Anti-RF antibody or functional fragment thereof and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an immune blocker antibody, an anti-rheumatoid factor (RF) antibody or a functional fragment thereof and application of the immune blocker antibody and the anti-rheumatoid factor (RF) antibody. The antibody prepared by the invention can be combined with a target antigen with high specificity, can effectively reduce and even eliminate rheumatoid factor endogenous interference after being added into an immunodetection kit, and is low in cost and high in stability.

Description

Technical Field

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

[0002] The present invention claims priority to application number 202311047160.7 filed on August 18, 2023, the entire contents of which are incorporated herein by reference. Background Art

[0003] Blockers are a general term for materials that can be used in the development of in vitro diagnostic reagents to reduce interference from proteins in patient samples or other factors in the test process, which may cause false positive or false negative test results.

[0004] In immunological detection tests, nonspecific binding can occur from (1) the binding of solid phase materials to non-target proteins and (2) the binding of endogenous interfering substances present in the patient's sample to antibodies in the detection reagent. In order to prevent nonspecific binding, it is required that during the development and production of in vitro diagnostic reagents, a blocking buffer be used after the solid phase material is coated to block any remaining unbound sites on the solid phase material. At the same time, other blocking agents are used during the preparation of the test sample to prevent endogenous interfering substances from binding to the antibodies in the detection reagent components. Endogenous interference includes rheumatoid factor, heterophilic antibodies, complement, autoantibodies, hyperlipidemia, jaundice, etc.; among them, rheumatoid factor and heterophilic antibodies are important interfering factors.

[0005] Rheumatoid factor (RF) is an antibody produced in the body due to infectious factors (bacteria, viruses, etc.) with denatured IgG (a type of antibody) as an antigen, so it is also called anti-antibody. Common rheumatoid factors include IgM, IgG, IgA and IgE. Rheumatoid factors are ubiquitous in the human body and play a certain physiological role. Rheumatoid factor is mainly present in the serum and synovial fluid of patients with rheumatoid arthritis, mainly in the form of IgM. It can bind to the Fc segment of IgG. The principle of RF interfering in the antigen-antibody immune response is as follows: Figure 2 In the absence of antigen, RF can bind to the capture antibody and the labeled antibody, causing a false positive. Therefore, adding a blocking agent for RF interfering substances during the development of the kit can pre-block the RF interfering substances. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-RF antibody or a functional fragment thereof, mainly targeting the IgM type, achieving specific binding to RF interfering substances, having a significant blocking effect on endogenous interference, and being able to effectively reduce or eliminate endogenous RF interference, thereby improving the accuracy of the immune kit. The antibody can be effectively applied to various immunoassay fields, such as chemiluminescence, immunochromatography, biochemistry, etc., which is convenient, fast and efficient.

[0007] The details are as follows:

[0008] The anti-RF antibody or its functional fragment has the following complementarity determining region:

[0009] CDR-VH1: D-X1-YM-X2, where X1 is N or T, and X2 is H or R;

[0010] CDR-VH2: RI-X1-PPNG-X2-TQCDP-X3-FQG, where X1 is D or N, X2 is N or C, and X3 is H or K;

[0011] CDR-VH3: GL-X1-YYGSSTD-X2-FDY, where X1 is L or I, and X2 is A or F;

[0012] CDR-VL1: KAS-X1-SVDYD-X2-DSYMN, where X1 is N or Q, and X2 is G or W;

[0013] CDR-VL2: A-X1-SDL-X2-S, where X1 is A or G, and X2 is N or Q;

[0014] and CDR-VL3:QQ-X1-NEYPY-X2, where X1 is S and X2 is S or T.

[0015] Furthermore, X2 of CDR-VH1 of the complementary determining region is H, X1 of CDR-VH2 is D, X2 of CDR-VH3 is F, X1 of CDR-VL1 is Q, X2 of CDR-VL2 is Q, and X1 of CDR-VL3 is S.

[0016] The CDR segment division described in the present invention adopts the Kabat algorithm.

[0017] "CDR" is used herein to refer to a "complementarity determining region" within an antibody variable sequence. The variable regions of the heavy and light chains each have three CDRs, starting from the N-terminus of the heavy or light chain.

[0018] The antigen binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 in the present invention). A polypeptide comprising a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 or CDR-VL3) may be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has shown that the amino acid residues of CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may be primarily responsible for the specificity of the antigen binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0019] The inventors of the present invention have found that when the mutation sites in each complementary determining region are the amino acid residues, the antibody has a better affinity for RF.

[0020] In an optional embodiment, X1 in the CDR-VH1 is T;

[0021] In an optional embodiment, X1 in the CDR-VH1 is N;

[0022] In an optional embodiment, X2 in the CDR-VH1 is H;

[0023] In an optional embodiment, X2 in the CDR-VH1 is R;

[0024] In an optional embodiment, X1 in the CDR-VH2 is D;

[0025] In an optional embodiment, X1 in the CDR-VH2 is N;

[0026] In an optional embodiment, X2 in the CDR-VH2 is C;

[0027] In an optional embodiment, X2 in the CDR-VH2 is N;

[0028] In an optional embodiment, X3 in the CDR-VH2 is H;

[0029] In an optional embodiment, X3 in the CDR-VH2 is K;

[0030] In an optional embodiment, X1 in the CDR-VH3 is L;

[0031] In an optional embodiment, X1 in the CDR-VH3 is 1;

[0032] In an optional embodiment, X2 in the CDR-VH3 is A;

[0033] In an optional embodiment, X2 in the CDR-VH3 is F;

[0034] In an optional embodiment, X1 in the CDR-VL1 is Q;

[0035] In an optional embodiment, X1 in the CDR-VL1 is N;

[0036] In an optional embodiment, X2 in the CDR-VL1 is G;

[0037] In an optional embodiment, X2 in the CDR-VL1 is W;

[0038] In an optional embodiment, X1 in the CDR-VL2 is G;

[0039] In an optional embodiment, X1 in the CDR-VL2 is A;

[0040] In an optional embodiment, X2 in the CDR-VL2 is N;

[0041] In an optional embodiment, X2 in the CDR-VL2 is Q;

[0042] In an optional embodiment, X1 in the CDR-VL3 is S;

[0043] In an optional embodiment, X2 in the CDR-VL3 is T;

[0044] In an alternative embodiment, X2 in said CDR-VL3 is S.

[0045] Preferably, each complementary determining region is selected from any one of the following mutation combinations:

[0046]

[0047]

[0048] Further, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NOs: 1-4 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NOs: 5-8 in sequence.

[0049] Furthermore, the antibody also comprises a constant region.

[0050] Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species origin 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.

[0051] Preferably, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0052] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. The antibody according to the present invention is preferably a goat, sheep, mouse, rabbit or rat antibody, a chimeric antibody or a further genetically engineered antibody, as long as the characteristic properties according to the present invention are retained. "Antibody fragment" comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include diabodies, single-chain antibody molecules and multispecific antibodies formed by antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; diabodies; and multispecific antibodies formed by antibody fragments.

[0053] On the other hand, the present invention discloses the anti-interference use of the antibody or its functional fragment in an immunoassay kit.

[0054] The "anti-interference" mentioned in the present invention means that the antibody or its functional fragment binds to RF in the sample, thereby removing the interference of RF in the immune detection reaction.

[0055] On the other hand, the present invention discloses a blocking agent, which comprises the antibody or a functional fragment thereof.

[0056] In another aspect, the present invention discloses a detection kit, which comprises the labeled antibody or the blocking agent.

[0057] Beneficial effects:

[0058] The present invention not only provides a monoclonal antibody (wild type) that binds to RF with high affinity, but also makes a variety of excellent performance mutations to the wild type, which can remove the interference of RF on antigen-antibody binding in immune detection, ensure the smooth progress of immune detection reaction, and improve the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 : The effect of mutation 3-2 on RF antigen purification, where the left side is the marker and the right side is the purified RF-Fc antigen.

[0060] Figure 2 : Schematic diagram of the principle of rheumatoid factor RF causing false positive interference in the double antibody sandwich method DETAILED DESCRIPTION

[0061] Specifically, in order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they 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.

[0062] 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. 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. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0063] As used herein, the terms "including," "comprising," "having," "may," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0064] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir and CC Black, 1989). ell, ed.); 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 (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0065] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0066] Example 1 Preparation of monoclonal antibodies

[0067] A. Expression and purification of rheumatoid factor RF immunogen

[0068] 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 end was purified using a His tag to obtain an IgM-Fc end 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, the RF immunogen was obtained by purification using a Ni excel column.

[0069] B. 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 2 mg / ml RF immunogen in equal volumes. Use the emulsified antigen to immunize 6-8 week old SPF female BALB / c mice, and inject 40 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks after the first immunization, mix the antigen protein with Freund's incomplete adjuvant and emulsify it, and inject 40 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. 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 lymphocytes of isolated immune mice were fused with cultured SP2 / 0 cells by PEG1500 or electrofusion. The fused cells were screened and cultured in HAT-1640 medium containing 20% ​​FBS serum. The culture medium was replaced after one week, and the culture supernatant was taken after 4 days of culture for positive clone screening. The positive wells were screened using RF immunogen. The wells with a higher ratio of ELISA positive value to cell number were selected for multiple subcloning. The ELISA plate was coated with RF immunogen. The culture supernatant of the subclone was taken to screen the monoclones that could show affinity under antigen coating conditions, and the monoclonal hybridoma cells with the highest affinity were selected from them. Finally, a hybridoma cell line with a high antibody titer that could secrete RF immunogen monoclonal antibodies was obtained, named RF-15#, which had good stability.

[0073] D. Production and purification of monoclonal antibodies

[0074] Two groups of 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL paraffin oil to suppress the immune response of mice. One week after the injection, 0.5 ml of RF-15# cells were injected into the peritoneal cavity of the mice. The number of cells was about 1×10 6 After two weeks, ascites was collected. The collected ascites was precipitated with ammonium sulfate and affinity purified with protein A to obtain the target antibody RF-15#.

[0075] E. Identification of monoclonal antibody subtypes 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 operations 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 3 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 3 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 antibody RF-15# is IgG1, and the light chain is Kappa.

[0080] According to the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells with good growth were collected, and total RNA of hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the operating method of Takara's SMARTer RACE manual, and the full-length sequence of the target antibody was amplified.

[0081] Example 2 Verification of antibody performance

[0082] 1. ELISA affinity test

[0083] Dilute the goat anti-mouse to 1ug / mL using coating solution (0.05M carbonate and bicarbonate buffer at pH = 9.5), add 100μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20. Dilute the antibody to 1ug / mL using diluent (1% BSA, 0.1% PBST), add 100μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Dilute the biotin-labeled RF-immunogen to 100 ng / mL with diluent, take out the ELISA plate incubated at 37°C, wash the plate three times, add 150 μL of the prepared biotin-labeled RF-immunogen diluent to the 96-well ELISA plate in row A, add 100 μL of the diluent to each well of the 96-well ELISA plate in rows B to H, take 50 uL of the antibody diluent in the wells of row A, and dilute it 3 times from row B, and incubate at 37°C for 30 minutes. Dilute the avidin labeled with horseradish peroxidase at 1:5000 with diluent, take out the ELISA plate and wash the plate three times, add 100 μL to each well of the 96-well ELISA plate, and incubate at 37°C for 30 minutes. Take out the ELISA plate and wash the plate three times, add the color developing solution, color at room temperature for 3 minutes, add 0.5 M sulfuric acid to terminate the reaction, and read the OD450 value on the plate reader.

[0084] The heavy chain variable region of antibody RF-15# in Example 1 is shown in SEQ ID NO:9, wherein the amino acid sequences of each complementary determining region on the heavy chain variable region are as follows:

[0085] CDR-VH1: DT(X1)-YMR(X2)

[0086] CDR-VH2: RIN(X1)-PPNGN(X2)-TQCDPH(X3)-FQG

[0087] CDR-VH3: GLL(X1)-YYGSSTDA(X2)-FDY

[0088] The light chain variable region is shown in SEQ ID NO: 10, wherein the amino acid sequences of the complementary determining regions on the light chain variable region are as follows:

[0089] CDR-VL1:KASN(X1)-SVDYDG(X2)-DSYMN

[0090] CDR-VL2:AG(X1)-SDLN(X2)-S

[0091] CDR-VL3:QQS(X1)-NEYPYS(X2)

[0092] Based on antibody RF-15#, mutations were performed at sites related to antibody activity in the complementary determining region, among which X1, X2, and X3 were all mutation sites.

[0093] Table 1 Mutation sites related to antibody activity

[0094]

[0095] Table 2 Antibody activity analysis data

[0096]

[0097] As can be seen from the table above, mutation 3 has the best activity effect. Therefore, mutation 3 is used as the backbone sequence to screen other mutation sites with better affinity. Some results are as follows:

[0098] Table 3 Mutation sites related to antibody affinity

[0099]

[0100]

[0101] RF immunogen 100ng / ml, measure the absorbance value of each mutation OD450, the results are as follows:

[0102] Table 4 Affinity testing of antibody mutations

[0103] Mutation 3-1 2.5223 Mutation 3-11 2.3758 Mutation 3-21 2.3141 Mutation 3-2 2.4213 Mutation 3-12 2.2219 Mutation 3-22 2.5053 Mutation 3-3 2.2998 Mutation 3-13 2.1964 Mutation 3-23 2.4789 Mutation 3-4 2.3313 Mutation 3-14 2.2842 Mutation 3-24 2.3218 Mutation 3-5 2.2859 Mutation 3-15 2.1697 Mutation 3-25 2.2457 Mutation 3-6 2.3835 Mutation 3-16 2.2511 Mutation 3-26 2.1895 Mutation 3-7 2.4576 Mutation 3-17 2.4012 Mutation 3-27 2.0869 Mutation 3-8 2.5137 Mutation 3-18 2.3321 Mutation 3-28 2.1542 Mutation 3-9 2.4249 Mutation 3-19 2.1538 Mutation 3-29 2.1891 Mutation 3-10 2.2536 Mutation 3-20 2.3217 Mutation 3-30 2.2674

[0104] It can be seen that the affinity of each mutation is higher.

[0105] 2. Stability determination

[0106] The concentration of the antibody of the present invention is 1 ug / mL, and the concentration of the RF immunogen is 100 ng / mL.

[0107] The antibodies were diluted in a defined buffer (PBS, 0.05% ProClin TM 300) was heat accelerated at 37℃ for 7 days, and the accelerated antibodies were evaluated by SDS-PAGE and ELISA indirect method, and compared with 4℃ to identify the long-term stability of the antibodies. In addition, the test results were displayed as the deviation between the 4℃ value and the accelerated value by repeated freezing and thawing at -20℃ for 5 times. The measurement results are as follows:

[0108] Table 5 Stability study

[0109]

[0110]

[0111] As can be seen from the above, mutations 3 to 3-30 can be stably stored at 4°C, and their properties remain stable when accelerated at 37°C for 0.5 months (the slight decrease in reactivity does not affect the performance of the reagent), ensuring the performance of the reagent after opening the bottle, thereby ensuring the accuracy and stability of the test results.

[0112] Example 3 Antibody Application Research

[0113] Because mutations 1 to 30 of RF-15# antibody have comparable performance, it can be inferred that all mutations have comparable application effects. The examples cannot exhaust all the effect experiments of antibodies, and this example selects mutations 3-18 for application research.

[0114] 1. ELISA affinity test

[0115] The RF immunogen was diluted to 1ug / mL using a coating solution (0.05M carbonate and bicarbonate buffer at pH = 9.5), and 100μL was added to each well of a 96-well ELISA plate, and coated overnight at 4°C. The plate was washed three times with PBS buffer containing 0.05% Tween-20. The antibody was diluted to 1ug / mL using a diluent (1% BSA, 0.1% PBST), and 100μL was added to each well of a 96-well ELISA plate, and incubated at 37°C for 30 minutes. The goat anti-mouse labeled with horseradish peroxidase was diluted 1:5000 using a diluent, and the ELISA plate was removed and washed three times, and 100μL was added to each well of a 96-well ELISA plate, and incubated at 37°C for 30 minutes. The ELISA plate was removed and washed three times, and a color developing solution was added, and the color was developed at room temperature for 3 minutes. 0.5M sulfuric acid was added to terminate the reaction, and the OD450 value was read on a plate reader. The results are shown in Table 6. Mutation 3-18 has good reactivity and linearity.

[0116] Table 6 Linearity test results

[0117]

[0118] 2. Evaluation of blocking performance of PGⅠ biochemical kit

[0119] Mutation 3-18 was prepared into a 50ug / ml working solution with reagent R1, and the reagent was placed in the R1 cavity of the reagent bottle after sufficient mixing. No mutation 3-18 was added to the blank group, and the reagent R2 working solution was placed in the R2 cavity of the reagent bottle. The blocking effect of mutation 3-18 in the PGⅠ biochemical kit on RF high-value serum was tested using a fully automatic biochemical analyzer. As shown in Table 7, mutation 3-18 had a significant blocking effect on clinical samples containing RF.

[0120] Table 7 Blocking results of biochemical clinical samples

[0121]

[0122]

[0123] 3. Evaluation of blocking performance of AMH chemiluminescence kit

[0124] Take a PD-10 desalting column, pour out the storage buffer in the PD-10 desalting column, and cut the bottom of the PD-10 desalting column with scissors. Place it vertically on the shelf, rinse the PD-10 desalting column with pure water twice, and rinse the PD-10 desalting column with biotin labeling buffer 1 5 times after the liquid in the column is drained; then rinse the PD-10 desalting column with biotin labeling buffer 2, repeat 5 times; add AMH antibody, and then add biotin labeling buffer 2; when there is no liquid flowing out of the bottom of the PD-10 desalting column, put the liquid collection tube at the bottom and add biotin labeling buffer 2 to elute and collect the antibody conjugate eluate. Dilute the above biotin-labeled AMH antibody with reagent buffer to a concentration of 3ug / ml and prepare 5ml. Add mutation 3-18 to 5ml of biotin-labeled antibody working solution to make its final concentration 20ug / mL. The preparation is R1 working solution. The blank group does not add mutation 3-18 antibody, which is R1 blank working solution. The blocking effect of RF-15# on clinical samples containing RF was tested by fully automatic chemiluminescence immunoassay. As shown in Table 8, mutations 3-18 have a good blocking effect on clinical samples containing RF, which can effectively reduce the impact of endogenous interfering substances RF and reduce false positives.

[0125] Table 8 Linearity test results

[0126]

[0127]

[0128] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. An anti-rheumatoid factor (RF) antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment comprises the following complementarity determining region: CDR-VH1: D-X1-YM-X2, where X1 is N or T, and X2 is H or R; CDR-VH2: RI-X1-PPNG-X2-TQCDP-X3-FQG, where X1 is D or N, X2 is N or C, and X3 is H or K; CDR-VH3: GL-X1-YYGSSTD-X2-FDY, where X1 is L or I, and X2 is A or F; CDR-VL1: KAS-X1-SVDYD-X2-DSYMN, where X1 is N or Q, and X2 is G or W; CDR-VL2: A-X1-SDL-X2-S, where X1 is A or G, and X2 is N or Q; and CDR-VL3:QQ-X1-NEYPY-X2, where X1 is S and X2 is S or T.

2. The anti-RF antibody or a functional fragment thereof according to claim 1, wherein: X2 of CDR-VH1 of the complementary determining region is H, X1 of CDR-VH2 is D, X2 of CDR-VH3 is F, X1 of CDR-VL1 is Q, X2 of CDR-VL2 is Q, and X1 of CDR-VL3 is S.

3. The anti-RF antibody or a functional fragment thereof according to claim 1, wherein: In the CDR-VH1, X1 is T; Preferably, in the CDR-VH1, X1 is N; Preferably, in the CDR-VH1, X2 is H; Preferably, in the CDR-VH1, X2 is R; Preferably, X1 in the CDR-VH2 is D; Preferably, in the CDR-VH2, X1 is N; Preferably, X2 in the CDR-VH2 is C; Preferably, X2 in the CDR-VH2 is N; Preferably, X3 in the CDR-VH2 is H; Preferably, X3 in the CDR-VH2 is K; Preferably, in the CDR-VH3, X1 is L; Preferably, in the CDR-VH3, X1 is I; Preferably, in the CDR-VH3, X2 is A; Preferably, X2 in the CDR-VH3 is F; Preferably, X1 in the CDR-VL1 is Q; Preferably, in the CDR-VL1, X1 is N; Preferably, X2 in the CDR-VL1 is G; Preferably, X2 in the CDR-VL1 is W; Preferably, in the CDR-VL2, X1 is G; Preferably, in the CDR-VL2, X1 is A; Preferably, X2 in the CDR-VL2 is N; Preferably, X2 in the CDR-VL2 is Q; Preferably, in the CDR-VL3, X1 is S; Preferably, X2 in the CDR-VL3 is T; Preferably, X2 in said CDR-VL3 is S.

4. The anti-RF antibody or a functional fragment thereof according to any one of claims 1 to 3, characterized in that: Each complementary determining region is selected from any one of the following mutation combinations:

5. The anti-RF antibody or a functional fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NOs: 1-4 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NOs: 5-8 in sequence.

6. The anti-RF antibody or a functional fragment thereof according to claim 5, characterized in that: The antibody further comprises a constant region; Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species origin 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.

7. The anti-RF antibody or a functional fragment thereof according to claim 6, wherein: The functional fragment is selected from any one of Fab, Fab', F(ab')2, Fv and scFv of the antibody.

8. Use of the antibody or its functional fragment as described in any one of claims 1 to 7 for anti-interference in an immunoassay kit.

9. 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 7.

10. A detection kit, characterized in that: The detection kit comprises the antibody or functional fragment thereof described in any one of claims 1 to 7 or the blocking agent described in claim 9.

Images