CN115677856A

CN115677856A - Anti-human IgM antibody and application thereof

Info

Publication number: CN115677856A
Application number: CN202110861408.8A
Authority: CN
Inventors: 孟媛; 钟冬梅; 唐丽娜
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2023-02-03
Anticipated expiration: 2041-07-29
Also published as: CN115677856B

Abstract

The invention belongs to the technical field of antibodies. In particular to an anti-human IgM antibody and application 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 the 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 antibody and application thereof

Technical Field

The invention belongs to the technical field of antibodies. More specifically, it relates to anti-human IgM antibodies and uses thereof.

Background

The immunoassay method based on antigen-antibody reaction has wide application, and is divided into different detection methods according to antibody markers, such as: enzyme linked immunosorbent assay, radioimmunoassay, chemiluminescence and the like. In clinical applications, the accuracy of the immunoassay results is often affected to various degrees by the interferents in the patient's serum, resulting in erroneous assay results. The interferents in the serum can be divided into endogenous interference and exogenous interference, wherein the exogenous interference comprises hemolysis, sample contamination, incomplete blood coagulation, sample stability, storage conditions and the like; endogenous interference includes Rheumatoid Factor (RF), heterophil Antibody (HA), autoantibody, complement, jaundice, high fat, etc.; among them, RF and HA are important interference factors. Studies have demonstrated that about 3% to 15% of healthy populations contain endogenous interferents, over 10% (30% to 40%) of patients have HA interference, and 10% to 40% have HAMA interference. Among endogenous interferences, RF and 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 ensuring the reliability of medical immunoassay results and guaranteeing the benefits of doctors and patients. The simplest and most effective method for eliminating RF interference and HA interference in immunodiagnosis 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 that is added to the immunoassay system to react with endogenous antibodies, thereby effectively preventing non-analyte mediated antibody bridging. Blockers can be divided into passive blockers and active blockers. Active blocking agents are specific for human immunoglobulins and can specifically, actively and efficiently neutralize components of interfering antibodies, thereby blocking the production of unintended binding, such as IIR, HBR, etc. in commercial reagents. The preparation can eliminate various heterophilic interferences, has specific binding force on heterophilic antibodies causing interferences, 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 high affinity (109L/mol) for HAAA. HBR is McAb of murine anti-human IgM. In the active blocking process, the effect of interference cancellation depends on the affinity of the active blocker for heterophilic antibodies. Due to the high affinity of active blockers, their blocking capacity in some assays is stronger than that of passive blockers.

Although a lot of blocker products exist in the market, certain performance defects exist, in addition, the usage amount of the blocker is very large, the mainstream blocker is imported mostly and has high price, so that the market demands a blocker with better performance and lower cost.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and defects of high cost and poor performance of the existing immune blocking agent, and provide 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 immune detection as or for preparing an immune blocking agent, and preparing an immune detection reagent or a kit so as to reduce or even eliminate endogenous interference, and has a blocking effect obviously better than that of a market blocking agent raw material.

The object of the present invention is to provide an anti-human IgM antibody or an antigen-binding fragment thereof, which antibody comprises the following heavy chain complementarity determining regions HCDRs:

the amino acid sequence is shown as HCDR1 shown in SEQ ID NO.1, the amino acid sequence is shown as HCDR2 shown in SEQ ID NO.2, and the amino acid sequence is shown as HCDR3 shown in SEQ ID NO. 3.

Another objective of the invention is to provide a nucleic acid, vector or cell related to the anti-human IgM antibody or an antigen-binding fragment thereof.

The invention also provides the application of the anti-human IgM antibody or the antigen binding fragment thereof, and the related nucleic acid, vector or cell thereof in immunodetection.

The invention also provides the application of the anti-human IgM antibody or the antigen binding fragment thereof, and related nucleic acid, vector or cell thereof as/in preparing an immune blocking agent.

The invention also provides an immune blocking agent which contains the anti-human IgM antibody or the antigen binding fragment thereof, the carrier, the nucleic acid or the cell.

The invention also provides a method for reducing/eliminating endogenous interference, wherein the immune blocker is added into an immune detection system.

The invention also provides an immunodiagnostic reagent/kit comprising the immune blocker.

Drawings

FIG. 1 is a graph showing the result of reducing SDS-PAGE of Ig-M-9G1RMb1 antibody prepared in example 1.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The present invention relates to anti-human IgM antibodies or antigen-binding fragments thereof, which antibodies comprise the following heavy chain complementarity determining regions HCDRs:

In some embodiments, the antibody further comprises the following light chain complementarity determining regions, LCDRs:

the amino acid sequence of the LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of the LCDR3 is 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 the antigen-binding fragment thereof is that the antibody has a remarkable blocking effect on endogenous interferences such as a false positive sample and an RF sample, 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 that comprises a portion or all of an antibody CDR 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 whole antibodies) for binding to a given epitope. Such fragments are selected from Fab (consisting of a complete light chain and Fd), fv (consisting of VH and VL), scFv (single chain antibody, connected by 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 high affinity for human IgM. In the detailed description of the inventionIn the formula, the antigen binding fragment of the anti-human IgM antibody can obviously block endogenous interferences such as a false positive sample, an RF sample and the like, and reduce or even eliminate the endogenous interferences.

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 to regions comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment thereof for the antigen or epitope that it recognizes. In a particular 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, which includes LCDR1, LCDR2, and LCDR1. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the Chothia and Lesk numbering scheme, and the 1997 New standardized numbering system introduced by Lefranc et al for all protein sequences of the immunoglobulin superfamily. Kabat et al was 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 the Kabat database, and the Kabat numbering scheme is generally considered to be a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR area, but other methods to mark CDR area 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 a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO. 11, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO. 12 or SEQ ID NO. 13.

The invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof.

The nucleic acid is typically RNA or DNA, and the nucleic acid molecule 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 acid is used when it is ligated to a vector.

The invention also relates to vectors containing said nucleic acids.

The invention also relates to a cell containing said nucleic acid or said vector.

The antibody or antigen binding fragment thereof, the nucleic acid, the vector or the cell, and the application thereof in immunodetection and the application thereof as/for preparing an immune blocking agent are all within the protection scope of the invention.

The invention also relates to an immune blocker containing the antibody or the antigen binding fragment thereof, the nucleic acid, the vector or the cell.

The invention also relates to a method for reducing/eliminating endogenous interference, wherein the immune blocker is added in an immune detection system. In a specific embodiment of the invention, the endogenous interference is rheumatoid factor interference or heterophile antibody interference.

The invention also relates to an immunodiagnostic reagent/kit comprising the immune blocker.

In some embodiments, the kit is an immunochromatographic assay kit, an enzyme-immune assay kit, a chemiluminescent kit, or an immunoturbidimetric assay kit.

In some embodiments, the kit may include a test strip or card onto which the liquid sample from the subject is placed, or an ELISA assay plate having a well in which a liquid sample from a single subject can be placed. In some embodiments, the kit may include a testing device configured for use in a flow cytometer, a bioanalyzer, a biosensor.

In some embodiments, the immune blockers 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 attached to a solid support, the solid support may preferably be a chromatographic medium such as a membrane, 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 market blocker raw material, the anti-human IgM antibody has good stability, low cost and high binding affinity with human IGM, has obvious blocking effect on endogenous interferences such as a false positive sample, an RF sample and the like, can reduce or even eliminate the endogenous interferences by directly adding the anti-human IgM antibody into a detection system when in use, further improves the accuracy of immunodetection, and is efficient and convenient; therefore, the antibody or the antigen binding fragment thereof, and the related nucleic acid, vector or cell thereof can be used as or used for preparing an immune blocking agent, and preparing an immune detection reagent or kit, and can be widely applied to the field of immune detection.

Embodiments of the present invention will be described in detail with reference to examples.

In the following examples, restriction enzymes, prime Star DNA polymerase, were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen corporation. Primer synthesis and gene sequencing were done by Invitrogen.

EXAMPLE 1 preparation of anti-human IgM antibody (Ig-M-9G 1RMb1 antibody)

1. Expression plasmid construction

(1) Ig-M-9G1RMb1 antibody gene preparation

mRNA is extracted from a hybridoma cell strain secreting an Ig-M-9G1RMb1 antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then is inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, 4 clones of heavy chain and light chain genes are respectively taken to clone after colonies are grown, and the clones are sent to a gene sequencing company for sequencing.

(2) Sequence analysis of variable region genes of Ig-M-9G1RMb1 antibody

The gene sequence obtained by the above sequencing was put in an IMGT antibody database for analysis and analyzed using VNTI11.5 software.

The genes amplified by the heavy chain primer pair and the light chain primer pair are determined to be correct through analysis; in the gene fragment amplified by the light chain primer pair, the VL gene sequence is 396bp, belongs to VkII gene family, and a leader peptide sequence of 57bp 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 a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(3) Construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

vector is a constructed Ig-M-9G1RMb1 antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, which is called 3.4A expression vector for short in the following; 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, two ends of the primers are respectively provided with HindIII and EcoRI restriction enzyme sites and protective bases, 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 gene fragment and the light chain gene fragment are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, 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) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Ig-M-9G1RMb1 antibody expression plasmid was diluted to 40. Mu.g/100. Mu.L with ultrapure water, and CHO cells were conditioned at 1.43X 10 ⁷ cells/mL are put in a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, sampling and counting are carried out on days 3, 5 and 7, and sampling and detecting are carried out on day 7.

Coating liquid (main component is NaHCO) ₃ ) Diluting human IgM to 1. Mu.g/mL, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing solution (Na as the main component) ₂ HPO ₄ NaCl) for 2 times, patting dry; add blocking solution (20% BSA +80% PBS), 120 μ L per well, 37 deg.C, 1h, pat dry; the diluted cell supernatant, 100. Mu.L/well, 37 ℃ was added for 30min (partial supernatant for 1 h). Meanwhile, the supernatant without the cell is used as a blank control; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole, the main components of the developing solution A are citric acid, sodium acetate, acetanilide and carbamide peroxide), adding a developing solution B (50 muL/hole, the main components of the developing solution B are citric acid, EDTA & 2Na, TMB and concentrated HCl), and carrying out 10min; adding stop solution (the main components of the stop solution are EDTA-2 Na and concentrated H) ₂ SO ₄ ) 50 μ L/well; OD readings were taken at 450nm (reference 630 nm) on the microplate reader.

The results showed that the OD of the reaction after the dilution of the cell supernatant by 1000 times was still greater than 1.0, and the OD of the reaction without the addition of the cell supernatant was less than 0.1, indicating that the antibodies generated after the transient transformation of the plasmid were active against human IGM.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mu L of Buffer, 100 mu g/tube of DNA, 10 mu L of PvuI enzyme and sterile water are supplemented to 500 mu L, and the mixture is subjected to enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25, followed by chloroform (aqueous phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water when ethanol is completely volatilized, and finally measuring the concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 40. Mu.g/100. Mu.L with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ Putting cells/mL into a centrifuge tube, mixing 100 mu L of plasmid and 700 mu L of cells, transferring into an electric rotating cup, electrically rotating, and counting the next day; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with the cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds are preserved and cultured in batches, and the cell density is adjusted to be 0.5 multiplied by 10 ⁶ cells/mL,2.2mL, at a cell density of 0.3X 10 ⁶ cell/mL, 2mL for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3. Preparation of Ig-M-9G1RMb1 antibody

(1) Cell expanding culture

After the cells were recovered, they were cultured in 125 mL-sized shake flasks, inoculated with 30mL Dynamis medium at a medium volume of 100%, and placed in a shaker at a rotation speed of 120r/min and a temperature of 37 ℃ with 8% carbon dioxide. Culturing for 72h, inoculating and expanding at inoculation density of 50 ten thousand cells/mL, calculating the expanding volume according to production requirements, and the culture medium is 100% Dynamis culture medium. Then carrying out propagation every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/mL.

(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 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Performing affinity purification by using a proteinA affinity chromatography column to obtain the Ig-M-9G1RMb1 antibody. Mu.g of Ig-M-9G1RMb1 antibody was subjected to reducing SDS-PAGE.

The reducing SDS-PAGE results of Ig-M-9G1RMb1 antibody are shown in FIG. 1, which shows two bands, one with an Mr of 50KD (heavy chain) and the other with an Mr of 28KD (light chain).

The amino acid sequence of HCDR1 of the 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 1 st amino acid I (IIe, isoleucine) of LCDR3 of the Ig-M-9G1RMb1 antibody was mutated to L (Leu, leucine) by conventional methods of introducing amino acids into site-directed mutations, and another antibody (Ig-M-9G 1RMb2 antibody) was constructed using the same methods as in step (3), step 2 and step 3 of step 1 of example 1.

The amino acid sequence of HCDR1 of the 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 assay for anti-human IgM antibody

The affinity of the Ig-M-9G1RMb1 antibody prepared in example 1, the affinity of the Ig-M-9G1RMb2 antibody prepared in example 2 and the market blocker starting material were analyzed by the following methods:

using AMC sensors, ig-M-9G1RMb1 antibody and Ig-M-9G1RMb2 antibody were diluted to 10. Mu.g/mL with PBST and human IGM was diluted with PBST in a gradient;

the operation flow is as follows: buffer 1 (PBST, main component Na) ₂ HPO ₄ + NaCl + TW-20) for 60s, immobilized antibody in antibody solution for 300s, buffer 2 (PBST, main component Na) ₂ HPO ₄ + NaCl + TW-20) for 180s, bound for 420s in antigen solution, dissociated for 1200s in buffer 2, incubated with 10mM pH 1.69 GLY solution and buffer 3 (PBST, na as the main component) ₂ HPO ₄ + NaCl + TW-20) to 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 obviously higher than that of the raw materials of the market blocking agent.

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 antibody	3.02E-12	9.41E+06	2.84E-05
				Ig-M-9G1RMb2 antibody	4.27E-12	1.42E+07	6.07E-05

Note: in table 1, KD represents the equilibrium dissociation constant, i.e. affinity; kon denotes the binding rate; kdis denotes the off-rate. The lower the KD value, the higher the affinity.

Example 4 measurement of blocking Performance of anti-human IgM antibody

1. Verification of blocking Performance on CTNI fluorescence platform

In CTNI fluorescence platform pairing detection, an experimental group respectively treats a sample pad 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 does not treat the sample pad; the samples (L1-L10) were individually tested.

The blocking effect of the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody on the CTNI fluorescence platform is shown in Table 2, and the result shows that the experimental group has obvious elimination effect on the false positive sample; the antibody Ig-M-9G1RMb1 and the antibody Ig-M-9G1RMb2 have obvious blocking effect on false positive samples, and the blocking effect is obviously better than that of market blocking agent raw materials.

TABLE 2

In Table 2, the T/C values indicate:

the sample to be detected is added into a sample adding port of the detection reagent card, under the action of lateral capillary, the sample to be detected firstly passes through the combination pad, and is specifically combined with the fluorescent group labeled antibody on the combination pad in an immunological manner, and the fluorescent group labeled antibody are respectively combined to form an antigen-antibody fluorescent complex, so that the antigen-antibody fluorescent complex is fixed in the T line. The C line is coated with a substance that reacts with the free fluorophore-labeled antibody, and when the free fluorophore-labeled antibody passes through the C line, it is immobilized in the C line by specific immunological binding to the substance on the C line. The fluorescence intensities of the two bands detected by the fluorescence immunoassay analyzer are represented by peak areas, and the T/C value is calculated by the calculation software of the fluorescence immunoassay analyzer. The reading value T/C of the instrument represents the ratio of the area of the T peak to the area of the C peak, and the higher the T/C is, the higher the activity is under the quality control sample and the positive sample; the lower the T/C under the false positive sample, the better the blocking effect; when the T/C value is less than 0.1, the sample is judged to be negative.

2. Verification of blocking Performance on GRP chemiluminescence platform

In the GRP chemiluminescence platform pairing detection, the Ig-M-9G1RMb1 antibody prepared in example 1, ig-M-9G1RMb2 antibody prepared in example 2 and a market blocker raw material with the concentrations of 100. Mu.g/mL, 30. Mu.g/mL, 10. Mu.g/mL and 5. Mu.g/mL are respectively added into a coating system in the concentration of an experimental group, and an RF sample 1 and an RF sample 2 are respectively detected without adding into a coating system in a blank control group.

The blocking effect of the Ig-M-9G1RMb1 antibody and the Ig-M-9G1RMb2 antibody on the GRP chemiluminescence platform is shown in Table 3, and the results show that the experimental group has obvious effect of eliminating the RF sample; the antibody Ig-M-9G1RMb1 and the antibody Ig-M-9G1RMb2 can have obvious blocking effect on an RF sample under very low concentration (such as 5 mu G/mL), and the blocking effect is obviously better than that of a market blocking agent raw material.

TABLE 3

The values in table 3 are OD values read by the chemiluminescence immunoassay analyzer, and the lower the OD value, the weaker the detection signal, indicating 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 ℃ C. (refrigerator), -80 ℃ C. (refrigerator) and 37 ℃ C. (incubator) for 21 days, and samples for 7 days, 14 days and 21 days were observed for their states, and the activity of the samples for 21 days was measured (the activity of the samples was examined using the results of the enzyme immunoassay OD).

The result of the stability test of the Ig-M-9G1RMb1 antibody is shown in Table 4, and the result shows that no obvious protein state change is seen in the antibody after the antibody is placed for 21 days under three examination conditions, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the anti-human IgM antibody prepared by the invention has high stability.

TABLE 4

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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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

1. An anti-human IgM antibody or an antigen-binding fragment thereof, wherein the antibody comprises the following heavy chain complementarity determining regions HCDRs:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody further comprises the following light chain complementarity determining regions (LCDRs):

the amino acid sequence is LCDR1 shown in SEQ ID NO.4, the amino acid sequence is LCDR2 shown in SEQ ID NO.5, and the amino acid sequence is LCDR3 shown in SEQ ID NO.6 or SEQ ID NO. 7.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein 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.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein 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;

optionally, the species of heavy and light chain constant regions are from cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, banisters, or humans.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO. 11, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO. 12 or SEQ ID NO. 13.

6. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

7. A vector comprising the nucleic acid of claim 6.

8. A cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, a nucleic acid according to claim 6, a vector according to claim 7 or a cell according to claim 8 in an immunoassay or as/in the preparation of an immune blocking agent.

10. An immune blocking agent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, or the cell according to claim 8.

11. A method of reducing/eliminating endogenous interference comprising adding the immune blocking agent of claim 10 to an immunoassay system.

12. An immunodiagnostic reagent/kit comprising an immune blocking agent according to claim 10.