In Table 2, the T/C values illustrate:
the sample to be detected is added into a sample adding port of the detection reagent card, and under the lateral capillary action, the sample to be detected passes through the binding pad and is subjected to specific immunological combination with the fluorescent group marked antibody on the binding pad, and the specific immunological combination is respectively combined to form an antigen-antibody fluorescent compound, so that the antigen-antibody fluorescent compound is fixed in a T line. The C line is coated with a substance which reacts with the free fluorescent group-labeled antibody, and when the free fluorescent group-labeled antibody passes through the C line, the free fluorescent group-labeled antibody can be specifically and immunologically combined with the substance on the C line, so that the free fluorescent group-labeled antibody is fixed in the C line. The fluorescence intensities of the two bands detected by the fluorescence immunoassay instrument are reflected in peak areas, and the T/C value is calculated by calculation software of the instrument itself. The instrument reading T/C represents the ratio of the area of the T peak to the area of the C peak, and under a quality control sample and a positive sample, the higher the T/C is, the higher the representative activity is; the lower the T/C under the pseudo-cation sample, the better the blocking effect is represented; when the T/C value is less than 0.1, the negative sample is judged.
2. Verification of blocking Performance on GRP chemiluminescent platform
In the GRP chemiluminescent platform pairing detection, the Ig-M-9G1RMb1 antibody prepared in example 1, the Ig-M-9G1RMb2 antibody prepared in example 2 and the market blocker raw materials with the concentrations of 100 mug/mL, 30 mug/mL, 10 mug/mL and 5 mug/mL are respectively added into a coating system, a blank control group coating system is not added, and an RF sample 1 and an RF sample 2 are respectively detected.
The blocking effects of the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody on the GRP chemiluminescent platform are shown in Table 3, and the results show that the experimental group has remarkable elimination effect on the RF sample; the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody have obvious blocking effect on RF samples at very low concentration (such as 5 mug/mL), and the blocking effect is obviously better than that of the market blocker raw material.
TABLE 3 Table 3
The values in Table 3 are the OD values read by the chemiluminescent immunoassay analyzer, and the lower the OD value, the weaker the detection signal, indicating that the better the blocking effect.
Example 5 stability assessment of anti-human IgM antibodies
The Ig-M-9G1RMb1 antibody prepared in example 1 and the Ig-M-9G1RMb2 antibody prepared in example 2 were placed at 4 ℃, -80 ℃, -37 ℃ (refrigerator), and 37 ℃ (incubator) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and activity detection was performed on the 21-day samples (the activity of the samples was examined using the results of the enzyme-free detection OD).
The results of the Ig-M-9G1RMb1 antibody stability test are shown in Table 4, and the results show that the antibodies do not have obvious protein state change after being placed for 21 days under three examination conditions, and the activity does not have a descending trend along with the increase of the examination temperature, so that the stability of the anti-human IgM antibody prepared by the invention is high.
TABLE 4 Table 4
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
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<213> Artificial sequence ()
<400> 4
Arg Ala Ser Gln Glu Ile Ser Gly Phe Leu Ser
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence ()
<400> 5
Ala Ala Ser Thr Leu Asp Ser
1 5
<210> 6
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 6
Ile Gln Tyr Thr Ser Phe Pro Leu
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial sequence ()
<400> 7
Leu Gln Tyr Thr Ser Phe Pro Leu
1 5
<210> 8
<211> 120
<212> PRT
<213> Artificial sequence ()
<400> 8
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Ser Ile Lys Asp Ala
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Lys Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Pro Leu Pro His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 9
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Phe
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Ile Gln Tyr Thr Ser Phe Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 10
<211> 107
<212> PRT
<213> Artificial sequence ()
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Phe
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Thr Ser Phe Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 11
<211> 444
<212> PRT
<213> Artificial sequence ()
<400> 11
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Ser Ile Lys Asp Ala
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Lys Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Pro Leu Pro His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys
210 215 220
Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val
245 250 255
Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270
Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro
290 295 300
Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
305 310 315 320
Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys
340 345 350
Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn
355 360 365
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro
370 375 380
Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser
385 390 395 400
Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala
405 410 415
Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His
420 425 430
His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
<210> 12
<211> 214
<212> PRT
<213> Artificial sequence ()
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Phe
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Ile Gln Tyr Thr Ser Phe Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 13
<211> 214
<212> PRT
<213> Artificial sequence ()
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Phe
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Thr Ser Phe Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210

Claims (15)

1. An anti-human IgM antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising:
HCDR1 with the amino acid sequence shown as SEQ ID NO.1, HCDR2 with the amino acid sequence shown as SEQ ID NO.2, and HCDR3 with the amino acid sequence shown as SEQ ID NO. 3;
the light chain variable region comprises: LCDR1 with the amino acid sequence shown as SEQ ID NO.4, LCDR2 with the amino acid sequence shown as SEQ ID NO.5, and LCDR3 with the amino acid sequence shown as SEQ ID NO.6 or SEQ ID NO. 7.
2. An anti-human IgM antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment thereof further comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9 or SEQ ID NO. 10.
3. The antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE or IgM, and the light chain constant region is a kappa chain or a lambda chain.
4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the species source of the heavy and light chain constant regions is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
5. The antibody or antigen-binding fragment thereof of claim 4, wherein the species source of the heavy and light chain constant regions is dairy cows.
6. The antibody or antigen-binding fragment thereof of claim 4, wherein the species source of the heavy and light chain constant regions is turkey or chicken.
7. An anti-human IgM antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain having an amino acid sequence as shown in SEQ ID No. 11 and a light chain having an amino acid sequence as shown in SEQ ID No. 12 or SEQ ID No. 13.
8. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.
9. A vector comprising the nucleic acid of claim 8.
10. A cell comprising the nucleic acid of claim 8 or the vector of claim 9.
11. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 7, a nucleic acid according to claim 8, a vector according to claim 9 or a cell according to claim 10 for the preparation of an immunoassay reagent or kit.
12. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 7, a nucleic acid according to claim 8, a vector according to claim 9 or a cell according to claim 10 for the preparation of an immune blocker.
13. An immune blocking agent comprising the antibody or antigen binding fragment thereof according to any one of claims 1 to 7, the nucleic acid according to claim 8, the vector according to claim 9 or the cell according to claim 10.
14. A method for reducing/eliminating endogenous interference, characterized in that the immunoblocker of claim 13 is added to an immunodetection system.
15. An immunodiagnostic reagent/kit comprising the immunoblocker of claim 13.
CN202110861408.8A 2021-07-29 2021-07-29 Anti-human IgM antibodies and uses thereof Active CN115677856B (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US6129914A (en) * 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
CN108712908A (en) * 2016-01-08 2018-10-26 梅迪托普生物科学有限公司 It is selfed len antibody
CN110642950A (en) * 2019-07-25 2020-01-03 熊浩 Humanized T cell activated V domain immunosuppressive factor antigen binding fragment

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Publication number Priority date Publication date Assignee Title
WO2020172601A1 (en) * 2019-02-21 2020-08-27 Elstar Therapeutics, Inc. Multifunctional molecules that bind to calreticulin and uses thereof

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US6129914A (en) * 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
CN108712908A (en) * 2016-01-08 2018-10-26 梅迪托普生物科学有限公司 It is selfed len antibody
CN110642950A (en) * 2019-07-25 2020-01-03 熊浩 Humanized T cell activated V domain immunosuppressive factor antigen binding fragment

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* Cited by examiner, † Cited by third party
Title
immunoglobulin heavy chain variable region, partial [Mus musculus];GenBank;GenBank;GenBank: AGN91406.1 *
抗人IgM抗体对鼻咽癌HNE-1细胞生物学特性的影响;周珊;刘彦婷;赵飞鹏;冯华君;涂晓敏;杨金亮;梁传余;覃纲;;重庆医学(第16期);11-15 *

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