Anti-RF antibody or functional fragment thereof and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-RF antibody or a functional fragment thereof and application thereof.The anti-RF antibody or the functional fragment thereof is stable in sequence and antibody performance, can be specifically combined with RF in the presence of endogenous interfering substances, has affinity to HAMAs, can reduce or eliminate interference of the RF and the HAMAs on antigen-antibody detection at the same time, and has the advantages that the anti-RF antibody or the functional fragment thereof is high in specificity, high in specificity and high in specificity. And the method is of great significance for obtaining a correct clinical diagnosis result.

Description

Technical Field

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

[0002] The present invention claims priority to application number 202311056555.3 filed on August 22, 2023, the entire contents of which are incorporated herein by reference. Background Art

[0003] With the rapid technological development of domestic in vitro diagnostic reagent manufacturers, more and more domestic in vitro diagnostic kits are being developed, and immunodiagnosis has become the mainstream diagnostic technology. In order to produce highly sensitive and highly specific reagents, the most critical factor is to select antibodies or antigens that have high specificity binding to target molecules. However, in actual immunoassays, there are a variety of endogenous interfering substances in patient samples: such as rheumatoid factor (RF), heterophilic antibodies (Id), human anti-animal antibodies (HAAA) and drugs and their metabolites, etc. These endogenous interfering substances can lead to false positive or false negative results in diagnosis. Therefore, the reagents must be improved and the endogenous interfering substances must be properly blocked to obtain accurate clinical results of diagnostic reagents.

[0004] Blocker is a general term for materials that can be used in the development of in vitro diagnostic reagents to reduce the impact of endogenous interfering substances from patient samples. Blockers are divided into passive blockers and active blockers. Passive blockers are IgG from heterologous animals that competitively bind to interfering antibodies. Active blockers are ingredients that can 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.

[0005] 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 are IgM, IgG, IgA and IgE. Rheumatoid factors are ubiquitous in the human body and play a certain physiological role. Rheumatoid factors are mainly present in the serum and synovial fluid of patients with rheumatoid arthritis, mainly IgM type. Human anti-mouse antibodies (HAMAs) are the anti-animal antibodies that cause the most trouble for immune detection, and are also the most concerned anti-animal antibodies to date, which is related to the widespread use of monoclonal antibody drugs. HAMAs are a type of anti-antibody, including IgG, IgM, IgA, and a small amount of IgE. There are idiotype antibodies, isotype antibodies and anti-anti-idiotype antibodies. Idiotype antibodies can bind to the Fab segment of immunoglobulins. 80% of HAMAs are anti-idiotype antibodies. The key to improving the performance of reagent products is to prepare active blockers to block the effects of endogenous interfering substances such as rheumatoid factor RF and human anti-mouse antibodies HAMAs on immunoassay reagents. Summary of the invention

[0006] The present invention provides an anti-RF antibody or a functional fragment thereof, which can specifically bind to RF and has affinity for HAMAs, and can simultaneously reduce or eliminate the interference of RF and HAMAs in antigen-antibody detection.

[0007] The details are as follows:

[0008] An anti-RF antibody or a functional fragment thereof and use thereof, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that 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.

[0009] Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

[0010] Preferably, the antibody or functional fragment thereof is characterized in that the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system,

[0011] Wherein, the HCDR1 is shown in SEQ ID NO:3;

[0012] The HCDR2 is shown in SEQ ID NO:4;

[0013] The HCDR3 is shown in SEQ ID NO:5;

[0014] The LCDR1 is shown in SEQ ID NO:6;

[0015] The LCDR2 is shown in SEQ ID NO:7;

[0016] And the LCDR3 is shown in SEQ ID NO:8.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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,

[0021] 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;

[0022] 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;

[0023] 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;

[0024] 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;

[0025] 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;

[0026] 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;

[0027] 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;

[0028] 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.

[0029] 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.

[0030] 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 .

[0031] Preferably, the antibody or its functional fragment further comprises a constant region;

[0032] Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0033] 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;

[0034] 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;

[0035] Optionally, the species origin of the constant region is mouse;

[0036] Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0037] The functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand from the contents of the disclosure that the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction splitting disulfide bonds. Based on the structure of the complete antibody disclosed in the disclosure, it is easy for those skilled in the art to obtain the above-mentioned functional fragments. The functional fragments of the above-mentioned antibodies can also be obtained by recombinant genetics techniques known to those skilled in the art or by, for example, automatic peptide synthesizers, such as those sold by Applied BioSystems.

[0038] On the other hand, the present invention also discloses that the antibody conjugate 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 RF, the method 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 antibody sequence disclosed in the present invention has stable antibody performance, can specifically bind to RF in the presence of endogenous interfering substances, and has affinity for HAMAs, and can simultaneously reduce or eliminate the interference of RF and HAMAs in antigen-antibody detection, thereby obtaining correct clinical diagnosis results. 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. 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 Preparation of mouse monoclonal antibodies against endogenous human interferors

[0067] 1. Preparation of immunogen

[0068] First, human serum is graded and precipitated with ammonium sulfate according to the different solubility of different proteins in a certain concentration of salt solution to separate the impurity protein and IgM antibody. Then, molecular sieves are used to separate antibodies of different molecular sizes according to the different pore sizes of the gel, and different types of antibodies in the ammonium sulfate crude extract are separated to obtain human IgM antibodies as immunogens.

[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 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 foot 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 foot injection or back subcutaneous 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] C. Cell fusion, positive hybridoma cell screening and subcloning

[0072] The antibody screening scheme is the key to successfully developing the antibodies of the present invention. Lymphocytes of isolated immune mice are fused with cultured SP2 / 0 cells by PEG1500 or electrofusion. The fused cells are cultured in HAT-1640 medium containing 20% ​​FBS serum for screening. 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 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 immunogens. The culture supernatant of the subclones 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 line with a higher antibody titer that can secrete anti-human IgM immunogen monoclonal antibodies is obtained, which is named Blocker-1.

[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 Blocker-1 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 (washing solution) containing 0.05% Tween-20.

[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 mouse monoclonal antibody against human endogenous interferons obtained was IgG1, and the light chain was 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 reversely 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

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

[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 regular 96-well ELISA plate. Use biotinylation reagent (finished commercial reagent) to modify the purified human IgM with biotin. Dilute the biotin-modified human IgM to the desired concentration (0.3ng / ml, 0.8ng / ml, 2.5ng / ml,…, 200ng / ml). Add a specific concentration of human IgM 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 (finished 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 the interaction between the antibody and the purified expressed human IgM were detected by enzyme-linked immunosorbent assay (ELISA) experiment. The horizontal axis in the figure indicates the concentration of the antibody, 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 detected interaction. The results show that the interaction between the antibody and human IgM is strong, and the antibody of the present invention is suitable for blocking endogenous interfering substances in humans.

[0085] Table 1 Linearity test results

[0086]

[0087]

[0088] Example 3 Application of Blocker-1 Antibody to fPSA Chemiluminescent Detection Kit

[0089] The Blocker-1 antibody reagent R1 was prepared into a 20ug / ml working solution, and after being fully mixed, the reagent was placed in the R1 chamber of the reagent bottle. The blank group did not add the Blocker-1 antibody, and the control group added the control antibody reagent R1 to prepare a 20ug / ml working solution; the reagent R2 working solution was placed in the R2 chamber of the reagent bottle, and the magnetic bead working solution was placed in the magnetic bead chamber. The blocking effect of the Blocker-1 antibody in the fPSA chemiluminescent kit on endogenous interference samples containing HAMAs and RF was tested using a fully automatic chemiluminescence immunoassay. As shown in Table 2, by comparing with the blank and control groups, it was found that the Blocker-1 antibody had a significant blocking effect on clinical samples containing HAMAs and RF, and the performance of the Blocker-1 antibody was significantly better than the control.

[0090] Table 2 Blocking results of chemiluminescent clinical samples

[0091]

[0092]

[0093] 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 made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Anti-RF antibodies or functional fragments thereof and use thereof, characterized in that: include: It comprises 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.

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. Preferably, the antibody or its functional fragment is 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.

3. The antibody or functional fragment thereof according to claim 1 or 2, 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.

4. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or its functional fragment according to any one of claims 1 to 3 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.

5. Use of the antibody or functional fragment thereof according to any one of claims 1 to 3 in the preparation of an immunoassay kit or in the preparation of an immunoblocking agent.

6. 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 3; Optionally, the concentration of the antibody in the blocking agent is 5-100 μg / ml.

7. 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 3, the antibody conjugate according to claim 4 or the blocking agent according to claim 6.

8. A method for reducing / eliminating endogenous interference, characterized in that: The antibody or functional fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 4, or the blocking agent according to claim 6 is added to the immunoassay system.

9. A method of immunoassay, characterized in that: The method comprises: The antibody or functional fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 4, or the blocking agent according to claim 6 is added to the immunoassay system.

10. A method for detecting RF, characterized in that: The method comprises: A) contacting the antibody or functional fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 4, the blocking agent according to claim 6, or the reagent or kit according to claim 7 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.

Images