Anti-human cd83 antibody and use thereof

Abstract

The application provides an anti-human CD83 antibody and application, the antibody includes a heavy chain variable region and a light chain variable region; amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.The anti-human CD83 antibody provides a light chain variable region gene and a heavy chain variable region gene of the anti-human CD83 monoclonal antibody, a hybridoma cell strain capable of producing CD83 specific antibody, and can be used for detecting human bacterial infection.

Description

Technical Field

[0001] This invention relates to an antibody for diagnostic testing, and more particularly to an anti-human CD83 antibody and its application. Background Technology

[0002] Anti-human CD83 monoclonal antibody is a 43 kDa single-chain type I transmembrane glycoprotein that specifically recognizes and binds to human CD83 molecules. It is primarily expressed on the surface of mature dendritic cells (DCs) and is an important marker of mature DCs, often expressed in membrane-bound or soluble forms. Membrane CD83 can be detected on various activated immune cells, and it can regulate maturation, activation, and homeostasis. Soluble CD83 is elevated in the serum of patients with autoimmune diseases and some hematologic malignancies and has immunosuppressive functions.

[0003] This antibody has wide applications in immunological research. Since CD83 is a specific marker of mature dendritic cells (DCs), anti-human CD83 monoclonal antibodies can be used to identify and sort DCs by methods such as flow cytometry and immunohistochemistry, and to study the development, differentiation, function, and role of DCs in immune responses.

[0004] In addition, the number and function of dendritic cells (DCs) in the body can be altered in certain diseases such as tumors, autoimmune diseases, and infectious diseases. Anti-human CD83 monoclonal antibodies can be used to detect the number and phenotype of DCs in the peripheral blood or tissues of patients, aiding in disease diagnosis, assessing disease progression, and evaluating treatment efficacy.

[0005] Flow cytometry is a powerful tool for detecting the expression of cell surface protein molecules at the single-cell level. With the help of fluorescently labeled anti-CD83 monoclonal antibodies, the presence or absence of fluorescence signals and changes in fluorescence intensity can reflect the expression and changes of CD83 on the surface of related cells in the human body. This can then be used for the auxiliary diagnosis, disease monitoring, efficacy evaluation, and prognosis of tumors, autoimmune diseases, infectious diseases, transplant rejection, and inflammatory diseases. 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 CD83 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 CD83 antibody, which includes a heavy chain variable region and a light chain variable region; 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 CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0008] Furthermore, the amino acid sequences of FR1, FR2, FR3, and FR4 in the heavy chain variable region are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The amino acid sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are 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 CD83 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 CD83 antibody in the preparation of a detection reagent for CD83 molecules.

[0014] The present invention also provides an immunoassay kit comprising the aforementioned anti-human CD83 antibody.

[0015] Compared with the prior art, the present invention has the following advantages: The anti-human CD83 antibody described in this invention provides the light chain variable region gene and heavy chain variable region gene of the anti-human CD83 monoclonal antibody, and a hybridoma cell line that can produce CD83-specific antibodies, which can be used for the detection of human bacterial infections. Attached Figure Description

[0016] Figure 1 This is an electrophoresis image of the purified anti-human CD83 antibody described in an embodiment of the present invention; Figure 2 This is an affinity detection diagram of the anti-human CD83 antibody described in an embodiment of the present invention; Figure 3 This is a detection graph of the activity of the anti-human CD83-PE antibody described in an embodiment of the present invention. 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 embodiments.

[0019] Example 1: Cloning of the human CD83 antigen sequence The lentiviral expression vector used in this invention was constructed by inserting the CD83 gene (encoding the human CD83 protein) into the multiple cloning site of pCDH-CMV-MCS-EF1-copGFP as the empty vector (GENE ID: NM_004233.4, https: / / www.ncbi.nlm.nih.gov / nuccore / NM_004233.4). Packaging vectors pMD2.G and psPAX2 were also prepared. The plasmids were extracted using a plasmid mini-extraction kit, and the obtained target plasmids were stored frozen at -20°C to avoid repeated freeze-thaw cycles.

[0020] Example 2: Preparation of Lentiviral Cells Carrying Human CD83 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 CD83-3T3.

[0022] (3) Flow cytometry analysis of lentivirus infection efficiency: CD83-3T3 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 positive rate of cells reaches more than 95%, they can be used to immunize animals.

[0023] Example 3 Animal Immunization Log-growth-phase CD83-3T3 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 CD83: 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 CD83 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 CD83 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 Affinity determination of anti-human CD83 antibody CD83-K562 cell assay: 10 μL of CD83-K562 cells were added to each tube. 6 Different amounts of CD83 antibody were added to each sample, and the samples were incubated at room temperature in the dark for 30 minutes. 2 ml of cold PBS buffer was added, the sample was resuspended, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 0.5 μg of APC-labeled mouse secondary antibody was added, and the samples were incubated at room temperature in the dark for 30 minutes. 2 ml of cold PBS buffer was added, the sample was resuspended, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 250 μL of PBS buffer was added, and the samples were analyzed by flow cytometry.

[0029] The principle of GraphPad analysis is: when using different concentrations of antibody for flow cytometry detection, the antibody concentration corresponding to half of the sum of the highest and lowest fluorescence intensities is determined.

[0030] like Figure 2 As shown, the average fluorescence intensity of the CD83 antibody prepared in this invention and the commercially available CD83 antibody on CD83-K562 transfected cells showed a completely consistent trend with antibody concentration, both reaching a peak at 0.50 μg / L, and the peak intensities were similar. Throughout the entire antibody concentration gradient, the two curves essentially overlapped, indicating that the binding activity of the self-screened antibody and the control antibody was comparable. The CD83 antibody prepared in this invention, at half the sum of its highest fluorescence intensity (36478) and lowest fluorescence intensity (306), corresponds to an antibody dosage of 0.23 μg / mL, which translates to an absolute dosage of 0.028 μg / T; the commercially available CD83 antibody, at half the sum of its highest fluorescence intensity (36008) and lowest fluorescence intensity (282), corresponds to an antibody dosage of 0.225 μg / mL, which translates to an absolute dosage of 0.027 μg / T. Therefore, it can be seen that the affinity of the CD83 antibody prepared by this invention is not significantly different from that of commercially available CD83 antibodies.

[0031] Example 7 APC labeling of anti-human CD83 antibody The anti-human CD83 engineered antibody was reduced using a reducing agent, mixed thoroughly with activated APC, stirred at 25°C, and reacted for 1 hour; then purified using an S200 increase purification column to obtain APC-labeled CD83 antibody.

[0032] Example 8: Activity assay of anti-human CD83-APC antibody Peripheral blood testing from healthy individuals: 100 μL of anticoagulated peripheral blood from healthy individuals was added to each tube, along with different amounts of antibody CD83-APC (1.0 μg, 0.5 μg, 0.25 μg, 0.13 μg, 0.06 μg, 0.03 μg), followed by antibodies CD45-PerCP and CD33-FITC. The mixture was incubated at room temperature in the dark for 30 minutes. 2 mL of hemolysin was added, and the mixture was reacted at room temperature in the dark for 10 minutes. The mixture was then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 2 mL of cold PBS buffer was added, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 300 μL of PBS buffer was added, and the mixture was analyzed by flow cytometry.

[0033] like Figure 3 As shown, the CD83-APC antibody prepared by this invention exhibits a consistent fluorescence intensity trend compared with the commercially available control CD83-APC antibody at various antibody dosage gradients. At high dosage levels, the average fluorescence intensity of the antibody prepared by this invention is significantly higher, indicating better binding ability. At medium and low dosage levels, the average fluorescence intensity of the two antibodies is highly overlapping, indicating comparable binding ability.

[0034] 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 CD83 antibody, characterized in that: The antibody includes a heavy chain variable region and a light chain variable region; 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 CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

2. The anti-human CD83 antibody according to claim 1, characterized in that: The amino acid sequences of FR1, FR2, FR3, and FR4 in the heavy chain variable region are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The amino acid sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively.

3. The anti-human CD83 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 encodes the anti-human CD83 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 CD83 antibody according to any one of claims 1-3 in the preparation of a detection reagent for CD83 molecules.

8. An immunoassay kit, characterized in that: The kit contains the anti-human CD83 antibody as described in any one of claims 1-3.

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