Monoclonal antibodies against natural rheumatoid factor RF and their applications

Abstract

The present invention provides a monoclonal antibody against natural rheumatoid factor RF and its application. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDRH1 with an amino acid sequence as shown in SEQ ID NO: 1, CDRH2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDRH3 with an amino acid sequence as shown in SEQ ID NO: 3. The light chain variable region comprises CDRL1 with an amino acid sequence as shown in SEQ ID NO: 4, CDRL2 with an amino acid sequence as shown in SEQ ID NO: 5, and CDRL3 with an amino acid sequence as shown in SEQ ID NO: 6. The present invention also provides a nucleic acid molecule encoding the monoclonal antibody, the application of the monoclonal antibody in the preparation of an immune blocking reagent for eliminating the interference of rheumatoid factor RF, and an immune blocking kit for eliminating endogenous interference comprising the monoclonal antibody. The monoclonal antibody of the present invention has high sensitivity and specificity and can be used in immunological detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a monoclonal antibody against natural rheumatoid factor (RF) and applications thereof. Background Art

[0002] In immunoassays, such as ELISA, CLIA, or lateral flow immunoassays, heterophilic antibodies (HA), human anti-mouse antibodies (HAMA), and rheumatoid factor (RF) can reduce the sensitivity and specificity of the test by nonspecifically binding to antibodies, leading to erroneous results and diagnoses. Endogenous interference is an unavoidable phenomenon in immunoassays, and eliminating interference is an important task in the development of immunoassay kits. Immunoblockers are a class of substances added to in vitro diagnostic reagent systems that can eliminate or reduce endogenous interference in immunoassays and improve the sensitivity and precision of the kit detection system. Adding immunoblockers is a simple and direct method to eliminate endogenous interference.

[0003] Immunoblockers are classified as passive and active. Rheumatoid factor, as one of the interfering factors, has a minimal effect when added to passive blockers. Therefore, it is necessary to develop immunoblockers with antibodies targeting rheumatoid factor to reduce false positive results caused by these interfering factors, actively block these interferences, and improve the sensitivity and precision of detection. Summary of the Invention

[0004] According to one aspect of the present invention, a monoclonal antibody against natural rheumatoid factor (RF) is provided, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDRH1 having an amino acid sequence as shown in SEQ ID NO: 1, CDRH2 having an amino acid sequence as shown in SEQ ID NO: 2, and CDRH3 having an amino acid sequence as shown in SEQ ID NO: 3. The light chain variable region comprises CDRL1 having an amino acid sequence as shown in SEQ ID NO: 4, CDRL2 having an amino acid sequence as shown in SEQ ID NO: 5, and CDRL3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0005] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0006] According to another aspect of the present invention, a nucleic acid molecule is provided, comprising a nucleotide sequence encoding the monoclonal antibody against natural rheumatoid factor (RF) as described above.

[0007] In some embodiments, the nucleotide sequences encoding CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the nucleotide sequences encoding CDRL1, CDRL2 and CDRL3 of the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0008] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 16.

[0009] According to another aspect of the present invention, there is provided a use of the monoclonal antibody against natural rheumatoid factor RF in the preparation of an immunoblocking reagent for eliminating interference from rheumatoid factor RF. The monoclonal antibody against natural rheumatoid factor RF is used for immunological detection to eliminate endogenous interference.

[0010] According to another aspect of the present invention, a detection kit for eliminating endogenous interference is provided, wherein the kit comprises the above-mentioned monoclonal antibody against natural rheumatoid factor (RF). DETAILED DESCRIPTION

[0011] The present invention, as set forth in the present invention and claims, does not specifically refer to the singular but includes the plural unless the context clearly indicates otherwise. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0012] Endogenous interference often results from cross-reactions between antibodies used in reagents and endogenous substances in patient samples. This type of interference is collectively referred to as endogenous interference. It falls into three main categories: Heterophile antibodies (HAs) are antibodies circulating in human peripheral blood that bind to undefined antigens. They are typically multispecific and have weak affinity. The degree and frequency of heterophile interference are related to their concentration, affinity, and the detection system. The second major category is human anti-animal antibodies (HAAAs), antibodies with activity against animal proteins, arising from both iatrogenic and non-iatrogenic sources. A common example is human anti-mouse antibodies (HAMAs). HAAAs from different species cross-react with each other, have specific antigens, and exhibit strong affinity. Human anti-animal antibodies have strong affinity and target specific antigens, while heterophile antibodies have weaker affinity for undefined antigens. The third major category is rheumatoid factor (RF). These autoantibodies target denatured IgG and are classified into five types: IgM, IgA, IgG, IgD, and IgE. 68%-80% are IgM. They bind poorly to native IgG and readily bind to denatured IgG from humans and animals or IgG in immune complexes. The interference is random and independent of RF concentration. RF binds to denatured IgG in vivo to form immune complexes that can activate complement or be engulfed by phagocytes. Phagocytic release of lysosomal enzymes, activated peptides, collagenase, prostaglandin E2, and other substances, coupled with cytokines and inflammatory adhesion molecules, induces tissue inflammatory damage, potentially leading to osteoarthritis and vasculitis. Rheumatoid factor is not exclusive to patients with rheumatoid arthritis. Rheumatoid arthritis, lupus erythematosus, diabetes, autoimmune diseases, etc. can cause rheumatoid factor RF to be found in the body of patients (heterophilic antibodies, autoantibodies, etc. can also be found). These special components have a certain adsorption effect during the reaction process, and are mostly caused by interference from certain antigenic substances in the body, resulting in false color reactions and false positives.

[0013] The most direct solution to these interfering factors is to add blockers to the sample to correct these endogenous interferences in the detection system and avoid the occurrence of inflated test values, false negative and / or false positive results.

[0014] Therefore, the present invention uses natural rheumatoid factor RF as an antigen and prepares corresponding monoclonal antibodies as blockers (for example, active blockers), which can eliminate or reduce endogenous interference in immunoassays and improve the sensitivity and accuracy of detection. Specifically, Balb / c mice are immunized with purchased natural rheumatoid factor RF, mouse spleen cells are taken and fused with myeloma cells, and hybridoma cells with high specificity are obtained through specific high-throughput screening. A large amount of mouse ascites is obtained by culture and re-immunization, and then a monoclonal antibody against natural rheumatoid factor RF with high titer, high purity, high sensitivity and high specificity is obtained through multi-step separation and purification. The anti-natural rheumatoid factor RF monoclonal antibody of this specification can be used to eliminate endogenous interference in immunological tests such as ELISA, CLLA, and lateral flow, and provides the required raw materials for immune blocking reagents (for example, active blockers). The immune blocking kit developed with this antibody as raw material has great application value. The anti-natural rheumatoid factor RF monoclonal antibody has a significant interference elimination effect, and can reduce the impact of interference by 10 times compared to samples without antibody addition; and its interference elimination effect is stronger than that of purchased antibodies.

[0015] The monoclonal antibody may include a heavy chain variable region and a light chain variable region. The heavy chain variable region includes CDRH1 with an amino acid sequence as shown in SEQ ID NO: 1, CDRH2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDRH3 with an amino acid sequence as shown in SEQ ID NO: 3. The light chain variable region includes CDRL1 with an amino acid sequence as shown in SEQ ID NO: 4, CDRL2 with an amino acid sequence as shown in SEQ ID NO: 5, and CDRL3 with an amino acid sequence as shown in SEQ ID NO: 6.

[0016] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0017] The present invention also provides a nucleic acid molecule. The nucleic acid molecule may include a nucleotide sequence encoding the above-mentioned monoclonal antibody against natural rheumatoid factor RF. In some embodiments, the nucleotide sequences encoding CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. The nucleotide sequences encoding CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively. In some embodiments, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:15, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:16.

[0018] The monoclonal antibodies against natural rheumatoid factor RF of the present invention can be used as immune blocking agents, for example, active blocking agents. In some embodiments, the monoclonal antibodies against natural rheumatoid factor RF can be used to eliminate endogenous interference in immunological tests such as ELISA, CLLA, and lateral flow.

[0019] The present invention also provides a detection kit for eliminating endogenous interference, for example, an immune blocking kit. The kit may include the aforementioned monoclonal antibody against natural rheumatoid factor (RF). The immune blocking kit developed using the monoclonal antibody against natural rheumatoid factor (RF) as raw material has excellent application value and exhibits high sensitivity and specificity.

[0020] Example

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0022] Preparation of monoclonal antibodies

[0023] Preparation of antigens.

[0024] Natural rheumatoid factor RF was used as an antigen and was purchased from Beijing Holmes Biotechnology Co., Ltd. with the product number HA129AN and a concentration of 10.0 mg / ml.

[0025] Preparation of monoclonal antibodies.

[0026] Healthy female Balb / c mice aged 6-8 weeks were selected and immunized according to a pre-specified immunization protocol. The natural rheumatoid factor (RF) purchased in the above steps was used as an immunogen to immunize Balb / c mice, and splenic lymphocytes from the successfully immunized mice were extracted. The lymphocytes were fused with mouse myeloma cells SP2 / 0 using cell fusion technology. After two rounds of subcloning screening, a hybridoma cell line that stably secreted monoclonal antibodies against natural rheumatoid factor (RF) was obtained, thereby obtaining monoclonal antibodies against natural rheumatoid factor (RF).

[0027] Animal immunization experiments were conducted using purchased antigens (natural rheumatoid factor RF).

[0028] The specific steps of animal immunization experiments include:

[0029] 1. Balb / c mice with the same mean body weight and age were randomly divided into two groups: the aluminum adjuvant (aluminum hydroxide adjuvant) group and the non-aluminum adjuvant group.

[0030] 2. Before the experiment, collect the pre-immune serum of each mouse (collect immune serum on the fifth day, draw blood through the eyeball, and draw an appropriate amount of blood to ensure the normal state of the mouse) as a negative control, and store the collected serum at -80℃.

[0031] 3. Preparation of the Aluminum Adjuvant (Aluminum Hydroxide Adjuvant) Group: Before immunization, each antigen was diluted to the corresponding dose (50 μg / mouse) in 75 μL PBS and mixed with alum adjuvant (i.e., aluminum hydroxide adjuvant) (1 mg / mouse) at a volume ratio of 3:1 antigen:adjuvant (i.e., 25 μl of adjuvant was added to 75 μl of immunogen diluent). The adjuvant was shaken well before use, and the injection adjuvant (25 μl) was slowly added dropwise to the immunogen solution. After the adjuvant and immunogen diluent were thoroughly mixed, the two were thoroughly mixed for 30 minutes to allow the adjuvant to effectively adsorb the antigen. Subsequent procedures were performed according to the animal immunization experimental procedures.

[0032] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL of PBS to the corresponding dose in the above table (i.e., 50 μg / mouse), and 100 μL of immunogen was added. Subsequent animal immunization experiments were performed according to the procedures.

[0033] 5. Subcutaneous injection at intervals of 2 weeks: The experimental design is a three-time immunization method, but blood is collected from the eyeball 7 days after each immunization injection, and part of the mouse supernatant is obtained by centrifugation. The serum antibody titer is first tested. 7 days after the last immunization, blood is collected from the heart to obtain the maximum amount of blood, centrifuged to obtain the supernatant, and stored at -80℃.

[0034] 6. Detect serum titer

[0035] A total of five mice were immunized, numbered A0, A1, A2, A3, and A4. After three immunizations, serum titers were measured. The test data are shown in Table 1 below.

[0036] Table 1 Antibody titer test data of 3 immune serum

[0037]

[0038] Immune spleen cells were fused with myeloma cell line SP2 / 0 cells, and the fused cells were selected using HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin, and thymidine). The fused cells were screened for positive results by ELISA and subcloned. Ascites fluid was collected from the selected positive monoclonal clones, and the ascites antibodies produced by the hybridoma cells were purified using a Protein A / G antibody purification column. The ELISA titer of the purified ascites antibodies was >1:128,000, and the purity was >90%. This result was confirmed by the "Enzyme-linked reaction ELISA to detect the binding activity of native rheumatoid factor RF" described in the next step.

[0039] ELISA to detect the binding activity of natural rheumatoid factor RF

[0040] 1. IgG antibody titer detection method

[0041] (1) Bottom plate coating: Dilute the antigen to be used with coating diluent to 3ug / ml, add 100μl of the prepared coating solution to each well, and place in a 4℃ refrigerator for 24h.

[0042] (2) After 24 hours, take the wells out of the refrigerator and place them at 37°C for 30 minutes. Then discard the liquid in the wells and wash the wells three times with washing solution, each time for 3 minutes.

[0043] (3) Blocking the enzyme-labeled reaction wells: Add 200 μl of 5% calf serum to each well and block at 37°C for 90 min. After blocking, wash the wells three times with washing solution, each time for 3 min.

[0044] (4) Add the sample to be tested: dilute the sample according to the required ratio, add the diluted sample to the enzyme-labeled reaction well, 100 μl per well, incubate at 37°C for 90 min; wash the well three times with washing solution, each time for 3 min.

[0045] (5) Add enzyme-labeled antibody: Add secondary antibody at an appropriate concentration according to the instructions; incubate at 37°C for 90 min, add 100 μl per well and wash as before.

[0046] (6) Add substrate solution: Add 100 μl of substrate to each well and incubate at 37°C in the dark for 15 to 30 minutes.

[0047] (7) Termination reaction: Add 50 μl of stop solution to each well to terminate the reaction and measure the experimental results within 20 minutes.

[0048] Detection of the binding activity of monoclonal antibodies to natural rheumatoid factor RF

[0049] (1) Cell fusion and clone screening data

[0050] A total of five rounds of fusion were completed, and the mice were numbered A0, A1, A2, A3, and A4.

[0051] A total of 18 positive wells were selected from the A0 mouse fusion screening for subcloning, ultimately resulting in three complete cell lines. After fusion screening, 72 positive clones with OD450 values ​​>2.2 were selected for titer testing using serial dilutions, followed by a second subcloning screening. Three cell lines were obtained, named A0-1 to A0-3.

[0052] A total of 16 positive wells were selected for subcloning in the A1 mouse fusion screening. After fusion screening, a total of 72 positive wells with OD450 values ​​>2.1 were selected for serial dilution titer detection. The second and third subcloning screenings were then performed, and finally two cell lines were completed, named A1-1 and A1-2.

[0053] A total of 16 positive wells were selected from the A2 mouse fusion screening for subcloning, followed by a second and third subclone screening, and ultimately two cell lines were obtained, named A2-1 and A2-2.

[0054] A total of 12 positive wells were selected from the A3 mouse fusion screening for subcloning, followed by a second and third subclone screening, and ultimately two cell lines were obtained, named A3-1 and A3-2.

[0055] A total of 4 positive wells were picked out from the A4 mouse fusion screening for subcloning, and then a second and third subcloning screening was performed, and finally a cell line was completed, named A4-1. After 5 cell fusions, a total of 10 cell lines were obtained.

[0056] (2) Ascites preparation and testing data

[0057] Three F1 mice were injected with each complete cell line, and a total of 10 ascites were prepared. The titer data of all ascites tests are shown in Table 2:

[0058] Table 2 Ascites antibody detection titer data

[0059] Dilution multiple A0-1 A0-2 A0-3 A1-1 A1-2 A2-1 A2-2 A3-1 A3-2 A4-1 100 2.127 2.254 2.081 1.455 1.670 2.081 1.815 2.210 2.014 2.043 500 1.873 2.132 1.754 1.439 1.452 1.813 1.581 1.864 1.821 1.946 2500 1.321 2.040 1.366 1.164 1.025 1.671 1.266 1.519 1.523 2.038 12500 0.773 1.967 0.847 0.896 0.983 1.014 0.861 1.179 1.174 1.993 62500 0.467 1.502 0.546 0.545 0.774 0.837 0.616 0.679 0.715 1.318 312500 0.301 0.948 0.279 0.145 0.374 0.438 0.415 0.391 0.461 0.749 1562500 0.166 0.423 0.177 0.055 0.107 0.192 0.246 0.194 0.195 0.306 PBS 0.124 0.195 0.178 0.061 0.101 0.053 0.126 0.054 0.060 0.056

[0060] (3) Exploration of antibody purification conditions and detection data

[0061] The above ascites was purified by 3.3% n-octanoic acid-thiamine precipitation method to obtain a total of 10 antibodies. The titer test data of all antibodies are shown in Table 3 below:

[0062] Table 3. Potency test data of all antibodies

[0063]

[0064]

[0065] The above data show that monoclonal antibodies A0-1, A2-2, A3-2, and A4-1 have good specific binding ability to the natural rheumatoid factor (RF) antigen. Therefore, the four blocking antibodies A0-1, A2-2, A3-2, and A4-1 were used to test for false positive RF specimens.

[0066] Application of monoclonal antibodies in products

[0067] Active blockers were verified by the immune colloidal gold platform. Specifically, the experimental group used the four blocker antibodies obtained in the above experiment to detect two clinical false-positive serum specimens (named RF1 and RF2) purchased from Hangzhou Dian Diagnostics. The original concentration of antibody A0-1 was 9.95 mg / ml, the original concentration of antibody A2-2 was 10.18 mg / ml, the original concentration of antibody A3-2 was 10.14 mg / ml, and the original concentration of antibody A4-1 was 10.3 mg / ml. During the experiment, the concentration of each blocker antibody was diluted to 0.4 mg / ml. The standard control group used commercial antibodies from a clinical diagnostic kit to detect false-positive serum specimens, and there was no blocker in the control group. In addition, the purchased blocker antibody 85000 (concentration of 0.4 mg / ml) was used as a positive control to compare the effects of the blocker antibody obtained by the present invention with the blocker antibody 85000. The above reagents were added to the sample, and then the results were tested using the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biotechnology Co., Ltd. The experimental data results are shown in Table 4.

[0068] Table 4 Detection results of RF false positive quality control products

[0069]

[0070] Note: L4-L8 represent the color depth levels of the test strips. Higher values ​​indicate darker colors and weaker false positive elimination. + / - indicates colors slightly darker or lighter than the specified level. Test values ​​correspond to the levels; higher values ​​indicate weaker false positive elimination.

[0071] The results showed that the standard control group, without a blocking antibody, had the highest color level and value, but no false-positive elimination effect. The four blocking antibodies demonstrated excellent false-positive elimination, effectively reducing both color level and test value. Furthermore, these blocking antibodies demonstrated superior blocking efficacy to the purchased blocking antibody 85000. These results demonstrate that after two rounds of scale-up production and purified antibody reassessment, the four blocking antibodies all met the criteria for false-positive elimination. False-positive test values ​​without a blocking agent were significantly higher. However, the addition of an active blocking agent significantly eliminated interference in two false-positive samples, 9233354 (RF1) and 9233353 (RF2), containing rheumatoid factor interference. This interference elimination was significantly superior to that of the commercially available blocking antibody 85000. The most effective antibody, A3-2, was designated anti-blocker-MAB7, which reduced the effects of interference by 10-fold.

[0072] Sequence analysis of the heavy chain variable region and light chain variable region of the monoclonal antibody anti-blocker-MAB7

[0073] Primers were designed to amplify the heavy chain variable region and light chain variable region genes of the monoclonal antibody anti-blocker-MAB7.

[0074] Take the hybridoma cell line (about 10 7 cells), and total RNA of the cells was extracted according to the instructions of the Trizol RNA extraction kit. The total RNA was used as a template for reverse transcription to synthesize the first chain cDNA. The above amplified products were used as templates for PCR amplification of the heavy chain variable region and light chain variable region genes of the antibody.

[0075] The heavy chain variable region (approximately 360 bp) and light chain variable region (approximately 330 bp) gene fragments of anti-blocker-MAB7 were recovered and sent to the company for sequencing. The sequencing results are as follows:

[0076] The nucleotide sequence (348 bp) encoding the heavy chain variable region of the monoclonal antibody anti-blocker-MAB7 is as follows: GTCAAACTGCAGCAGTCAGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTAGCACTAGCTACAACCCATCTCTCCAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTGGCTAACTGGGTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 15).

[0077] The sequence encoding the heavy chain variable region of CDRH1 is: GGCTACTCAATCACCAGTGATTATGCC (SEQ ID NO: 9).

[0078] The sequence encoding the heavy chain variable region of CDRH2 is: ATAAGCTACAGTGGTAGCACT (SEQ ID NO: 10).

[0079] The sequence encoding the heavy chain variable region of CDRH3 is: GCAAGATTGGCTAACTGGGTCTTTGACTAC (SEQ ID NO: 11).

[0080] The amino acid sequence of the heavy chain variable region (116aa) is shown below:

[0081] VKLQQSGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTSYNP SLQSRISITRDTSKNQFFLQLNSVTTEDTATYYCARLANWVFDYWGQGTTLTVSS (SEQ ID NO: 7).

[0082] The amino acid sequence of CDRH1 in the heavy chain variable region is: GYSITSDYA (SEQ ID NO: 1).

[0083] The amino acid sequence of CDRH2 in the heavy chain variable region is: ISYSGST (SEQ ID NO: 2).

[0084] The amino acid sequence of CDRH3 in the heavy chain variable region is: ARLANWVFDY (SEQ ID NO: 3).

[0085] The nucleotide sequence (312 bp) encoding the light chain variable region of the monoclonal antibody anti-blocker-MAB7 is as follows: GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAAAATATTTACAGTTTTTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTTTATGCTGCAACAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTATGAAGATTTTGGGAGTTATTACTGTCAACATTTTCGGGGTACTCCGTACACGTTCGGAGGGGGGACCAGGCTG (SEQ ID NO: 16).

[0086] The sequence encoding the light chain variable region of CDRL1 is: GAAAATATTTACAGTTTT (SEQ ID NO: 12).

[0087] The sequence encoding the light chain variable region of CDRL2 is: GCTGCAACA (SEQ ID NO: 13).

[0088] The sequence encoding the light chain variable region of CDRL3 is: CAACATTTTCGGGGTACTCCGTACACG (SEQ ID NO: 14).

[0089] The amino acid sequence of the light chain variable region (104aa) is shown below:

[0090] DIQMTQSPASLSVSVGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYAATNLADGVPSR FSGSGSGTQYSLKINSLQYEDFGSYYCQHFRGTPYTFGGGTRL (SEQ ID NO: 8).

[0091] The amino acid sequence of CDRL1 in the light chain variable region is: ENIYSF (SEQ ID NO: 4).

[0092] The amino acid sequence of CDRL2 in the light chain variable region is: AAT (SEQ ID NO: 5).

[0093] The amino acid sequence of CDRL3 in the light chain variable region is: QHFRGTPYT (SEQ ID NO: 6).

[0094] The monoclonal antibody against natural rheumatoid factor RF disclosed in the present invention has beneficial effects including but not limited to: (1) the monoclonal antibody against natural rheumatoid factor RF obtained by the present invention has high titer, high purity, high sensitivity and high specificity; (2) the monoclonal antibody against natural rheumatoid factor RF of the present invention can be used to eliminate endogenous interference in immunological tests such as ELISA, CLLA, and lateral flow, and provides the required raw materials for immune blocking reagents (e.g., active blockers). It should be noted that different embodiments may produce different beneficial effects. In different embodiments, the beneficial effects that may be produced may be any one or a combination of the above, or any other possible beneficial effects.

[0095] Those skilled in the art will appreciate that the above embodiments are intended only to illustrate the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and variations made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against natural rheumatoid factor RF, characterized in that It includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes CDRH1 with an amino acid sequence as shown in SEQ ID NO: 1, CDRH2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDRH3 with an amino acid sequence as shown in SEQ ID NO: 3; and The light chain variable region includes CDRL1 shown in SEQ ID NO: 4, CDRL2 shown in SEQ ID NO: 5, and CDRL3 shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against natural rheumatoid factor RF according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequences encoding CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the nucleotide sequences encoding CDRL1, CDRL2 and CDRL3 of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

16.

6. An active blocker for clearing immune interference of rheumatoid factor RF, characterized in that: The invention comprises the monoclonal antibody against natural rheumatoid factor RF according to claim 1 or 2.

7. Use of the monoclonal antibody against natural rheumatoid factor (RF) according to claim 1 or 2 or the active blocker according to claim 6 in the preparation of a detection kit.

8. The use according to claim 7, characterized in that The detection kit includes one or more of a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay kit, a chemiluminescence kit, and an immunoturbidimetric detection kit.

9. A detection kit, characterized in that: The kit comprises the monoclonal antibody against natural rheumatoid factor RF according to claim 1 or 2 or the active blocking agent according to claim 6.

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