Anti-human CD42b antibody and application

Abstract

The invention provides an anti-human CD42b antibody and application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, the CDR1, the CDR2 and the CDR3 of the heavy chain variable region are amino acid sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively; the CDR1, the CDR2 and the CDR3 of the light chain variable region are amino acid sequences as shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively. The anti-human CD42b antibody provided by the invention provides an anti-human CD42b monoclonal antibody light chain variable region gene and a heavy chain variable region gene, and a hybridoma cell strain capable of generating a CD42b specific antibody, and can be used for detection of human and megakaryocyte / platelets.

Description

Technical Field

[0001] This invention relates to an antibody for diagnostic testing, and more particularly to an anti-human CD42b antibody and its application. Background Technology

[0002] The cell surface CD42b antigen is a 145kD glycoprotein called GPIbα. It covalently binds with CD42c to form GPIb. The CD42b antigen is expressed on the surface of platelets and megakaryocytes. The CD42b / c heterodimer forms a complex with CD42a and d, acting as a receptor for vWF and thrombin, mediating platelet adhesion, and participating in platelet-thrombin interactions.

[0003] CD42b / GPIbα is a megakaryocyte-platelet lineage-specific marker. Its expression begins in the early stages of hematopoietic stem cell differentiation into the megakaryocyte lineage and is highly expressed on mature platelets. Platelets are primarily involved in hemostasis and thrombosis. When endothelial cells are damaged, platelets bind to von Willebrand factor (vWF) via glycoprotein Ibα (GPIbα, the major ligand-binding subunit of the GPIb-IX-V complex), adhere to collagen via GPVI, and subsequently aggregate through fibrinogen-αIIbβ3 interaction. At sites of vascular injury, platelets also interact with leukocytes, releasing prothrombotic and pro-inflammatory molecules, thereby exacerbating the thrombotic inflammatory state—a pathological process in which inflammation and thrombosis are interrelated. Platelet-leukocyte adhesion is mainly mediated by the interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1, and the interaction between GPIbα and neutrophil αMβ2 integrin. GPIbα can bind to a variety of molecules, including vWF, thrombin, P-selectin, and αMβ2 integrin, enabling platelets to participate in various disease processes. Of particular note is the interaction between GPIbα and αMβ2, which stabilizes platelet-leukocyte binding, thereby promoting the occurrence and development of thrombotic inflammatory diseases.

[0004] Abnormal CD42b / GPIbα expression is almost entirely reflected in platelets, primarily leading to two major categories of diseases: hemorrhagic diseases and thrombotic diseases. When CD42b expression is absent or its function is impaired, platelets cannot bind to vWF via GPIbα, resulting in severely compromised initial adhesion at the site of vascular injury. This is mainly seen in Bernard-Soulier syndrome (BSS, macroplatelet syndrome), clinically manifesting as spontaneous mucosal bleeding, skin ecchymosis, or purpura. In women, it can also cause menorrhagia, persistent bleeding after trauma or surgery, etc. Even when CD42b expression is normal, its function can be overactivated under pathological conditions, leading to pathological thrombosis.

[0005] In megakaryocytic leukemia, the pathogenesis involves malignant transformation of hematopoietic stem / progenitor cells during differentiation into megakaryocytes, leading to differentiation arrest or unlimited proliferation. CD42b, as a specific marker of the megakaryocytic lineage, plays a crucial role in the subtyping of megakaryocytic leukemia. Summary of the Invention

[0006] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes an anti-human CD42b antibody and its application.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: The present invention also provides an anti-human CD42b antibody, which includes a heavy chain variable region and a light chain variable region; The CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0008] Furthermore, the FR1, FR2, FR3, and FR4 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; The FR1, FR2, FR3, and FR4 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody 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.

[0010] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned anti-human CD42b antibody.

[0011] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0012] The present invention also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned expression vector.

[0013] The present invention also provides the application of anti-human CD42b antibody in the preparation of detection reagents for CD42b molecules.

[0014] The present invention also provides an immunoassay kit comprising the aforementioned anti-human CD42b antibody.

[0015] Compared with the prior art, the present invention has the following advantages: The anti-human CD42b antibody described in this invention provides the light chain variable region gene and heavy chain variable region gene of the anti-human CD42b monoclonal antibody, and can produce a hybridoma cell line that produces CD42b-specific antibodies, which can be used for the detection of human megakaryocytes / platelets. Attached Figure Description

[0016] Figure 1 This is an electrophoresis image of the purified anti-human CD42b antibody described in an embodiment of the present invention; Figure 2 This is a detection graph of the activity of the anti-human CD42b antibody described in an embodiment of the present invention; Figure 3 This is an affinity detection diagram of the anti-human CD42b antibody described in an embodiment of the present invention; Figure 4 This is a comparison chart of the activity of the anti-human CD42b-PE antibody described in the embodiments of the present invention with that of a commercially available control. Detailed Implementation

[0017] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0018] The present invention will be described in detail below with reference to the embodiments.

[0019] Example 1: Cloning of the human CD42b antigen sequence The lentiviral expression vector used in this project was constructed by inserting the CD42b gene (encoding the human CD42b protein) into the multiple cloning site of pCDH-CMV-MCS-EF1-copGFP as the empty vector (GENE ID: NM_000173.7, https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000173.7). Packaging vectors pMD2.G and psPAX2 were also prepared. Plasmids were extracted using a plasmid mini-extraction kit, and the obtained target plasmids were stored at -20°C to avoid repeated freeze-thaw cycles.

[0020] Example 2: Preparation of Lentiviral Coating and Immune Cells Carrying Human CD42b Antigen Sequence (1) Lentiviral coating: Collect 293T cells with normal cell morphology in the proliferation phase and spread them evenly in a cell culture dish; the next day, use three pre-extracted plasmids and transfection reagents to mix in a certain ratio; gently and evenly add the plasmid-transfection reagent mixture after incubation to the 293T cells, avoiding blowing the cells up; after culturing for 48-72 hours, collect the supernatant to obtain the virus; then use a high-speed centrifuge to concentrate the virus; use the concentrated virus directly for subsequent infection experiments, or aliquot it into small volumes and store it in a -80℃ freezer.

[0021] (2) Lentiviral infection of 3T3 cells: 3T3 cells in the logarithmic growth phase were seeded into 24-well culture plates and cultured overnight in an incubator; the next day, concentrated lentivirus was added to the well plates containing cells, and polybrene was added to promote infection. The cells were gently mixed and placed in an incubator for culture; a few days later, the GFP fluorescence intensity of 3T3 cells was observed under a filter. After obtaining the ideal brightness, the cells were cultured and expanded, and the cells were named CD42b-3T3.

[0022] (3) Flow cytometry analysis of lentivirus infection efficiency: CD42b-3T3 cells expanded after infection were collected, and the positive rate data of the cells were obtained by using antibody labeling and flow cytometry detection; when the cell positive rate reaches more than 95%, it can be used to immunize animals.

[0023] Example 3 Animal Immunization Log-growth-phase CD42b-3T3 cells were collected as immunogens and immunized 4-5 week old female BALB / c mice via intraperitoneal injection. Booster immunizations were administered at 3 and 5 weeks after the initial immunization. Serum titers of the mice were then measured.

[0024] Example 4: Cell Fusion and Screening (1) Three days before fusion, mice were given a shock immunization via tail vein injection. On the day of fusion, mice with high immune titers were selected, and the spleens of the mice were carefully removed in a biosafety cabinet. The spleens were then ground and the spleen cells were collected. At the same time, SP2 / 0 cells in good growth condition during the logarithmic growth phase were collected. The SP2 / 0 cells and spleen cells were mixed in a certain ratio to prepare for fusion. The fusion process was carried out in a 37°C water bath environment. PEG was used to fuse the cells. After fusion, the cells were resuspended in a special culture medium and the cells were plated in a flat-bottomed 96-well plate and placed in a cell culture incubator for culture.

[0025] (2) Screening hybridoma cells and subclones that secrete antibodies specifically targeting CD42b: When the cell clones are large enough under a microscope, the supernatant of the corresponding wells is taken for flow cytometry detection; when a positive well is screened, the well is enlarged and cultured and retested. If the test result is still positive, a first round of subcloning is performed using the limiting dilution method. If the test result is negative, it is discarded; clones that are still positive after the first round of subcloning are subjected to a second round of subcloning; clones that are positive after the second round of subcloning are subjected to a third round of subcloning. Generally, at least three rounds of subcloning are performed. All clones obtained from the three rounds of subcloning are positive clones, that is, a stable hybridoma cell line is obtained.

[0026] Example 5: Preparation and purification of anti-human CD42b antibody (1) Production of monoclonal antibodies in mice: Collect hybridoma cells in good growth condition and inject them into mice via intraperitoneal injection; collect ascites fluid when the mouse abdomen is significantly distended; freeze the collected ascites fluid at -20℃.

[0027] (2) Purification of anti-human CD42b antibody: The ascites fluid collected in the previous step was purified using a Protein A affinity column. After equilibrating the column with PBS, the sample was loaded and eluted with glycine at pH 3.5. The purified antibody was then replaced with PBS using a G25 column. The solution was then aliquoted and stored at -20°C. The purification electrophoresis results are shown in the figure. Figure 1 As shown.

[0028] Example 6: Activity Assay of Anti-human CD42b Antibody Peripheral blood assay in healthy individuals: Add 100 μL of the supernatant plasma from anticoagulated peripheral blood of healthy individuals (600 rpm, centrifuged for 10 minutes) to each tube, and fix with 300 μL of 2% formaldehyde solution for 10 minutes. Wash with 2 mL of 0.01 M PBS (pH 7.2) buffer, centrifuge at 1600 rpm for 5 minutes, and discard the supernatant. Repeat washing once. Resuspend in PBS buffer to 1 mL. Add different amounts of CD42b antibody and incubate at room temperature in the dark for 30 minutes; add 2 mL of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 0.5 μg of APC-labeled mouse secondary antibody, incubate at room temperature in the dark for 30 minutes; add 2 mL of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 250 μL of PBS buffer and analyze by flow cytometry.

[0029] The results are as follows Figure 2As shown, the CD42b antibody at 0.5 μg (per sample) reached a maximum average fluorescence intensity of 38114, which is the optimal reaction concentration for CD42b. When the antibody concentration is higher than the optimal concentration, the antigen-antibody reaction exhibits a hook effect; when the antibody concentration is lower than the optimal concentration, the average fluorescence intensity shows a decreasing trend.

[0030] Example 7: Affinity determination of anti-human CD42b antibody Peripheral blood assay in healthy individuals: Add 100 μL of the supernatant plasma from anticoagulated peripheral blood of healthy individuals (600 rpm, centrifuged for 10 minutes) to each tube, and fix with 300 μL of 2% formaldehyde solution for 10 minutes. Wash with 2 mL of 0.01 M PBS (pH 7.2) buffer, centrifuge at 1600 rpm for 5 minutes, and discard the supernatant. Repeat washing once. Resuspend in PBS buffer to 1 mL. Add different amounts of CD42b antibody and incubate at room temperature in the dark for 30 minutes; add 2 mL of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 0.5 μg of APC-labeled mouse secondary antibody, incubate at room temperature in the dark for 30 minutes; add 2 mL of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 250 μL of PBS buffer and analyze by flow cytometry.

[0031] The results are as follows Figure 3 As shown, the affinity detection results of the CD42b antibody show that the fluorescence intensity first increases and then decreases with decreasing antibody concentration. GraphPad analysis revealed that the average fluorescence intensity reached a maximum of 7172 at an antibody concentration of 0.037 μg (per sample), and then stabilized at a concentration of 0.00046 μg (per sample). Based on curve fitting, the affinity of the CD42b antibody described in this invention is determined to be 0.455 μg / mL.

[0032] Example 8: PE labeling of anti-human CD42b antibody The anti-human CD42b engineered antibody was reduced using a reducing agent, mixed evenly with activated PE, stirred at 25°C, and reacted for 1 hour; then purified using an S200 increase purification column to obtain PE-labeled CD42b antibody.

[0033] Example 9: Comparison of anti-human CD42b-PE activity with commercially available reference standard Peripheral blood assay in healthy individuals: Add 100 μL of the supernatant plasma from anticoagulated peripheral blood of healthy individuals (600 rpm, centrifuged for 10 minutes) to each tube, and fix with 300 μL of 2% formaldehyde solution for 10 minutes. Wash with 2 mL of 0.01 M PBS (pH 7.2) buffer, centrifuge at 1600 rpm for 5 minutes, and discard the supernatant. Repeat washing once. Resuspend in PBS buffer to 1 mL. Add different amounts of antibody CD42b-PE (1.0 μg, 0.5 μg, 0.25, 0.13 μg, 0.06 μg, 0.03 μg), and incubate at room temperature in the dark for 30 minutes; add 2 mL of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 250 μL of PBS buffer and analyze by flow cytometry. Control assays were performed simultaneously.

[0034] The results are as follows Figure 4 As shown, the activity detection results of CD42b-PE antibody show that the fluorescence intensity first increases and then decreases as the antibody concentration decreases from high to low; at the same time, the fluorescence intensity of each dilution is significantly higher than that of the commercially available control.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-human CD42b antibody, characterized in that: The antibody includes a heavy chain variable region and a light chain variable region; The CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

2. The anti-human CD42b antibody according to claim 1, characterized in that: The FR1, FR2, FR3, and FR4 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The FR1, FR2, FR3, and FR4 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively.

3. The anti-human CD42b antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody 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.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule contains a nucleotide sequence encoding the anti-human CD42b antibody as described in any one of claims 1-3.

5. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule as described in claim 4.

6. A cell, characterized in that: The cell contains the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. The use of the anti-human CD42b antibody according to any one of claims 1-3 in the preparation of a detection reagent for CD42b molecules.

8. An immunoassay kit, characterized in that: The kit contains the anti-human CD42b antibody as described in any one of claims 1-3.

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