Anti-cd38 recombinant rabbit monoclonal antibodies and uses thereof

Abstract

The present application relates to the technical field of antibodies, in particular to anti-CD38 recombinant rabbit monoclonal antibody and application thereof.The anti-CD38 recombinant rabbit monoclonal antibody provided by the present application has high specificity and high sensitivity in combination with CD38 protein molecules, can specifically recognize and detect the expression of CD38 protein on cells, is positively highly expressed in the detection of CD38 protein, and is found through immunohistochemical detection of various different tissues that the antibody can be applied to the detection and screening fields of IHC, ELISA, immunoblotting, antibody chip preparation, flow cytometry and the like, and is favorable for obtaining accurate evaluation and detection results.Compared with the existing anti-CD38 antibody, the CD38 recombinant rabbit monoclonal antibody of the 645J4B5 clone of the present application is good in specificity, strong in positive signal and the like, is easier to score in IHC staining, is more accurate in the detection and differentiation of cancer, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to anti-CD38 recombinant rabbit monoclonal antibody and its applications. Background Technology

[0002] CD38 protein has a molecular weight of 34 kDa, belongs to the type II transmembrane glycoprotein, and is a member of the ADP-ribonuclease superfamily. It is also known as ADP-ribosylcyclase / cyclic ADP-ribohydrolase 1, or NAD(+) nucleotide enzyme. CD38 is widely expressed on the surface of various immune cells, such as activated T cells, B cells, plasma cells, and natural killer cells, playing a role in immune cell development, activation, proliferation, and immune regulation. As an immune cell receptor, it participates in cell migration regulation, signal transduction, and receptor-mediated adhesion, helping immune cells better recognize and respond to foreign pathogens.

[0003] However, the binding specificity and sensitivity of commercially available CD38 antibodies to CD38 protein molecules need to be improved. Their recognition in IHC staining is poor, which is not conducive to detection and differentiation, thus limiting the application of commercially available CD38 antibodies.

[0004] Because CD38 protein has a homologous protein, CD157, non-specific cross-reactivity can easily occur during immunohistochemical detection. Furthermore, some tumor cells (such as CD38-negative myeloma) exhibit a lack of CD38 protein expression, requiring the use of other markers (CD138) for complementary detection. Therefore, it is necessary to develop monoclonal antibodies with higher specificity to avoid cross-reactivity. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a recombinant rabbit monoclonal antibody against CD38 and its application. The present invention provides a monoclonal antibody that can specifically recognize CD38 protein and its preparation method, which has high sensitivity and strong specificity and can avoid cross-reaction in detection.

[0006] This invention provides an anti-CD38 recombinant rabbit monoclonal antibody, characterized in that it comprises:

[0007] (1) The amino acid sequences of the three CDR regions of its heavy chain are shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of the three CDR regions of its light chain are shown in SEQ ID NO:4, 5 and 6, respectively; or

[0008] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (1), and which has the same or similar function as the amino acid sequence shown in (1); or

[0009] (3) A CDR that has at least 90% sequence identity with the amino acid sequence shown in (1) or (2).

[0010] In some embodiments, the amino acid sequences of the three CDR regions of the heavy chain variable region are shown in SEQ ID NO: 1, 2 and 3;

[0011] The amino acid sequences of the three CDR regions of the light chain variable region are shown in SEQ ID NO: 4, 5 and 6.

[0012] In some embodiments,

[0013] (4) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7, and its light chain variable region has the amino acid sequence shown in SEQ ID NO:8; or

[0014] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (4), and which has the same or similar function as the amino acid sequence shown in (4); or

[0015] (6) An amino acid sequence that is at least 90% homologous to the sequence described in (4) or (5).

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

[0017] The present invention provides a nucleic acid molecule encoding the aforementioned anti-CD38 recombinant rabbit monoclonal antibody.

[0018] In some embodiments, the nucleic acid molecule has:

[0019] (7) Its heavy chain has the nucleotide sequence shown in SEQ ID NO:9, and its light chain has the nucleotide sequence shown in SEQ ID NO:10; or

[0020] (8) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (7); or

[0021] (9) A sequence having at least 90% homology to the nucleotide sequence described in (7) or (8); or

[0022] (10) The complementary sequence of the nucleotide sequence described in (7), (8) or (9).

[0023] In some embodiments, the heavy chain has the nucleotide sequence shown in SEQ ID NO:9, and the light chain has the nucleotide sequence shown in SEQ ID NO:10.

[0024] This invention provides biological materials, including at least one of the following ① to ③:

[0025] ① An expression vector containing the nucleic acid molecule;

[0026] ② Transform or transfect the host cells containing the expression vector;

[0027] ③ Culturing the host cells to obtain a culture containing the anti-CD38 recombinant rabbit monoclonal antibody.

[0028] In some embodiments, the present invention also provides an expression module comprising the nucleic acid, the expression module comprising a promoter, a terminator, and the nucleic acid molecule described in the present invention.

[0029] In some embodiments, the expression module may further include an expression module formed by combining one or more nucleic acid molecules described in this invention in tandem, fusion expression or other feasible ways, and this invention does not limit this.

[0030] In some embodiments, the present invention also provides a transcription unit, which refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included flanking or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, transmembrane signal peptides, and homologous recombination sites operatively linked to a nucleic acid sequence, such as enhancers of promoters, ITR sequences, polyA, MIS signal peptides, etc.

[0031] In some embodiments, the host cells described in this invention may be derived from plants, animals, microorganisms, 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.

[0032] In some embodiments, the transformation method includes chemical transformation and electrotransformation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0033] The present invention provides labeled antibodies, including markers and the aforementioned anti-CD38 recombinant rabbit monoclonal antibody.

[0034] In some embodiments, the markers include chemical markers and biomarkers;

[0035] The chemical markers include isotopes and / or chemical drugs;

[0036] The biomarkers include biotin, avidin, or enzymes.

[0037] In some embodiments, the enzyme includes horseradish peroxidase or alkaline phosphatase.

[0038] The present invention provides a conjugate comprising a conjugation medium and the aforementioned anti-CD38 recombinant rabbit monoclonal antibody.

[0039] In some embodiments, the coupling medium includes a solid medium or a semi-solid medium.

[0040] In some embodiments, the coupling medium is selected from colloidal gold, polystyrene sheets, or beads.

[0041] This invention provides the use of at least one of the following (I-V) in the preparation of immunoassay reagents:

[0042] I. The aforementioned anti-CD38 recombinant rabbit monoclonal antibody;

[0043] II. The aforementioned nucleic acid molecules;

[0044] III. The aforementioned biomaterials;

[0045] IV. The labeled antibody mentioned above;

[0046] V. The aforementioned coupling.

[0047] This invention provides an immunoassay reagent, comprising at least one of the following i to v:

[0048] i. The anti-CD38 recombinant rabbit monoclonal antibody;

[0049] ii. The aforementioned nucleic acid molecules;

[0050] iii. The aforementioned biomaterials;

[0051] iv. The labeled antibody;

[0052] v. The aforementioned coupling.

[0053] The present invention provides a method for immunoassay, comprising detecting a sample using the immunoassay reagent and / or kit.

[0054] The recombinant rabbit monoclonal antibody against CD38 provided by this invention exhibits high specificity and sensitivity in binding to the CD38 protein molecule. It can specifically recognize and detect the expression of CD38 protein on cells, showing a high positive expression when detecting CD38 protein. Immunohistochemical assays in various tissues have revealed that this antibody can be applied to immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody chip preparation, flow cytometry, and other detection and screening fields, facilitating accurate assessment and detection results. Compared with existing anti-CD38 antibodies, the recombinant rabbit monoclonal antibody against CD38 of clone 645J4B5 of this invention, due to its high specificity and strong positive signal, is easier to score in IHC staining, making it more accurate for detecting and differentiating cancers, and suitable for widespread application. Attached Figure Description

[0055] Figure 1 The images show the immunohistochemical detection results of the 645J4B5 anti-CD38 monoclonal antibody prepared in this invention and a commercially available antibody in appendix, lung squamous cell carcinoma, and tonsil tissues. The concentration of the 645J4B5 anti-CD38 monoclonal antibody used was 1 μg / mL. Figure a shows the immunohistochemical detection results of the 645J4B5 cloned CD38 antibody in appendix tissue; Figure b shows the immunohistochemical detection results of the commercially available antibody in appendix tissue (Figures a and b have the same magnification); Figure c shows the immunohistochemical detection results of the 645J4B5 cloned CD38 antibody in lung squamous cell carcinoma tissue; Figure d shows the immunohistochemical detection results of the commercially available antibody in lung squamous cell carcinoma tissue (Figures c and d have the same magnification); Figure e shows the immunohistochemical detection results of the 645J4B5 cloned CD38 antibody in tonsil tissue; Figure f shows the immunohistochemical detection results of the commercially available antibody in tonsil tissue (Figures e and f have the same magnification).

[0056] Figure 2 A statistical chart showing the titer test results of the 645J4B5 anti-CD38 monoclonal antibody of the present invention and commercially available antibodies at eight concentration gradients is presented.

[0057] Figure 3 The Western blotting results of the 645J4B5 anti-CD38 monoclonal antibody of the present invention as a primary antibody verify its ability to recognize CD38 protein. In this paper, MOLT-4 represents human acute lymphoblastic leukemia cell lysate, Raji represents human lymphoma cell lysate, RPMI 8226 represents human multiple myeloma cell lysate, and CEM represents human acute lymphoblastic leukemia cell lysate. Detailed Implementation

[0058] This invention provides a recombinant rabbit monoclonal antibody against CD38 and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted 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 obviously make modifications or appropriate alterations and combinations to 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.

[0059] The present invention proposes an anti-CD38 recombinant rabbit monoclonal antibody and its application, aiming to address the problem that currently available commercially available CD38 antibodies have insufficient binding specificity and sensitivity to CD38 protein molecules, resulting in poor recognition in IHC staining and hindering detection and differentiation. The invention also relates to the nucleotide sequence encoding the anti-CD38 recombinant rabbit monoclonal antibody, the recombinant plasmid or expression vector, the preparation method, and the application of the anti-CD38 recombinant rabbit monoclonal antibody in CD38 protein detection methods or devices.

[0060] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0061] In a first aspect, the present invention provides an anti-CD38 recombinant rabbit monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0062] This anti-CD38 recombinant rabbit monoclonal antibody (CD38 rabbit-derived antibody) can be used for immunohistochemical detection, and can identify and detect the expression of CD38 protein on tumor cells or immune cells with high specificity and high sensitivity.

[0063] The anti-CD38 monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the anti-CD38 recombinant rabbit monoclonal antibody provided by this invention was produced through rabbit hybridoma fusion screening and eukaryotic expression in 293 cells. In preparing the anti-CD38 monoclonal antibody, the antigen used to immunize the rabbits (New Zealand white rabbits) was a synthetic polypeptide, the amino acid sequence of which is shown in SEQ ID NO:19, and it was obtained through artificial chemical synthesis. After immunizing the rabbits, cell fusion and clonal screening were performed to obtain a positive hybridoma cell line that could efficiently secrete monoclonal antibodies. Molecular cloning technology was used to obtain the nucleotide sequences encoding the heavy chain and light chain amino acid sequences of the antibody. The nucleotide sequences were constructed on a eukaryotic expression vector, transfected into the 293 cell line using a transfection reagent, and the cell supernatant was collected and purified by protein A column affinity chromatography to obtain the rabbit monoclonal antibody. Immunohistochemical detection showed that the antibody specifically recognizes the CD38 protein.

[0064] The anti-CD38 monoclonal antibody can recognize recombinant CD38 antigen protein and CD38 molecules on tumor cells and immune cells; the anti-CD38 monoclonal antibody can also be used in immunohistochemical pathological diagnostic agents.

[0065] Secondly, the present invention provides a coding gene for encoding the above-mentioned anti-CD38 recombinant rabbit monoclonal antibody.

[0066] Preferably, the encoding gene includes a DNA sequence as shown in SEQ ID NO:9 or a complementary sequence thereto, for encoding the heavy chain variable region of the anti-CD38 recombinant rabbit monoclonal antibody; and a DNA sequence as shown in SEQ ID NO:10 or a complementary sequence thereto, for encoding the light chain variable region of the anti-CD38 recombinant rabbit monoclonal antibody.

[0067] Thirdly, the present invention provides a nucleic acid molecule comprising a coding gene for encoding the aforementioned anti-CD38 recombinant rabbit monoclonal antibody.

[0068] Fourthly, the present invention provides an expression vector or recombinant plasmid comprising the above-described nucleic acid molecule.

[0069] Fifthly, the present invention provides a host cell that has been transformed or transfected with the above-mentioned expression vector or recombinant plasmid.

[0070] In a sixth aspect, the present invention provides a method for preparing a recombinant rabbit monoclonal antibody against CD38, wherein the above-mentioned expression vector or recombinant plasmid is used to transform or transfect the above-mentioned host cells, the transformed or transfected cells are cultured, the cell supernatant is collected and purified, and the recombinant rabbit monoclonal antibody against CD38 is obtained.

[0071] Seventhly, the application of the aforementioned anti-CD38 recombinant rabbit monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid in the preparation of a CD38 protein molecule detection device. The detection device includes, but is not limited to, reagent kits, antibody chips, etc.

[0072] Eighthly, the present invention also provides a CD38 detection kit, which includes the above-mentioned anti-CD38 recombinant rabbit monoclonal antibody and immunohistochemical detection reagent.

[0073] Preferably, the CD38 detection kit comprises: anti-CD38 recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, 3,3'-diaminophenylhydrazine concentrate, 3,3'-diaminophenylhydrazine buffer, hematoxylin and blueing solution.

[0074] The immunohistochemical testing process includes dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, 3,3'-diaminophenylhydrazine (DAB) staining, counterstaining, dehydration, mounting, and microscopic examination.

[0075] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0076] Example 1

[0077] This embodiment describes the preparation and screening of anti-CD38 recombinant rabbit monoclonal antibodies, including the following steps:

[0078] (1) Antigen preparation

[0079] The amino acid sequence of the CD38 antigen is shown below:

[0080] ETVLARCVKYTEIHPEMR (SEQ ID NO: 19)

[0081] The aforementioned polypeptide sequence was selected based on the analysis of the CD38 molecular sequence, considering the structure, antigenicity, hydrophilicity / hydrophobicity of the constituent amino acids, and secondary structure of the CD38 protein molecule. The polypeptide with the sequence shown in SEQ ID NO:19 was artificially synthesized and used as an antigen for immunizing rabbits. During immunization, the polypeptide with the sequence shown in SEQ ID NO:19 was conjugated with hemocyanin (KLH) and used as the CD38 antigen to immunize rabbits.

[0082] (2) Immunity

[0083] The CD38 antigen obtained in step (1) was mixed with complete Freund's adjuvant (volume ratio 1:1) and emulsified. Multiple New Zealand white rabbits were immunized by subcutaneous injection. Two weeks later, the CD38 antigen containing the above sequence (the polypeptide shown in SEQ ID NO:19) was emulsified with incomplete Freund's adjuvant (volume ratio 1:1) for a second and third immunization. After the three immunizations, blood was collected and serum titers were determined by serial dilution using ELISA. The rabbit with the highest antibody titer against the SEQ ID NO:19 antigen was selected for the next step of cell fusion.

[0084] (3) Cell fusion

[0085] Prepare mouse-derived sp2 / 0 myeloma cells in advance, ensuring they are in the logarithmic growth phase at the time of fusion. Immune rabbit spleens were used to prepare a single-cell suspension of lymphocytes. The rabbit spleen lymphocytes were mixed with the myeloma cells, and 50% PEG1500 was added. IMDM medium was added, and after centrifugation and discarding the supernatant, the cells were gently resuspended and mixed in HAT medium. The volume was adjusted to 800 mL, and the cells were aliquoted into 96-well plates and incubated at 37°C with 5% CO2. The fused cell status was observed 6-9 days after fusion. The medium was changed with HT, and the cells were continued to be incubated at 37°C with 5% CO2.

[0086] (4) Screening and cloning

[0087] Seven to ten days after fusion, clonal cells were screened using an ELISA test with CD38 antigen (SEQ ID NO:19). The corresponding cell line numbers were labeled, and the cells in the positive wells were subjected to limiting dilution until the entire 96-well plate showed a positive ELISA result. Stable monoclonal lines with high positive values ​​were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies, recorded as 645J4B5.

[0088] (5) Perform antibody sequencing on the selected hybridoma cell lines.

[0089] Total RNA was isolated from 645J4B5 hybridoma cells according to the instructions of the TriZol RNA extraction reagent. Following the instructions of the TIANScript first-strand cDNA synthesis kit, the total RNA was reverse transcribed into cDNA. The nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were amplified using specific primers (heavy chain variable region primers, VH-F: AGACTGGGCTGCGCTGGCTTC (SEQ ID NO: 20), VH-R: GTGAGGGTGCCCGAG (SEQ ID NO: 21); light chain variable region primers: VK-F: ATGGACAYGAGGGCCCCCACTC (SEQ ID NO: 22), VK-R: GGTGGGAAGATGAGGACAGTAGG (SEQ ID NO: 23)). These nucleotide sequences were then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk1) for cell transfection.

[0090] (6) Cell transfection and screening

[0091] Prepare 293 cells for transfection in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 24-well plates at a density of 1.5 mL per well (3 × 10⁻⁶ cells / well). 6 per mL.

[0092] The eukaryotic expression vector was mixed with polyethyleneimine (PEI) at a mass ratio of 1:6 and added to the prepared 293 cells. The cells were then cultured in a shaker at 37°C and 5% CO2. After 3-5 days of culture, the transfected cell supernatant was subjected to ELISA to screen for positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunohistochemical detection. A positive immunohistochemical test confirmed that the detected antibody sequence was correct.

[0093] (7) Preparation and purification of monoclonal antibodies on cells

[0094] The confirmed positive expression vector was used to transfect a large number of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography with protein A to obtain antibodies with a purity >95%. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4-8°C.

[0095] Finally, the heavy chain variable region nucleotide sequence of the 645J4B5 anti-CD38 recombinant rabbit monoclonal antibody is shown in SEQ ID NO:9, and the light chain variable region nucleotide sequence of the anti-CD38 recombinant rabbit monoclonal antibody is shown in SEQ ID NO:10.

[0096] cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggaatcgacctcagtaactttgcaataaactgggtccgccaggctccagggaaggggctggaatggatcggaatcactactgatagtggaaacatatactac gcgagctgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggatctgaaaatctccagtccgacaaccgaggacacggccacctatttctgtggcagatttggttatactagtggacggaacttgtggggccaaggcaccctggtcaccgtctcctca (SEQ ID NO:9)

[0097] gatgttgtgatgacccagactccatcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtcagagcatttacagctacttatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgaagcatccaaactggcctc tggggtcccatcgcggttcagcggcagtggatctgggacagagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaaactacgttacggttagtagttatggtgctgctttcggcggagggaccgaggtggtggtcaaa (seq ID NO:10)

[0098] The obtained base sequence was translated into an amino acid sequence and analyzed. The amino acid sequence of the heavy chain variable region of the 645J4B5 anti-CD38 recombinant rabbit monoclonal antibody was shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region of the anti-CD38 recombinant rabbit monoclonal antibody was shown in SEQ ID NO:8.

[0099] The amino acid sequence of the heavy chain variable region is as follows:

[0100] QSVEESGGRLVTPGTPLTLTCTASGIDLSNFAINWVRQAPGKGLEWIGITTDSGNIYYASWAKGRFTISKTSSTTVDLKISSPTTEDTATYFCGRFGYTSGRNLWGQGTLVTVSS (SEQ ID NO:7)

[0101] FR1 region amino acid sequence: QSVEESGGRLVTPGTPLTLTCTASGIDLS (SEQ ID NO:11)

[0102] CDR1 region amino acid sequence: NFAIN (SEQ ID NO:1)

[0103] FR2 region amino acid sequence: WVRQAPGKGLEWIG (SEQ ID NO:12)

[0104] CDR2 region amino acid sequence: ITTDSGNIYYASWAKG (SEQ ID NO:2)

[0105] The amino acid sequence of the FR3 region is: RFTISKTSSTTVDLKISSPTTEDTATYFCGR (SEQ ID NO:13)

[0106] CDR3 region amino acid sequence: FGYTSGRNL (SEQ ID NO:3)

[0107] FR4 region amino acid sequence: WGQGTLVTVSS (SEQ ID NO:14)

[0108] The amino acid sequence of the light chain variable region is as follows:

[0109] DVVMTQTPSSVSEPVGGTVTIKCQASQSIYSYLSWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISDLECADAATYYCQNYVTVSSYGAAFGGGTEVVVK (SEQ ID NO: 8)

[0110] FR1 region amino acid sequence: DVVMTQTPSSVSEPVGGTVTIKC (SEQ ID NO:15)

[0111] CDR1 region amino acid sequence: QASQSIYSYLS (SEQ ID NO:4)

[0112] FR2 region amino acid sequence: WYQQKPGQPPKLLIY (SEQ ID NO:16)

[0113] CDR2 region amino acid sequence: EASKLAS (SEQ ID NO:5)

[0114] FR3 region amino acid sequence: GVPSRFSGSGSGTEFTLTISDLECADAATYYC (SEQ ID NO:17)

[0115] CDR3 region amino acid sequence: QNYVTVSSYGAA (SEQ ID NO:6)

[0116] FR4 region amino acid sequence: FGGGTEVVVK (SEQ ID NO:18)

[0117] Example 2

[0118] This example demonstrates immunohistochemical detection using anti-CD38 recombinant rabbit monoclonal antibody as the primary antibody. The method is as follows:

[0119] (1) Sample preparation: The tissue sections of human appendix, lung squamous cell carcinoma and tonsils that have been fixed in formalin and embedded in paraffin are baked in a constant temperature oven at 60℃ for 1-2 hours and stored for later use.

[0120] (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 min each time;

[0121] (3) Hydration of sections: The sections were hydrated by soaking in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes in sequence, and then rinsed twice with purified water for 3 minutes each time.

[0122] (4) Antigen retrieval: It is recommended to use the high temperature heat retrieval method for 3 min (if using an automatic retrieval instrument, the high temperature retrieval at 98℃ for 20 min can be set). After the slides are naturally cooled to room temperature, the tissue to be tested is circled with an immunohistochemical pen and rinsed twice with purified water for 3 min each time.

[0123] (5) Inactivation of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocking agent to completely cover the tissue, incubate at room temperature for 10 min, rinse twice with purified water for 3 min each time, and rinse once with phosphate buffer (PBST).

[0124] (6) Primary antibody incubation: Add 100 μL of 1 μg / mL 645J4B5 anti-CD38 recombinant rabbit monoclonal antibody to completely cover the tissue, incubate in a 37℃ incubator for 1 h, and wash with PBST 3 times for 5 min each time;

[0125] (7) Secondary antibody incubation: Perform secondary antibody incubation according to the instructions of the DAB staining solution kit of the secondary antibody staining system used. After incubation, rinse the slides with PBST 3 times for 5 minutes each time, and rinse with purified water once.

[0126] (8) DAB staining: Prepare DAB staining solution according to the instructions of the DAB staining solution kit. Drop an appropriate amount of the prepared DAB staining solution to completely cover the tissue. Stop staining when the color does not deepen. Rinse 3 times with purified water.

[0127] (9) Hematoxylin counterstaining: Counterstain the sections according to the operating steps and suggestions in the instructions of the hematoxylin manufacturer, and rinse with PBST or tap water to return to blue;

[0128] (10) Dehydration and clearing: Soak in 70%, 85%, 95%, and 100% graded alcohol in sequence for 3 min each time; clear with xylene twice for 5 min each time;

[0129] (11) Mounting: Mount the sample with neutral resin.

[0130] Depend on Figure 1The results showed that CD38 protein specifically stained the nuclear cells of human appendix, lung squamous cell carcinoma, and tonsil tissues. Furthermore, the staining effect of the recombinant rabbit monoclonal antibody against CD38 of clone 645J4B5 was better than that of the commercially available CD38 antibody (purchased from ABCAM, catalog number: ab108403), with a deeper staining color. This indicates that the recombinant rabbit monoclonal antibody against CD38 of clone 645J4B5 of this invention, due to its high specificity and strong positive signal, is easier to evaluate in IHC staining, has high identification accuracy, and is more accurate in detecting and differentiating cancers.

[0131] Example 3

[0132] This example describes the determination of the affinity of the 645J4B5 anti-CD38 recombinant rabbit monoclonal antibody for CD38. The determination method is as follows:

[0133] (1) Remove the labeled CD38 peptide (SEQ ID NO:1) from 4℃ and allow it to return to room temperature. Dilute to a concentration of 1 μg / mL and add 100 μL / well to a 96-well microplate and incubate overnight at 4℃. Then block with 2% bovine serum albumin (BSA) overnight at 4℃.

[0134] (2) The CD38 recombinant rabbit monoclonal antibody cloned by 645J4B5 was diluted to an initial concentration of 0.5 μg / mL, and then serially diluted 2-fold, with a total of 8 concentration gradients for comparison;

[0135] (3) Add the diluted anti-CD38 recombinant rabbit monoclonal antibody at 100 μL / well to a 96-well microplate containing peptides, cover with a sealing film, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium.

[0136] (4) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0137] (5) Dilute horseradish peroxidase (HRP) labeled goat anti-rabbit IgG according to the instructions for use of the secondary antibody, add 100 μL / well to the microplate, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium.

[0138] (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0139] (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution at 100 μL / well and react at room temperature for 6 minutes;

[0140] (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development;

[0141] (9) Read the OD value at 450 nm using an ELISA reader, organize the data, and analyze the results as shown in Table 1 below. Figure 2As shown.

[0142] Table 1

[0143]

[0144] The results showed that, in eight concentration gradient experiments, the anti-CD38 recombinant rabbit monoclonal antibody of the 645J4B5 clone of the present invention had a strong affinity for CD38 protein molecules and high sensitivity. It could still achieve a high OD value under low antibody concentration conditions, which could save experimental and detection costs.

[0145] Example 4

[0146] This example demonstrates the detection using Western blotting with the anti-CD38 recombinant rabbit monoclonal antibody 645J4B5 as the primary antibody. The method is as follows:

[0147] (1) Activate the polyvinylidene fluoride (PVDF) membranes containing MOLT-4, Raji, RPMI 8226, and CEM cell lysates by activating with methanol for 1 min, washing the membrane twice with pure water, and then washing it three times with TBST; Blocking: Place the membrane in a blocking solution prepared with 5% bovine serum albumin (BSA) and shake at room temperature for 2 h; TBST is a commonly used washing buffer suitable for experiments such as immunoblotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and surfactant Tween-20.

[0148] (2) Primary antibody incubation: Dilute the 645J4B5 antibody to a concentration of 0.5 μg / mL, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking.

[0149] (3) Remove the membrane and wash it in TBST solution 3 times (2×5min+1×10min).

[0150] (4) Secondary antibody incubation: HRP-anti-rabbit IgG was diluted with FG solution at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 hour;

[0151] (5) Remove the membrane strip and wash it in TBST solution 4 times (3×5min+1×8min).

[0152] (6) Substrate: Mix equal volumes of luminol / enhancer solution and hydrogen peroxide solution diluted 5 times with pure water in the same container, add the membrane strip, and incubate for 2 min;

[0153] (7) Exposure: Place the film in the dark box and expose the X-ray film for different time periods according to the fluorescence intensity; then perform the operation in the order of 1 min development, 1 min cleaning, and 1 min fixing, and finally clean and dry; the results are as follows Figure 3 As shown.

[0154] The theoretical molecular weight of CD38 protein is around 34 kDa. However, due to glycosylation modifications and other factors, the actual measured molecular weight is greater than the theoretical value. Figure 3 In this context, MOLT-4 represents human acute lymphoblastic leukemia cell lysis, Raji represents human lymphoma cell lysis, RPMI 8226 represents human multiple myeloma cell lysis, and CEM represents human acute lymphoblastic leukemia cell lysis. Figure 3 The results showed that CD38 positive bands appeared in all four lysis buffers, with the band position around 45 kDa, which is greater than the theoretical value. This means that the 645J4B5 anti-CD38 recombinant rabbit monoclonal antibody can specifically recognize CD38 protein in MOLT-4, RPMI 8226, Raji, and CEM cells in the swimming lane. This indicates that the 645J4B5 clone of the present invention can recognize CD38 protein with high specificity.

[0155] 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 recombinant rabbit monoclonal antibody against CD38, characterized in that, The amino acid sequences of the three CDR regions of its heavy chain are shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of the three CDR regions of its light chain are shown in SEQ ID NO:4, 5 and 6, respectively.

2. The anti-CD38 recombinant rabbit monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

8.

3. A nucleic acid molecule encoding the anti-CD38 recombinant rabbit monoclonal antibody as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of its heavy chain is shown in SEQ ID NO:9, and the nucleotide sequence of its light chain is shown in SEQ ID NO:

10.

5. A biomaterial, characterized in that, Includes at least one of the following ① to ③: ① An expression vector comprising the nucleic acid molecule of claim 3 or 4; ② Transformation or transfection of host cells with the expression vector described in ①; ③ Cultivate the host cells described in ② to obtain a culture containing the anti-CD38 recombinant rabbit monoclonal antibody.

6. A labeled antibody, characterized in that, Includes markers and the anti-CD38 recombinant rabbit monoclonal antibody as described in claim 1 or 2.

7. The labeled antibody according to claim 6, characterized in that, The markers include chemical markers and biological markers; The chemical markers include isotopes; The biomarkers include biotin, avidin, or enzymes.

8. The application of at least one of the following (I-IV) in the preparation of immunoassay reagents: I. The anti-CD38 recombinant rabbit monoclonal antibody as described in claim 1 or 2; II. The nucleic acid molecule as described in claim 3 or 4; III. The biomaterial as described in claim 5; IV. The labeled antibody as described in claim 6 or 7.

9. An immunoassay reagent, characterized in that, Includes at least one of the following i to iv: i. The anti-CD38 recombinant rabbit monoclonal antibody as described in claim 1 or 2; ii. The nucleic acid molecule as described in claim 3 or 4; iii. The biomaterial as described in claim 5; iv. The labeled antibody as described in claim 6 or 7.

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