Anti-human IgM antibody or functional fragment thereof and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-human IgM antibody or a functional fragment thereof and application of the anti-human IgM antibody or the functional fragment thereof, and discloses an anti-human IgM antibody or an antigen binding part antibody thereof, which has high affinity and stable performance on IgM, can block endogenous interferent rheumatoid factors, and has great significance on obtaining correct diagnosis results.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-human IgM antibody or a functional fragment thereof and an application thereof.

[0002] The present invention claims priority to application number 202311044958.6 filed on August 18, 2023, the entire contents of which are incorporated herein by reference. Background Art

[0003] In immunoassays, the presence of a variety of heterophilic antibodies and rheumatoid factors (RF) in patient samples can reduce the sensitivity and specificity of the test by nonspecific binding to antibodies, resulting in false positive or false negative results in the diagnosis. It is reported that specific interference can occur in 30-40% of patient samples. To solve this problem, during the reagent development test, it was found that the correct use of blockers can help improve the sensitivity and specificity of the test. Blockers are special biological materials that, when added to immunoassay reagents, can block interferences that appear during the test. They are divided into passive blockers and active blockers. Among them, passive blockers are IgG from heterologous animals that competitively bind to interfering antibodies. Active blockers are ingredients that are added to specifically, actively, and efficiently neutralize interfering antibodies, thereby blocking unexpected binding. According to market feedback, the amount of passive blockers used is large, but the effect is not obvious. Combined with current market demand, active blockers have become the preferred choice.

[0004] Rheumatoid factor (RF) is an antibody produced in the body due to infectious factors (bacteria, viruses, etc.) with denatured IgG as antigen, so it is also called anti-antibody. Common rheumatoid factors include IgM, IgG, IgA and IgE. Rheumatoid factors are widely present in the human body and play a certain physiological role. Rheumatoid factors are mainly present in the serum and joint fluid of patients with rheumatoid arthritis, mainly IgM type. The preparation of active blockers to block the influence of rheumatoid factors on immunoassay reagents has become the key to improving the performance of reagent products. Summary of the invention

[0005] The present invention provides an antibody with high recognition reactivity to human IgM, which can specifically bind to IgM and reduce or eliminate the interference of RF on antigen-antibody detection.

[0006] The details are as follows:

[0007] An anti-human IgM antibody or a functional fragment thereof, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1 to HCDR3 are amino acid sequences consistent with HCDR1 to HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, and the LCDR1 to LCDR3 are amino acid sequences consistent with LCDR1 to LCDR3 of the light chain variable region shown in SEQ ID NO:2.

[0008] Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

[0009] Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system,

[0010] Wherein, the HCDR1 is shown in SEQ ID NO:3;

[0011] The HCDR2 is shown in SEQ ID NO:4;

[0012] The HCDR3 is shown in SEQ ID NO:5;

[0013] The LCDR1 is shown in SEQ ID NO:6;

[0014] The LCDR2 is shown in SEQ ID NO:7;

[0015] And the LCDR3 is shown in SEQ ID NO:8.

[0016] As used herein, the terms "variable region" and "CDR" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to the regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment to its recognized antigen or epitope.

[0017] Herein, the heavy chain variable region (heavy chain complementary determining region) is represented by "HCDR", which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by "LCDR", which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. 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 the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present disclosure uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0018] Herein, "framework region" or "FR" region includes heavy chain framework region and light chain framework region, and refers to the region excluding CDR in antibody heavy chain variable region (which can be expressed as VH) and light chain variable region (which can be expressed as VL); wherein, the heavy chain framework region is expressed as "HFR" and can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region is expressed as "LFR" and can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0019] Preferably, the antibody or functional fragment thereof further comprises the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region,

[0020] Wherein, the HFR1 is an amino acid sequence as shown in EQ ID NO:9 or an amino acid sequence having at least 80% homology thereto;

[0021] The HFR2 is an amino acid sequence as shown in EQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto;

[0022] The HFR3 is an amino acid sequence as shown in EQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto;

[0023] The HFR4 is an amino acid sequence as shown in EQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto;

[0024] The LFR1 is an amino acid sequence as shown in EQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto;

[0025] The LFR2 is an amino acid sequence as shown in EQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto;

[0026] The LFR3 is an amino acid sequence as shown in EQ ID NO: 15 or an amino acid sequence having at least 80% homology thereto;

[0027] The LFR4 is an amino acid sequence as shown in EQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto.

[0028] In this article, the heavy chain variable region is obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0029] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or their functional fragments provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the above-mentioned corresponding framework regions (SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 or 16). In an optional embodiment, the antibody or its functional fragment has a KD of ≤10 -7 M, KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M or KD≤10 -11 Binds human IgM with an affinity of .

[0030] Preferably, it further comprises a constant region;

[0031] Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0032] Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0033] Optionally, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans;

[0034] Optionally, the species origin of the constant region is mouse.

[0035] Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0036] The functional fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents of the disclosure that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the disclosure, a person skilled in the art can easily obtain the above functional fragments.

[0037] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0038] On the other hand, the present invention also discloses an antibody conjugate, which comprises the antibody or its functional fragment and a conjugated part conjugated thereto;

[0039] Optionally, the coupling moiety is selected from one or more of a purification tag or a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / avidin, a spin label;

[0040] Optionally, the coupling moiety is selected from a solid phase carrier, such as magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

[0041] On the other hand, the present invention discloses a nucleic acid, characterized in that the nucleic acid encodes the antibody or a functional fragment thereof.

[0042] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. DNA nucleic acids are used when they are linked to a vector.

[0043] Some embodiments of the present disclosure also provide vectors containing the above-mentioned nucleic acid molecules.

[0044] Some embodiments of the present disclosure also provide cells containing the above-mentioned vector.

[0045] Some embodiments of the present disclosure also provide a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cells as described above, and isolating and purifying the antibody or the functional fragment thereof from the culture product.

[0046] Based on the amino acid sequence of the antibody or its functional fragment in the present disclosure, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment in the present disclosure, it belongs to the protection scope of the present disclosure.

[0047] On the other hand, the present invention discloses the use of the antibody or its functional fragment in the preparation of an immunoassay kit or in the preparation of an immunoblocking agent.

[0048] In another aspect, the present invention discloses a blocking agent, which includes the antibody or a functional fragment thereof;

[0049] Optionally, the concentration of the antibody in the blocking agent is 5-100 μg / ml.

[0050] In alternative embodiments, the concentration of the antibody in the blocking agent is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / ml.

[0051] On the other hand, the present invention discloses a detection reagent or a kit, wherein the reagent or the kit comprises the antibody or a functional fragment thereof, the antibody conjugate or the blocking agent.

[0052] In an optional embodiment, the blocking agent included in the kit may be in the form of a liquid solution, in the form attached to a solid support, or in the form of a dry powder. When the blocking agent is a liquid solution, the liquid solution may be an aqueous solution. When the immunoblocking agent is in the form attached to a solid support, the preferred solid support may be a chromatographic medium such as a film, a test strip, a plastic bead or a plate, or a microscope slide. When the blocking agent is a dry powder, the powder may be reconstituted by adding an appropriate solvent.

[0053] On the other hand, the present invention discloses a method for reducing / eliminating endogenous interference, which comprises adding the antibody or its functional fragment, the antibody conjugate, or the blocking agent to the immunoassay system.

[0054] In another aspect, the present invention discloses a method for immunoassay, comprising:

[0055] The antibody or its functional fragment, the antibody conjugate, or the blocking agent is added to the immunoassay system.

[0056] In another aspect, the present invention discloses a method for detecting IgM, comprising:

[0057] A) contacting the antibody or functional fragment thereof, the antibody conjugate, the blocking agent or the reagent or kit with the sample from the subject under conditions sufficient for a binding reaction to perform a binding reaction; and

[0058] B) Detection of immune complexes produced by the binding reaction.

[0059] Beneficial Effects

[0060] The anti-IgM antibodies provided by the present disclosure have high affinity, high reactivity, high sensitivity or specificity for human IgM. They have good blocking and immune interference elimination effects when used for immunoassays, and provide an important source of blocking agent raw materials for immunodiagnosis. Therefore, the anti-IgM antibodies, immunoassay reagents and kits provided by the present disclosure all have excellent practical performance and broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The antibody gel map disclosed in the present invention, wherein the left side is the marker and the right side is the purified antibody reduction band;

[0062] Figure 2 The results of the enzyme-linked immunosorbent assay (ELISA) experiment were used to detect the interaction between the antibody and recombinantly expressed and purified human IgM-Fc. DETAILED DESCRIPTION

[0063] In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be described clearly and completely below. If no specific conditions are specified in the examples or embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0065] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0066] Example 1 IgM antibody preparation

[0067] 1. Preparation of immunogen

[0068] Human IgM was digested with pepsin in a sodium citrate buffer solution at 25-37°C, pH=3.5 for 2 hours, and the obtained Fc terminus was purified using a His tag to obtain an IgM-Fc terminus with a purity greater than 75%, which was constructed into a pcDNA3.1(+) vector and then transfected into 1ug / mL HEK293 cells. After culturing for 5 days, it was purified using a Ni excel column to obtain a human IgM-Fc immunogen.

[0069] 2. Mouse immunization and antibody detection

[0070] Select 5 6-8 week old SPF female BALB / c mice, mix and emulsify Freund's complete adjuvant and human IgM-Fc at a concentration of 2 mg / ml in equal volumes. Use the emulsified antigen to immunize 6-8 week old SPF female BALB / c mice, and inject 40 μg of the immunogen into each mouse by subcutaneous injection. Two weeks after the first immunization, mix the antigen protein with Freund's incomplete adjuvant and emulsify it, and inject 40 μg of the antigen protein into each mouse by subcutaneous injection or back injection. Two weeks later, collect blood through the tail vein, collect the supernatant by centrifugation, and test the serum titer by ELISA. Immunize and test the serum titer every two weeks. After two immunizations, the serum titer is as high as 2.0 or above after a million-fold dilution. Screening serum titer 10 6 Lymph was taken from the above mice to separate lymphocytes for cell fusion.

[0071] 3. Cell fusion, positive hybridoma cell screening and subcloning

[0072] The antibody screening scheme is the key to successfully developing the antibody of the present invention. Lymphocytes of isolated immune mice are fused with cultured SP2 / 0 cells by PEG1500 or by electrofusion. The fused cells are cultured in HAT-1640 medium containing 20% ​​FBS serum for screening and culture. The culture medium is replaced after one week, and the culture supernatant is taken after culturing for another 4 days for positive clone screening. Positive wells are screened using human IgM-Fc immunogen. Wells with a higher ratio of ELISA positive value to cell number are selected for multiple subcloning. ELISA plates are coated with human IgM-Fc immunogen. The culture supernatant of the subclone is taken to screen monoclones that can show affinity under antigen coating conditions, and the monoclonal hybridoma cells with the highest affinity are selected from them, and finally a hybridoma cell strain with a higher antibody titer that can secrete human IgM-Fc immunogen monoclonal antibodies is obtained, which is named IgM-mab and has good stability.

[0073] 4. Production and purification of monoclonal antibodies

[0074] A group of 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL paraffin oil to suppress the immune response of the mice. One week after the injection, 0.5 ml of IgM-mab hybridoma cells were injected intraperitoneally into the mice. The number of cells was about 1×10 6 After two weeks, the ascites was collected. The collected ascites was precipitated with ammonium sulfate and purified with protein G affinity to obtain the target antibody.

[0075] 5. Monoclonal antibody subtype identification and gene sequence cloning

[0076] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions. The specific procedures were as follows:

[0077] (1) Dilute the capture antibody to 1 μg / mL with coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (plate washing solution).

[0078] (2) Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) at a ratio of 1:3000 with diluent.

[0079] (3) After washing the plate three times with the plate washing solution, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three times again, add the color developing solution. After about 5 minutes (depending on the strength of the reaction), add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance value. After identification, the heavy chain subtype of the anti-human IgM-Fc antibody obtained is IgG1, and the light chain is Kappa. According to the antibody subtype results, the antibody gene sequence was cloned using a method based on the RACE technology route. Hybridoma cells with good growth status were collected, and the total RNA of the hybridoma cells was obtained using a total RNA extraction kit. According to the operating procedures of Takara's SMARTer RACE instructions, the mRNA was reverse transcribed into cDNA, and the full-length sequence of the target antibody was amplified.

[0080] Example 2 Antibody Performance Verification

[0081] (1) Interaction between antibodies and human IgM-Fc

[0082] This example verifies the binding performance of the antibody of the present invention through the interaction between the antibody and human IgM-Fc.

[0083] The specific operation steps are as follows: Use coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5) to dilute the antibody to 1ug / mL and coat it on a conventional 96-well ELISA plate. Use biotinylation reagent (ready-made commercial reagent) to modify the human IgM-Fc purified after recombinant expression. Dilute the biotin-modified human IgM-Fc to the target concentration (0.3ng / ml, 0.8ng / ml, 2.5ng / ml,…, 200ng / ml). Add a specific concentration of human IgM-Fc to the ELISA wells coated with the target antibody, add 100μl to each well and incubate at 37℃ for 30min. After washing the plate with plate washing solution (150mM NaCl solution containing 0.05% Tween-20), add 1:5000 diluted streptavidin-horseradish peroxidase conjugate (ready-made commercial reagent), add 100μl to each well and incubate at 37℃ for 15min. Finally, the cells were incubated with ELISA colorimetric solution and the OD450 absorbance was detected using an enzyme-labeled instrument.

[0084] The results are shown in Table 1 and Figure 2 As shown, the experimental results of using enzyme-linked immunosorbent assay (ELISA) to detect the interaction between antibodies and recombinantly expressed and purified human IgM-Fc. The horizontal axis in the figure indicates the concentration of human IgM-Fc used, and the vertical axis indicates the detection result of the ELISA experiment, that is, the absorbance value (OD450) detected by the spectrophotometer at 450nm. The higher the value of OD450, the stronger the interaction detected. The results show that the interaction between the antibody and human IgM-Fc is strong, and the antibody of the present invention is suitable for detecting human IgM-Fc.

[0085] Table 1 Linearity test results

[0086]

[0087]

[0088] Example 3 Application of IgM-mab antibody to cTnl biochemical detection kit

[0089] The IgM-mab antibody was prepared into a 50ug / ml working solution with reagent R1, and after being fully mixed, the reagent was loaded into the R1 cavity of the reagent bottle. The blank group did not add IgM-mab antibody, and the reagent R2 working solution was loaded into the R2 cavity of the reagent bottle, and the magnetic bead working solution was loaded into the magnetic bead cavity. Since the RF interfering substance is mainly human IgM type, it is applied to the blocking effect on RF. The blocking effect of IgM-mab antibody in the cTnl biochemical kit on RF high-value serum was tested with a fully automatic biochemical analyzer. As shown in Table 2, by comparing with the blank control group, it was found that IgM-mab antibody had a significant blocking effect on clinical samples containing RF.

[0090] Table 2 Blocking results of biochemical clinical samples

[0091]

[0092]

[0093] Example 4 Application of IgM-mab Antibody to CK-MB Immunochromatographic Detection

[0094] (1) Treatment of nitrocellulose membrane

[0095] a. Preparation of test line

[0096] Prepare 0.5g / mL CK-MB coated antibody with PBS, take 0.5ul and draw a detection line in the middle of the nitrocellulose membrane.

[0097] b. Drying

[0098] Bake in a drying oven at 37°C for 2 h.

[0099] (2) Preparation of sample pad

[0100] Cut the glass cellulose membrane into strips of 20*30cm each. For one group, prepare the IgM-mab antibody with a buffer solution of 1 mg / mL, take 6 mL and soak it on the sample pad, put it in a drying oven and bake it at 45℃ for 2h for later use. For another group, no antibody is added as a blank control.

[0101] (3) Preparation of absorbent pad

[0102] Cut the absorbent paper into strips of 30*2.7cm each.

[0103] (4) Assembly

[0104] The plastic backing, sample pad and absorbent pad are common materials in the art, and the sample pad, nitrocellulose membrane and absorbent pad are tightly overlapped on the plastic backing at one time. The attached intermediate is cut into a 0.5 cm wide test strip using a chopper.

[0105] (5) How to use the test strips

[0106] a. Mix the sample to be tested and the CK-MB capture antibody-fluorescent microsphere complex at a ratio of 4:1;

[0107] b. Add 60ul of the mixed solution to the sample well of the test strip prepared above;

[0108] c. The sample mixture is chromatographed until it reaches the test line of the test strip and binds to the CK-MB coated antibody.

[0109] (6) Kit performance verification

[0110] The experiment was conducted according to the above (5). The results are shown in Table 3. The difference in T value indicates that when the clinical sample containing RF is tested, the lower the T value is than the blank. When the sample pad contains IgM-mab antibody, it can effectively block the interference of RF on the lateral flow immunochromatography reagent, and can effectively reflect the real good test results.

[0111] Table 3 Blocking results of immunochromatographic clinical samples

[0112]

[0113]

[0114] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An anti-human IgM antibody or a functional fragment thereof, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1-HCDR3 are amino acid sequences consistent with HCDR1-HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, and the LCDR1-LCDR3 are amino acid sequences consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:

2.

2. The antibody or functional fragment thereof according to claim 1, characterized in that: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

3. The antibody or functional fragment thereof according to claim 2, characterized in that: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system, Wherein, the HCDR1 is shown in SEQ ID NO:3; The HCDR2 is shown in SEQ ID NO:4; The HCDR3 is shown in SEQ ID NO:5; The LCDR1 is shown in SEQ ID NO:6; The LCDR2 is shown in SEQ ID NO:7; And the LCDR3 is shown in SEQ ID NO:

8.

4. The antibody or functional fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or its functional fragment further comprises the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region, Wherein, the HFR1 is an amino acid sequence as shown in EQ ID NO:9 or an amino acid sequence having at least 80% homology thereto; The HFR2 is an amino acid sequence as shown in EQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The HFR3 is an amino acid sequence as shown in EQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The HFR4 is an amino acid sequence as shown in EQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR1 is an amino acid sequence as shown in EQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR2 is an amino acid sequence as shown in EQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto; The LFR3 is an amino acid sequence as shown in EQ ID NO: 15 or an amino acid sequence having at least 80% homology thereto; The LFR4 is an amino acid sequence as shown in EQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto; Preferably, the antibody or its functional fragment further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region; Optionally, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans; Optionally, the species origin of the constant region is mouse; Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

5. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or its functional fragment according to any one of claims 1 to 4 and a conjugated part conjugated thereto; Optionally, the coupling moiety is selected from one or more of a purification tag or a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / avidin, a spin label; Optionally, the coupling moiety is selected from a solid phase carrier, such as magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

6. Use of the antibody or functional fragment thereof according to any one of claims 1 to 4 in the preparation of an immunoassay kit or in the preparation of an immunoblocking agent.

7. A blocking agent, characterized in that The blocking agent comprises the antibody or a functional fragment thereof according to any one of claims 1 to 4; Optionally, the concentration of the antibody in the blocking agent is 5-100 μg / ml.

8. A detection reagent or kit, characterized in that: The reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 4, the antibody conjugate according to claim 5 or the blocking agent according to claim 7.

9. A method for reducing / eliminating endogenous interference, characterized in that: The antibody or functional fragment thereof according to any one of claims 1 to 4, the antibody conjugate according to claim 5, or the blocking agent according to claim 7 is added to the immunoassay system.

10. A method of immunoassay, characterized in that: The method comprises: The antibody or functional fragment thereof according to any one of claims 1 to 4, the antibody conjugate according to claim 5, or the blocking agent according to claim 7 is added to the immunoassay system.

11. A method for detecting IgM, characterized in that: The method comprises: A) contacting the antibody or functional fragment thereof according to any one of claims 1 to 4, the antibody conjugate according to claim 5, the blocking agent according to claim 7, or the reagent or kit according to claim 8 with a sample from the subject under conditions sufficient for a binding reaction to occur to perform a binding reaction; and B) Detection of immune complexes produced by the binding reaction.

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