CN115746131A - Monoclonal antibody for resisting natural rheumatoid factor RF and application thereof - Google Patents

Abstract

The invention provides a monoclonal antibody for resisting natural rheumatoid factor RF and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and a CDRH3 with an amino acid sequence shown as SEQ ID NO. 3. The light chain variable region comprises a CDRL1 shown by an amino acid sequence SEQ ID NO. 4, a CDRL2 shown by an amino acid sequence SEQ ID NO. 5 and a CDRL3 shown by an amino acid sequence SEQ ID NO. 6. The invention also provides a nucleic acid molecule for coding the monoclonal antibody, application of the monoclonal antibody in preparing 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 has higher sensitivity and specificity, and can be applied to immunological detection.

Description

Monoclonal antibody for resisting natural rheumatoid factor RF and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a monoclonal antibody for resisting natural rheumatoid factor RF and application thereof.

Background

In immunoassays, such as ELISA, CLIA or lateral flow immunoassays, xenotropic antibodies (HA), human anti-mouse antibodies (HAMA) and Rheumatoid Factor (RF) can reduce the sensitivity and specificity of the assay by non-specific binding to the antibody, leading to erroneous results and diagnosis. Endogenous interference is a phenomenon which cannot be bypassed in immunoassay, and interference elimination is an important task in the research and development of immunoassay kits. The immune blocking agent is a substance added into an in-vitro diagnostic reagent system, can eliminate or reduce endogenous interference in immunoassay, and improves the sensitivity and precision of a kit detection system. The addition of an immune blocker is a straightforward way to eliminate endogenous interference.

The immune blocking agent is divided into a passive blocking agent and an active blocking agent, wherein the rheumatoid factor is used as one of interference factors, and the effect is small when the passive blocking agent is added. Therefore, there is a need to develop an immune blocking agent against antibodies against rheumatoid factor to reduce the false positive results caused by these interferences, so as to actively block such interferences and improve the sensitivity and precision of detection.

Disclosure of Invention

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

In some embodiments, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 8.

According to another aspect of the invention, a nucleic acid molecule is provided. The nucleic acid molecule comprises a nucleotide sequence encoding the anti-natural rheumatoid factor RF monoclonal antibody of claim 1 or 2.

In some embodiments, the nucleotide sequences of CDRH1, CDRH2, and CDRH3 encoding the heavy chain variable region are shown in SEQ ID NOs 9, 10, and 11, respectively, and the nucleotide sequences of CDRL1, CDRL2, and CDRL3 encoding the light chain variable region are shown in SEQ ID NOs 12, 13, and 14, respectively.

In some embodiments, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 15 and the nucleotide sequence encoding the light chain variable region is set forth in SEQ ID NO. 16.

According to still another aspect of the invention, the application of the monoclonal antibody against the natural rheumatoid factor RF in preparing an immune blocking reagent for eliminating the interference of the rheumatoid factor RF is provided. The monoclonal antibody of the anti-natural rheumatoid factor RF is used for immunological detection for eliminating endogenous interference.

According to a further aspect of the invention, there is provided a test kit for eliminating endogenous interference. The kit comprises the anti-natural rheumatoid factor RF monoclonal antibody.

Detailed Description

As used herein, the terms "a," "an," "the," and/or "the" are not intended to be inclusive and include the plural as well, unless the context clearly indicates otherwise. In general, the terms "comprises" and "comprising" merely indicate that steps and elements are included which are explicitly identified, that the steps and elements do not form an exclusive list, and that a method or apparatus may include other steps or elements.

Endogenous interference is often due to cross-reactivity of the antibodies used in the reagents with endogenous substances in the patient's sample, and is collectively referred to as endogenous interference. The three main categories include: the first broad category is: heterophilic Antibodies (HA) are antibodies against undefined antigens present in human peripheral circulation, and are generally multispecific and less aviphilic antibodies. The degree and frequency of heterophilic antibody interference is related to its concentration, affinity and detection system. The second broad category is: human anti-Animal antibodies (HAAA), antibodies having activity against Animal proteins, both iatrogenic and non-iatrogenic. The common is human anti-mouse antibody (HAMA), HAAA of different species are crossed with each other, and the antibody is specific and has stronger affinity. The human anti-animal antibody has stronger affinity and is specific to the antigen; heterophilic antibodies are not specific for the antigen and have a weak affinity. The third major category is: rheumatoid Factor (RF), which is an autoantibody with denatured IgG as a target antigen, includes IgM, igA, igG, igD and IgE pentatypes, 68% -80% of which are IgM types, has poor binding capacity with natural IgG, is easy to bind with denatured IgG of human and animals or IgG in immune complexes, has random interference effect and is irrelevant to RF concentration. RF binds to denatured IgG in vivo to form an immune complex that activates complement or is phagocytosed by phagocytic cells. The substances such as lysosomal enzyme, activated peptide, collagenase, prostaglandin E2, etc. released from phagocytes cause tissue inflammatory injury in the presence of cytokines and inflammatory adhesion molecules, and can cause osteoarthritis and vasculitis in patients. The rheumatoid factor is not a specific factor for patients with rheumatoid arthritis. Rheumatoid arthritis, lupus erythematosus, diabetes, autoimmune diseases and the like can enable patients to find rheumatoid factors RF (also can find heterophilic antibodies, autoantibodies and the like) in vivo, and the special components have certain adsorption effect in the reaction process and are mostly false positive caused by false color reaction generated by interference of certain antigen substances in vivo.

The most direct solution to these interference factors is to add a blocking agent to the sample to correct these endogenous interferences in the detection system and to avoid false high detection values, false negative and/or false positive results.

Therefore, the invention takes the natural rheumatoid factor RF as an antigen, prepares the corresponding monoclonal antibody as a blocking agent (such as an active blocking agent), can eliminate or reduce endogenous interference in immunoassay, and improves the sensitivity and accuracy of detection. Specifically, a purchased natural rheumatoid factor RF is used for immunizing a Balb/c mouse, spleen cells of the mouse are taken to be fused with myeloma cells, hybridoma cells with high specificity are obtained through specific high-throughput screening, a large amount of mouse ascites is obtained through culture and re-immunization, and then the monoclonal antibody of the 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 can be used for eliminating endogenous interference in immunological detection such as ELISA, CLLA, lateral flow and the like, and provides a required raw material for an immune blocking reagent (for example, an active blocking agent). The immune blocking kit developed by taking the antibody as a raw material has good application value. The anti-natural rheumatoid factor RF monoclonal antibody has the obvious interference elimination effect, and can reduce the interference effect by 10 times compared with a sample without the antibody; and more interference-eliminating effect than that of the purchased antibody.

The monoclonal antibody may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and a CDRH3 with an amino acid sequence shown as SEQ ID NO. 3. The light chain variable region comprises a CDRL1 with an amino acid sequence shown as SEQ ID NO. 4, a CDRL2 with an amino acid sequence shown as SEQ ID NO. 5 and a CDRL3 with an amino acid sequence shown as SEQ ID NO. 6.

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

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

The monoclonal antibody against natural rheumatoid factor RF of the present invention can be applied to an immune blocking reagent, for example, an active blocking agent. In some embodiments, monoclonal antibodies against native rheumatoid factor RF may be used to eliminate endogenous interference in immunological assays such as ELISA, CLLA, lateral flow, and the like.

The invention also provides a detection kit for eliminating endogenous interference, such as an immune blocking kit. The kit can comprise the monoclonal antibody for resisting the natural rheumatoid factor RF. The immune blocking kit developed by taking the monoclonal antibody of the anti-natural rheumatoid factor RF as a raw material has good application value and high sensitivity and specificity.

Examples

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Preparation of monoclonal antibodies

And (4) preparing an antigen.

Natural rheumatoid factor RF was purchased as AN antigen from Holmes (Holmes) Biotechnology Inc. of Beijing, cat # HA129AN at a concentration of 10.0mg/ml.

And (3) preparing a monoclonal antibody.

Healthy female Balb/c mice of 6-8 weeks of age were selected for immunization injection according to a pre-specified immunization schedule. And (3) taking the natural rheumatoid factor RF purchased in the step as immunogen to immunize Balb/c mice, and extracting the spleen lymphocytes of the mice successfully immunized. Lymphocytes were fused with mouse myeloma cells SP2/0 by cell fusion techniques. Obtaining the hybridoma cell strain which stably secretes the monoclonal antibody of the anti-natural rheumatoid factor RF after two rounds of subcloning and screening, thereby obtaining the monoclonal antibody of the anti-natural rheumatoid factor RF.

Animal immunization experiments were performed using the purchased antigen (natural rheumatoid factor RF).

The animal immunization experiment comprises the following specific steps:

1. balb/c mice with consistent body weight and week age mean were randomly divided into 2 groups, an aluminum adjuvant (aluminum hydroxide adjuvant) group and an aluminum adjuvant-free group.

2. Before the experiment, serum before each mouse immunization was collected (immune serum was collected on the fifth day, blood was collected through eyeballs, and appropriate amount of blood was collected to ensure the normal state of the mice), which was used as a negative control, and the collected serum was stored at-80 ℃.

3. The formulation of the adjuvant added with aluminum (aluminum hydroxide adjuvant) is as follows: before immunization, each antigen was diluted separately to the corresponding dose in 75 μ L PBS (50 ug/mouse) and mixed with alum adjuvant (i.e., aluminum hydroxide adjuvant) (1 mg/mouse) in a volume of antigen: adjuvant =3:1 (i.e., 25ul adjuvant was added to 75ul immunogen dilution); shaking the adjuvant before use, and slowly dripping the injection adjuvant (25 ul) into the immunogen solution; after the adjuvant and the immunogen diluent were mixed thoroughly, both were mixed thoroughly for 30 minutes. Allowing the adjuvant to effectively adsorb the antigen; the subsequent steps are carried out according to the experimental operation of the immune animals.

4. Group without aluminum adjuvant: the antigen was diluted in 100. Mu.L of PBS to the corresponding dose in the above table (i.e., 50 ug/mouse), 100. Mu.L of immunogen, and then the procedure was followed for the immunized animal experiment.

5. Subcutaneous injections were performed at 2 week intervals: the experiment is designed in a 3-time immunization mode, but eyeball blood is taken 7 days after each immunization injection, partial mouse supernatant is obtained by a centrifugation method, the serum antibody titer is firstly detected, the heart blood is taken 7 days after the last immunization, the maximum blood volume is taken, and the supernatant is obtained by centrifugation and stored at-80 ℃.

6. Detecting serum titer

A total of 5 mice were immunized, numbered A0, A1, A2, A3, A4 in that order. After 3 immunizations, the serum titers were determined. The test data are shown in table 1 below.

TABLE 1 data of the antibody titer in immune sera

Fusing immune splenocytes with myeloma cell line SP2/0, screening fused cells by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymidine), and performing ELISA positive screening and subcloning on the fused cells; and (3) taking ascites from the screened positive monoclonal, purifying the ascites antibody generated by the hybridoma cells by using a Protein A/G antibody purification column, wherein the ELISA titer of the purified ascites antibody is more than 128,000, the purity is more than 90%, and the result is determined by the binding activity of the natural rheumatoid factor RF identified by enzyme-linked reaction ELISA detection described in the following step.

Enzyme-linked immunosorbent assay (ELISA) for detecting and identifying binding activity of natural Rheumatoid Factor (RF)

1. IgG antibody titer detection mode

(1) Coating the bottom plate: the antigen was diluted to 3ug/ml with coating diluent, and 100. Mu.l of the prepared coating solution was added to each well, and the mixture was placed in a refrigerator at 4 ℃ for 24 hours.

(2) After 24h, taking out the mixture from the refrigerator, placing the mixture at 37 ℃ for balancing for 30min, and then removing liquid in the holes; washing with washing solution for 3 times, each for 3min.

(3) And (3) sealing the enzyme-labeled reaction hole: adding 200ul of calf serum 5% into each well, sealing at 37 deg.C for 90min, and washing with washing solution for 3 times (each time for 3 min).

(4) Adding a sample to be detected: diluting the sample according to a required proportion, adding the diluted sample into an enzyme-labeled reaction hole, wherein each hole is 100 mu l, and placing the sample at 37 ℃ for 90min; washing with washing solution for 3 times, each time for 3min.

(5) Adding an enzyme-labeled antibody: adding a secondary antibody with a proper concentration according to the instruction; the wells were washed at 37 ℃ for 90min with 100. Mu.l/well as before.

(6) Adding a substrate solution: the substrate is added in an amount of 100. Mu.l per well, and is placed at 37 ℃ for 15-30 min in the dark.

(7) And (3) terminating the reaction: the reaction was stopped by adding 50. Mu.l of stop solution to each well and the results were measured within 20 min.

Detecting the binding activity of the monoclonal antibody for identifying the natural rheumatoid factor RF

(1) Cell fusion and clonal screening data

The fusion was completed in 5 rounds, and the mouse numbers were A0, A1, A2, A3, and A4 in order.

A0 mice are subjected to fusion screening to pick out 18 positive holes for subcloning, and finally 3 cell strains are completely obtained; through fusion screening, 72 positive clones with OD450 value greater than 2.2 are selected for detecting titer by multiple dilution, and then secondary subclone screening is carried out. 3 cell lines were obtained, designated A0-1 to A0-3, respectively.

A1 mouse fusion screening selects 16 positive holes for subcloning, 72 positive holes with OD450 value more than 2.1 are selected for carrying out multiple dilution to detect titer through fusion screening, and secondary and third subcloning screening are carried out to complete 2 cell strains finally, which are respectively named as A1-1 and A1-2.

A2 mice are subjected to fusion screening to pick out 16 positive holes for subcloning, and then secondary and third subcloning screening are carried out, so that 2 cell strains are finally completed and are respectively named as A2-1 and A2-2.

A3 mouse fusion screening picks up 12 positive holes for subcloning, and then secondary and third subcloning screening are carried out, finally 2 cell strains are completely selected and named as A3-1 and A3-2 respectively.

A4 mice are subjected to fusion screening, 4 positive holes are selected for subcloning, and then secondary and third subcloning screening are carried out, so that 1 cell strain is finally completed and named as A4-1. After 5 cell fusions, a total of 10 cell lines were obtained.

(2) Ascites preparation and test data

3F 1 mice were individually cultured for each complete cell line, and 10 ascites were prepared, and the titer data for all ascites tests are shown in Table 2 below:

TABLE 2 ascites antibody test titer data

Dilution factor	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

(3) Antibody purification condition exploration and detection data

The ascites fluid is purified by a 3.3% caprylic acid-thiamine precipitation method to obtain 10 antibodies in total, and the titer detection data of all the antibodies are shown in the following table 3:

TABLE 3 titer test data for all antibodies

The data show that the monoclonal antibodies A0-1, A2-2, A3-2 and A4-1 have good specific binding capacity to the natural rheumatoid factor RF antigen. Therefore, four blocker antibodies, A0-1, A2-2, A3-2, A4-1, were used for the testing of RF false positive specimens.

Application of monoclonal antibody in product

The active blocking agent was verified by an immune colloidal gold platform. Specifically, the experimental group was performed by using 4 blocking agent antibodies obtained from the above experiments to detect 2 clinical false positive serum samples (designated as RF1 and RF 2) purchased from the changzhou dean diagnosis. The original concentration of antibody A0-1 was 9.95mg/ml, the original concentration of antibody A2-2 was 10.18mg/ml, the original concentration of antibody A3-2 was 10.14mg/ml, and the original concentration of antibody A4-1 was 10.3mg/ml. The concentration of each blocker antibody was diluted to 0.4mg/ml during the experiment. The standard control group is used for detecting a false positive serum sample by adopting a commercial antibody of a clinical diagnosis kit, and no blocking agent exists in the control group. In addition, an externally purchased blocking agent antibody 85000 (at a concentration of 0.4 mg/ml) was used as a positive control to compare the effects of the blocking agent antibody obtained by the present invention and the blocking agent antibody 85000. The above reagents were applied and the results were measured using POCT test instrument ACG1000 (ID-a 003) from hangzhou An Xu, biotechnology, ltd, and the results of the experimental data are shown in table 4.

TABLE 4 detection results of RF false positive quality control

Note: L4-L8 represent the light color level of the strip of the test strip, and the higher the numerical value, the darker the color, and the weaker the false positive eliminating effect. And +/-indicates a color that is slightly darker or lighter than the level. The detection value corresponds to the level, and the higher the value, the weaker the effect of eliminating the false positive.

According to the results, the standard control group has no blocker antibody, the color grade and the numerical value are highest, and the false positive eliminating effect is avoided. The four blocker antibodies have better effect of eliminating false positive, the color grade and the detection value are effectively reduced, and simultaneously the blocking effect of the blocker antibodies is better than that of the purchased blocker antibody 85000. The results show that after four blocker antibodies are produced by two rounds of amplification, the purified antibodies are evaluated again, and the false positive eliminating effect is qualified. The false positive detection value without adding the blocking agent is obviously higher, after adding the active blocking agent, the interference elimination effect on 2 cases of false positive specimens 9233354 (RF 1) and 9233353 (RF 2) containing rheumatoid factor interference is obvious, and the interference elimination effect is obviously better than that of the blocking agent antibody 85000 on the market. The antibody A3-2 with the best effect is named anti-blocker-MAB7, and the antibody can reduce the influence of interference by 10 times.

Carrying out sequence analysis on heavy chain variable region and light chain variable region of monoclonal antibody anti-blocker-MAB7

Designing a primer for amplifying genes of a heavy chain variable region and a light chain variable region of a monoclonal antibody anti-blocker-MAB 7.

Taking hybridoma cell strain (about 10) of monoclonal antibody anti-blocker-MAB7 in logarithmic growth phase ⁷ Individual cells) are extracted according to the instructions of the Trizol RNA extraction kit, total RNA is used as a template to carry out reverse transcription to synthesize a first chain of cDNA, and the amplified product is used as the template to carry out PCR amplification on the heavy chain variable region gene and the light chain variable region gene of the antibody.

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

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

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

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

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

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

VKLQQSGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTSYNPSLQSRISITRDTSKNQFFLQLNSVTTEDTATYYCARLANWVFDYWGQGTTLTVSS(SEQ ID NO:7)。

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

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

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

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

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

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

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

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

DIQMTQSPASLSVSVGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQYEDFGSYYCQHFRGTPYTFGGGTRL(SEQ ID NO:8)。

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

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

The amino acid sequence of CDRL3 of the light chain variable region is as follows: QHFRGTPYT (SEQ ID NO: 6).

The monoclonal antibody against natural rheumatoid factor RF disclosed by the invention brings about the following beneficial effects, but not limited to: (1) The monoclonal antibody for resisting the natural rheumatoid factor RF has high titer, high purity, high sensitivity and high specificity; (2) The monoclonal antibody of the invention can be used for eliminating endogenous interference in immunological detection such as ELISA, CLLA, lateral flow and the like, and provides a required raw material for an immune blocking reagent (such as an active blocking agent). It is to be noted that different embodiments may produce different advantages, and in different embodiments, any one or combination of the above advantages may be produced, or any other advantages may be obtained.

It should be understood by those skilled in the art that the above examples are only illustrative and not limiting of the present invention. Any modification, equivalent replacement, and variation made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A monoclonal antibody against natural rheumatoid factor RF, comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises a CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and a CDRH3 with an amino acid sequence shown as SEQ ID NO. 3; and

the light chain variable region comprises a CDRL1 with an amino acid sequence shown as SEQ ID NO. 4, a CDRL2 with an amino acid sequence shown as SEQ ID NO. 5 and a CDRL3 with an amino acid sequence shown as SEQ ID NO. 6.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 8.

3. A nucleic acid molecule comprising a nucleotide sequence encoding the anti-natural rheumatoid factor RF monoclonal antibody of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequences encoding CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown as 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 as SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, respectively.

5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 15 and the nucleotide sequence encoding the light chain variable region is set forth in SEQ ID NO. 16.

6. An active blocking agent for eliminating immune interference of rheumatoid factor RF, which comprises the monoclonal antibody against natural rheumatoid factor RF of 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 a detection kit.

8. The use of claim 7, wherein the detection kit comprises one or more of a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay detection kit, a chemiluminescent kit, and an immunoturbidimetric detection kit.

9. The use according to claim 7, wherein the monoclonal antibody or active blocker of native rheumatoid factor RF is used in an immunological assay for eliminating the interference of rheumatoid factor RF in endogenous interference.

10. 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 blocker according to claim 6.