CN102250910B - A human soluble B7-DC quantitative detection kit - Google Patents

Abstract

The invention discloses a kit capable of quantitatively detecting soluble B7-DC. The kit comprises a horse radish peroxidase marker, tetramethyl benzidine serving as a reaction substrate, bovine serum albumin, an elisa plate, a wash solution and a stop solution, and is characterized by further comprising a coated antibody, standard proteins and a detection antibody, wherein the coated antibody is a mouse anti-human B7-DC monoclonal antibody 8F2; and the detection antibody is a mouse anti-human B7-DC monoclonal antibody 10D6. The kit capable of quantitatively detecting soluble B7-DC has high specificity, can be used for accurately and quantitatively analyzing the concentrations of soluble B7-DC protein factors in liquids such as human cell culturing supernatant, blood serum, blood plasma, hydrothorax and the like, and can be applied to clinical differential diagnosis and treatment effects of asthma.

Description

A human soluble B7-DC quantitative detection kit

technical field

The invention relates to a kit capable of quantitatively detecting human soluble B7-DC, in particular to a kit for quantitative detection prepared by using two strains of specific mouse anti-human B7-DC monoclonal antibodies 8F2 and 10D6 and B7-DCIg fusion protein An enzyme-linked immunoassay kit for analyzing soluble B7-DC factor, the quantitative detection system can be applied in the fields of differential diagnosis and curative effect judgment of asthma.

Background technique

Many receptor/ligand interactions are known to be involved in the induction, establishment and regulation of antigen-specific immune responses. In order to effectively activate a T cell response, at least two signals are usually required. In addition to the T cell antigen receptor (TCR) recognizing the MHC-antigen complex on the antigen-presenting cell (APC) to provide the first signal, the antigen-specific signal, T cells need to interact with costimulatory molecules expressed by APC The second signal generated after that is the co-stimulatory signal. If there are only antigen-specific signals but lack of co-stimulatory signals, T cells will appear in a state of anergy or immune tolerance, and even apoptosis. It can be seen that co-stimulatory signals are essential for the expansion, differentiation and biological effects of T cell clones. Therefore, the first signal determines the specificity of T cell activation, while the second signal determines whether the T cell-mediated immune response can be effectively carried out.

In recent years, molecular biology techniques have been widely used in immunology research, and co-stimulatory molecules have been continuously discovered. According to their structures, these co-stimulatory molecules can be divided into two categories: one is the tumor necrosis factor/tumor necrosis factor receptor superfamily, including CD40/CD154, CD27/CD27L, CD30/CD30L, 4-1BB/4-1BBL and Fas/FasL etc. The other is the immunoglobulin superfamily, such as B7/CD28, LAF1/ICAM-1, ICOS-GL50, PD-1/B7-DC, BTLA/HVEM, and CD2/LFA-3. These co-stimulatory molecules signal through receptor/ligand interactions. Receptors/ligands are generally expressed on the surface of different immune cells, usually one is persistently expressed and the other is characterized by inducible expression. At different stages of the immune response, these molecules participate in signal transduction in their own unique and related ways, and jointly regulate the effective initiation, appropriate response and timely termination of the body's immune response.

PD-1 (CD279) is an immunoglobulin superfamily type I transmembrane glycoprotein composed of 288 amino acids, which is considered to be related to apoptosis and named programmed death-1 (PD-1) . Studies have found that PD-1 molecules are mainly inductively upregulated on the surface of T, B, and NK cells, and immature T, B cells are also weakly expressed at specific stages of development in the thymus and bone marrow. Amino acid residues can interact with downstream signal transduction molecules such as SHP-1 and SHP-2 to inhibit further activation of immune cells. B7-DC is one of the two ligands of PD-1. Like other members of the B7 family, the B7-DC molecule contains an IgV-like region, an IgC-like region, a transmembrane region and a short and conserved cell Tail of pulp area. The human B7-DC gene is located at 9p24.2, which can encode 247 amino acids, and its full-length cDNA is 1.7kb. Human B7-DC mRNA is highly expressed in placenta, liver cancer cells, breast cancer cells, and neuroblasts, but low in spleen, lymph nodes, thymus, and fibroblasts. B7-DC is not only expressed in parenchymal organ tissues, but also moderately expressed in some antigen-presenting cells, such as: activated T cells and dendritic cells. The distribution of B7-DC is relatively limited, mainly expressed in activated monocyte-macrophages and dendritic cells (DCs). DCs induced by IL-4 and GM-CSF from peripheral monocytes can highly express B7-DC. A large number of studies have shown that the interaction between B7-DC and PD-1 on activated T cells can significantly inhibit the biological function of effector T cells, and changes in the expression of PD-1 or B7-DC will lead to PD-1/PD-L Abnormalities in the inhibitory pathways lead to hyperimmune/hypoimmune diseases.

PD-1/PD-L interaction inhibits the expression of Bcl-xL and inhibits the expression of various transcription factors in effector T cells, such as GATA-3, Tbet and Eomes. In vitro studies using B lymphoma cell lines transfected with PD-1 or FcγRII-PD-1 fusion protein genes have shown that PD-1 can inhibit the stimulatory signal of the B cell receptor, thereby reducing B cell activation. Cross-linking of BCR with PD-1 can inhibit Ca ²⁺ influx and phosphorylation of tyrosine residues of downstream signal activation molecules such as syk, PI3K, phospholipase-3 and vav. Interestingly, this inhibitory effect does not require the participation of the tyrosine residue located in the ITIM activation inhibitory motif at the N-terminal of the PD-1 cytoplasmic region, but the combination of the C-terminal tyrosine residue and SHP-2 tyrosine A result of acid phosphatase binding. In addition, PD-1 can also inhibit the phosphorylation of tyrosine residues of mitogen-activated protein kinase (MAPK), a further downstream signal activation molecule, to inhibit the proliferation of B lymphoma cell lines. In addition, the research results using the Jurkat cell line as a model also show that the cross-linking of TCR and PD-1 can cause the phosphorylation of SHP-2 and recruit it to the cytoplasmic region of PD-1.

PD-1/B7-DC can inhibit the proliferation of T cells and the production of cytokines under antigen stimulation, and can antagonize the positive signal mediated by CD28/B7. The inhibitory effect of the combination of B7-DC and its receptor PD-1 on T cells depends on the strength of TCR and CD28 transmission signals, and enhancing the transmission of TCR and CD28 can counteract the inhibitory effect of PD-1/B7-DC. The relative levels of positive and negative signals determine the threshold of the immune response. Therefore, in the absence of the interaction between B7-DC and PD-1, the threshold of TCR inhibitory signal required for T cell activation is lowered, which is prone to cause autoimmune diseases. As a negative co-stimulatory signal, B7-DC plays an important role in both the breadth and depth of the body's immune response, participates in the activation of lymphocytes, and plays an irreplaceable role in immune tolerance and immune injury. PD-1/B7-DC pathway, as a negative regulatory mode, is highly expressed in many non-lymphoid tissues and plays an important role in the induction of peripheral tolerance and autoimmune diseases.

Bronchial asthma is prevalent all over the world, especially in developed countries, and its prevalence is increasing year by year. Its incidence is mainly related to type I allergy caused by cellular and molecular immune dysfunction. Asthma is an airway involving a variety of cells including airway inflammatory cells and structural cells (such as eosinophils, mast cells, lymphocytes, neutrophils, airway epithelial cells, smooth muscle cells, etc.) and cellular components Chronic inflammatory disease. This chronic inflammation is associated with airway hyperresponsiveness, usually causing widespread and variable reversible airflow limitation, and causing recurrent episodes of wheezing, shortness of breath, chest tightness, and/or coughing, mostly at night and/or in the early morning occur.

Asthma is a chronic airway inflammation involving a variety of cells, in which T lymphocytes play a leading role in initiating and regulating lung inflammatory responses. its effect function. Under normal circumstances, there are only a small number of T cells in the lungs, and a large number of T cells increase during the inflammatory response. Helper T cells are divided into Th1 and Th2 groups according to their functions. The Th2 type mainly secretes IL-4 (promotes the synthesis of IgE by B cells), IL-5 (promotes the growth and differentiation of eosinophils), IL-9 (promotes the differentiation of mast cells), and IL-13 (promotes mucus secretion, induces airway hyperresponsiveness), promote the occurrence of allergic inflammation, and are considered to be the main regulatory cells that cause asthma. Th1 type has antagonistic effect on Th2 cells by secreting IFN-γ. Therefore, T cells play an important role in the airway inflammation of asthma, and their activation requires two signals, one is the specificity of TCR/CD3 binding to the MHC-antigen peptide complexes on the surface of antigen-presenting cells (APCs) Antigen stimulation signal; the other is non-specific antigen stimulation signal, which is what we often call co-stimulation signal. Only by recognizing these two signals can T cells activate, proliferate, and function. If they lack co-stimulatory signals, they will enter a state of non-response, or even apoptosis.

PD-1/B7-DC can inhibit the proliferation of T cells and the production of cytokines under antigen stimulation, so it plays an important role in the immune response of asthma. Matsumoto found that PD-L2 was highly expressed in lung dendritic cells and macrophages of sensitized mice, and anti-PD-L2 antibodies were administered during the antigen challenge phase, resulting in increased AHR and increased production of Th2 cytokines. Akbari et al established a mouse model of asthma and found that the lack of PD-L2 expression led to increased AHR and airway inflammation in mice, and then proposed that the expression of PD-L2 has a protective effect in the initiation and progression of asthma.

Studies have found that many co-stimulatory molecules can exist in two forms of cell membrane and soluble, including CD40, OX40L, 4-1BBL, CD86, CD30 and CTLA-4. Soluble molecules can be formed by cleavage of membrane-type molecules on cells by proteolytic enzymes, such as: B7-H3 and s4-1BBL; they can also be produced by specific mRNA encoding, such as: sCD86 and sCTLA-4. The expression level of many soluble co-stimulatory molecules has clinical diagnostic value, for example, soluble CD40L (sCD40L) is activated CD4 ⁺ T cells and platelet surface membrane type CD40L molecule cleavage, sCD40L level is abnormal in the serum of some autoimmune diseases Serum levels of sCD40L in patients with essential thrombocythemia and arteriosclerosis were also higher. Soluble CD30 molecules are also expressed at high levels in the serum of certain tumors and AIDS (AIDS) patients. It has been shown that soluble molecules in the body have the same biological functions as molecules on the membrane surface. On the one hand, molecules on the cell membrane can mediate co-stimulatory signals through direct receptor/ligand interactions; on the other hand, soluble proteins Factors can participate in blood circulation like cytokines and play an important regulatory role in immune response. They can not only affect adjacent cells but also bind to receptors on the surface of distant cells, thus participating in the occurrence and development of diseases. The breadth and depth go far beyond the molecules on the surface of the cell membrane. So far, due to the lack of effective detection methods, there have been no research reports on soluble B7-DC (sB7-DC) at home and abroad.

Contents of the invention

The object of the present invention is to provide a kit capable of quantitatively detecting soluble B7-DC.

In order to achieve the above-mentioned purpose of the invention, the technical solution adopted in the present invention is: a kit capable of quantitatively detecting soluble B7-DC, comprising: the horseradish peroxidase marker, the reaction substrate tetramethylbenzidine, bovine serum albumin Protein, microtiter plate, washing solution and stop solution, the kit also includes: coating antibody, standard protein and detection antibody, wherein, the coating antibody is a mouse anti-human B7-DC monoclonal antibody, and its heavy The chain has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 5; the light chain of the monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing sequence, or the amino acid sequence shown in SEQ ID NO: 6; the detection antibody is a mouse anti-human B7-DC monoclonal antibody, and its heavy chain has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence table, or SEQ ID The amino acid sequence shown in NO: 7; the light chain of the monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 8.

In a preferred technical scheme, the coating antibody is a mouse anti-human B7-DC monoclonal antibody 8F2, which is secreted by a mouse anti-human PD-L2 molecule monoclonal antibody hybridoma cell line SIPD with the preservation number CGMCC No. 4789 -A monoclonal antibody prepared from -L2 (8F2); the detection antibody is a mouse anti-human B7-DC monoclonal antibody 10D6, obtained from a hybridoma that secretes a mouse anti-human PD-L2 molecular monoclonal antibody with a preservation number of CGMCC No. 4790 Monoclonal antibody produced by cell line SIPD-L2 (10D6).

In the above technical scheme, the preservation information of the hybridoma cell line SIPD-L2 (8F2) is: preservation number: CGMCC No. 4789, classification and name: hybridoma cell line secreting mouse anti-human PD-L2 molecular monoclonal antibody, and preservation date : April 27, 2011, depository unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, deposit address: No. 13, North Yiyi, Zhongguancun, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; hybridoma cell line SIPD-L2 (10D6 )’s deposit information is: deposit number: CGMCC No. 4790, classification name: hybridoma cell line secreting mouse anti-human PD-L2 monoclonal antibody, deposit date: April 27, 2011, depository unit: China Microbial Bacteria General Microbiology Center of Species Preservation Management Committee, deposit address: No. 13, North Zhongguancun, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

In the above technical solution, the standard protein is the standard protein B7-DCIg protein, that is, the B7-DCIg protein in the common prior art.

In the above technical solution, the lotion is selected from: 0.002M imidazolium salt solution containing 0.02%~0.2% Tween-20 by volume fraction or PBS solution with pH7.4, preferably 0.02% volume fraction Tween-20 (Tween-20) in PBS solution at pH 7.4.

In the above technical solution, the stop solution is selected from: 2M sulfuric acid or 1% HCl solution; preferably 2M sulfuric acid.

In addition to the monoclonal antibody against B7-DC molecules on the cell surface of the present invention, the above kit may also include a blood sample collection device and related solvents, buffers and standards.

The above-mentioned kit for quantitative detection of soluble B7-DC can quantitatively analyze the expression level of soluble B7-DC factors in human serum, plasma and tissue fluid such as pleural effusion and ascites; it can be applied in the differential diagnosis and treatment effect of clinical asthma.

The present invention also claims to protect a nucleotide sequence as shown in SEQ ID NO:1.

The present invention also claims to protect a nucleotide sequence as shown in SEQ ID NO:2.

The present invention also claims to protect a nucleotide sequence as shown in SEQ ID NO:3.

The present invention also claims to protect a nucleotide sequence as shown in SEQ ID NO:4.

The present invention also claims to protect a mouse anti-human B7-DC monoclonal antibody 8F2, the heavy chain of the monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence table, or shown in SEQ ID NO: 5 Amino acid sequence; the light chain of the monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 6.

The present invention also claims to protect a mouse anti-human B7-DC monoclonal antibody 10D6, the heavy chain of the monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence table, or shown in SEQ ID NO: 7 Amino acid sequence; the light chain of the monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 8.

The present invention also claims to protect a hybridoma cell strain, the hybridoma cell strain of Longantibody is a hybridoma cell strain SIPD-L2 (8F2) secreting mouse anti-human B7-DC molecular monoclonal antibody, and its preservation number is CGMCC No. 4789.

At the same time, the present invention claims to protect a hybridoma cell line, the long antibody hybridoma cell line is a hybridoma cell line SIPD-L2 (10D6) secreting mouse anti-human B7-DC molecular monoclonal antibody, and its preservation number is CGMCC No. 4790.

Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

1. The kit capable of quantitatively detecting soluble B7-DC of the present invention has good specificity, and can accurately quantitatively analyze the concentration of soluble B7-DC protein factors in liquids such as human cell culture supernatant, serum, plasma, and pleural effusion; It is used in the differential diagnosis and treatment effect of clinical asthma.

2. The kit capable of quantitatively detecting soluble B7-DC of the present invention has good sensitivity and can maintain a good linear relationship within the concentration range of 3.125-200ng/ml sB7-DC.

Description of drawings

The preservation information of the hybridoma cell line SIPD-L2 (8F2) is: preservation number: CGMCC No. 4789, classification name: hybridoma cell line secreting mouse anti-human PD-L2 molecule monoclonal antibody, preservation date: April 2011 On the 27th, preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation address: No. 13, Zhongguancun North Yiyi, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the preservation information of the hybridoma cell line SIPD-L2 (10D6) is : Preservation number: CGMCC No. 4790, classification and naming: hybridoma cell line secreting mouse anti-human PD-L2 monoclonal antibody, deposit date: April 27, 2011, depository unit: China Microorganism Culture Collection Management Committee General Microbiology Center, preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 13, North 1st Road, Zhongguancun, Haidian District, Beijing.

Fig. 1 is the identification figure of antibody in embodiment one;

Fig. 2 is the specificity analysis figure of antibody in embodiment one;

Fig. 3 is the result figure of antigenic epitope competition experiment in embodiment one;

Fig. 4 is the result figure of the specificity analysis of human soluble B7-DC ELISA kit in embodiment three;

Fig. 5 is the linear detection standard curve of human soluble B7-DC ELISA kit in embodiment four;

6 is a diagram showing the expression levels of sB7-DC in the peripheral serum of asthmatic patients in the attack phase and stable phase in Example 4;

Fig. 7 is a graph showing the dynamic changes of sB7-DC before and after treatment of asthmatic patients in Example 4.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Embodiment one:

(1) Reagents and materials: calf serum Hyclone Company (USA); add calf serum 100ml, L-glutamine 0.15g, NaHCO ₃ 2.0g, pyruvate to each liter of RPMI1640 or DMEM medium (Gibco, USA) Sodium 0.11g, glucose 3.6g, HEPES 4.766g, 2-mercaptoethanol 10.0ml; HAT, HT selection medium Dilute the 50-fold concentration of HAT, HT selection medium (Sigma, USA) with RPMI1640 or DMEM complete medium is the working concentration; Freund'adjuvant (Freund'adjuvant, Sigma, USA); cell fusion agent polyethylene glycol (PEG1500); Protein G affinity layer suction column (Pharmacia, Sweden); Pristane (Sigma, USA). Cell culture flasks and plates (Nunc, Denmark); CO2 incubator, centrifuge (Jouan, France), inverted microscope (O1ympus, Japan), flow cytometry (Coulter, USA); human B7-DC gene cell line L929 /B7-DC (built by me). All cell lines were tested and found no mycoplasma contamination; 6-8 weeks old female Balb/c mice (Shanghai Experimental Animal Center).

(2) Cultivation of cell lines: Using RPMI1640 medium containing 10% FCS, L929/B7-DC and L929/mock gene transfected cell lines were regularly pressurized with resistance screening drug Zeocin (500 μg/ml) to maintain Stable high expression of the target gene product.

(3) Animal immunization: Collect well-grown L929/B7-DC cells, wash them twice with PBS, add mitomycin solution (50μg/100μl/1×107), mix well, place at 37°C for 45min, After washing with PBS three times, re-suspend with 0.3-0.4ml of normal saline, fully emulsify with the same amount of IFA, and inject into the neck subcutaneously and intraperitoneally (1×10 ⁷ /body); and after the initial immunization Immunization was performed again in the third and fifth weeks, and the number of cells was changed to 8×10 ⁶ /body. The pretreatment of cells and the site of injection were the same as above. 4 to 5 days before fusion, another intraperitoneal injection of 5×10 ⁶ /body was used to strengthen immunity.

(4) Preparation of myeloma cells: 10-14 days before fusion, resuscitate mouse myeloma cells SP2/0, subculture with DMEM medium containing 10% FCS, and wait until the cells grow vigorously, have good shape, and the cell viability is greater than 95% can be used for fusion. Adjust the cell concentration to 3×10 ⁵ /ml with fresh medium 24-36 hours before fusion.

(5) Preparation of feeder cells: 1 day before fusion, take Balb/c mice, enucleate the eyeballs to kill, rinse with tap water, place in 75% ethanol for 10 minutes, and then fix them on a dissection rack, open the mouse chest cavity along the sternum, Cut the thymus, place it in a 200-mesh steel mesh, move the screen into a plate containing 10ml of basal medium, grind the thymus, make it a single cell and suspend in the medium; draw a little cell suspension and count, 1000rpm, Centrifuge for 8 minutes, adjust the cell concentration to 3~4×10 ⁵ /ml with complete medium, add 0.1ml cell suspension (equivalent to 3~4×10 ⁴ /well) in each well of a 96-well culture plate, CO ₂ cultured in an incubator.

(6) Preparation of spleen cells from immunized mice: The mice after booster immunization were sacrificed according to the above method, the mice were treated, the abdominal cavity was opened, the spleen was removed from the left rear abdomen, and a single spleen cell was obtained by grinding. Use trypan blue solution to count the nuclei of living cells, centrifuge (1000rpm×8min), wash twice, suspend the precipitated cells in 20ml RPMI1640, and place them in an incubator for later use.

(7) Cell fusion and selective culture: Prewarm RPMI1640 or DMEM basal medium and PEG solution in a 37°C water bath. Collect 2×10 ⁷ well-grown SP2/0 cells, mix them with 1×10 ⁸ spleen cells in a 50ml transparent plastic centrifuge tube, wash twice with RPMI1640, centrifuge at 1000rpm for 8min, discard the supernatant, flick with your fingers At the bottom of the tube, the two kinds of pelleted cells were thoroughly mixed into a paste. Put the plastic tube in a thermos cup at 37°C, draw 1ml of PEG solution, gently insert the pointed pipette into the bottom of the cell suspension, add it at a constant speed within 1min, stir gently while adding, and then let it stand in a water bath for 90s . Then add pre-warmed serum-free DMEM, stand at room temperature for 5 minutes, centrifuge (800rpm×10min), and discard the supernatant. Gently resuspend the pelleted cells in 40ml DMEM containing 2% HAT and 15% FCS. After mixing, add it dropwise to 96-well culture plate containing feeder cells, 100 μl/well, culture at 37°C, 5% CO ₂ , replace half of the medium in 3 to 4 days, change to HT medium after 10 days, and switch to HT medium after 2 weeks DMEM medium containing 15% FCS.

(8) Screening of hybridoma cells: When the cell clones cover 1/3~1/4 of the culture wells, the supernatant can be aspirated and screened by indirect immunofluorescence labeling. The well-grown L929/B7-DC cells were washed with PBS, distributed into flow tubes (5×10 ⁵ /tube), and the culture supernatant of hybridoma cells (50 μl/tube) was added. React at 4°C for 30 minutes, wash twice with PBS containing 1% calf serum, add PE-labeled goat anti-mouse IgG secondary antibody and react for 30 minutes at 4°C, analyze with FACS after washing, and use L929/mock cells as negative control cells strain.

The 293T/B7-DC gene-transfected cells stably expressing human B7-DC were screened and identified, and the positive clones were cloned and cultured for 3 consecutive times, and finally 2 strains capable of continuously secreting specific anti-human B7-DC were obtained Molecular hybridoma cell lines were named 10D6 and 8F2, and the results of flow cytometry showed that the monoclonal antibody could specifically recognize human B7-DC, but not combined with 293T/mock gene transfected cells (Figure 1).

(9) Cloning culture of hybridoma cells: Use the limiting dilution method to accurately count the cells in the antibody-positive wells, then use the HAT selection medium to gradiently dilute to 50 cells/ml and 10 cells/ml, add 100 μl/well to the Incubate in 96-well culture plates containing feeder cells at 37°C, 5% CO ₂ . Change the medium in due course and re-screen in time according to the established screening method for positive clones according to the growth status of the cells. Select cells with high antibody titer, single clonal growth, and good morphology to continue cloning until the positive rate of antibody secretion is greater than 95%; expand the culture and freeze in liquid nitrogen in time.

(10) Preparation of monoclonal antibody: Monoclonal antibody was produced by in vivo induction in ascites. Take 6-8 weeks old female Balb/c mice and inject 0.5ml of Pristane intraperitoneally. One week later, intraperitoneally inject hybridoma cells at 1×10 ⁷ per mouse, and at the same time inject an equal-volume mixture of Pristane and Freund’s semizole 0.2ml per mouse into the other abdominal cavity, and gently massage the mouse’s abdomen to disperse the hybridoma cells in the abdominal cavity middle. Harvest the ascites after 5-10 days, centrifuge the supernatant and store it at -80°C.

(11) Antibody purification: thaw the ascites, centrifuge to remove the clot, precipitate the antibody protein with a saturated (NH ₄ ) ₂ SO ₄ solution, and reconstitute with PBS. (NH ₄ ) ₂ SO ₄ was removed by dialysis, and the sample was eluted with a protein G affinity layer and then eluted with pH 2.8 glycine-hydrochloric acid. Collect the protein elution peak and adjust the pH to 7.0 with Tris-Base at pH 9.0. After dialyzing PBS, use a UV spectrophotometer to measure the content of antibody protein. The formula for calculating the concentration is: antibody protein content (mg/ml) = OD280× 1.55-OD260×0.76, result: the concentrations of monoclonal antibodies 8F2 and 10D6 were 1.55mg/ml and 1.87mg/ml, respectively.

(12) Monoclonal antibody titer determination: divide well-grown L929/B7-DC cells into flow tubes, 5×10 ⁵ /tube. Add hybridoma cell culture supernatant, ascites and purified antibody protein gradient dilution (20 μl/tube), react at 4°C for 30 minutes, add goat anti-mouse IgG-PE after washing with PBS, and react at 4°C for 30 minutes. After washing, it was analyzed by flow cytometry, and the titer of the antibody was determined by the maximum dilution factor when the same positive rate and fluorescence intensity appeared.

(13) Type identification of monoclonal antibody: Take hybridoma cell culture supernatant and dilute at 1:50 and ascitic fluid at 1:20000. Take 0.2ml of the diluent and add it to a small test tube containing the subclass qualitative reagent, place it at room temperature for 1min, wait for it to dissolve naturally, mix gently, gently insert the subclass qualitative test strip into the tube, after 5~10min, the test paper On one side, a blue protein band corresponding to the name of the mAb heavy chain appears, which is the mouse Ig subclass to which the antibody belongs; on the other side, a blue protein band corresponding to the name of the mAb light chain appears. That is, the light chain type of the mAb; the monoclonal antibody is sequenced, and the heavy chain of the mouse anti-human B7-DC monoclonal antibody 8F2 is measured as the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, or as SEQ ID The amino acid sequence shown in NO: 5; the light chain of the monoclonal antibody 8F2 is shown in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 6; the detection antibody is The heavy chain of the mouse anti-human B7-DC monoclonal antibody 10D6 has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 7; the light chain of the monoclonal antibody 10D6 The nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 8.

(14) Specific identification of monoclonal antibodies: react L929/mock, L929/B7-H1, L929/B7-DC and L929/B7-H3 transfected cells with the obtained anti-human B7-DC monoclonal antibody for 30 minutes After washing with PBS, PE-labeled goat anti-mouse IgG secondary antibody was added, and the reaction was continued for 30 min. After washing, FACS was used to analyze the fluorescence intensity of cell markers. A commercial anti-human B7-DC monoclonal antibody (MIH18) was used as a positive control. The obtained monoclonal antibodies 10D6 and 8F2 were immunofluorescently labeled with mock, B7-DC, B7-H1 and B7-H3 gene transfected cells, and the results showed that the monoclonal antibodies could specifically recognize human B7-DC, but not with The combination of mock, B7-H1 and B7-H3 transfected cells (Figure 2).

(15) Biotin-labeled monoclonal antibody: Take 200 μg of anti-human B7-DC monoclonal antibody (10D6) to be labeled, add it to dialysis bag containing 1ml of 0.01M PH=9.3 carbonate buffer solution, and dialyze at 4°C overnight to equilibrate. Add 40 μl of BNHS-DMSO solution with a concentration of 1 mg/ml, react at room temperature in the dark for 4 hours, then dialyze in PBS with pH 7.2 at 4°C for 72 hours, and store at -20°C after aliquoting.

(16) Antibody competition experiment: Wash L929/B7-DC cells (5×10 ⁵ /tube) with PBS, add monoclonal antibody (2 μg per tube), incubate at 4°C for 30 minutes, add biotin-labeled monoclonal antibody after washing , and then incubated at 4°C for 30min, after washing, added streptavidine-PE, incubated at 4°C for 30min and washed, then analyzed by flow cytometry, and positive and negative control groups were set at the same time.

The results showed that different concentrations of 10D6 could not effectively block the binding of Biotin-labeled 8F2 to L929/B7-DC. Therefore, the epitope recognized by 10D6 is different from that of 8F2 (Fig. 3).

The above two hybridoma cell lines were preserved, and the preservation information of the hybridoma cell line SIPD-L2 (8F2) is: preservation number: CGMCC No. 4789, classification name: secretion of mouse anti-human PD-L2 molecule monoclonal antibody hybridization Tumor cell line, preservation date: April 27, 2011, preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation address: No. 13, North Zhongguancun, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; hybridoma cells The preservation information of strain SIPD-L2 (10D6) is: preservation number: CGMCC No. 4790, classification name: hybridoma cell line secreting mouse anti-human PD-L2 molecule monoclonal antibody, preservation date: April 27, 2011, Preservation Unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, Preservation Address: No. 13, North Yiyi Road, Zhongguancun, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

Embodiment two:

A kit capable of quantitatively detecting soluble B7-DC, comprising the following components:

(1) Coating antibody: mouse anti-human B7-DC monoclonal antibody (8F2), 30μg/tube, 1 tube;

(2) Standard protein: B7-DC Ig protein (R&D), 25ng/tube, 1 tube;

(3) Detection antibody: mouse anti-human B7-DC monoclonal antibody (Biotin-10D6), 10 μg/tube, 1 tube;

(4) Streptavidin-HRP (Sigma-Aldrich), 1μl//tube, 1 tube;

(5) TMB (Sigma-Aldrich), 10ml;

(6) BSA (Shanghai Sangong), 2g/100ml, 30ml;

(7) ELISA plate (Costar), 1 piece;

(8) Lotion: 10×PBS, 100 ml; Tween-20, 0.5 ml;

(9) Stop solution: 2M H ₂ SO ₄ , 5 ml;

Storage conditions:

Embodiment three:

Analysis of the specificity of the human soluble B7-DC ELISA kit described in Example 2:

B7.1Ig, OX40Ig, B7-DCIg and B7-H4Ig were serially diluted to different concentrations. Anti-human B7-DC monoclonal antibody (8F2) was adjusted to 3 μg/ml with carbonate buffer (0.01M CBS, pH9.3) to coat the ELISA detection plate, overnight at 4°C. Wash 3 times with PBS (containing 0.1% Tween 20), and block with 2% BSA for 1 h at room temperature. After washing with PBS for 3 times, the above-mentioned diluted commercial protein was added, reacted at room temperature for 2 hours, and washed with PBS for 3 times. Then, add biotin-labeled monoclonal antibody biotin-10D6 (1 μg/ml, 100 μl/well), continue to react at room temperature for 1 hour, wash with PBS for 3 times, add Streptavidin-HRP (1:3000, 100 μl/well), and react for 1 hour at room temperature , washed 6-8 times with PBS, then added HRP reaction substrate TMB (100μl/well), reacted at room temperature for 10-15min, stopped the reaction with 2mol/L H ₂ SO ₄ , measured OD ₄₅₀ with a microplate reader, and set 3 for each sample multiple holes.

The specificity analysis results are shown in Figure 4.

Embodiment four:

Quantitative analysis of the concentration of soluble B7-DC protein factors in human serum liquid using the kit for quantitatively detecting soluble B7-DC described in Example 2

(1) Sample handling:

Serum samples: 2500 rpm×10 min, store at -20°C, avoid repeated freezing and thawing.

(2) Operation steps:

1. Prepare the coating antibody 8F2 with 0.01M CBS (pH9.6) buffer solution to make a coating antibody working solution with a concentration of 2.5μg/ml, add 100μl/well to the ELISA plate, and let stand overnight at 4°C;

2. Discard the coating solution, wash the plate 3 times with 0.01M PB; add 1% BSA 200μl/well, block at room temperature for 1h;

取7个EP管，每管加入100μl稀释液并置于EP管架上；

将100μl 250ng/ml B7-H1Ig纯品蛋白加到第一个EP管，混匀后再取100μl到第二个EP管进行倍比稀释，依此类推至第7管。血清样品按100μl/孔加入ELISA板；

检测抗体Biotin-10D6用抗体稀释液调整至终浓度为0.5μg/ml； 3. Discard the blocking solution and wash the plate 3 times with 0.01M PB; use PBS (containing 0.5% BSA) as the sample diluent to prepare the sample for measurement. Standard protein:

Take 7 EP tubes, add 100 μl of diluent to each tube and place on the EP tube rack;

Add 100μl 250ng/ml pure B7-H1Ig protein to the first EP tube, mix well, then transfer 100μl to the second EP tube for doubling dilution, and so on to the seventh tube. Serum samples were added to the ELISA plate at 100 μl/well;

The detection antibody Biotin-10D6 was adjusted to a final concentration of 0.5 μg/ml with antibody diluent;

4. Add 100 μl/well of standard substance and sample to be tested, and react at room temperature for 2 hours;

5. Wash the plate 3 times with 0.01M PB after discarding the sample; add detection antibody Biotin-10D6. Streptavidin-HRP (1:3000), 100 μl/well, react at room temperature for 1 hour;

6. Discard the reaction solution and wash the plate 5-6 times with 0.01M PB (containing 0.2% Tween20); add Streptavidin-HRP (1:3000), 100μl/well, and react at room temperature for 1h;

7. Discard the reaction solution and wash the plate 8-10 times with 0.01M PB (containing 0.2% Tween20);

8. Add the reaction substrate TMB, 100 μl/well, and react in the dark at room temperature for 10-15 minutes; 2M H ₂ SO ₄ to terminate;

9. Use a microplate reader to select the OD450 nm wavelength to measure the OD reading of each detection well of the ELISA plate;

(3) Result analysis:

1. The OD value of the standard product uses the GraphPad Software software to list the most suitable function equation with the best fitting curve;

2. Substitute the OD value of the sample/control well into the equation and multiply it by the corresponding dilution factor to calculate the corresponding concentration of the sample and control well;

3. Take the average value of multiple wells for the sample well;

(4) Examples of experimental applications:

1. Raw data:

2. Curve equation:

y = 0.0035x + 0.1276

^R2 = 0.9964

3. Prepare the standard curve as shown in Figure 5:

(5) Accuracy analysis:

method:

1. Intra-batch accuracy analysis: In the same experiment, three samples with known concentrations (100, 50, 25 ng/ml) were set up in 20 duplicate wells respectively, and sB7-DC was detected to analyze the accuracy of the kit ;

2. Inter-batch accuracy analysis: In this different laboratory, three samples with known concentrations (100, 50, 25ng/ml) were tested for sB7-DC to analyze the accuracy of the kit;

result:

Conclusion: The coefficient of variation CV<10%, confirming that the detection methods have good accuracy.

(6) Experiment example:

1. Analyze the expression level of sB7-DC in the peripheral serum of asthmatic patients in attack period and stable period, as shown in Figure 6;

2. Analyze the dynamic changes of sB7-DC in asthmatic patients before and after treatment, as shown in Figure 7.

In summary, the ELISA kit for specific determination of soluble B7-DC of the present invention can be used to diagnose and stage asthma, and evaluate the effect of asthma patients during clinical treatment. The expression of sB7-DC exists in the serum of asthmatic patients, and the expression level increases with the improvement of treatment until it reaches the level of healthy people. In addition to the monoclonal antibody against B7-DC molecules on the cell surface of the present invention, the described kit may also include a blood sample collection device and related solvents, buffers and standards.

Nucleotide and/or Amino Acid Sequence Listing

the

<110> Soochow University

<120> A human soluble B7-DC quantitative detection kit

<160> 8

<170> PatentIn version 3.5

the

<210> 1

<211> 420

<212> DNA

<213> Unknown

the

<400> 1

gtgcagctgc agcagtcagg agctgagctg atgaagcctg gggcctcagt gaagatatcc 60

tgcaaggcta ctggctacac attcagtagc tactggatag agtgggtaaa gcagaggcct 120

ggacatggcc ttgagtggat tggagagatt ttacctggaa gtggtagtac taactacaat 180

gagaagttca agggcaaggc cacattcact gcagatacat cctccaacac agcctacatg 240

caactcagca gcctgacatc tgaggactct gccgtctatt actgtgcaag aagcgattac 300

tacggtagtc tgtactactt tgactactgg ggccaaggca ccactctcac agtctcctca 360

gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc 420

the

<210> 2

<211> 344

<212> DNA

<213> Unknown

the

<400> 2

cagattgcga tgacccagtc tccagcaatc atgtctgcat ctccaggggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 120

acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cacccacgtt cggagggggg 300

accaagctgg aaataaaacg ggctgatgct gcaccaactg tatc 344

the

<210> 3

<211> 420

<212> DNA

<213> Unknown

the

<400> 3

gtcaagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatgtcc 60

tgcaaggctt ctggatacac cttcattgac tactacatga agtgggtgaa gcagagccat 120

gaaaagagcc ttgagtggat tggagatatt aatcctaaca atggtgatac tttctacaac 180

cagaagttca aggacaaggc cacattgact gtagacaaat cctccagcac agcctacatg 240

cagctcaaca gcctgacatc tgaggactct gcagtctatt actgtgcaag ggaatctagg 300

tacgccgcct ggtttgaatg ctggggccaa gggactctgg tcactgtctc tgcagccaaa 360

acaacaccccc catcagtcta tccactggcc cctgggtgtg gagatacaac tggttcctcc 420

the

<210> 4

<211> 381

<212> DNA

<213> Unknown

the

<400> 4

gatgacccag tctccaaatt catgtccact tcactaggag acagagtcag tttcgcctgc 60

aaggccagtc aggatgtggg tcctgctgta gcctggtgtc aagagaaacc aggacaatct 120

cctaaactac tgattactg ggcatccacc cggcacactg gagtccctga tcgcttcaca 180

ggcagtggat ctgggacaga tttcactctc accattatca atgtgcagtc tgaagacttg 240

gcagattatt tctgtcagca atatagcaac tatccgtaca cgttcggagg ggggaccaaa 300

ctggaaataa aacgggctga tgctgcacca actgtatcaa tctctagagg atccccgggt 360

accgagctcg aattcactgg c 381

the

<210> 5

<211> 140

<212> PRT

<213> Unknown

the

<400> 5

VQLQQSGAEL MKPGASVKIS CKATGYTFSS YWIEWVKQRP GHGLEWIGEI LPGSGSTNYN 60

EKFKGKATFT ADTSSNTAYM QLSSSLTSEDS AVYYCARSDY YGSLYYFDYW GQGTTLTVSS 120

AKTTAPSVYP LAPVCGDTTG 140

the

<210> 6

<211> 115

<212> PRT

<213> Unknown

the

<400> 6

QIAMTQSPAI MSASPGEKVT MTCSASSSVS YMHWYQQKSG TSPKRWIYDT SKLASGVPAR 60

FSGSGSGTSY SLTISSMEAE DAATYYCQQW SSNPPTFGGG TKLEIKRADA APTVS 115

the

<210> 7

<211> 140

<212> PRT

<213> Unknown

the

<400> 7

VKLQQSGPEL VKPGASVKMS CKASGYTFID YYMKWVKQSH EKSLEWIGDI NPNNGDTFYN 60

QKFKDKATLT VDKSSSTAYM QLNSLTSEDS AVYYCARESR YAAWFECWGQ GTLVTVSAAK 120

TTPPSVYPLA PGCGDTTGSS 140

the

<210> 8

<211> 127

<212> PRT

<213> Unknown

the

<400> 8

DDPVSKFMST SLGDRVSFAC KASQDVGPAV AWCQEKPGQS PKLLIYWAST RHTGVPDRFT 60

GSGSGTDFTL TIINVQSEDL ADYFCQQYSN YPYTFGGGTK LEIKRADAAP TVSISRGSPG 120

TELEFTG 127

Claims (5)

1. mouse-anti people B7-DC monoclonal antibody 8F2 is characterized in that, the heavy chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:5; The light chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:6.

2. mouse-anti people B7-DC monoclonal antibody 10D6 is characterized in that, the heavy chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:7; The light chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:8.

3. hybridoma cell strain is characterized in that, described grand antibody hybridoma cell strain is the hybridoma cell strain SIPD-L2 of secretion mouse anti human B7-DC molecule monoclonal antibody, and its preserving number is CGMCC No. 4789.

4. hybridoma cell strain is characterized in that, described grand antibody hybridoma cell strain is the hybridoma cell strain SIPD-L2 of secretion mouse anti human B7-DC molecule monoclonal antibody, and its preserving number is CGMCC No. 4790.

5. but the test kit of a detection by quantitative solubility B7-DC, comprise: this horseradish peroxidase-labeled, reaction substrate tetramethyl benzidine, bovine serum albumin, enzyme plate, standard substance albumen, washing lotion and stop buffer, it is characterized in that, described test kit also comprises: coated antibody and detection antibody, wherein, described coated antibody is the described mouse-anti people of claim 1 B7-DC monoclonal antibody 8F2; Described detection antibody is the described mouse-anti people of claim 2 B7-DC monoclonal antibody 10D6.

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