IgM antibody as well as preparation method and application thereof

Abstract

The invention provides an IgM antibody as well as a preparation method and application thereof, and relates to the technical field of biology. The rheumatoid factor IgM antibody (RF-IgM antibody) provided by the invention can specifically recognize an FC fragment of denatured IgG, can be used as a raw material of an RF detection reagent quality control product, and can improve the screening efficiency of the raw material, reduce the difficulty of batch-to-batch difference control of the raw material and improve the sensitivity and accuracy of detection; the antibody can be used for an RF IgM detection kit, has good specificity and stability, facilitates clinical operation, greatly saves the cost, and greatly controls the batch difference between quality control products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an IgM antibody and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology, with symmetrical, multi-articular, chronic proliferative synovitis as the main clinical features. Repeated attacks lead to progressive destruction of the affected joints and adjacent tissues, often causing deformities and dysfunction of the damaged joints, and can cause damage to target organs and systems such as the heart, kidneys, eyes, respiratory system, and blood system. The prevalence of RA is close to 1%, the highest among middle-aged women. It is the most widespread immune-mediated inflammatory joint disease in the world and has gradually become one of the main causes of labor loss and disability in the domestic population.

[0003] Rheumatoid factor (RF) is an autoantibody that uses the FC fragment of denatured IgG as its target antigen. RF can activate complement or be engulfed by phagocytes after combining with denatured IgG in the body to form immune complexes. Lysosomal enzymes, activated peptides, collagenase, prostaglandin E2 and other substances released by phagocytes, with the participation of cytokines and inflammatory adhesion molecules, cause tissue inflammatory damage, which can cause osteoarthritis and vasculitis in patients. RF is mainly 19S IgM, and there are also 7S IgG and IgA. IgM is considered to be an important serological indicator for the diagnosis of RA. IgM titer > 80IU / mL and accompanied by severe joint dysfunction usually indicate a poor prognosis. At the same time, the content of IgM RF is not closely related to the activity of RA. The content of IgG RF is closely related to synovitis, vasculitis and extra-articular symptoms in RA patients. The detection of IgG RF in synovial fluid is more meaningful than that in serum. IgA RF is an indicator of RA clinical activity and has a significant correlation with bone destruction. It has been reported that IgG RF and IgARF show a certain pattern in the serum of RA patients. The appearance of IgG RF and IgA RF is always accompanied by an increase in the IgM RF content. The presence of IgM RF alone often indicates a better prognosis for the patient, and the increase of multiple RF subtypes indicates a poor prognosis.

[0004] At present, the commonly used methods for determination are latex agglutination, enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry. Among them, latex agglutination has been widely used because of its simplicity and ease. This method mainly detects IgM RF, while IgG RF is difficult to detect with this method. However, this method is easily affected by factors such as blood lipids, has many interfering factors, poor repeatability, and can only perform qualitative or semi-quantitative determination, which has obvious limitations and defects in the diagnosis of RA. The advantages of immunoturbidimetry are high sensitivity, good repeatability, fast reaction speed, and easy automation and standardization. ELISA can determine the subtype of RF, which is helpful for judging the prognosis of the disease.

[0005] In the current field of RF in vitro diagnostic kits, quality control products mainly come from clinical high-value serum as raw materials, which are extremely difficult to collect and difficult to control batch differences. In addition, there are no commercialized specific recombinant antibodies for reference, which has seriously affected the production of RF quality control products.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The first object of the present invention is to provide a rheumatoid factor IgM antibody to solve the above technical problems.

[0008] The second object of the present invention is to provide a nucleotide.

[0009] The third object of the present invention is to provide a carrier.

[0010] A fourth object of the present invention is to provide a cell.

[0011] The fifth object of the present invention is to provide a method for preparing rheumatoid factor IgM antibody.

[0012] The sixth object of the present invention is to provide a use of a rheumatoid factor IgM antibody as a quality control product in the preparation of a rheumatoid factor IgM antibody detection product.

[0013] The seventh object of the present invention is to provide a kit for detecting rheumatoid factor IgM antibodies.

[0014] In order to achieve the above objectives, the following technical solutions are adopted:

[0015] In a first aspect, the present invention provides a rheumatoid factor IgM antibody selected from antibody A, antibody B, antibody C or antibody D;

[0016] The antibody A comprises light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NOs. 1, 36, and 2, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NOs. 3 to 5;

[0017] The antibody B comprises light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL, whose amino acid sequences are shown in SEQ ID NOs. 6, 37, and 7, respectively, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH, whose amino acid sequences are shown in SEQ ID NOs. 8 to 10, respectively;

[0018] The antibody C comprises light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NOs. 11, 38, and 12, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NOs. 13 to 15;

[0019] The antibody D comprises light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are shown in SEQ ID NOs. 16, 39, and 17, respectively, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NOs. 18 to 20, respectively.

[0020] As a further technical solution, the antibody A comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are sequentially shown in SEQ ID NOs. 21 to 22;

[0021] The antibody B comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 23 to 24;

[0022] The antibody C comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are sequentially shown in SEQ ID NOs. 25 to 26;

[0023] The antibody D comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 27-28.

[0024] As a further technical solution, the heavy chain constant region of the rheumatoid factor IgM antibody is selected from the human IgM constant region.

[0025] As a further technical solution, the light chain constant region of the rheumatoid factor IgM antibody is selected from the human κ chain constant region.

[0026] In a second aspect, the present invention provides a nucleotide comprising a nucleotide sequence encoding the rheumatoid factor IgM antibody.

[0027] In a third aspect, the present invention provides a vector carrying the nucleotide.

[0028] In a fourth aspect, the present invention provides a cell, wherein the cell carries the nucleotide, or contains the vector, or expresses the rheumatoid factor IgM antibody.

[0029] In a fifth aspect, the present invention provides a method for preparing rheumatoid factor IgM antibodies, which are obtained by fermenting the cells described above.

[0030] In a sixth aspect, the present invention provides a use of a rheumatoid factor IgM antibody as a quality control product in the preparation of a rheumatoid factor IgM antibody detection product.

[0031] In a seventh aspect, the present invention provides a kit for detecting rheumatoid factor IgM antibodies, wherein the quality control product of the kit is the rheumatoid factor IgM antibodies.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The rheumatoid factor IgM antibody (RF-IgM antibody) provided by the present invention can specifically recognize the FC fragment of denatured IgG, has good stability, and can be used as a raw material for RF detection reagent quality control products, greatly improving the screening efficiency of raw materials and greatly reducing the difficulty of controlling the batch difference of raw materials.

[0034] 2. The present invention utilizes RF-IgM antibody as the raw material of RF detection reagent quality control product, thereby improving the sensitivity and accuracy of detection.

[0035] 3. The recombinant antibody human RF IgM prepared by the present invention can be used in RF IgM detection kits, and has good specificity and stability, which not only facilitates clinical operations, but also greatly saves costs and greatly controls the batch difference between quality control products. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1The non-reducing electrophoresis patterns of purified RF-IgM antibodies of five strains A, B, C, D, and E;

[0038] Figure 2 This is the reduction electrophoresis pattern of five purified RF-IgM antibodies, A, B, C, D, and E. DETAILED DESCRIPTION

[0039] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0040] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0041] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain antigen binding sites. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also called hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held together closely by FRs to form the variable region. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0042] The term "vector" refers to a nucleic acid delivery vehicle into which a nucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.

[0043] The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0044] In a first aspect, the present invention provides a rheumatoid factor IgM antibody selected from antibody A, antibody B, antibody C or antibody D;

[0045] The antibody A comprises light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NOs. 1, 36, and 2, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NOs. 3 to 5;

[0046] The antibody B comprises light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL, whose amino acid sequences are shown in SEQ ID NOs. 6, 37, and 7, respectively, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH, whose amino acid sequences are shown in SEQ ID NOs. 8 to 10, respectively;

[0047] The antibody C comprises light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NOs. 11, 38, and 12, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NOs. 13 to 15;

[0048] The antibody D comprises light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are shown in SEQ ID NOs. 16, 39, and 17, respectively, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NOs. 18 to 20, respectively.

[0049] The amino acid sequences of the various complementarity determining regions are as follows:

[0050]

[0051]

[0052] In some optional embodiments, the antibody A comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 21 to 22:

[0053] LPVLTQPASVSGSPGQSITISCSGSSSDVGSYNLVSWYQQYPGKAPK LLISEVTKRPSGVSDRFSGSSKNTASLTISGLQADDEADYYCSSFAGITTL VLFGGGTKVTVL (SEQ ID NO. 21);

[0054] QVQLVQSGAEVKKTGESLKISCKGSGYSFTSYWIGWVRQMPGKGL EWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYY CARGSSWNYSRLPVWGQGTLVTVSS (SEQ ID NO. 22).

[0055] The antibody B comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 23 to 24:

[0056] DIQMTQSPSTLSASEGDRVTITCRASQSISSQLAWYQQKPGKAPKLLI YKASTLESGVPSRFSGSGSGTEFTLTISSVQPDDFASYYCQHFKGYPLTFG GGTKLEIK(SEQ ID NO.23);

[0057] QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGL EWVANIKQDGSEKYYVDSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAV YYCARETSMGAFDIWGQGTMVTVSS (SEQ ID NO. 24).

[0058] The antibody C comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 25 to 26:

[0059] DIQMTQSPSTLSASEGDRVTITCRASSSDFGSTCLLAWYQQKPGKAP KLLIYKDTTLESGVPSRFSGSGSGTEFTLTISSVQPDDFASYYCSFAKGYT LVFGGGTKLEIK(SEQ ID NO. 25);

[0060] QVQLVQSGGGLVQPGGSLRLSCAASGFTFTSSWMSWVRQAPGKGL EWVANGSSWDGSKYYVDSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAV YYCGSSWNSMGALPVWGQGTMVTVSS (SEQ ID NO. 26).

[0061] The antibody D comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 27 to 28:

[0062] DIQMTQSPSTLSASEGDRVTITCRASVGSYQSLAWYQQKPGKAPKL LIYKGYTLESGVPSRFSGSGSGTEFTLTISSVQPDDFASYYCKGYPFAGIFG GGTKLEIK(SEQ ID NO. 27);

[0063] QVQLVQSGGGLVQPGGSLRLSCAASFTSYTFTSSMSWVRQAPGKGL EWVANFTFSNSMYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCETSMGGALPWGQGTMVTVSS (SEQ ID NO. 28).

[0064] In some optional embodiments, the heavy chain constant region of the rheumatoid factor IgM antibody is selected from the human IgM constant region.

[0065] In some optional embodiments, the light chain constant region of the rheumatoid factor IgM antibody is selected from the human κ chain constant region.

[0066] In a second aspect, the present invention provides a nucleotide comprising a nucleotide sequence encoding the rheumatoid factor IgM antibody.

[0067] In a third aspect, the present invention provides a vector carrying the nucleotide.

[0068] In a fourth aspect, the present invention provides a cell, wherein the cell carries the nucleotide, or contains the vector, or expresses the rheumatoid factor IgM antibody.

[0069] In a fifth aspect, the present invention provides a method for preparing rheumatoid factor IgM antibodies, which are obtained by fermenting the cells described above.

[0070] The preparation method is simple and efficient, and a large amount of rheumatoid factor IgM antibodies can be obtained through fermentation.

[0071] In a sixth aspect, the present invention provides a use of a rheumatoid factor IgM antibody as a quality control product in the preparation of a rheumatoid factor IgM antibody detection product.

[0072] The rheumatoid factor IgM antibody provided by the present invention can specifically recognize the FC fragment of denatured IgG, has good stability, and can be used in an RF IgM detection kit, which not only facilitates clinical operation, but also greatly saves costs and greatly controls the batch difference between quality control products.

[0073] In a seventh aspect, the present invention provides a kit for detecting rheumatoid factor IgM antibodies, wherein the quality control product of the kit is the rheumatoid factor IgM antibodies.

[0074] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0075] Embodiment 1:

[0076] Peripheral blood mononuclear cell cDNA acquisition

[0077] TRIzol reagent was used to culture autoimmune and allergic PBMC (D001201116) from Rubai Biotechnology from 5×10 6 Total RNA was extracted from PBMCs, and cDNA was obtained by reverse transcription using 1 μg RNA and SuperScript VILO cDNA Synthesis Kit according to the instructions provided with the kit.

[0078] name Volume / Mass RNA 1ug Olig(dT)20(50uM) or Random primers 1ul Annealing buffer 1ul RNase / DNase-free buffer water Make up the volume to 8ul

[0079] 65℃, 5min; quickly put in ice bath for at least 1min.

[0080] name Volume / Mass 2*frist-strand reaction Mix 10ul Superscript III / RNase out enzyme Mix 2ul

[0081] Reverse transcription PCR procedure

[0082] Oligo(dT)23 reverse transcription reaction parameters:

[0083] Temperature(℃) Time (min) 50 50 85 5

[0084] Random (random primer) reverse transcription reaction parameters:

[0085] Temperature(℃) Time (min) 25 5-10 50 50 85 5

[0086] Embodiment 2:

[0087] Construction of human phage library

[0088] The human rheumatoid factor (RF) SCFV primers were mixed in equal proportions to amplify the light chain variable region and heavy chain variable region genes from cDNA. The kit gel recovered a band of about 350 bp (VH and VL were both 350 bp). The SCFV was spliced ​​together according to the following reaction system.

[0089] name Volume / Mass VL-linker 300ng VH-linker 350ng SCFV-DF(10uM) 1.5ul SCFV-DR(10uM) 1.5ul 2x PrimerStar 25ul High pressure water Make up to 50ul system

[0090] PCR reaction parameters:

[0091]

[0092] The PCR product was run on an electrophoresis gel, and a fragment of about 750 bp was recovered as SCFV. The target fragment SCFV and the vector Pcomb3xss were digested with sfiⅠ, and the Pcomb3xss digestion reaction solution was treated with CIP. The above digestion reaction solution was run on electrophoresis, gel-recovered, and the target gene fragment SCFV and the vector Pcomb3xss after digestion were ligated, run on an electrophoresis gel, and the ligation product was recovered by gel-recovery. The ligation product was transformed into TG1 competent cells, coated on a 2YT-AG large plate, cultured overnight, and the strains were collected and preserved. At the same time, the bacterial solution was diluted to 10 -5 , 10 -6 , 10 -7 , spread on 2YT-AG plates and culture overnight to detect the reservoir capacity.

[0093] Take 100ul of bacterial strain and inoculate it into 100ml liquid culture medium, culture it until the OD600 value of the bacterial solution reaches 0.4-0.6, add the pre-prepared M13K07 at 1:20, amplify and culture overnight, precipitate and collect phages to obtain a phage library.

[0094] Embodiment 3:

[0095] Panning, screening and identification

[0096] Rheumatoid factor (RF) SCFV was washed: the immunotube was coated with natural IgG denatured antigen, blocked with PBS containing 4% skim milk, and 1x10 12 The phage library was combined for 1 hour, washed, and eluted with pH3 0.15mol / LArgHAC (arginine acetate), 1.6w / v% peptone, 1w / v% yeast extract, and then eluted with 50mmol / LTris containing 20mmol / L trypsin. Except for the titer of the eluted phage, all the others were infecting 5mL TG1, standing for 0.5h, adding 20ml 2YT-AG medium until the OD600 value was about 0.5, adding pre-helper phage M13K07 at 1:20, amplifying and culturing overnight, and collecting phages by precipitation. The first round of selection was completed.

[0097] The second, third and fourth rounds of screening were completed in sequence to enrich high-affinity phage antibodies. The immune tubes were coated with various recombinant proteins, and the subsequent steps were the same as the first round of selection.

[0098] Screening for specific antibodies: After the phages from the third round of panning infect TG1, they are spread on plates and cultured overnight. Single clones are picked and cultured in 5 96-well 2mL deep-well plates. When the OD600 value of the bacterial solution is around 0.5, the bacterial solution is preserved, and then the helper phage M13K07 is added, and the solution is allowed to stand at 37℃ for 0.5h, and then the phage is induced at 28℃ and 220rpm overnight.

[0099] The natural IgG denatured antigen was coated on the ELISA plate, blocked with PBS containing 4% skim milk, and then 50 μL of phage was added. After washing, the diluted anti-M13 HRP antibody was added. TMB was used for color development. The phages that showed strong positive color development by ELISA were sequenced to obtain the antibody sequence.

[0100] The amino acid sequence of the light chain CDR1 region of the A strain antibody is SEQ ID NO.1: SSDVGSYNL, the amino acid sequence of the CDR2 region is SEQ ID NO.36: EVT, and the amino acid sequence of the CDR3 region is SEQ ID NO.2: SSFAGITTLVL; the heavy chain CDR1 sequence is SEQ ID NO.3: GYSFTSYW, the CDR2 sequence is SEQ ID NO.4: IYPGDSDT, and the CDR3 sequence is SEQ ID NO.5: ARGSSWNYSRLPV;

[0101] The amino acid sequences of the light chain variable region and the heavy chain variable region of the A strain antibody are the light chain variable region and the heavy chain variable region shown in SEQ ID NOs. 21 to 22, respectively;

[0102] The amino acid sequence of the light chain CDR1 region of the B strain antibody is: SEQ ID NO.6: QSISSQ, the amino acid sequence of the CDR2 region is SEQ ID NO.37: KAS, and the amino acid sequence of the CDR3 region is SEQ ID NO.7: QHFKGYPLT; the heavy chain CDR1 sequence is SEQ ID NO.8: GFTFSSYW, the amino acid sequence of the CDR2 region is SEQ ID NO.9: IKQDGSEK, and the amino acid sequence of the CDR3 region is SEQ ID NO.10: ARETSMGAFDI;

[0103] The amino acid sequences of the light chain variable region and the heavy chain variable region of the B strain antibody are the light chain variable region and the heavy chain variable region shown in SEQ ID NOs. 23 to 24, respectively;

[0104] The amino acid sequence of the light chain CDR1 of the C strain antibody is SEQ ID NO.11: SSDFGSTCL, the amino acid sequence of CDR2 is SEQ ID NO.38: KDT, and the amino acid sequence of CDR3 is SEQ ID NO.12: SFAKGYTLV, respectively; the amino acid sequence of the heavy chain CDR1 is SEQ ID NO.13: GFTFTSSW, the amino acid sequence of CDR2 is SEQ ID NO.14: GSSWDGSK, and the amino acid sequence of CDR3 is SEQ ID NO.15: GSSWNSMGALPV;

[0105] The amino acid sequences of the light chain variable region and the heavy chain variable region of the C strain antibody are the light chain variable region and the heavy chain variable region shown in SEQ ID NOs. 25 to 26, respectively;

[0106] The amino acid sequence of the light chain CDR1 of the D strain antibody is SEQ ID NO.16: VGSYQS, the amino acid sequence of CDR2 is SEQ ID NO.39: KGY, the amino acid sequence of CDR3 is SEQ ID NO.17: KGYPFAGI, the amino acid sequence of the heavy chain CDR1 is SEQ ID NO.18: FTSYTFTSS, the amino acid sequence of CDR2 is SEQ ID NO.19: FTFSNSM, and the amino acid sequence of CDR3 is SEQ ID NO.20: ETSMGGALP;

[0107] The amino acid sequences of the light chain variable region and the heavy chain variable region of the D strain antibody are the light chain variable region and the heavy chain variable region shown in SEQ ID NOs. 27 to 28, respectively;

[0108] The amino acid sequence of CDR1 of the light chain of the E strain antibody is SEQ ID NO.29: FGSTSDF, the amino acid sequence of CDR2 is SEQ ID NO.40: YPL, the amino acid sequence of CDR3 is SEQ ID NO.30: FAGHFKAG, the amino acid sequence of CDR1 of the heavy chain is SEQ ID NO.31: FSSYTFSMG, the amino acid sequence of CDR2 is SEQ ID NO.32: SWDDGS, and the amino acid sequence of CDR3 is SEQ ID NO.33: GSSWNETSMGYP.

[0109] The amino acid sequences of the light chain variable region and the heavy chain variable region of the E strain antibody are the light chain variable region and the heavy chain variable region shown in SEQ ID NOs. 34 to 35, respectively;

[0110] DIQMTQSPSTLSASEGDRVTITCRASFGSTSDFLAWYQQKPGKAPKL LIYYPLTLESGVPSRFSGSGSGTEFTLTISSVQPDDFASYYCFAGHFKAGFGGGTKLEIK(SEQ ID NO.34);

[0111] QVQLVQSGGGLVQPGGSLRLSCAASFSSYTFSMGMSWVRQAPGKG LEWVANSWDDGSYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCGSSWNETSMGYPWGQGTMVTVSS (SEQ ID NO. 35).

[0112] Embodiment 4:

[0113] Construction, expression and purification of rheumatoid factor (RF) IgM recombinant antibody

[0114] The VH and VL of the antibody were spliced ​​with the sig-MFc-PCMV3 vector containing the human IgM constant region sequence and the pcdna3.4.2 vector containing the human Cκ constant region sequence by PCR; VL-pCDNA3.2 and VH-sig-MFc-PCMV3 plasmids were co-transfected into 293 cells to obtain RF-IgM expression supernatant. RF-IgM was purified by SPL. Figure 1 This is the non-reduced protein electrophoresis diagram of purified RF-IgM antibodies of strains A, B, C, D, and E. Figure 2The following are the reduced protein electrophoresis diagrams of purified RF-IgM antibodies of strains A, B, C, D, and E. Non-reduced electrophoresis refers to the antibody being treated with SDS, and reduced electrophoresis refers to the antibody being treated with SDS and 100mM DTT, which can open the disulfide bonds between the light and heavy chains of the antibody. The purity of antibodies A, B, C, D, and E was 95%, 97%, 98%, 97%, and 97%, respectively, as analyzed by Image J software.

[0115] Example 5

[0116] Rheumatoid Factor (RF) IgM Control Potency and Linearity

[0117] The recombinant A, B, C, D and E antibodies were verified by ELISA reactivity and added to the recombinant antibody buffer to prepare quality control products. The ingredients are as follows:

[0118] Components Element RF-IgM recombinant antibody RF Antibody Buffer

[0119] Potency:

[0120]

[0121] According to the dilution ratio, the concentrations of the recombinant A, B, C, D, and E antibody stock solutions were 8934.43, 8985.23, 8925.45, 9356.78, and 6937.27 IU / ml, respectively. The E antibody titer was relatively low.

[0122] Dilution linearity:

[0123] Recombinant A, B, C, D, and E strain antibody quality control products RF-IgM were prepared to a concentration close to the upper limit of the linear range of the Antu Biotechnology RF-IgM Kit (20240625) (about 300 IU / mL), and then diluted to a concentration close to the lower limit of the linear range (10 IU / mL) by multiple ratios. The theoretical concentration was compared with the actual concentration. The results are as follows:

[0124]

[0125] Correlation coefficients of the quality control products of the four recombinant antibodies A, B, C, and D 2 >0.99, good dilution linearity, E recombinant antibody quality control product correlation coefficient R 2 <0.99, dilution linearity is poor.

[0126] Example 6

[0127] Thermal stability of rheumatoid factor (RF) IgM quality control

[0128] The recombinant antibody quality control products of strains A, B, C, D, and E (1 and 2) and the purified natural human RF-IgM antibody quality control product (F) were placed at 37°C for 10 days, and the signal retention rate of the quality control material raw materials was measured using the company's self-produced kit. The results are shown in the table:

[0129]

[0130] The results in the table show that after 10 days of heat acceleration at 37°C, the signal retention rates of the quality control products of RF-IgM recombinant antibodies of strains A, B, C, and D were all greater than 90%, and the signal retention rates of the quality control products of strain E antibody and purified natural human RF-IgM antibody were both less than 90%, indicating that the quality control products of RF-IgM recombinant antibodies of strains A, B, C, and D have good stability, meet clinical needs, and have excellent thermal stability.

[0131] Example 7

[0132] Rheumatoid Factor (RF) IgM Control Specificity

[0133] The natural and denatured human IgG, natural and denatured human IgM, and natural and denatured human IgA antigens were coated on the ELISA plate at a concentration of 2ug / ml, respectively, with CB buffer, and coated overnight at 4°C for 12-16h. The coated antigens were discarded the next day, and the plates were washed twice with PBST, patted dry, and 150ul of cascin sealing solution was added to each well and sealed at 37°C for 2h. The sealing solution was discarded, the plates were patted dry, and placed in a 37°C constant temperature incubator to dry for 8h for use.

[0134] Add 50ul diluted HRP-labeled A, B, C, D, E antibodies (5ug / ml) to each well of the ELISA plate coated with human natural and denatured IgG, IgM, and IgA, incubate at 37°C for 30min, wash 5 times with PBST, add 100ul TMB substrate solution to each well, incubate at 37°C for 10min, add 50ul stop solution to each well, and immediately read the absorbance value at 450nm using an ELISA reader. The results are as follows:

[0135]

[0136] The table shows that the recombinant RF-IgM antibodies of strains A, B, C, and D react only with denatured human IgG, and have no reaction with natural IgG, natural and denatured IgM, and natural and denatured IgA, with good specificity. The recombinant antibody of strain E has a weak cross reaction with denatured human IgM / human IgA, with poor specificity.

[0137] Conclusion: It has been verified that recombinant RF-IgM antibodies A, B, C, and D can be used as quality control products for RF antibody IgM detection kit (magnetic particle chemiluminescence method). They can be used as alternative raw materials for reagent development and optimization of RF antibody detection kits.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rheumatoid factor IgM antibody, characterized in that: Selected from Antibody A, Antibody B, Antibody C or Antibody D; The antibody A comprises light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NOs. 1, 36, and 2, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NOs. 3 to 5; The antibody B comprises light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL, whose amino acid sequences are shown in SEQ ID NOs. 6, 37, and 7, respectively, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH, whose amino acid sequences are shown in SEQ ID NOs. 8 to 10, respectively; The antibody C comprises light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NOs. 11, 38, and 12, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NOs. 13 to 15; The antibody D comprises light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are shown in SEQ ID NOs. 16, 39, and 17, respectively, and heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NOs. 18 to 20, respectively.

2. The rheumatoid factor IgM antibody according to claim 1, characterized in that The antibody A comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 21 to 22; The antibody B comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 23 to 24; The antibody C comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are sequentially shown in SEQ ID NOs. 25 to 26; The antibody D comprises a light chain variable region and a heavy chain variable region whose amino acid sequences are shown in SEQ ID NOs. 27-28.

3. The rheumatoid factor IgM antibody according to claim 1, characterized in that The heavy chain constant region of the rheumatoid factor IgM antibody is selected from human IgM constant region.

4. The rheumatoid factor IgM antibody according to claim 1, characterized in that The light chain constant region of the rheumatoid factor IgM antibody is selected from the human κ chain constant region.

5. A nucleotide, characterized in that The nucleotide comprises a nucleotide sequence encoding the rheumatoid factor IgM antibody according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector carries the nucleotide according to claim 5.

7. A cell, characterized in that The cell carries the nucleotide according to claim 5, or contains the vector according to claim 6, or expresses the rheumatoid factor IgM antibody according to any one of claims 1-4.

8. The method for preparing the rheumatoid factor IgM antibody according to any one of claims 1 to 4, characterized in that: Obtained by fermentation of the cell according to claim 7.

9. Use of the rheumatoid factor IgM antibody according to any one of claims 1 to 4 as a quality control product in the preparation of a rheumatoid factor IgM antibody detection product.

10. A kit for detecting rheumatoid factor IgM antibodies, characterized in that: The quality control product of the kit is the rheumatoid factor IgM antibody according to any one of claims 1 to 4.

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