An anti-CD79B protein monoclonal antibody and related products and uses thereof

Abstract

The present invention relates to an anti-CD79B protein monoclonal antibody and related products and uses thereof, belonging to the field of biomedicine technology. The anti-CD79B protein monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region; the light chain comprises a light chain variable region, and the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; the antibody has high specificity and sensitivity, can specifically identify cells expressing CD79B protein, is not only suitable for immunological detection, but also can effectively prevent the occurrence of false positive and false negative phenomena in immunological detection, and is conducive to further improving the accuracy of detection and diagnosis results.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-CD79B protein monoclonal antibody and uses thereof. Background Art

[0002] CD79B (also known as Ig-β) is a core component of the B cell antigen receptor (BCR) complex. It forms a heterodimer with CD79a (Ig-α) through a disulfide bond and is jointly anchored on the surface of B cells. This protein is a single transmembrane receptor. Its extracellular region contains an Ig-like V-type domain, which is responsible for binding to antigens; the intracellular region contains an immunoreceptor tyrosine inhibitory motif (ITIM), which is involved in the regulation of signal transduction. The expression of CD79B precedes immunoglobulin heavy chain gene rearrangement and CD20, and disappears when B cells differentiate into plasma cells. Therefore, it is a specific marker for the early development of B cells. This structural feature makes it play a key role in B cell development, activation, antigen recognition, and initiation of downstream signals.

[0003] In normal B cells, CD79B and CD79a mediate BCR signal transduction together. After antigen binding, the ITIM motif of CD79B is phosphorylated and activated by kinases such as Lyn, recruiting molecules such as SYK and CARD11 to activate the PI3K / AKT and NF-κB pathways. The former regulates cell metabolism and survival, while the latter promotes proliferation and inflammatory response. In addition, CD79B supports B cell survival by maintaining BCR surface expression, and its loss can lead to B cell development stagnation. In lymphoma, abnormalities of CD79B are mainly manifested as mutations and dysregulated expression. For example, approximately 20%-21% of diffuse large B-cell lymphomas (DLBCL) have CD79B mutations, most of which are located in the intracellular ITAM domain (such as the Y196 site), leading to persistent activation of the BCR signal. In chronic lymphocytic leukemia (CLL), the CD79B positivity rate is approximately 18%-20%, and is associated with chromosome 12 trisomy and CD22 / FMC7 positivity, suggesting that its expression may be related to disease subtype differentiation.

[0004] At present, CD79B antibodies can be prepared through various methods such as hybridoma technology and phage display technology. For example, some research institutions have used genetic engineering technology to humanize antibodies, reducing immunogenicity and improving the safety and effectiveness of their application in humans. In the process of immunohistochemical staining, existing technologies usually use automated staining instruments and standard staining procedures, which are conducive to improving the consistency and accuracy of staining; at the same time, standardized interpretation methods and scoring systems have also been established for immunohistochemical staining of CD79B, which helps pathologists evaluate the results more accurately.

[0005] However, for existing CD79B antibodies, although CD79B is relatively specifically expressed in B cells, in some special cases, such as some tumors or lesions of non-B cell origin, abnormal expression or cross-reaction may occur, resulting in false positive results. For example, in some rare tumor types or cases of abnormal cell differentiation, non-specific CD79B expression may occur, affecting the accuracy of diagnosis. At the same time, for some lesions or tumor cells with low expression of CD79B, weak staining or undetectable staining may occur, resulting in false negative results. For example, in some early B-cell lymphomas or cases with high heterogeneity of tumor cells, the CD79B expression level of some cells is low, which is easy to be missed, affecting the accuracy of diagnosis. Therefore, how to avoid the problem of false positives and false negatives of CD79B antibodies in immunological testing needs to be solved urgently. Summary of the invention

[0006] In order to solve the above technical problems, the first aspect of the present invention provides an anti-CD79B protein monoclonal antibody, which has high specificity and sensitivity and can specifically identify cells expressing CD79B protein. It is not only suitable for immunological detection, but also can effectively prevent the occurrence of false positive and false negative phenomena, and can significantly improve the accuracy of detection and diagnosis. The main concept is:

[0007] An anti-CD79B protein monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region; the light chain comprises a light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. Compared with the existing CD79B antibodies, the anti-CD79B protein monoclonal antibody provided in this scheme has higher specificity and sensitivity, can specifically identify cells expressing CD79B protein, can effectively prevent the occurrence of false positives and false negatives in immunological detection, can solve the shortcomings of the existing CD79B antibodies, and can further improve the accuracy of detection and diagnosis results.

[0008] Furthermore, the amino acid sequences of FR1, FR2, FR3, and FR4 of the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0009] Furthermore, the amino acid sequences of FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO:8.

[0011] Furthermore, the amino acid sequence of the light chain is shown in SEQ ID NO:16.

[0012] The second aspect of the present invention also provides a detection reagent for detecting CD79B protein, comprising the anti-CD79B protein monoclonal antibody. Since the anti-CD79B protein monoclonal antibody has higher specificity and sensitivity, it can specifically identify cells expressing CD79B protein, and can be applied to the detection reagent for detecting CD79B protein, which is conducive to achieving better detection results.

[0013] The third aspect of the present invention also provides a kit for detecting CD79B protein, comprising the anti-CD79B protein monoclonal antibody or the detection reagent.

[0014] The fourth aspect of the present invention provides the use of the anti-CD79B protein monoclonal antibody in immunological detection. Since the anti-CD79B protein monoclonal antibody has high specificity and sensitivity, it can specifically identify cells expressing CD79B protein and is suitable for immunological detection.

[0015] The fifth aspect of the present invention provides the use of the anti-CD79B protein monoclonal antibody in immunohistochemical detection. Since the anti-CD79B protein monoclonal antibody has high specificity and sensitivity, it can specifically identify cells expressing CD79B protein, which is not only suitable for immunohistochemical detection, but also can effectively reduce the probability of false positives and false negatives in immunological detection, which is conducive to more accurate diagnostic results.

[0016] The sixth aspect of the present invention provides the use of the anti-CD79B protein monoclonal antibody in ELISA detection. Since the anti-CD79B protein monoclonal antibody has high specificity and sensitivity, it can specifically identify cells expressing CD79B protein, making the anti-CD79B protein monoclonal antibody also suitable for ELISA detection.

[0017] The seventh aspect of the present invention provides the use of the anti-CD79B protein monoclonal antibody in chemiluminescent detection. Since the anti-CD79B protein monoclonal antibody has high specificity and sensitivity, it can specifically identify cells expressing CD79B protein, making the anti-CD79B protein monoclonal antibody also suitable for chemiluminescent detection.

[0018] Compared with the prior art, the anti-CD79B protein monoclonal antibody and related products and uses provided by the present invention have high specificity and sensitivity, can specifically identify cells expressing CD79B protein, are not only suitable for immunological detection, but also can effectively prevent the occurrence of false positive and false negative phenomena in immunological detection, and are conducive to further improving the accuracy of detection and diagnosis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The figure is a comparison of immunohistochemical staining results of a diffuse large B-cell lymphoma, wherein the left figure uses LBP2-CD79B prepared by the present invention; the right figure uses commercially available CD79B (Abcam, EPR6861).

[0021] Figure 2 The diagram is a comparison of immunohistochemical staining results of salivary gland tissues, wherein the left diagram uses LBP2-CD79B prepared by the present invention, and the right diagram uses commercially available CD79B (Abcam, EPR6861).

[0022] Figure 3 This is the kinetic curve of the interaction between LBP2-CD79B monoclonal antibody and antigen.

[0023] Figure 4 This is the kinetic curve of the interaction between commercially available CD79B (Abcam, EPR6861) antibody and antigen.

[0024] Figure 5 It is a comparison chart of immunoblotting detection results, wherein the left picture uses commercially available CD79B (Abcam, EPR6861), and the right picture uses LBP2-CD79B prepared by the present invention. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0026] Example 1

[0027] This embodiment provides a method for preparing an anti-CD79B protein monoclonal antibody, comprising the following steps:

[0028] Step 1: Prepare antigen.

[0029] In this example, the antigen is the extracellular region (AA 29-159) of recombinant human CD79B (Uniport code: P40259, in the UniProt database, detailed information such as its amino acid sequence, domain, and functional annotation can be queried according to this number), and the region where the antibody binds to the CD79B antigen is the extracellular region (AA 29-159) of the CD79B antigen, and the antigen sequence is:

[0030] ARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKD

[0031] For ease of description, in this example, the recombinant human CD79B antigen is labeled as huCD79B.

[0032] Step 2: Immunization.

[0033] The CD79B antigen obtained in step 1 was mixed with complete Freund's adjuvant and emulsified, and multiple rabbits were immunized by subcutaneous injection for three times. After three immunizations, blood was collected and serum titers were determined using ELISA and immunohistochemistry; the rabbit with the highest antibody titer against CD79B antigen was selected as the target animal for the next step of single B cell screening.

[0034] In this example, for the huCD79B antigen, similar sequences in the same family were compared, highly specific regions were selected as antigens, and highly immunogenic haptens were coupled to enhance the immune response of experimental rabbits, thereby increasing the possibility of obtaining specific antibodies.

[0035] Step 3: Single B cell screening and sequencing.

[0036] The spleen of the target animal was taken, and the B cells that secreted specific antibodies were separated by antigen coating and adsorption. After culture, the supernatant of B cells was taken, and the B cells that could secrete antibodies binding to CD79B were identified by ELSIA method. These B cells were lysed and total RNA (ribonucleic acid) was obtained. The cDNA (complementary DNA) was obtained using a reverse transcription kit, and the reverse transcription product was used as a template for PCR (polymerase chain reaction). The antibody encoding heavy chain variable region (VH) sequence and light chain variable region (VL) sequence were amplified using the corresponding primers. The reaction procedure was as follows: VH: 95℃, 5min; 95℃, 30s; 70℃, 30s; 72℃, 1 min; 72℃, 10min for 35 cycles of amplification, VL: 95℃, 5min; 95℃, 30s; 55℃, 30s; 72℃, 1min; 72℃, 10min for 35 cycles of amplification, and the amplified products were detected by 1% agarose gel electrophoresis, and then a single target band was selected for gel recovery. The target band recovered from the gel and the mammalian cell expression vector containing the constant region gene were transformed into TOP10 competent cells by homologous recombination, cultured at 37°C for 12 hours, and single clones were picked for sequencing. The sequencing results were analyzed by existing software to screen the expression plasmids of VH and VL sequences that met the sequence characteristics of rabbit antibodies.

[0037] In this embodiment, single B cell sequencing technology is used to improve the efficiency of screening high-affinity antibodies and the success rate of cloning. Since B cells cannot survive for a long time in vitro, even if ideal antibodies are produced, they will be lost due to the death of B cells. Traditional antibody screening methods mostly use hybridoma technology to fuse B cells with myeloma cells to obtain the ability to immortalize. However, the defect of this technology is that the success rate of fusion is generally only about 5%, and there is also the possibility that hybridoma cells will reject the two cells and eventually lose the antibody information. Single B cell sequencing technology uses B cell separation equipment to sort single B cells into culture dishes, and uses nutrient-rich cell culture media rich in growth factors that can stimulate B cell growth. It can screen B cells that can express high-affinity antibodies with extremely low loss rates and perform DNA sequencing. The DNA sequence can be preserved or regenerated forever through vector cloning technology.

[0038] Step 4: Preparation and purification of monoclonal antibodies on cells.

[0039] The confirmed positive expression vectors were transfected into a large number of cells, and after continued culture for 3-5 days, the cell suspension was collected, the supernatant was taken after centrifugation, and the supernatant was purified by affinity chromatography using protein A to obtain a high-purity antibody. Finally, the purified anti-CD79B recombinant rabbit monoclonal antibody was measured for concentration and packaged, labeled as LBP2-CD79B (anti-CD79B protein monoclonal antibody), and stored in a 4°C refrigerator.

[0040] The anti-CD79B recombinant rabbit monoclonal antibody obtained in this example was sequenced, which includes a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region and a heavy chain constant region; the light chain includes a light chain variable region and a light chain constant region, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the amino acid sequences of FR1, FR2, FR3, and FR4 in the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, as shown in Table 1, and the amino acid sequence of the complete heavy chain is shown in SEQ ID NO:8, as shown in Table 2.

[0041] Table 1 Sequences of the heavy chain variable region (VH) of anti-CD79B protein monoclonal antibodies

[0042]

[0043] Table 2 Sequences of heavy chains in anti-CD79B protein monoclonal antibodies

[0044]

[0045] The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; the amino acid sequences of FR1, FR2, FR3 and FR4 in the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, as shown in Table 3; the amino acid sequence of the complete light chain is shown in SEQ ID NO:16, as shown in Table 4.

[0046] Table 3 Sequences of light chain variable regions (VL) in anti-CD79B protein monoclonal antibodies

[0047]

[0048] Table 4 Sequences of light chains in anti-CD79B protein monoclonal antibodies

[0049]

[0050] Example 2

[0051] This example is an immunohistochemical detection using anti-CD79B recombinant rabbit monoclonal antibody as the primary antibody, and the method is as follows:

[0052] (1) Sample slice preparation: Formalin-fixed, paraffin-embedded Jurkat cells, Daudi cells, diffuse large B-cell lymphoma, and various normal tissue slices were baked in a 60°C incubator for 1-2 h and stored for later use.

[0053] (2) Use an automatic repair device to dewax, hydrate, and repair the sections. During this process, the temperature is preferably controlled at 95°C and the time is preferably controlled at 30 minutes.

[0054] (3) Soak in a hydrogen peroxide tank for 10 minutes for blocking, then take it out and rinse it with pure water three times. Then circle the tissue to be tested with an immunohistochemistry pen, rinse it with pure water twice, and soak it in phosphate buffered saline (PBST) for 2-3 minutes.

[0055] (4) Primary antibody incubation: Add 100 μL of anti-CD79B recombinant rabbit monoclonal antibody to completely cover the tissue. Incubate at room temperature for 1 h, and then rinse with PBST three times, 3 min each time.

[0056] (5) Secondary antibody incubation: Incubate with secondary antibody for 30 min according to the instructions of the DAB staining solution kit of the secondary antibody staining system used, and rinse with PBST three times, 3 min each time.

[0057] (6) DAB color development: Prepare DAB color development solution according to the kit instructions, add an appropriate amount of DAB color development solution to completely cover the tissue, incubate for 10 min, and rinse with pure water 3 times, 3 min each time.

[0058] (7) Hematoxylin counterstaining: Counterstain the sections for 2 min according to the hematoxylin manufacturer's instructions and recommendations, and then incubate in PBST for 5 min to blue.

[0059] (8) Dehydration and transparency: Soak in alcohol for 2-3 minutes and air dry.

[0060] (9) Sealing: Seal the sample with neutral gum.

[0061] (10) Perform slice scanning.

[0062] (11) Result analysis and statistics.

[0063] Immunohistochemical staining results are usually divided into: positive and negative. Positive expression must be in specific antigenic sites of cells and tissues to be considered positive. In the case of clear tissue staining distribution and accurate cell localization, the staining results can be further divided according to the difference in staining intensity, as follows:

[0064] 1. The sample is weakly positive; marked as “+”.

[0065] 2. The sample is moderately positive; marked as "++".

[0066] 3. The sample is highly positive; marked as "+++".

[0067] 4. The sample is negative and marked as “-”.

[0068] The CD79B monoclonal antibody of the present invention and the commercially available antibody CD79B (Abcam, EPR6861) were used to simultaneously test 69 cases of diffuse large B-cell lymphoma and the test results were compared. The whole test process was designed in a double-blind manner, and the immunohistochemistry of CD79B was statistically analyzed. The statistical results are shown in the following table:

[0069] Table 5 Statistics of immunohistochemistry results of CD79B

[0070]

[0071] First, the results in Table 5 show that the staining of LBP2-CD79B is accurately positioned, the staining is clear, there is no non-specific staining, and the background is clean. In immunohistochemical detection, the positive rate is comparable to that of commercially available antibodies; at the same time, there are 13 cases where the positive intensity of LBP2-CD79B monoclonal antibodies is higher than that of commercially available antibodies, indicating that the sensitivity of LBP2-CD79B monoclonal antibodies is higher than that of commercially available antibodies.

[0072] second, Figure 1 The results of immunohistochemical staining of a diffuse large B-cell lymphoma are compared. The left picture uses LBP2-CD79B of the present invention; the right picture uses commercially available CD79B (EPR6861). Figure 1 It can be seen that for the same diffuse large B-cell lymphoma tissue, the staining intensity of the LBP2-CD79B monoclonal antibody of the present invention is significantly stronger than that of the commercially available CD79B (EPR6861).

[0073] Third, normal tissue chip test results: The normal tissue chip includes 24 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed surgical specimens.

[0074] The 24 normal tissue samples included tonsils, appendix, lung, kidney, thyroid, pancreas, liver, stomach, testis, endometrium, cervix, fallopian tube, breast, placenta, prostate, large intestine, small intestine, thymus, spleen, salivary gland, esophagus, brain, striated muscle, and skin.

[0075] The results showed that the commercially available antibody CD79B (EPR6861) showed nonspecific staining in pancreatic, large intestine and salivary gland tissues, resulting in false positive results; while the LBP2-CD79B prepared by the present invention did not show nonspecific staining in pancreatic, large intestine and salivary gland tissues, indicating that the LBP2-CD79B monoclonal antibody of the present invention has higher specificity than commercially available antibodies, can effectively reduce the probability and risk of false positives, and is conducive to improving the accuracy of diagnosis.

[0076] For example, Figure 2 The following is a comparison of immunohistochemical staining results of salivary gland tissues. The left picture uses LBP2-CD79B of the present invention, and the right picture uses commercially available CD79B (EPR6861). Since CD79B is a B lymphocyte-specific antigen, it should not be expressed in salivary gland tissues. Figure 2 It can be seen that the LBP2-CD79B monoclonal antibody prepared by the present invention did not stain salivary gland tissue and did not produce false positive results; while the commercially available CD79B (EPR6861) stained salivary gland tissue, resulting in false positive results, proving that the LBP2-CD79B prepared by the present invention has better specificity than similar antibodies currently sold commercially.

[0077] Example 3

[0078] This example is an affinity determination of anti-CD79B recombinant rabbit monoclonal antibody, and the method is as follows:

[0079] The ability of antibodies to bind to antigens was characterized using Biacore SPR, using a proteinA chip (channel 1 was the reference channel and channel 2 was the ligand channel). The antibody was fixed to channel 2 of the proteinA chip at a concentration of 10 μg / ml, with a binding time of 30 seconds and a flow rate of 10 ul / min.

[0080] In this example, human CD79B antigen was used as the analyte, and the antigen sample ID was huCD79B. The LBP2-CD79B monoclonal antibody of the present invention was used as the antibody, and the antibody was diluted to 125 nM, 62.5 nM, 31.25 nM, 5.63 nM, 7.81 nM, 3.91 nM, 1.95 nM, 0.98 nM and 0 nM using running buffer, respectively.

[0081] In this embodiment, the analyte flows through channel 1 and channel 2, the binding time is 60s, the dissociation time is 90s, and the flow rate is 30ul / min. After the analyte is loaded, the chip surface is regenerated using a glycine buffer with a pH of 1.5. Before each analyte is loaded, the ligand needs to be fixed first, and the chip surface is regenerated with a regeneration solution after the analyte is loaded.

[0082] Biacore SPR was used to determine the affinity of the CD79B antibody to the human CD79B antigen at the above different concentrations, and the corresponding analysis software was used to analyze the binding and separation using a 1:1 kinetic model. The results are shown in Table 6 and Figure 3 shown.

[0083] At the same time, a control group was set up. The antibody in the control group was commercially available CD79B (Abcam, EPR6861). The other test parameters and conditions were the same as above. The results are shown in Table 6 and Figure 4 As shown, Figure 3 and Figure 4 This is the original spectrum determined from the data in Table 6.

[0084] Table 6 Comparison of affinity determination results

[0085]

[0086] In Table 6, KD represents the dissociation constant, which reflects the comprehensive index of affinity. The lower the KD value, the higher the affinity between molecules, that is, a lower concentration is required to achieve a certain degree of binding, which means that the two molecules are more likely to combine to form a complex and are more stable. Kon represents the binding rate constant, which reflects the speed of antibody-antigen binding. The larger its value, the more efficient the binding process. Kdis represents the dissociation rate constant, which reflects the speed of complex dissociation. It is a core parameter for measuring the stability of antibody-antigen complexes. The smaller its value, the more stable the complex and the higher the affinity. Therefore, from the data comparison in Table 6, it can be seen that the affinity of the LBP2-CD79B monoclonal antibody in the present invention to the human CD79B antigen is much higher than the affinity of the commercially available CD79B (Abcam, EPR6861) to the human CD79B antigen, indicating that the LBP2-CD79B monoclonal antibody in the present invention has higher sensitivity.

[0087] Example 4

[0088] This example is a Western blotting test using the anti-CD79B recombinant rabbit monoclonal antibody LBP2-CD79B as the primary antibody, and the method is as follows:

[0089] (1) A polyvinylidene fluoride (PVDF) membrane of Jurkat / Daudi cell lysate was activated with methanol for 1 min, washed 3 times with TBST, and blocked with 5% skim milk for 1 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.

[0090] (2) Primary antibody incubation: Place the blocked PVDF membrane in the diluted LBP2-CD79B antibody solution and incubate at 4°C overnight. After the incubation, remove the membrane and wash it 5 times with TBST, each time for 5 minutes.

[0091] (3) Secondary antibody incubation: Place the cleaned PVDF membrane in a diluted HRP-anti-rabbit IgG (1:10,000) solution and shake at room temperature for 1 hour. After the incubation is complete, remove the membrane and wash it 5 times with TBST, each time for 5 minutes.

[0092] (4) Prepare the developer according to the instructions of the Immobilon Western Development Kit and evenly drip it onto the membrane. Use the GelView+6000ProⅡ multifunctional imaging workstation to develop the membrane according to the instructions. The results are as follows: Figure 5 As shown, the right picture uses LBP2-CD79B prepared by the present invention, and the left picture uses commercially available CD79B (Abcam, EPR6861)

[0093] The theoretical molecular weight of CD79B protein is about 26 kDa. Due to phosphorylation modification of CD79B, the actual measured molecular weight will be greater than the theoretical value. Figure 5 Daudi is a positive cell line and Jurkat is a negative cell line. The CD79B monoclonal antibody prepared by the present invention and the commercially available CD79B (Abcam, EPR6861) antibody have no bands in Jurkat in the immunoblotting results, while the Daudi bands are of the same size, indicating that the anti-CD79B recombinant rabbit monoclonal antibody of the present invention can recognize CD79B protein with high specificity.

[0094] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0095] Sequence Listing

[0096] SEQ ID NO:1

[0097] IDLSTNVMR 9

[0098] SEQ ID NO:2

[0099] GSMLISGNIY 10

[0100] SEQ ID NO:3

[0101] TRGRL 5

[0102] SEQ ID NO:4

[0103] QSLEESGGRL VTPGGSLTLT CTVSG 25

[0104] SEQ ID NO:5

[0105] WVRQAPGKGL EWI 13

[0106] SEQ ID NO:6

[0107] YASWAKGRLT ISKTSSTTVD LKMTSLTAAD TATYFC 36

[0108] SEQ ID NO:7

[0109] WGQGTLVTVS S 11

[0110] SEQ ID NO:8

[0111] METGLRWLLL VAVLKGVQCQ SLEESGGRLV TPGGSLTLTC TVSGIDLSTN VMRWVRQAPG 60

[0112] KGLEWIGSML ISGNIYYASW AKGRLTISKT SSTTVDLKMT SLTAADTATY FCTRGRLWGQ 120

[0113] GTLVTVSSGQ PKAPSVFPLA PCCGDTPSST VTLGCLVKGY LPEPVTVTWN SGTLTNGVRT 180

[0114] FPSVRQSSGL YSLSSVVSVT SSSQPVTCNV AHPATNTKVD KTVAPSTCSK PTCPPPELLG 240

[0115] GPSVFIFPPK PKDTLMISRT PEVTCVVVDV SQDDPEVQFT WYINNEQVRT ARPPLREQQF 300

[0116] NSTIRVVSTL PIAHQDWLRG KEFKCKVHNK ALPAPIEKTI SKARGQPLEP KVYTMGPPRE 360.

[0117] ELSSRSVSLT CMINGFYPSD ​​ISVEWEKNGK AEDNYKTTPA VLDSDGSYFL YSKLSVPTSE 420

[0118] WQRGDVFTCS VMHEALHNHY TQKSISRSPG K 451

[0119] SEQ ID NO:9

[0120] QSVWANNYLA

[0121] SEQ ID NO:10

[0122] IASASTLAS

[0123] SEQ ID NO:11

[0124] CLGSYDCSSA DCNA 14

[0125] SEQ ID NO:12

[0126] QVLTQTPSSV SAAVGGTVTI NCQSS

[0127] SEQ ID NO:13

[0128] WYQQKPGQPP KIL

[0129] SEQ ID NO:14

[0130] GVSSRFKGSG SGTQFTLTIS DVQCDDAATY Y

[0131] SEQ ID NO:15

[0132] FGGGTEVVVK

[0133] SEQ ID NO:16

[0134] MDTRAPTQLL GLLLLWLPGA TFAQVLTQTP SSVSAAVGGT VTINCQSSQS VWANNYLAWY 60

[0135] QQKPGQPPKI LIASASTLAS GVSSRFKGSG SGTQFTLTIS DVQCDDAATY YCLGSYDCSS 120

[0136] ADCNAFGGGT EVVVKGDPVA PTVLIFPPAA DQVATGTVTI VCVANKYFPD VTVTWEVDGT 180

[0137] TQTTGIENSK TPQNSADCTY NLSSTLTLTS TQYNSHKEYT CKVTQGTTSV VQSFNRGDC 239

Claims

1. An anti-CD79B protein monoclonal antibody, characterized in that: It comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region; the light chain comprises a light chain variable region, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

2. The anti-CD79B protein monoclonal antibody according to claim 1, characterized in that: The amino acid sequences of FR1, FR2, FR3 and FR4 of the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

3. The anti-CD79B protein monoclonal antibody according to claim 1, characterized in that: The amino acid sequences of FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

4. The anti-CD79B protein monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO:8; the amino acid sequence of the light chain is shown in SEQ ID NO:

16.

5. A detection reagent for detecting CD79B protein, characterized in that: It comprises the anti-CD79B protein monoclonal antibody described in any one of claims 1-4.

6. A kit for detecting CD79B protein, characterized in that: It comprises the anti-CD79B protein monoclonal antibody according to any one of claims 1 to 4, or the detection reagent according to claim 5.

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