An antigenic polypeptide, antibodies made therefrom and their use in detecting cd8

Abstract

The application discloses an antigen polypeptide, an antibody prepared from the antigen polypeptide and application of the antibody in detection of CD8. Specifically, the application provides an antigen polypeptide which is derived from an N-terminal extracellular region of CD8a and the N-terminal of the antigen polypeptide comprises an additional cysteine. The antigen polypeptide is located at the N-terminal of the CD8a molecule, and after immunization of rabbits, polyclonal anti-CD8 antibodies obtained after purification can be applied to immunofluorescence technology, and single cell membrane structures can be detected. The polyclonal anti-CD8 antibodies of the application have advantages of clear epitope and stronger luminescence signal in human tonsil frozen tissues compared with polyclonal anti-CD8 antibodies of an immunogen located at the C-terminal.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antigenic polypeptide, an antibody prepared therefrom, and its application in the detection of CD8. Background Technology

[0002] Human CD8 (Cluster 8 of Differentiation) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). CD8 binds to the major peptide-histocompatibility complex (pMHC) on antigen-presenting cells, leading to antigen-specific activation of naive CD8+ T cells or effector CD8+ T cells. Because appropriately activated and uninhibited CD8+ cytotoxic T lymphocytes (CTLs) can recognize tumor-associated antigens and kill tumor cells, CTLs are an important component of the immune system.

[0003] Under natural conditions, CD8 typically forms dimers, which can have two structures. The first is a heterodimer (CD8αβ) composed of one α chain and one β chain, encoded by the leu-2a and leu-2b genes, respectively. The second is a homodimer (CD8αα) composed of two α chains, both encoded by the leu-2a gene. The α chain is approximately 34-37 kDa, while the β chain is 32 kDa. The CD8α chain is commonly used as an immunogen for anti-CD8 antibodies.

[0004] CD8 molecules are distributed on the surface of some T cells and thymocytes. Methods for detecting the localization of CD8 molecules in cells or tissues include immunofluorescence (ICC) and immunohistochemistry (IHC), but there is still a need to develop antibodies that can be used in immunofluorescence (IF). CD8a is a single transmembrane protein; currently, the immunogens for anti-CD8 antibodies include T cells, the full-length CD8a protein, or intracellular peptides. Anti-CD8 antibodies are usually polyclonal antibodies obtained through immunization, or monoclonal antibodies obtained through further screening.

[0005] Anti-CD8 antibodies obtained using T cells or full-length CD8a protein as immunogens target sequences that are not well understood. Antibodies obtained using intracellular CD8a fragments as immunogens are more suitable for IHC in paraffin sections. Therefore, there is a need to develop anti-CD8 antibodies for IF in frozen tissues. Summary of the Invention

[0006] To address the shortcomings of existing anti-CD8 antibody immunoepitopes, such as unclear epitopes and limitations in IF (immunofluorescence) techniques for frozen tissues, this invention provides an antigenic peptide, an antibody prepared from it, and its application in CD8 detection. Specifically, the antigenic peptide provided by this invention is derived from the N-terminal extracellular region of CD8a, and the polyclonal antibody obtained after immunization exhibits a stronger signal in immunofluorescence applications than the peptide located at the C-terminus. The anti-human CD8 rabbit polyclonal antibody obtained by immunizing animals with the antigenic peptide provided by this invention, after purification, can be applied to the immunofluorescence detection of human tonsil tissue, detecting single-cell membrane structures.

[0007] A first aspect of the present invention provides an antigenic polypeptide derived from the N-terminal extracellular region of CD8a, wherein the N-terminus of the antigenic polypeptide includes an additional cysteine ​​residue.

[0008] In some embodiments, the antigenic polypeptide comprises the amino acid sequence shown in SEQ ID NO:1. Preferably, the amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO:1.

[0009] A second aspect of the present invention provides a derived polypeptide that, upon enzymatic hydrolysis, can generate an antigenic polypeptide as described in the first aspect of the present invention, or the derived polypeptide has a tag sequence added to the N-terminus or C-terminus of the antigenic polypeptide as described in the first aspect of the present invention.

[0010] A third aspect of the present invention provides a fusion protein comprising an antigenic polypeptide as described in the first aspect of the present invention or a derived polypeptide as described in the second aspect of the present invention. Preferably, a carrier protein, such as KLH or OVA, is coupled to the N-terminus of the antigenic polypeptide or the derived polypeptide.

[0011] A fourth aspect of the present invention provides an isolated nucleic acid molecule that encodes an antigenic polypeptide as described in the first aspect of the present invention, a derived polypeptide as described in the second aspect of the present invention, or a fusion protein as described in the third aspect of the present invention.

[0012] A fifth aspect of the present invention provides a cell comprising nucleic acid molecules as described in the fourth aspect of the present invention. Preferably, the cell is a T cell or an antigen-presenting cell. More preferably, the antigen-presenting cell includes macrophages, dendritic cells, syndactyl cells, Langham cells, and B cells, etc.

[0013] A sixth aspect of the present invention provides an anti-CD8 antibody, which is obtained by immunizing an animal with an antigenic polypeptide as described in the first aspect of the present invention, a derived polypeptide as described in the second aspect of the present invention, or a fusion protein as described in the third aspect of the present invention, and specifically targets the antigenic polypeptide as described in the first aspect of the present invention. Preferably, the animal is a mammal, such as a rabbit.

[0014] In some embodiments, the anti-CD8 antibody is a monoclonal antibody or a polyclonal antibody.

[0015] The seventh aspect of the present invention provides the use of antigenic peptides as described in the first aspect of the present invention, derivative peptides as described in the second aspect of the present invention, fusion proteins as described in the third aspect of the present invention, cells as described in the fifth aspect of the present invention, and anti-CD8 antibodies as described in the sixth aspect of the present invention in ELISA, WB, flow cytometry, immunofluorescence, immunohistochemistry detection and / or preparation of detection reagents.

[0016] The eighth aspect of the present invention provides a method for detecting CD8, wherein an anti-CD8 antibody as described in the sixth aspect of the present invention is contacted with a sample, and the presence of CD8 in the sample is characterized.

[0017] The sample described in this invention may be derived from tissues or organs, such as epithelial tissue, connective tissue, nerve tissue, and muscle tissue; and the organ may be an organ containing a single-cell membrane structure, such as the tonsil.

[0018] The ninth aspect of the present invention provides the use of an antigenic polypeptide as described in the first aspect of the present invention or a derivative polypeptide as described in the second aspect of the present invention in the preparation of tumor-specific T cells or antigen-presenting cells, the preparation of TCR-T cells, the preparation of tumor diagnostic reagents and / or the preparation of medicaments for the prevention or treatment of tumors.

[0019] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0020] The reagents and raw materials used in this invention are all commercially available.

[0021] The positive and progressive effects of this invention are as follows:

[0022] This invention provides a polypeptide for preparing anti-CD8 antibodies and its application in ELISA and IF detection. The polypeptide is located at the N-terminus of the CD8a molecule. The polyclonal anti-CD8 antibody obtained after immunizing rabbits, after purification, can be used in immunofluorescence technology to detect single-cell membrane structures. The polyclonal anti-CD8 antibody of this invention has advantages such as a clear epitope and a stronger luminescent signal in frozen human tonsil tissue compared to polyclonal anti-CD8 antibodies with the immunogen located at the C-terminus. Attached Figure Description

[0023] Figure 1 The extracellular region structure of CD8a and the location of the polypeptide are shown in dark.

[0024] Figure 2 A negative control for immunofluorescence detection of human tonsil tissue.

[0025] Figure 3 Immunofluorescence detection of human tonsil tissue for which monoclonal antibody ant527_351_B06 (reference PMID:18047641) was obtained through immunoscreening of the extracellular region.

[0026] Figure 4 Immunofluorescence detection of human tonsil tissue immunized with CD8a C-terminal peptide to obtain polyclonal anti-CD8 antibodies.

[0027] Figure 5 Immunofluorescence detection of human tonsil tissue immunized with the N-terminal peptide of CD8a to obtain polyclonal anti-CD8 antibodies.

[0028] Figure 6 A magnified view of the immunofluorescence detection of human tonsil tissue immunized with the N-terminal peptide of CD8a to obtain polyclonal anti-CD8 antibodies.

[0029] Figure 7 Image J immunofluorescence signal analysis of three anti-CD8 antibodies. Detailed Implementation

[0030] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0031] The amino acid sequence of the antigenic polypeptide (SEQ ID NO:1)

[0032] CPKAAEGLDTQRFSGKRLGDTFVLTLSD

[0033] The amino acid sequence of human CD8a (SEQ ID NO:2)

[0034] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVIT LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

[0035] The underlined portion represents the transmembrane region.

[0036] Example 1: Conjugation and purification of CD8a peptide

[0037] This embodiment provides a newly generated antigenic polypeptide with the amino acid sequence SEQ ID NO:1. The antigenic polypeptide is selected from human CD8a, and its amino acid sequence is SEQ ID NO:2. The position of the antigenic polypeptide in CD8a is as follows: Figure 1 As shown in dark.

[0038] The antigenic peptide was modified by adding a cysteine ​​residue to its N-terminus for conjugation to the carrier protein KLH used for immunization. The antigenic peptide was conjugated and purified with maleamide-activated mcKLH according to the kit instructions. All solvents and consumables were supplied by Thermo Scientific. TM Imject TM Provided in the Maleimide-Activated mcKLH Centrifugation Kit (Catalog No.: 77666).

[0039] The specific steps for coupling are as follows:

[0040] 1. Add 200 μL of ultrapure water to mcKLH to prepare a solution with a concentration of 10 mg / mL.

[0041] 2. Add 200 μL of Conjugation Buffer to 2 mg of the antigenic polypeptide.

[0042] 3. Immediately mix the antigen peptide with mcKLH and incubate at room temperature (22-26℃) for 2 hours to obtain the conjugated sample.

[0043] The specific steps for desalting and purification after coupling are as follows:

[0044] 1. Preparation of eluent: Add 10 mL of ultrapure water to the Imject Purification Buffer Salts.

[0045] 2. Balancing the desalting column: Unscrew the knob at the bottom of the centrifugal desalting column, centrifuge at 1000g for 2 minutes, add 1mL purification buffer, centrifuge at 1000g for 2 minutes, and you will get a balanced desalting column.

[0046] 3. Sample desalting and purification: Replace with a new receiving tube, add 500 μL of the coupled sample to the equilibrated desalting column, and centrifuge at 1000g for 2 min. The new receiving tube contains the desalted and purified antigen peptide KLH-conjugated product, which can be used for immunization.

[0047] Example 2 Animal Immunization

[0048] In this embodiment, the immunization was performed by a CRO company, with each antigenic peptide immunizing two rabbits.

[0049] 1. Collect 1 ml of blood from the ears of two rabbits and use the collected serum as a control. Immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen in 1.5 ml of PBS, mix 1.5 ml of antigen with 1.5 ml of complete adjuvant, and shake on a shaker for at least 15 minutes until it reaches a water-in-oil state; immunize each rabbit with 1.5 ml of the antigen on its back.

[0050] 2. Two weeks later, administer a second immunization. Each rabbit is immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant. Dilute 1 mg of antigen in 1 ml of PBS, mix 1 ml of antigen with 1 ml of incomplete adjuvant, and shake on a shaker for at least 15 minutes until it reaches a water-in-oil state. Immunize 1 ml of the antigen on the back of each rabbit.

[0051] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, and 1 mg of antigen was diluted in 1 ml of PBS. The mixture was 1 ml of antigen + 1 ml of incomplete adjuvant and shaken on a shaker for more than 15 minutes until it reached a water-in-oil state. Each rabbit was immunized with 1 ml of the antigen on its back.

[0052] 4. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, and 1 mg of antigen was diluted in 1 ml of PBS. The mixture was 1 ml of antigen + 1 ml of incomplete adjuvant and shaken on a shaker for more than 15 minutes until it reached a water-in-oil state. 1 ml of the mixture was immunized on the back of each rabbit.

[0053] 5. One week later, take 1ml of blood from the ear and test the titer with ELISA. If the titer reaches 1:100,000 or higher, then perform a shock immunization.

[0054] 6. For shock immunization, immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen in 1.5 ml of PBS, mix 1.5 ml of antigen + 1.5 ml of complete adjuvant, shake on a shaker for more than 15 minutes until it reaches a water-in-oil state; immunize each rabbit with 1.5 ml on its back.

[0055] 7. Three days later, blood was collected from the ears of each rabbit. 5 ml of blood was collected from each rabbit to test the titer. The titer met the expectations (ELISA titer reached 1:100,000 or higher). Whole blood was collected in anticoagulant tubes and serum was separated.

[0056] Example 3: Antiserum titer ELISA

[0057] Post-immunotherapy titer was tested using OVA-conjugated antigen peptides. The reagents used for OVA conjugation were Thermo Scientific. TM Imject TM The specific operation and purification protocol for maleimide-activated ovalbumin (OVA catalog number: 77126) is as described in Example 1.

[0058] The specific steps for ELISA detection of antiserum titer are as follows:

[0059] 1. Dilute the OVA-conjugated peptides to 0.1 μg / mL with PBS, add 100 μL to each well of the immunoassay plate, and incubate overnight at 4°C.

[0060] 2. Wash the microplate with PBST 3 times, 300 μl per well, and pat off any remaining liquid on a paper towel on the last wash.

[0061] 3. Add 300 μl of 2% BSA blocking solution to each well and incubate at 37°C for 1 h.

[0062] 4. Wash the microplate with PBST 3 times, 300 μl per well, and pat off any remaining liquid on a paper towel on the last wash.

[0063] 5. Dilute the antiserum with PBS at 22,000, 64,000, and 128,000 times, respectively, add 100 μL to each well of the immunoassay plate, and incubate at 37°C for 1 h.

[0064] 6. Wash the microplate with PBST 6 times, 300 μl per well, and pat off any remaining liquid on a paper towel on the last wash.

[0065] 7. Add 100 μl of Anti-rabbit Antibody (HRP) diluted in PBS to each well and incubate at room temperature for 1 h.

[0066] 8. Wash the microplate with PBST 6 times, 300 μl per well, and pat off any remaining liquid on a paper towel on the last wash.

[0067] 9. Remove solutions A and B from the TMB Substrate Solution Set from 4°C, allow them to return to room temperature, mix them in equal proportions to prepare an appropriate amount of colorimetric solution, add 100 μl to each well of the microplate, and incubate in the dark for 10 min.

[0068] 10. Add 50 μl of 1M HCl to each well to stop the colorimetric reaction, and read the OD450 value using a microplate reader.

[0069] The test results are shown in Table 1. The potency after the four immunizations was greater than 128,000.

[0070] Table 1. OD450 values ​​of immunized rabbits

[0071] blank 0.045 Rabbit 1 after four vaccinations 22000 2.539 2.494 Rabbit 1 after four vaccinations 64000 2.228 2.183 Rabbit 1 after four vaccinations 128000 1.980 1.935 Rabbit 1 before immunization 22000 0.046 0.001 Rabbit 1 before immunization 64000 0.044 -0.001 Rabbit 1 before immunization 128000 0.045 0.000 Rabbit 2 after four exemptions 22000 1.560 1.515 Rabbit 2 after four exemptions 64000 2.058 2.013 Rabbit 2 after four exemptions 128000 2.060 2.015 Rabbit 2 before immunization 22000 0.042 -0.003 Rabbit 2 before immunization 64000 0.044 -0.001 Rabbit 2 before immunization 128000 0.047 0.002

[0072] Example 4: Affinity chromatography purification of antiserum

[0073] The magnetic beads (Yisheng Biotechnology, 36417ES03) required for affinity chromatography (protein A) were washed with PBS. The serum was added to the equilibrated magnetic beads for penetration (or incubated at 4℃ for 1-2 hours, mixing every 10 minutes). The beads were washed with PBS (at least 3 times, 5 column volumes). Elution was performed with elution buffer (100mM glycine + 20mM arginine, pH adjusted to 3.0 with acetic acid). Protein concentration was detected using an ELISA reader in IgG mode. The elution buffer was neutralized with 1M Tris-HCl at pH 9.0, and the solution was dialyzed into PBS to obtain the purified polyclonal anti-CD8 antibody after protein A purification. The antibody was aliquoted, flash-frozen in liquid nitrogen, and stored at -80℃.

[0074] Example 5: Immunofluorescence detection of polyclonal anti-CD8 antibodies

[0075] Purchased human tonsil sections were placed on glass slides and immersed in methanol, then incubated at -20°C for 30 minutes. They were then removed and air-dried. The sections were washed 1-2 times with washing buffer (PBS + 0.1% Triton X-100), and 100 μL of blocking buffer (PBS + 0.1% Triton X-100 + 5% serum (sheep serum and bovine serum mixed 1:1; sheep serum, Gibco, 16210064; bovine serum, Gibco, 26050070)) was added. The sections were incubated at room temperature for 15-20 minutes. The blocking buffer was then aspirated, and the polyclonal anti-CD8 antibody (1 mg / mL anti-CD8 antibody diluted 1000 times with blocking buffer) was immediately added. The sections were incubated at room temperature for 45 minutes. The anti-CD8 antibody solution was then aspirated, and the sections were washed 3 times with the same washing buffer, incubating for 1 minute each time at room temperature. The diluted anti-Rabbit IgG Alexa Fluor was then immediately added. TM The slides were incubated with Thermo 488 fluorescent secondary antibody (A-21206) at room temperature for 30 minutes. The slides were washed three times with the above washing solution, incubating for 1 minute each time at room temperature. The slides were then washed three times consecutively with nuclease-free water. After air-drying, the slides were observed under a microscope. Results showed that the polyclonal anti-CD8 antibody obtained from this polypeptide immunization could detect single-cell membrane structures in the immunofluorescence assay of human tonsils, such as… Figure 2 As shown.

[0076] Example 6 Image J Immunofluorescence Analysis

[0077] Open the original immunofluorescence images of the three anti-CD8 antibodies using ImageJ software. Figure 3 , Figure 4 , Figure 5Convert the image to 8-bit grayscale (Image>Type>8-bit) and invert it (Edit>Invert). Select the unit of measurement (Analyze>Set scale) as pixels and the measurement items (Analyze>Set measurements) as Area and Integrated density. Check Limit to threshold and click OK. Outline the target area in the image (i.e.,...) Figure 6 The measurement (Analyze>Measure) is performed on the area. The signal values ​​for each area are displayed in the Results section. The measured area (Area) and signal value (IntDen) are recorded, and the unit signal value (Average) is calculated. The statistical results are shown in Table 2, and the statistical graph is shown below. Figure 7 As shown.

[0078] Table 2. Measurement area and signal value of different anti-CD8 antibodies

[0079] NC 19320 579.164 0.029977 ECD monoclonal antibody 19872 625.625 0.031483 C-end multi-antibody 18216 1110.054 0.060938 N-terminal multi-antibody 18144 3253.732 0.179328

[0080] Immunofluorescence analysis using Image J showed that polyclonal anti-CD8 antibodies obtained after immunization with antigenic peptides derived from the N-terminal extracellular region of CD8a exhibited stronger signals than those of other peptides in immunofluorescence applications.

Claims

1. An antigenic polypeptide, characterized in that, The amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO:

1.

2. A derived polypeptide, characterized in that, The derived polypeptide is an antigen polypeptide with a tag sequence added to its C-terminus as described in claim 1.

3. A fusion protein, characterized in that, It is obtained by conjugating a carrier protein at the N-terminus of the antigenic polypeptide as described in claim 1 or the derived polypeptide as described in claim 2.

4. The fusion protein as described in claim 3, characterized in that, The carrier protein is KLH or OVA.

5. An isolated nucleic acid molecule, characterized in that, It encodes the antigenic polypeptide as described in claim 1, the derived polypeptide as described in claim 2, or the fusion protein as described in claim 3 or 4.

6. A cell, characterized in that, It contains the nucleic acid molecule as described in claim 5.

7. The cell as described in claim 6, characterized in that, The cells are T cells or antigen-presenting cells.

8. An anti-CD8 antibody, characterized in that, It is obtained by immunizing animals with the antigenic peptide as described in claim 1, the derived peptide as described in claim 2, or the fusion protein as described in claim 3 or 4, and specifically targets the antigenic peptide as described in claim 1; The animal in question is a rabbit; and the anti-CD8 antibody is a polyclonal antibody.

9. The use of the anti-CD8 antibody as described in claim 8 in the preparation of reagents for ELISA, Western Blot, flow cytometry, immunofluorescence, and immunohistochemistry for detecting CD8.

10. A method for detecting CD8, characterized in that, The anti-CD8 antibody as described in claim 8 is used to contact the sample, and the presence or absence of CD8 in the sample is characterized. The method described herein is not for diagnostic purposes.

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