An enzyme-linked immunoassay kit for detecting soluble b7-h5 protein content

Abstract

This invention discloses an enzyme-linked immunosorbent assay (ELISA) kit for detecting and analyzing the content of soluble B7-H5 protein. The kit comprises two anti-human B7-H5 monoclonal antibodies, B7-H5-2E5 and B7-H5-7B10. B7-H5-2E5 includes a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO.1; the amino acid sequence of the variable region of the light chain is SEQ ID NO.2. B7-H5-7B10 also includes a heavy chain and a light chain, with the amino acid sequence of the variable region of the heavy chain being SEQ ID NO.3; the amino acid sequence of the variable region of the light chain of B7-H5-7B10 is SEQ ID NO.4. Furthermore, this invention also discloses the uses of this kit. The ELISA kit of this invention can effectively detect the content of soluble B7-H5. Using this kit, it was found that healthy individuals have a certain baseline level of soluble B7-H5 protein in their serum. The soluble B7-H5 content in the serum of patients with colorectal cancer, lung cancer, and gastric cancer is significantly elevated and is correlated with the clinical stage and pathology of the patients. The detection of soluble B7-H5 content in the body fluids of cancer patients has potential clinical value.

Description

[0001] This application is a divisional application of patent application No. 202111536301.2, filed on December 15, 2021, entitled "An enzyme-linked immunosorbent assay kit for detecting the content of soluble B7-H5 protein and its application therein". Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to an enzyme-linked immunosorbent assay kit for quantitative analysis of soluble B7-H5 protein content and its applications. Background Technology

[0003] B7-CD28 family members mediate co-stimulatory signaling, playing a crucial role in T cell activation and immune tolerance regulation. With advancements in research on the functions of co-stimulatory molecules, this area has become a new hot topic in immunological research. Recent studies have revealed that B7-CD28 family members, including B7-H1 (PD-L1), B7-H2 (PD-L2), B7-H3, B7-H4, B7H5 (VISTA), and B7-H6, are abnormally expressed in the tumor microenvironment and participate in tumor immune escape, playing a significant role in tumor development and prognosis.

[0004] B7-H5 is one of the negatively regulatory molecules in the B7 family. Studies have shown that B7-H5 is highly expressed in various malignant tumor tissues, including colorectal cancer, non-small cell lung cancer, and gastric cancer, and is closely related to the clinical stage and prognosis of patients. Furthermore, literature reports that B7-H5 plays a crucial role in the pathogenesis and progression of autoimmune diseases such as arthritis, systemic lupus erythematosus, and psoriasis. However, current detection methods for B7-H5 are still lacking. Summary of the Invention

[0005] To overcome the aforementioned deficiencies of the prior art, the present invention provides an enzyme-linked immunosorbent assay kit for quantitative analysis of soluble B7-H5 protein content and its application, thereby making up for the shortcomings of the prior art.

[0006] The present invention first provides a monoclonal antibody that can bind to the B7-H5 protein, wherein the monoclonal antibody contains two anti-human B7-H5 monoclonal antibodies, B7-H5-2E5 and B7-H5-7B10.

[0007] The anti-human B7-H5 monoclonal antibody B7-H5-2E5 comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain variable region (mVH) of B7-H5-2E5 is SEQ ID NO.1:

[0008] DVKLQESGPGLVKPSQSLSLTCTVTGYSIASDYTWNWIRQFPGNKLEWMGYIDYSGATFYNPSLKSRISIIRDTSKNQFFLQLNSVTTGDTATYYCTRGFYYYGSDYWGQGTTLTVSS;

[0009] The amino acid sequence of the light chain variable region (mVL) of B7-H5-2E5 is SEQ ID NO.2:

[0010] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHNNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSHVPSTFGGGTKLEIK;

[0011] The aforementioned anti-human B7-H5 monoclonal antibody is a B7-H5-7B10 antibody, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain variable region (mVH) of the B7-H5-7B10 is SEQ ID NO.3:

[0012] EVQLKQSGGPGLVAPSQSLSITCTVSGFSLTNHGVQWVRQSPGKGLEWLGVIWGDGNRNYHSALMSRLSINKDNSKSQVFLKLNRLQTDDTATYFCAKERRLGYYGEYDVMDYWGQGTSVTVSS;

[0013] The amino acid sequence of the light chain variable region (mVL) of B7-H5-7B10 is SEQ ID NO.4:

[0014] DIVMTQSHKFMSTSVGDRVTITCKASQGVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIGNVQSEDLADYFCQQYSSSPFTFGSGTKLEIK.

[0015] The monoclonal antibody provided by this invention is used to prepare a kit for detecting soluble B7-H5 protein.

[0016] Another aspect of the present invention provides an enzyme-linked immunosorbent assay kit for detecting the content of soluble B7-H5, comprising B7-H5-2E5 and / or B7-H5-7B10 anti-human B7-H5 monoclonal antibody;

[0017] The purpose of the enzyme-linked immunosorbent assay kit is to detect the content of soluble B7-H5 protein in patients with tumors, autoimmune diseases, etc.

[0018] This invention also provides a method for detecting the content of soluble B7-H5 protein in patients with tumors or autoimmune diseases for non-disease treatment purposes.

[0019] The enzyme-linked immunosorbent assay (ELISA) kit of the present invention can effectively detect the content of soluble B7-H5. Using this kit, it was found that the serum of healthy individuals contains a certain baseline level of soluble B7-H5 protein (~350 pg / ml). The content of soluble B7-H5 in the serum of patients with colorectal cancer, lung cancer, and gastric cancer is significantly elevated and is correlated with the clinical stage and pathology of the patients. The detection of soluble B7-H5 content in the body fluids of tumor patients has potential clinical value. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the detection of soluble B7-H5 protein content in a sample using the enzyme-linked immunosorbent assay kit of the present invention.

[0021] Figure 2 Graph showing the serum soluble B7-H5 content of patients with rectal cancer, lung cancer and gastric cancer detected by monoclonal antibody. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0023] Example 1: Preparation of hybridoma cells against human B7-H5 monoclonal antibody

[0024] 1.1 Immunization in mice

[0025] BALB / c mice were immunized five times with the B7-H5 fusion recombinant protein. The first three immunizations were spaced 21 days apart. The antibody titer in the mice's blood was measured 5-7 days after the fourth immunization. If the titer was good, a fifth booster immunization was performed.

[0026] 1.2 Culture of trophoblast cells

[0027] 1.2.1 The spleens of 7-8 week old BALB / c mice were removed in a sterile laminar flow hood. A single-cell suspension was obtained by grinding the spleen through a 200-mesh sterile sieve.

[0028] 1.2.2 The single-cell suspension was washed twice with RPMI-1640 medium, and the cells were resuspended in an appropriate amount of RPMI-1640 medium containing 15% FBS. The cell suspension was then evenly dropped into a 96-well culture plate using a sterile Pasteur glass pipette, with a volume of about 80 μL per well. The cells were cultured overnight in a cell culture incubator.

[0029] 1.3 Cell Fusion and Screening

[0030] 1.3.1 Spleens were removed from 7-8 week old BALB / c mice in a sterile laminar flow hood. A single-cell suspension was obtained by grinding the cells through a 200-mesh sterile sieve. The cells were then resuspended twice by centrifugation at 1400 rpm for 5 min with 1640 basal culture medium preheated to 37°C.

[0031] 1.3.2 The collected spleen cells and sp2 / 0 cells in the logarithmic growth phase were mixed in a centrifuge tube at a ratio of approximately 5:1. Preheated RPMI-1640 basal medium was added and mixed thoroughly. The mixture was then centrifuged once at 1400 rpm for 5 min. The supernatant was discarded and the bottom of the tube was gently tapped with a finger to mix the two types of cells.

[0032] 1.3.3 Add 1 ml of preheated PEG solution to the two types of mixed cells, and gently shake the centrifuge tube continuously to accelerate the fusion of the two types of cells.

[0033] 1.3.4 After fusion is complete, add 14 ml of RPMI-1640 basal medium to terminate the fusion process. Centrifuge and discard the supernatant.

[0034] 1.3.5 Resuspend the confluent cells in RPMI-1640 whole medium, then use a Parshall glass plate to transfer approximately 80 μl of the cells into each well of a 96-well culture plate containing the feeder cells. Incubate in an incubator and change the medium with HT medium every 3-4 days. Afterward, take a small amount of supernatant from each well for ELISA detection.

[0035] 1.3.6 Select wells with high antibody detection values ​​for subcloning. Perform ELISA detection after each subcloning. Usually, it takes 3-4 subclonings to obtain cells with a stable antibody-secreting phenotype. Cryopreserve the cells after the last subcloning.

[0036] Example 2: Determination of the light and heavy chain variable region sequence of anti-human B7-H5 monoclonal antibody

[0037] The method for determining the variable regions of the heavy and light chains of anti-human B7-H5 monoclonal antibodies includes the following steps:

[0038] 2.1 Obtaining cDNA from hybridoma cells

[0039] RNA was obtained from the target hybridoma cells and reverse transcribed into cDNA using reverse transcription technology. The heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cells were then cloned by PCR using specifically designed upstream and downstream primers.

[0040] 2.2 The heavy chain variable region (mVH) and light chain variable region (mVL) were ligated to the pJET cloning vector, respectively. Subsequently, the ligation products were transformed into DH5α competent cells, and the transformed bacterial culture was evenly spread on LB solid medium.

[0041] 2.3 Select colonies with clear edges and good growth on LB solid medium for sequencing identification.

[0042] 2.4 Based on the sequencing results, candidate light and heavy chain variable region sequences were retained. The variable region sequences of the light and heavy chains that could be ligated to the expression vector were cloned again by PCR. The variable region sequences were then ligated to the expression vector, and the ligation product was transformed into DH5α. The transformed bacterial culture was evenly spread on LB solid medium and incubated overnight.

[0043] 2.5 Select well-growing bacteria for sequencing and compare the two sequencing results to obtain transformed bacteria with the correct sequence. After expansion culture, plasmid extraction is performed.

[0044] 2.6 The expression vector containing the heavy and light chain variable region genes of the Dangclone antibody was co-transfected into eukaryotic expression cells 293.

[0045] 2.7 293 cells were cultured in suspension using serum-free SFM4Transfx-293 medium without L-Glutamine. The medium was replaced with serum-free medium at transfection. FreeStyle TM 293 Expression Medium.

[0046] 2.7 The ELISA kit was used to detect the supernatant containing the target antibody, and the results were good.

[0047] The heavy chain variable region of the anti-human B7-H5-2E5 monoclonal antibody was obtained using the method described above.

[0048] DVKLQESGPGLVKPSQSLSLTCTVTGYSIASDYTWNWIRQFPGNKLEWMGYIDYSGATFYNPSLKSRISIIRDTSKNQFFLQLNSVTTGDTATYYCTRGFYYYGSDYWGQGTTLTVSS;

[0049] B7-H5-2E5 monoclonal antibody light chain variable region:

[0050] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHNNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSHVPSTFGGGTKLEIK;

[0051] B7-H5-7B10 monoclonal antibody heavy chain variable region

[0052] EVQLKQSGGPGLVAPSQSLSITCTVSGFSLTNHGVQWVRQSPGKGLEWLGVIWGDGNRNYHSALMSRLSINKDNSKSQVFLKLNRLQTDDTATYFCAKERRLGYYGEYDVMDYWGQGTSVTVSS;

[0053] B7-H5-7B10 monoclonal antibody light chain variable region

[0054] DIVMTQSHKFMSTSVGDRVTITCKASQGVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIGNVQSEDLADYFCQQYSSSPFTFGSGTKLEIK

[0055] This invention extracts the variable regions of the heavy and light chains of an anti-human B7-H5 monoclonal antibody from hybridoma cells. Based on sequencing results, candidate light and heavy chain variable region sequences are retained, and the light and heavy chain variable region sequences matching the expression vector are amplified by PCR. The PCR product is then ligated into the expression vector pretreated with double enzyme digestion. The expression vector containing the target monoclonal antibody's light and heavy chain variable regions is co-transfected into eukaryotic expression cell line 293. The supernatant collected after culture contains the target antibody, indicating that the obtained heavy and light chain variable region sequences are correct.

[0056] Example 3: Detection method of ELISA kit for detecting soluble B7-H5 protein content

[0057] 3.1 Components of the reagent kit

[0058] The ELISA kit of the present invention comprises the B7-H5 coated antibody (B7-H5-2E5) prepared in Example 1 coated on an enzyme-linked immunosorbent assay (ELISA) plate, the biotinylated B7-H5 detection antibody (B7-H5-7B10), the soluble B7-H5 protein standard (R&D Systems), horseradish peroxidase (HRP), sample diluent, washing buffer (PBST), chromogenic reagent (TMB), and stop solution.

[0059] 3.2 Sample Collection and Processing

[0060] 3.2.1 Collect a batch of serum from patients with colorectal cancer, lung cancer and gastric cancer in the hospital, aliquot and store at -80℃, and avoid repeated freeze-thaw cycles.

[0061] 3.3.2 Before testing serum samples, allow the samples to equilibrate at room temperature for half an hour and shake them to mix. Take an appropriate amount of patient serum and dilute it 50 times with sample diluent.

[0062] 3.3 Method for determining the content of soluble B7-H5 protein

[0063] 3.3.1 Dilute the coating antibody B7-H5-2E5 (1 μg / ml) with coating buffer (Na2CO3 and NaHCO3), then add the coating antibody to a 96-well microplate (100 μl / well) and incubate overnight at 4°C.

[0064] 3.3.2 On the second day, wash the plate three times with washing buffer (PBST), then add 100 μl of 3% bovine serum blocking buffer to each well and block at 37°C for 1 h.

[0065] 3.3.3 Remove the blocking solution, add 100 μl of diluted serum sample to be tested and serially diluted soluble B7-H5 protein standard (starting from 2 ng / ml for comparative dilution, a total of 7 concentrations were set, with the last blank containing sample dilution as a blank control, and 3 replicates for each gradient), and incubate at room temperature with shaking at 75 rpm for 1 h.

[0066] 3.3.4 Wash the plate three times with PBST washing buffer, add biotin-labeled detection antibody B7-H5-7B10 (100 μl / well), and incubate at room temperature with shaking at 75 rpm for 1 h.

[0067] 3.3. Wash the plate three times with PBST washing buffer, add horseradish peroxidase (HRP, 100 μl / well), and incubate at room temperature with shaking at 75 rpm for 1 h.

[0068] 3.3.5 Wash the plate six times with PBST washing buffer, add TMB chromogenic solution (100 μl / well), incubate at room temperature with shaking for 10 min, then add stop solution (100 μl / well) to stop the color reaction, and measure the OD450 value of each well using a microplate reader.

[0069] 3.3.6 Plot a standard curve with the concentration of B7-H5 standard multiplied by the sample dilution factor (×50) on the ordinate and the corresponding measured OD450 value on the abscissa (e.g., Figure 1 The calculation formula is obtained, and the content of soluble B7-H5 in the sample is calculated based on the OD450 value of the sample to be tested.

[0070] The reagent kit of this invention, according to methodological verification, can achieve the following indicators:

[0071] Standard curve linearity: R² = 0.9990; limit of detection ≤ 7.285 pg / ml.

[0072] 3.3.7 The enzyme-linked immunosorbent assay (ELISA) kit of the present invention can effectively detect the content of soluble B7-H5. Using this kit, it was found that healthy human serum contains a certain baseline level of soluble B7-H5 protein (~350 pg / ml), while the content of soluble B7-H5 in the serum of patients with colorectal cancer, lung cancer, and gastric cancer is significantly elevated. Figure 2 The content of soluble B7-H5 in the body fluids of cancer patients is correlated with the clinical stage and pathology of the patients and has potential clinical value.

[0073] Since there are currently no enzyme-linked immunosorbent assay (ELISA) kits for detecting soluble B7-H5 content on the domestic and international markets, it is evident that the kit of this invention represents a significant breakthrough for subsequent data collection and other tasks.

Claims

1. A monoclonal antibody against human B7-H5 protein, characterized in that, The monoclonal antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO.1 and the amino acid sequence of the variable region of the light chain is SEQ ID NO.

2.

2. The use of the monoclonal antibody according to claim 1 in the preparation of a kit for detecting soluble B7-H5 protein.

3. An enzyme-linked immunosorbent assay (ELISA) kit for detecting the content of soluble B7-H5, characterized in that, The test kit contains the monoclonal antibody as described in claim 1.

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