Monoclonal antibodies against rib-p0 and uses thereof

Abstract

The present application relates to the biomedical technical field, especially to anti-Rib-P0 monoclonal antibody and application thereof. The present application obtains anti-Rib-P0 monoclonal antibody with good universality and stability through phage display technology and screening, and the monoclonal antibody can be used in the immunological detection kit with Rib-P0 antibody as a detection target, and quality control of anti-Rib-P0 antibody.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to monoclonal antibodies against Rib-P0 and their applications. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a common chronic autoimmune disease. It is characterized by the production of autoantibodies, leading to skin lesions and organ dysfunction, affecting organs including the heart, liver, kidneys, lungs, skin, blood vessels, joints, and nervous system.

[0003] Anti-Rib-P0 antibodies target the P0 protein on the large subunit of the 60S ribosome in the cytoplasm. They are specific autoantibodies for autoimmune diseases such as systemic lupus erythematosus (SLE) and are almost undetectable in other autoimmune diseases. SLE often takes several years from onset to diagnosis. Although it is not currently curable, early diagnosis is crucial for slowing its progression. Autoantibodies associated with SLE mainly include anti-dsDNA antibodies and anti-Sm antibodies. Other antibodies include anti-ribosomal P protein, anti-PCNA, anti-U1-nRNP, SS-A, and SS-B. The positivity rate of AntiRib-P IgG in SLE patients ranges from 5% to 46%, making it a specific antibody for SLE.

[0004] The detection of Rib-P0 antibodies primarily relies on medical laboratory testing. The purpose of quality control in medical laboratories is to detect and control changes in the precision and accuracy of routine laboratory work, thereby improving the consistency of specimen testing in routine operations and ensuring the accurate issuance of test reports. Developing a quality control product suitable for clinical laboratories, third-party medical examination centers, and provincial and municipal clinical testing centers to evaluate the quality of specimens tested for Rib-P0 antibodies is crucial for ensuring the reliability and accuracy of Rib-P0 antibody test results.

[0005] Currently, the preparation of anti-Rib-P0 quality control products faces numerous severe challenges, making it difficult to obtain products with good quality control performance. Firstly, in the antibody production stage, the complex structure and weak immunogenicity of the Rib-P0 antigen make it extremely difficult to obtain high-affinity, high-specificity antibodies using traditional immunoassay methods. Low-quality antibodies not only reduce the detection sensitivity of the quality control products but also easily trigger non-specific reactions, leading to biased test results. Secondly, diagnostic specificity is insufficient. Traditional detection methods, such as ELISA and immunoblotting, are susceptible to cross-reaction interference, resulting in a high false-positive rate. Furthermore, sensitivity and throughput limitations exist; complex biological samples, such as matrix interference in whole blood, reduce the signal-to-noise ratio, affecting the detection of low-concentration antibodies. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a monoclonal antibody against Rib-P0 and its application.

[0007] This invention provides a monoclonal antibody against Rib-P0.

[0008] The amino acid sequence of its heavy chain variable region CDR1 is at least one of those shown in SEQ ID NO:1 and / or SEQ ID NO:9;

[0009] The amino acid sequence of its heavy chain variable region CDR2 is at least one of those shown in SEQ ID NO:2 and / or SEQ ID NO:10;

[0010] The amino acid sequence of its heavy chain variable region CDR3 is at least one of those shown in SEQ ID NO:3 and / or SEQ ID NO:11;

[0011] The amino acid sequence of its light chain variable region CDR1 is at least one of those shown in SEQ ID NO:4 and / or SEQ ID NO:12;

[0012] The amino acid sequence of its light chain variable region CDR2 is at least one of those shown in SEQ ID NO:5 and / or SEQ ID NO:13;

[0013] The amino acid sequence of its light chain variable region CDR3 is at least one of those shown in SEQ ID NO:6 and / or SEQ ID NO:14.

[0014] Furthermore, the monoclonal antibody described in this invention,

[0015] The heavy chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:3;

[0016] The heavy chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO:9, CDR2 with an amino acid sequence as shown in SEQ ID NO:10, and CDR3 with an amino acid sequence as shown in SEQ ID NO:11.

[0017] The light chain variable region includes CDR1 with the amino acid sequence shown in SEQ ID NO:4, CDR2 with the amino acid sequence shown in SEQ ID NO:5, and CDR3 with the amino acid sequence shown in SEQ ID NO:6; and / or

[0018] The light chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO:12, CDR2 with an amino acid sequence as shown in SEQ ID NO:13, and CDR3 with an amino acid sequence as shown in SEQ ID NO:14.

[0019] Furthermore, the monoclonal antibody described in this invention,

[0020] The amino acid sequence of the heavy chain variable region is at least one of those shown in SEQ ID NO:7 and / or SEQ ID NO:15;

[0021] The amino acid sequence of the light chain variable region is at least one of those shown in SEQ ID NO:8 and / or SEQ ID NO:16.

[0022] This invention involves screening human serum containing human anti-Rib-P0 monoclonal antibodies, constructing a phage display library, and further screening to obtain monoclonal antibodies 7D8, 14D2, and 6D10. Stability and universality tests were then conducted. Experimental results showed that monoclonal antibodies 7D8 and 14D2 exhibited good universality and stability. Monoclonal antibody 7D8 showed better reactivity and stability across different manufacturers' kits. Furthermore, considering the differences in antigen exposure sites among different manufacturers, quality control samples were prepared by mixing the two antibodies at a concentration ratio of 2:3, 1:1, or 3:2. This ensured that the prepared quality control samples were receptive to most mainstream manufacturers. In the specific embodiments of this invention, a 1:1 ratio was not used.

[0023] The monoclonal antibody of the present invention has a constant region selected from at least one of IgG, IgA, IgD, IgM and / or IgE; in a specific embodiment of the present invention, it is an IgG subtype; specifically, a human IgG1 subtype.

[0024] The present invention provides nucleic acids, including nucleic acids encoding the monoclonal antibodies described herein.

[0025] The nucleic acid described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0026] In this invention, the nucleic acid encoding the nucleic acid may be optimized or unoptimized. These optimizations include, but are not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0027] The present invention also provides a transcription unit for the nucleic acid, wherein the transcription unit refers to a DNA sequence from the start of the promoter to the end of the terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator, and these regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence, such as enhancers of promoters, poly(A) signals, etc.

[0028] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid described herein.

[0029] Furthermore, the vector backbone of this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit this. The viral vector includes adenovirus vectors, adeno-associated virus (AAV) vectors, retroviral vectors, or lentiviral vectors, etc. The bacteriophage vector includes phage particles and helper vectors, and the phage particles include, but are not limited to, pBluescript II-KS(+), pcomb3XSS, pCANTAB5E, or pKK233.3. The animals include mammals and non-mammals, and the mammalian expression vectors include, but are not limited to, pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, and pColdII. The fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, and pAUR23.

[0030] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in model animals or mammalian cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by this invention can be constructed in various phage-associated vectors, for example, whose backbone vector may be pcomb3XSS.

[0031] This invention provides a host cell that is transfected or transformed with a recombinant vector as described in this invention or whose genome is integrated with a nucleic acid as described in this invention.

[0032] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0033] The host cells provided by this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention is not limited thereto. This invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, thereby enabling the transformed host cells to replicate the protein-encoding vector or express the desired protein.

[0034] The present invention provides a reagent containing a monoclonal antibody against Rib-P0, comprising at least one of the monoclonal antibodies described in the present invention or monoclonal antibodies obtained by culturing host cells as described in the present invention, and excipients.

[0035] Furthermore, the excipients include: buffer solutions, preservatives, protective agents, surfactants, chelating agents, antioxidants, and thickeners; the buffer solutions include: phosphate buffer, Tris buffer, etc.; the preservatives include phenol, formaldehyde, sodium azide, etc.; the protective agents include sugars (sucrose, trehalose), amino acids and / or proteins, etc.; the surfactants include Tween, Span, etc.; the chelating agents include ethylenediaminetetraacetic acid (EDTA), etc.; the antioxidants include vitamin E, ascorbic acid, DTT, β-mercaptoethanol, etc.; and the thickeners include carboxymethyl cellulose, etc.

[0036] The reagent described in this invention contains the anti-Rib-P0 monoclonal antibody described in this invention, which can be used in Rib-P0 detection as a quality control material for quality control of anti-Rib-P0 antibodies.

[0037] This invention provides the application of at least one of the following (I) to (VI) in Rib-PO detection:

[0038] I) The monoclonal antibody described in this invention;

[0039] II) The nucleic acid described in this invention;

[0040] III) The recombinant vector described in this invention;

[0041] IV) The host cell described in this invention;

[0042] V) Monoclonal antibodies obtained from host cells as described in this invention;

[0043] VI) The reagents described in this invention.

[0044] This invention utilizes phage display technology and screening to obtain a monoclonal antibody against Rib-P0 with good versatility and stability. This monoclonal antibody can be used in immunoassay kits targeting Rib-P0 antibodies, replacing clinical positive samples and serving as a quality control material for third-party testing laboratories, thus saving testing costs. Furthermore, the antibody described in this invention is human-derived, and compared to human-mouse chimeric antibodies, it is closer in nature to natural samples, avoiding some matrix effect problems.

[0045] This invention provides a Rib-PO detection kit, comprising a Rib-PO immunoassay reagent, a negative control, and at least one of the following: a) to c)

[0046] a) The monoclonal antibody described in this invention;

[0047] b) Monoclonal antibodies obtained by culturing the host cells described in this invention;

[0048] c) The reagents described in this invention.

[0049] This invention obtains a monoclonal antibody against Rib-P0 with good universality and stability through phage display technology and screening. The monoclonal antibody can be used for quality control of anti-Rib-P0 antibodies in immunoassay kits that use Rib-P0 antibodies as the detection target. Attached Figure Description

[0050] Figure 1 The image shows an agarose gel electrophoresis diagram of the PCR product of the scFv gene, where 1 is the light chain variable region and 2 is the heavy chain variable region.

[0051] Figure 2 The purified antibody was shown by SDS-PAGE. Detailed Implementation

[0052] This invention provides a monoclonal antibody against Rib-P0 and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0053] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecule & Larbiology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0054] An antibody is a protein or polypeptide sequence that binds to a specific antigen, which is a protein or polypeptide sequence expressed on the cell membrane or secreted extracellularly that can be recognized by the antibody. Antibodies can be antibodies containing an Fc structure, or they can be single-chain antibodies (ScFv), single- or double-domain (VH or VHH) antibodies.

[0055] VL is the light chain of ScFv, VH is the heavy chain sequence of ScFv, and CDR is the complementarity-determining region of the antibody. The light chain and heavy chain each have three subregions: CDR1, CDR2, and CDR3. The CDR region recognizes and binds to specific antigenic determinants by interacting with the antigen. Its sequence diversity determines the specificity and affinity of the antibody.

[0056] Those skilled in the art should know that there are multiple ways to define a Complementary Determination Region (CDR). The LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 described in this application are defined by the IMGT system. LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are collectively referred to as Complementary Determination Regions (CDRs). Besides the IMGT system, different CDR definition methods can also be combined (CDR1 and CDR2 are defined by one CDR method, and CDR3 by another). Specific different CDR definition systems may include:

[0057] Kabat definition system: The amino acid numbering in this definition method follows the Kabat numbering system;

[0058] Chothia definition system: The amino acid numbering in this definition method follows the Chothia numbering system;

[0059] MacCallum definition system: The amino acid numbering in this definition method follows the MacCallum numbering system;

[0060] The amino acid numbering in the IMGT system definition method shall be based on the IMGT numbering system;

[0061] AHo definition system: The amino acid numbering system in this definition method shall prevail.

[0062] Because CDRs have different definitions, those skilled in the art can define CDRs according to different methods used in LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of this application, thereby obtaining CDRs that are sequence-differentiated from the same combination of heavy chain variable regions and light chain variable regions as in this application. Those skilled in the art should understand that even if the sequences of CDR combinations obtained from the antibodies of this application through different definitions are different from those of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of this application, antibodies containing such CDR combinations are still undoubtedly covered within the scope of this application.

[0063] In a specific embodiment of the present invention, the antibody includes a light chain, a heavy chain, and a corresponding CDR region as shown below:

[0064] CDR1 of the variable region of the 7D8 heavy chain monoclonal antibody: GYTFTSYA (SEQ ID NO:1);

[0065] The CDR2 of the variable region of the 7D8 heavy chain monoclonal antibody:IIAGNGNT (SEQ ID NO:2);

[0066] CDR3 of the variable region of the 7D8 heavy chain monoclonal antibody: VRGSSRGRAFDI (SEQ ID NO:3);

[0067] CDR1 of the variable region of the 7D8 light chain monoclonal antibody: QSISSY (SEQ ID NO:4);

[0068] CDR2 of the variable region of the 7D8 light chain monoclonal antibody: AAS (SEQ ID NO:5);

[0069] CDR3 of the variable region of the 7D8 light chain monoclonal antibody: QQSYSTPVT (SEQ ID NO:6);

[0070] Monoclonal antibody 7D8 heavy chain variable region: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAIHWVRQAPGQRLEWLGWIIAGNGNTKYSQNFQGRVTITRDTSASTAYMELSSLRSQDTAVYYCVRGSSRGRAFDIWGQGTMVTVSS (SEQ ID NO:7);

[0071] Monoclonal antibody 7D8 light chain variable region: DIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPVTFGQGTRLEIK (SEQ ID NO:8);

[0072] CDR1 of the variable region of the heavy chain of monoclonal antibody 14D2: GYTFTSYA (SEQ ID NO:9);

[0073] CDR2 of the variable region of the heavy chain of monoclonal antibody 14D2:IIAGNGNT (SEQ ID NO:10);

[0074] CDR3 of the variable region of the heavy chain of monoclonal antibody 14D2:VRGSSRGRAFDI (SEQ ID NO:11);

[0075] CDR1 of the variable region of the light chain of monoclonal antibody 14D2: QSISNY (SEQ ID NO:12);

[0076] CDR2 of the variable region of the light chain of monoclonal antibody 14D2:AAS (SEQ ID NO:13);

[0077] CDR3 of the light chain variable region of monoclonal antibody 14D2:QQSHSSPIT (SEQ ID NO:14);

[0078] Monoclonal antibody 14D2 heavy chain variable region: QVQLVQSWAEVKKPGASVKVSCKASGYTFTSYAIHWVRQAPGQRLEWLGWIIAGNGNTKYSQNFQGRVTITRDTSASTAYMELSSLRSQDTAVYYCVRGSSRGRAFDIWGQGTMVTVSSK (SEQ ID NO:15);

[0079] Monoclonal antibody 14D2 light chain variable region: AIRLTQSPSSLPASVGDRVTITCRASQSISNYLNWYQQKSGKAPKLLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSSPITFGQGTRLEIK (SEQ ID NO:16);

[0080] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0081] Example 1: Screening of positive human serum samples

[0082] The Anti-Rib-PO-IgG assay kit was used to detect 10 human serum samples (20 ml / sample). 20 μL of sample was diluted with 380 μL of diluent, and 10 μL of the diluted sample was added to 20 μL of magnetic beads and 90 μL of sample dilution. After incubation for 15 min, the sample was magnetically washed, and then 100 μL of enzyme conjugate was added. After incubation for 17 min, the sample was magnetically washed again, and then the substrate was added to detect the signal value. The test results are shown in Table 1. One positive sample (9 samples) was detected. Lymphocytes were isolated from the patient's whole blood, and total RNA was extracted from the lymphocytes using the Trizol method. cDNA was then synthesized via reverse transcription.

[0083] Table 1. Screening results of 10 human serum samples

[0084]

[0085] Example 2: Antibody gene library construction and phage screening

[0086] 1. SCFV gene splicing

[0087] PCR was used to amplify the variable regions of the light and heavy chains of the antibody, respectively. The PCR reaction procedure is shown in Figure 2. The PCR products were recovered by 1% agarose gel electrophoresis. Figure 1 The amplified light chain variable region and heavy chain variable region were spliced ​​together using overlap-PCR to form scFv. The product was recovered by 1% agarose gel electrophoresis and stored at -20°C.

[0088] Table 2. PCR amplification program

[0089]

[0090] 2. Construction and screening of phage single-chain antibody libraries

[0091] The phage vector pcomb3XSS and the purified ScFv fragment were digested with SfiI to construct the recombinant plasmid HGFc-PCMV3. The recombinant plasmid was electroporated into TG1 competent cells to construct an immune single-chain antibody library, and a primary phage single-chain antibody library was prepared. The primary phage single-chain antibody library underwent three rounds of enrichment and screening to obtain a specific phage single-chain antibody library with high affinity and strong specificity. 300 single clones were selected, and the phage supernatant was prepared. Positive clones were identified using Phage-ELISA to obtain positive sequences. The antibodies in the positive phages were numbered 7D8, 14D2, and 6D10.

[0092] Example 3: Expression and purification of the recombinant monoclonal antibody of the present invention

[0093] The heavy chain antibody gene and light chain antibody gene were homologously recombined with HGFc-PCMV3 and HCL-PCMV3 (containing the antibody constant region sequence) vectors, respectively, after KasI / BamHI double digestion, to obtain expression vectors for the human whole antibody sequence. The recombinant whole antibody plasmid was transformed into competent DH5α cells, and positive clones were selected for sequencing and plasmid extraction. The extracted plasmid was then transfected into HEK293 cells. After 48 hours of transfection, antibody expression was detected by ELISA. Cells were then fed with feed, and the cell supernatant was collected 7 days after transfection to obtain the target protein supernatant.

[0094] Cell supernatant was purified using SPA to obtain the Rib-P0 recombinant antibody. The antibody was stored in 10mM PBS pH 7.2 buffer, adjusted to a concentration of 2 mg / ml, and then detected by SDS-PAGE. Figure 2 As shown, the purity of the purified antibody exceeds 92%.

[0095] Example 4 Universality Evaluation of Recombinant Human Rib-P0 Antibody

[0096] The antibodies were diluted 100, 500, 1000, and 5000 times with negative serum (containing 0.1% P300 preservative), and then tested on mainstream Rib-P0 antibody IgG assay kits on the market (the kits mentioned are: Yahuilong Anti-Rib-P0 Anti-IgG Assay Kit; Haooubo 18-Item Autoantibody Profile Assay Kit; Oumeng Rib-P0 Assay Kit; and Antu Anti-Rib-P0 Anti-IgG Assay Kit). The results are shown in Tables 3-6. 6D10 showed poor universality, while 7D8 + 14D2 mixed at a molar ratio of 1:1 showed better reactivity on kits from different manufacturers.

[0097] Table 3. Reactivity of Rib-P0 recombinant antibody 7D8 on kits from different manufacturers

[0098]

[0099] Table 4. Reactivity of Rib-P0 recombinant antibody 14D2 on kits from different manufacturers

[0100]

[0101] Table 5. Reactivity of Rib-P0 recombinant antibody 6D10 on kits from different manufacturers

[0102]

[0103] Table 6. Results of Rib-P0 recombinant antibody 7D8 + 14D2 mixed at a molar ratio of 1:1 at different...

[0104] Reactivity on the manufacturer's reagent kit

[0105]

[0106] Different manufacturers use different live materials and antigen coating processes in their reagent kits, resulting in differences in the sites of antigen exposure. By using two antibodies in combination to prepare quality control samples, it can be ensured that the prepared quality control samples can react with samples from mainstream manufacturers.

[0107] Example 5: Stability evaluation of Rib-P0 recombinant antibody

[0108] The antibodies were diluted separately with negative serum (containing 0.1% P300 preservative), and their stability at 4°C after opening, accelerated stability at 37°C, and freeze-thaw stability at -20°C were assessed. Corresponding antibodies stored at 4°C–8°C were used as controls. The results were detected using the Antu anti-Rib-P0 antibody IgG assay kit and are shown in Tables 7 and 8 below.

[0109] Table 7. Stability test of Rib-P0 recombinant antibody 7D8

[0110]

[0111] Table 8. Stability test of Rib-P0 recombinant antibody 14D2

[0112]

[0113] After the two antibodies were prepared into quality control products using serum, all stability assessment indicators were good.

[0114] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against Rib-P0, characterized in that, These are monoclonal antibodies 7D8 and 14D2; The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody 7D8 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3. The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody 7D8 is shown in SEQ ID NO:4, the amino acid sequence of CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

6. The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody 14D2 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is shown in SEQ ID NO:10, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

11. The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody 14D2 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

14.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 7D8 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. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 14D2 is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

3. The monoclonal antibody according to claim 2, characterized in that, Its constant region is selected from IgG.

4. Nucleic acid, characterized in that, Includes nucleic acids encoding the monoclonal antibodies according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that, It includes a vector backbone and the nucleic acid as described in claim 4.

6. A host cell, characterized in that, Transfection or transformation of the recombinant vector as described in claim 5 or its genome integrated with the nucleic acid as described in claim 4.

7. A reagent containing a monoclonal antibody against Rib-P0, characterized in that, It includes at least one of the monoclonal antibodies according to any one of claims 1 to 3 or monoclonal antibodies obtained by culturing the host cells according to claim 6, and excipients.

8. The use of at least one of the following (I) to (VI) in the preparation of the Rib-PO detection kit: I) The monoclonal antibody according to any one of claims 1 to 3; II) The nucleic acid as described in claim 4; III) The recombinant vector as described in claim 5; IV) The host cell as described in claim 6; V) Monoclonal antibodies obtained by culturing the host cells as described in claim 6; VI) The reagent as described in claim 7.

9. A Rib-P0 detection kit, characterized in that, Includes Rib-PO immunoassay reagent, negative control and at least one of the following: a) to c) a) The monoclonal antibody according to any one of claims 1 to 3; b) Culturing the host cells as described in claim 6 to obtain monoclonal antibodies; c) The reagent according to claim 7.

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