A b7-h3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification

Abstract

This invention discloses a method for detecting B7-H3 protein based on signal conversion and multiplex isothermal amplification of nucleic acids. The detection method involves adding the sample to be tested, an anti-B7-H3 antibody-DNA1 / DNA3 complex, an anti-B7-H3 antibody-DNA2 conjugate, a hairpin probe assembly, deoxyribonucleotide triphosphate, nicking endonuclease, DNA polymerase, and a fluorescent dye to a detection system buffer. After reaction, fluorescence detection is performed. This method enables rapid detection of B7-H3 protein. It uses fluorescence as the detection signal, exhibits high specificity and sensitivity, and achieves a single-tube, one-step detection method with only one sample addition, eliminating the need for opening the tube for cleaning.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a method for detecting B7-H3 protein based on signal conversion and nucleic acid multiplex isothermal amplification. Background Technology

[0002] B7-H3, a newly discovered member of the B7 family, was discovered in dendritic cells in 2001 by Chapvol et al. (Chapoval AI, Ni J, Lau JS, et al. B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production[J]. Nat Immunol, 2001, 2(3): 269-274.). It is widely expressed on activated T cells, B cells, monocytes, DC cells, and some tumor cells. B7-H3 has a dual role as a co-stimulatory / co-inhibitory immune checkpoint molecule. Existing studies have shown that B7-H3 can promote the activation of CD4+ and CD8+ T cells and increase the expression of cytokines such as IFN-γ by binding to unknown receptors on T cells, while there are also reports of inhibiting T cell activation. The B7-H3 molecule exists in both membrane-bound and soluble forms. Previous studies have confirmed the abnormally high expression of membrane-bound B7-H3 under inflammatory conditions, and subsequent experiments have also found significantly increased levels of sB7-H3 in patients with sepsis. Therefore, both membrane-bound and soluble B7-H3 molecules may be important inflammatory markers. However, no research has reported on whether there are functional differences or correlations between the two types of B7-H3 molecules. Furthermore, the abnormal expression of co-stimulatory molecules in the peripheral blood of patients with hereditary spastic paraplegia (HSP) is considered a key factor in the excessive activation of T lymphocytes in the body's immune system. Research on the role of B7-H3 molecules in this disease course has not yet been reported. Most B7-H3 detection relies on immune-related detection techniques, such as ELISA and immunohistochemistry. While these methods are highly sensitive, they are time-consuming, labor-intensive, and costly. Therefore, researchers have been striving to find rapid, sensitive, low-cost, and easy-to-use protein detection tools.

[0003] In recent years, many signal sensors have been used to convert the protein signal to be detected into a sequence-specific output DNA signal, which is then further amplified using DNA signal amplification methods. For example, Ghadiri's team designed a new nucleic acid detection strategy that amplifies the signal by converting the target nucleic acid into a pre-designed output DNA (Picuri J M, Frezza BM, Ghadiri M R. Universal translators for nucleic acid diagnosis[J]. J Am Chem Soc, 2009, 131(26):9368-9377.). In related technologies, a molecular translator based on binding-induced DNA strand displacement has also been developed for homogeneous detection of proteins, including platelet-derived growth factor BB (PDGF BB) and prostate-specific antigen (PSA). These detection methods all involve singlet nucleic acid signal amplification, resulting in weak amplification capacity and low detection sensitivity. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification. After converting the protein signal into a DNA signal using a signal converter, multiplex nucleic acid amplification is performed using exponential amplification reaction (EXPAR) and dendritic hybridization chain reaction (D-HCR). This method greatly improves detection sensitivity, is highly efficient, and requires only one sample loading throughout the entire process.

[0005] According to one aspect of the present invention, a method for detecting B7-H3 protein is provided, the detection method comprising:

[0006] S1: Add the sample to be tested, anti-B7-H3 antibody-DNA1 / DNA3 complex, anti-B7-H3 antibody-DNA2 conjugate, hairpin probe set, deoxyribonucleotide triphosphate, nicking endonuclease, DNA polymerase and fluorescent dye to the detection system buffer; the detection buffer contains: 40-60mM potassium acetate, 10-30mM trihydroxyaminomethane acetate, 5-15mM magnesium acetate, and 50-150μg / mL recombinant albumin.

[0007] S2: After reacting at 35-40℃ for 80-100 minutes, fluorescence detection is performed.

[0008] In some embodiments of the present invention, the anti-B7-H3 antibody-DNA1 / DNA3 complex is obtained by mixing and incubating an anti-B7-H3 antibody-DNA1 conjugate and DNA3; the nucleotide sequence of the DNA3 is:

[0009] DNA3: 5'-GATACGGCTGAGG CCTACGTACGAA-3' (SEQ ID NO. 3).

[0010] In some embodiments of the present invention, the preparation steps of the anti-B7-H3 antibody-DNA1 / DNA3 complex include: adding the anti-B7-H3 antibody-DNA1 conjugate and DNA3 to the detection system buffer, incubating at 50-60°C for 3-7 minutes, then allowing it to cool naturally to 20-30°C, and then incubating for another 100-140 minutes to obtain the anti-B7-H3 antibody-DNA1 / DNA3 complex.

[0011] In some specific embodiments of the present invention, the detection buffer contains: 50 mM potassium acetate, 20 mM trihydroxyaminomethane acetate, 10 mM magnesium acetate, and 100 μg / mL recombinant albumin.

[0012] In some specific embodiments of the present invention, the CAS number of the trihydroxyaminomethane acetate is 6850-28-8.

[0013] In some embodiments of the present invention, the anti-B7-H3 antibody-DNA1 conjugate is obtained by conjugating DNA1 with a B7-H3 monoclonal antibody in a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; the nucleotide sequence of the DNA1 is as follows:

[0014] DNA1: 5'-NH2-C6-TTTTTTTTTTTTTTTTCGTACGTAGG-3' (SEQ ID NO. 1).

[0015] In this context, NH2-C6 represents amino modification, where NH2 is an amino group and C6 is the 6th carbon group at the glycosyl position of the nucleic acid.

[0016] In some embodiments of the present invention, the preparation steps of the anti-B7-H3 antibody-DNA1 conjugate include: conjugating DNA1 with the anti-B7-H3 monoclonal antibody in a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0017] In some embodiments of the present invention, the preparation steps of the anti-B7-H3 antibody-DNA1 conjugate are as follows: EDC and NHS are mixed in water and reacted; the above mixed solution is added to carbonate buffer containing anti-B7-H3 antibody and DNA1 and reacted, the solution is centrifuged and rinsed with water; the supernatant is discarded, and the precipitate is resuspended with rCutSmar buffer; the final protein concentration is determined using the Pierce BCA protein detection kit.

[0018] In some embodiments of the present invention, the anti-B7-H3 antibody-DNA2 conjugate is obtained by conjugating DNA2 with a B7-H3 monoclonal antibody in a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; the nucleotide sequence of the DNA2 is as follows:

[0019] DNA2: 5'-CCTACGTACGAATTTTTTTTTTTTTTTT-C6-NH2-3' (SEQ ID NO. 2).

[0020] In this context, C6-NH2 represents amino modification, where NH2 is an amino group and C6 is the 6th carbon group at the glycosyl position of the nucleic acid.

[0021] In some embodiments of the present invention, the preparation steps of the anti-B7-H3 antibody-DNA2 conjugate are the same as those of the preparation steps of the anti-B7-H3 antibody-DNA1 conjugate described above, except that DNA1 is replaced with an equal amount of DNA2, while other components and steps remain unchanged.

[0022] In some embodiments of the present invention, the hairpin probe set comprises hairpin probe 1, hairpin probe 2, and hairpin probe 3; the nucleotide sequences of hairpin probes 1 to 3 are as follows:

[0023] Hairpin probe 1: 5'-TTTGATACGGCTGCGTCGTAGGCCTCAGCCGTATCAAA-3' (SEQ ID NO.4);

[0024] Hairpin probe 2: 5'-TTTGATACGGCTGTGTCGTAGACCATGCTACGACACAGCCGT-3' (SEQ ID NO. 5);

[0025] Hairpin Probe 3:

[0026] 5'-CTACGACACAGCCGTATCAAACTACGACACAGCCGTATCAAAGCTGTGTCGTAGACGGCTGTGTCGTAGCATGGT-3' (SEQ ID NO. 6).

[0027] In some embodiments of the present invention, the nicking endonuclease is Nb.BbvCI nicking endonuclease.

[0028] In some embodiments of the present invention, the DNA polymerase is Bst 2.0 warmstart DNA polymerase.

[0029] In some embodiments of the present invention, the fluorescent dye is SYBR Green I.

[0030] In some embodiments of the present invention, the detection system of the detection method is as follows:

[0031]

[0032] In some specific embodiments of the present invention, the detection system of the detection method is as follows:

[0033]

[0034]

[0035] In some embodiments of the present invention, the fluorescence detection can be performed using conventional detection methods in the art, such as a quantitative PCR instrument, a fluorescence spectrophotometer, a fluorescence microplate reader, or by visual detection under excitation light of a specific wavelength.

[0036] In some specific embodiments of the present invention, the fluorescence intensity detection method is the endpoint method, and the equipment used is a fluorescence spectrophotometer.

[0037] In some specific embodiments of the present invention, the excitation peak in the fluorescence detection is 480 nm.

[0038] In some specific embodiments of the present invention, the emission peak recorded in the fluorescence detection is 524 nm.

[0039] A second aspect of the present invention provides a detection reagent comprising the anti-B7-H3 antibody-DNA1 / DNA3 complex, the anti-B7-H3 antibody-DNA2 conjugate, and the hairpin probe assembly described in the first aspect of the present invention.

[0040] In some embodiments of the present invention, the detection reagent also contains an auxiliary agent.

[0041] In some embodiments of the present invention, the adjuvants include: deoxyribonucleotide triphosphates (dNTPs), Nb.BbvCI nicking endonuclease, Bst 2.0 warmstart DNA polymerase, 1×SYBR Green I fluorescent dye, and detection system buffer.

[0042] A third aspect of the present invention provides a detection kit comprising the detection reagent described in the second aspect.

[0043] A fourth aspect of the present invention provides the application of the detection reagents described in the second aspect and the detection kits described in the third aspect of the present invention in the detection of B7-H3 protein.

[0044] In some embodiments of the present invention, the application includes the detection of B7-H3 protein in vivo and / or in vitro.

[0045] The beneficial effects of this invention are as follows:

[0046] 1. This invention provides a method for detecting B7-H3 protein, which is simple to operate and can achieve rapid detection of B7-H3 protein within two hours.

[0047] 2. The protein detection method in this invention uses fluorescence as the detection signal and can be combined with equipment such as real-time quantitative PCR instrument, fluorescence spectrophotometer, and fluorescence microplate reader to achieve the determination. It can also be visualized under excitation light of a specific wavelength.

[0048] 3. The method in this invention has good specificity for detecting B7-H3 protein, is not easily affected by other proteins, and can specifically identify B7-H3 protein from similar proteins (such as B7 family proteins such as B7-H1, B7-H2, B7-H4, B7-H5, B7-H6, etc.).

[0049] 4. The detection method in this invention has high sensitivity and can achieve quantitative detection of B7-H3 protein. The detection limit of this method can reach 100 fg / mL, and the linear range is 100 fg to 1 μg / mL.

[0050] 5. The detection method of the present invention can realize single-tube one-step detection, one sample addition, and no need for steps such as opening the cap for cleaning in the middle. Attached Figure Description

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0052] Figure 1 This is a schematic diagram illustrating the detection principle of B7-H3 protein based on signal conversion and nucleic acid multiplex isothermal amplification in an embodiment of the present invention.

[0053] Figure 2 The fluorescence emission spectra (510nm-600nm) of B7-H3 protein at different concentrations are shown in the embodiments of the present invention.

[0054] Figure 3The linear range of B7-H3 protein determination based on signal conversion and nucleic acid multiple isothermal amplification in this embodiment of the invention;

[0055] Figure 4 The figures show the fluorescence intensity of different proteins and the control group in the embodiments of the present invention. Figure A is an actual exposure image of different proteins and the control group, and Figure B is a fluorescence signal intensity image of different proteins and the control group when the emission peak is 524 nm.

[0056] Figure 5 This is a scatter plot of the recovery test of B7-H3 protein in 20% serum and saliva in the embodiments of the present invention;

[0057] Figure 6 This is a comparison of the results of the B7-H3 protein detection method and the control detection method (ELISA) in the clinical specimens in this embodiment of the invention;

[0058] Figure 7 This is a scatter plot of fluorescence intensity under different concentration gradients in an embodiment of the present invention. Detailed Implementation

[0059] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0060] Unless otherwise specified, all other experimental materials and reagents used in the following examples are consumables and reagents that are routinely available from commercial sources.

[0061] A method for detecting B7-H3 protein based on signal conversion and multiplex isothermal amplification of nucleic acids

[0062] This embodiment presents a method for detecting B7-H3 protein based on signal conversion and nucleic acid multiplex isothermal amplification. The specific method steps include:

[0063] 1. Preparation of anti-B7-H3 antibody-DNA1 and anti-B7-H3 antibody-DNA2 conjugates

[0064] DNA1 and DNA2 were conjugated with anti-B7-H3 monoclonal antibody via a mixed solution system of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), respectively. The specific steps are as follows:

[0065] (1) Mix 20.6 mg EDC and 11.5 mg NHS in 1 mL of water and react for 10 minutes. Take 10 μL of the EDC and NHS mixture and add it to 200 μL of carbonate buffer (pH 11.0) containing 13.5 mg / mL anti-B7-H3 monoclonal antibody (purchased from Merck Ltd.) and 10 μM DNA1. React for 2 hours.

[0066] (2) Centrifuge the above solution after reaction at 4000 rpm for 5 minutes, add 200 μL of water and centrifuge twice at 4000 rpm for 5 minutes;

[0067] (3) Discard the supernatant and resuspend the precipitate in rCutSmar buffer (purchased from New England Biotechnology Co., Ltd.) to a final volume of 200 μL to obtain the anti-B7-H3 antibody-DNA1 conjugate;

[0068] (4) The final protein concentration was determined using the Pierce BCA protein assay kit.

[0069] In this method, the anti-B7-H3 antibody-DNA2 conjugate can be prepared by replacing the DNA1 in the carbonate buffer containing 10 μM DNA1 with an equal amount of DNA2, while keeping other components and steps unchanged.

[0070] 2. Preparation of the detection system buffer

[0071] Prepare a buffer solution containing: 50 mM potassium acetate, 20 mM trihydroxymethyl acetate, 10 mM magnesium acetate, and 100 μg / mL recombinant albumin (purchased from New England Biotechnology Co., Ltd.).

[0072] The preparation steps of the detection system buffer are as follows: 410 mg of anhydrous potassium acetate, 410 mg of trihydroxyaminomethane acetate and 10 mg of recombinant albumin are added sequentially to 100 mL of deionized water. After thorough mixing, the solution is filtered through a sterile filter membrane with a pore size of 0.22 μm and stored at 4 °C for later use.

[0073] 3. Preparation of anti-B7-H3 antibody-DNA1 / DNA3 complex

[0074] Add 3 μM of anti-B7-H3 antibody-DNA1 conjugate and 2 μM of DNA3 to the above detection system buffer, incubate at 55°C for 5 minutes, then allow to cool naturally to 25°C, and incubate for another 120 minutes to obtain the anti-B7-H3 antibody-DNA1 / DNA3 complex.

[0075] 4. Detection of B7-H3 protein fluorescence

[0076] The reaction system was prepared in a laboratory environment at room temperature (25±2℃) (as shown in Table 1), and then the reaction was carried out at 37℃ for 90 minutes before fluorescence detection was performed. In this invention, the endpoint method was used for detection. The fluorescence spectrophotometer used in the fluorescence detection was purchased from PerkinElmer Instruments Ltd.

[0077] Table 1 Detection reaction system

[0078]

[0079]

[0080] Among them, the anti-B7-H3 monoclonal antibody was purchased from Merck Ltd., DNA1, DNA2, DNA3, hairpin probe 1, hairpin probe 2, and hairpin probe 3 were synthesized by Sangon Biotech Ltd., Bst 2.0 warmstart DNA polymerase and Nb.BbvCI nicking endonuclease were purchased from New England Biotech Ltd., and dNTPs were purchased from Sangon Biotech Ltd.

[0081] The nucleotide sequences of DNA1, DNA2, DNA3, H1, H2, and H3 are shown below:

[0082] DNA1: 5'-NH2-C6-TTTTTTTTTTTTTTTTTCGTACGTAGG-3' (SEQ ID NO. 1);

[0083] DAN2: 5'-CCTACGTACGAATTTTTTTTTTTTTTTT-C6-NH2-3' (SEQ ID NO. 2);

[0084] DNA3: 5'-GATACGGCTGAGG CCTACGTACGAA-3 (SEQ ID NO. 3);

[0085] Hairpin probe 1 (H1):

[0086] 5'-TTTGATACGGCTGCGTCGTAGGCCTCAGCCGTATCAAA-3' (SEQ ID NO. 4);

[0087] Hairpin probe 2 (H2):

[0088] 5'-TTTGATACGGCTGTGTCGTAGACCATGCTACGACACAGCCGT-3' (SEQ ID NO. 5);

[0089] Hairpin probe 3 (H3):

[0090] 5'-CTACGACACAGCCGTATCAAACTACGACACAGCCGTATCAAAGCTGTGTCGTAGACGGCTGTGTCGTAGCATGGT-3' (SEQ ID NO. 6).

[0091] The sequences of DNA1 and DNA2 are modified with amino groups, specifically by linking the NH2 group to the 6th carbon group (C6 group) of the glycosyl group of the corresponding nucleic acid.

[0092] The technical principle of the detection method of the present invention is as follows:

[0093] like Figure 1 As shown in the detection principle diagram, the protein signal transducer consists of target recognition elements (anti-B7-H3 antibody-DNA1 conjugate and anti-B7-H3 antibody-DNA2 conjugate) and a signal output element (DNA3): DNA1 conjugated with the anti-B7-H3 antibody first hybridizes with DNA3 to form a stable DNA1 / DNA3 dimer structure. DNA3 is designed to be positioned at its 5' end ( Figure 1 There is a nicking endonuclease recognition site near the green area in the image.

[0094] The DNA2 sequence was designed to be complementary to the DNA1 sequence.

[0095] Based on the presence or absence of the target protein B7-H3, the following two cases apply:

[0096] (1) In the presence of the target protein B7-H3, the same B7-H3 protein binds to two antibodies conjugated to DNA1 and DNA2, assembling DNA1 and DNA2 together in the conjugate, thereby increasing their local effective concentration. This process raises the melting temperature of intramolecular hybridization (DNA1 and DNA2), which is beneficial for the strand substitution reaction between DNA2 and DNA3. As a result, the output DNA3 is released to further amplify the detected protein signal.

[0097] (2) In the absence of target protein B7-H3, the strand substitution activity between DNA2 and DNA3 is poor at 37℃, and the ability of competing DNA2 to release output DNA3 is extremely limited, so no protein signal can be released.

[0098] Subsequently, the three hairpin probes added to the detection system, H1, H2, and H3, were subjected to multiple signal amplification using exponential isothermal amplification (EXPAR) and dendritic hybridization chain reaction (D-HCR), respectively.

[0099] The two scenarios corresponding to the presence or absence of the aforementioned target protein B7-H3 are as follows:

[0100] (1) In the presence of the target protein B7-H3, the hairpin probe outputs DNA3, which recognizes and partially complements H1 to form a double-stranded DNA structure. This partially complementary double-stranded DNA contains a complete nicking endonuclease recognition site near its 5' end. EXPAR is initiated in the presence of nicking enzyme and polymerase. NB.BbvCI nicking endonuclease, Bst2.0 warmstart DNA polymerase, and dNTPs generate a large amount of single-stranded DNA (D-HCR triggered DNA) in the cycle of nicking, polymerization, and strand displacement. The resulting ssDNA recognizes and partially hybridizes with H2, causing a conformational change in the hairpin probe and opening the stem structure to form the H2-ssDNA intermediate. The H2-ssDNA intermediate then exposes the complementary sequence to H3, thereby generating the ssDNA-H2-H3 multimer. Simultaneously, the generated ssDNA-H2-H3 multimer exposes two complementary sequences that bind to H2. This intermediate can then bind to both H2 molecules and open the H3 binding sites, amplifying the signal as the hairpin structure continuously opens. Finally, a large DNA product composed of double strands is generated, which, after binding to the fluorescent dye SYBR Green I, can be detected by recording the fluorescence signal using a fluorescence spectrophotometer.

[0101] (2) In the absence of target protein B7-H3, the hairpin probe cannot convert the protein signal into a DNA signal through the signal converter, thus it cannot generate a large amount of large double-stranded DNA and ultimately cannot generate a fluorescent signal.

[0102] Sensitivity evaluation of the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification:

[0103] This embodiment tests the sensitivity of the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification described in this application. The specific process is as follows:

[0104] With other experimental conditions unchanged, the detection method was the same as the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification described above. The B7-H3 protein concentration gradient was set at 0 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, and 10 μg / mL. The human B7-H3 protein used was purchased from Merck Ltd. The corresponding wavelengths and fluorescence intensities were measured.

[0105] Fluorescence emission spectroscopy results are as follows Figure 2 As shown, where Figure 2 The X and Y axes represent wavelength and fluorescence intensity, respectively. As the concentration of the target protein increases, the fluorescence intensity at 524 nm gradually increases. The excitation peak is at 495 nm, and the recorded emission peak is at 524 nm.

[0106] Linear range results are as follows Figure 3 As shown, using the logarithms of eight concentration gradients (100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL) as the independent variable X, and the fluorescence intensity at 524 nm as the dependent variable Y, a linear regression equation was plotted. The obtained linear regression equation is y = 20.4 + 42.81x, with a correlation coefficient R. 2 =0.997, indicating that the detection method based on signal conversion and nucleic acid multiplex isothermal amplification shows good linearity in the range of 100 fg / mL-1 μg / mL for B7-H3 protein. The limit of detection (LOD) is 100 fg / mL, calculated as the concentration obtained by converting the fluorescence signal of the negative control at 524 nm, which is three times the fluorescence signal of the negative control. Based on the molecular weight of human B7-H3 (CD276) of 49837.82 (49.83 kDa), the minimum detectable amount is 200 aM.

[0107] Specificity evaluation of the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification:

[0108] This embodiment verifies the specificity of the B7-H3 protein detected by the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification in this application. The specific process is as follows:

[0109] Under otherwise unchanged experimental conditions, the detection method was the same as described above for B7-H3 protein detection based on signal conversion and nucleic acid multiplex isothermal amplification. The following proteins were detected: B7-H3 protein, B7 family-related proteins B7-H1, B7-H2, B7-H4, B7-H5, and B7-H6, and a negative control (NC) group. Different proteins and the negative control were grouped, with the target protein concentration in the NC group being 0 μg / mL, and the concentration in the other groups being 10 μg / mL. The human B7-H1, B7-H2, B7-H4, B7-H5, and B7-H6 proteins used were purchased from Merck Ltd. In the negative control NC, an equal volume of deionized water replaced the protein in the standard solutions of the other proteins; the remaining components were consistent with the composition of the standard solutions of the other proteins.

[0110] The results are as follows Figure 4As shown in Figure A, the proteins detected, from left to right, correspond to B7-H3 protein, B7 family-related proteins such as B7-H1, B7-H2, B7-H4, B7-H5, and B7-H6, and the negative control NC. The fluorescence signal intensity of B7-H3 protein is significantly higher than that of the other groups. In Figure B, the X and Y axes of the quantitative value graph represent the target protein category and the fluorescence intensity at 524 nm, respectively. Comparing the fluorescence intensities of different groups, it can be found that the fluorescence signal of B7-H3 protein is significantly higher than that of B7 family-related proteins such as B7-H1, B7-H2, B7-H4, B7-H5, and B7-H6, and the fluorescence intensity of the NC group, with a signal difference of up to 10 times or more. The experimental results in both figures are consistent, indicating that the detection method described in this invention can specifically detect B7-H3 protein and has strong specificity for similar proteins.

[0111] Evaluation of protein recovery efficiency of a B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification:

[0112] This embodiment verifies the protein recovery effect in the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification in this application. The specific process is as follows:

[0113] Under the condition that other experimental conditions remain unchanged, the detection method refers to the B7-H3 protein detection method based on signal conversion and nucleic acid multiple isothermal amplification described above. 20% serum and saliva samples with B7-H3 protein concentrations of 100 ng / mL, 1 ng / mL, and 10 pg / mL were pre-set. The same detection method was used to detect samples of different concentrations, and the results were compared with the concentrations of samples with added non-serum and non-saliva to calculate the corresponding protein recovery amount and recovery rate.

[0114] Table 1. B7-H3 protein recovery test in 20% serum and saliva, respectively.

[0115] sample Standard concentration Protein recovery Recovery rate Relative standard deviation 20% serum 100ng / mL 106.9 ng / mL 106.9％ 2.62％ 20% serum 1ng / mL 1.054 ng / mL 105.4％ 4.64％ 20% serum 10 pg / mL 9.057 pg / mL 90.57％ 8.20％ saliva 100ng / mL 89.42 ng / mL 89.42％ 3.32％ saliva 1ng / mL 1.096 ng / mL 109.6％ 3.25％ saliva 10 pg / mL 11.22 pg / mL 112.2％ 8.28％

[0116] All data in the table represent the average of five measurements.

[0117] The results are as follows Figure 5 As shown in Table 1, in the protein recovery experiment, the recovery rates of 20% of serum and saliva samples with pre-set B7-H3 protein concentrations of 100 ng / mL, 1 ng / mL, and 10 pg / mL were between 89.42% and 112.2%, with relative standard deviations within 8.28%. These results indicate that the detection method is stable in 20% of serum and saliva samples and is not affected by complex components in the samples, showing potential for clinical application.

[0118] Comparison of B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification with existing detection technologies:

[0119] This embodiment verifies the difference in detection performance between the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification described in this application and the existing detection technique ELISA (ELISA kit purchased from Ruixin Biotechnology Co., Ltd.). The test results for soluble B7-H3 protein in 18 clinical serum samples (from Dalang Hospital, Dongguan City) were analyzed. The specific process is as follows:

[0120] The method for detecting B7-H3 protein based on signal conversion and multiple isothermal amplification of nucleic acids is described in Example 1. The control method is performed according to the instructions of the ELISA kit from Ruixin Biotechnology Co., Ltd. The soluble B7-H3 protein content of 18 clinical specimens was detected.

[0121] Test results as follows Figure 6 As shown, the detection method of this application yielded results that were basically consistent with the control method, with good clinical relevance. The linear regression equation was obtained as y = -1.907 + 1.032x, and the correlation coefficient R0 was [value missing]. 2 =0.9881. Therefore, the detection method of the present invention has high clinical application value.

[0122] Stability assessment of a B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification:

[0123] This embodiment verifies the stability of the B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification in this application. The specific process is as follows:

[0124] Prepare the anti-B7-H3 antibody-DNA1 / DNA3 complex, anti-B7-H3 antibody-DNA2 conjugate, hairpin probe kit, and other detection components. Mix the detection components according to the above detection method to prepare the detection reagent. After storing at 4℃ for 0, 1, 3, 6, 9, 12, and 15 days, detect six gradient concentrations of B7-H3 protein standard reagent at concentrations of 0 fg / mL, 100 fg / mL, 10 pg / mL, 1 ng / mL, 100 ng / mL, and 1 μg / mL, respectively. Under the same experimental conditions, the detection method refers to the steps in the above-described B7-H3 protein detection method based on signal conversion and nucleic acid multiplex isothermal amplification.

[0125] Depend on Figure 7 As shown, after storage at 4℃ for 0-15 days, the fluorescence intensity of the six gradients of the above-mentioned detection reagent showed no significant difference, and the fluorescence intensity was less than 10% in all cases. This result indicates that the detection reagent prepared in this invention exhibits good stability within 15 days.

[0126] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for detecting B7-H3 protein, characterized in that, The detection method includes the following steps: S1: Add the sample to be tested, anti-B7-H3 antibody-DNA1 / DNA3 complex, anti-B7-H3 antibody-DNA2 conjugate, hairpin probe set, deoxyribonucleotide triphosphate, nicking endonuclease, DNA polymerase and fluorescent dye to the detection system buffer; the detection system buffer contains: 40-60mM potassium acetate, 10-30mM trihydroxyaminomethane acetate, 5-15mM magnesium acetate, and 50-150μg / mL recombinant albumin; S2: After reacting at 35-40℃ for 80-100 minutes, fluorescence detection is performed; The nucleotide sequences of DNA1, DNA2, and DNA3 are shown below: DNA1: 5'-NH2-C6-TTTTTTTTTTTTTTTTCGTACGTAGG-3'; DNA2: 5'-CCTACGTACGAATTTTTTTTTTTTTTTT-C6-NH2-3'; DNA3: 5'-GATACGGCTGAGG CCTACGTACGAA-3'; The hairpin probe set consists of hairpin probe 1, hairpin probe 2, and hairpin probe 3; the nucleotide sequences of hairpin probes 1 to 3 are as follows: Hairpin probe 1: 5'-TTTGATACGGCTGCGTCGTAGGCCTCAGCCGTATCAAA-3'; Hairpin probe 2: 5'-TTTGATACGGCTGTGTCGTAGACCATGCTACGACACAGCCGT-3'; Hairpin Probe 3: 5'-CTACGACACAGCCGTATCAAACTACGACACAGCCGTATCAAAGCTGTGTCGTAGACGGCTGTGTCGTAGCATGGT-3'.

2. The detection method according to claim 1, characterized in that, The anti-B7-H3 antibody-DNA1 / DNA3 complex was obtained by mixing and incubating an anti-B7-H3 antibody-DNA1 conjugate and DNA3.

3. The detection method according to claim 2, characterized in that, The anti-B7-H3 antibody-DNA1 conjugate was obtained by conjugating DNA1 with B7-H3 monoclonal antibody in a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

4. The detection method according to claim 1, characterized in that, The anti-B7-H3 antibody-DNA2 conjugate was obtained by conjugating DNA2 with B7-H3 monoclonal antibody in a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

5. The detection method according to claim 1, characterized in that, The detection system of the detection method is as follows:

6. A detection reagent, characterized in that, The detection reagent contains the anti-B7-H3 antibody-DNA1 / DNA3 complex, the anti-B7-H3 antibody-DNA2 conjugate, and the hairpin probe set as described in claim 1.

7. A test kit, characterized in that, The test kit contains the test reagent as described in claim 6.

8. The application of the detection reagent of claim 6 or the detection kit of claim 7 in the detection of B7-H3 protein.

9. The application according to claim 8, characterized in that, The applications include the detection of B7-H3 protein in vivo and / or in vitro.

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