Application of serum SIRT3 as pulmonary arterial hypertension biomarker

Abstract

The invention discloses application of a silence information regulation factor SIRT3 in preparation of a pulmonary arterial hypertension detection reagent and further provides a pulmonary arterial hypertension detection kit, and pulmonary arterial hypertension can be diagnosed by detecting the SIRT3 level in serum.

Description

Technical Field

[0001] The present invention belongs to the field of clinical medical diagnostic tools, and specifically relates to the application of silent information regulator 3 (SIRT3) in the preparation of pulmonary arterial hypertension detection reagents, and also relates to a kit for detecting pulmonary arterial hypertension. Background Art

[0002] Pulmonary arterial hypertension (PAH) is called the "cancer" of the pulmonary circulatory system and has a high mortality rate. Especially for idiopathic pulmonary arterial hypertension (IPAH), if not treated with drugs, the survival period is only 3 years. However, as the international guidelines lower the pulmonary artery systolic pressure in the pulmonary arterial hypertension standard to 20 mmHg, the base of this group of people has increased. Among pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension and chronic thromboembolic pulmonary arterial hypertension (CTEPH) are important categories. Currently, the main means of diagnosing pulmonary arterial hypertension are right heart catheterization and echocardiography. Right heart catheterization is an invasive procedure that requires hospitalization for examination, which increases the burden on patients. In addition, patients cannot monitor pulmonary artery pressure multiple times in a short period of time through right heart catheterization. In addition, echocardiography estimates pulmonary artery pressure through the activity of the tricuspid valve leaflets, and its results are not accurate enough. In many grassroots hospitals, due to the limitations of echocardiography doctors, pulmonary artery systolic pressure cannot be calculated. Therefore, there is an urgent need to develop serum markers to monitor pulmonary artery pressure.

[0003] The literature (Research progress on the role of SIRT3 in the pathogenesis of pulmonary hypertension. Bao Boxin, et al. Chinese Journal of Medical Science, May 2023, Vol. 13, No. 9, pp. 63-66) reported that the silent information regulator 3 (SIRT3) located in mitochondria plays a certain role in regulating the formation and treatment of pulmonary hypertension and may become a new target for the treatment of pulmonary hypertension. The literature (The role of SIRT3 in cardiovascular disease. Xue Xiaofan, et al. The role of SIRT3 in cardiovascular disease. Advances in Physiological Sciences, 2019 (04): 256-258.) reported that SIRT3 plays a protective role in various cardiovascular diseases such as resisting oxidative stress response and improving endothelial cell function.

[0004] The Silent Information Regulator Sirtuins (SRIT) family is a class of enzymes classified as nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases. Among them, SIRT3, as a mitochondrial protein, plays a key role in regulating cell fate by regulating the acetylation of most mitochondrial lysines. SIRT3 has recently been found to be closely related to the proliferation and transformation of pulmonary artery smooth muscle cells. In addition, SIRT3 can regulate the important function of the proinflammatory response of cardiomyocytes by regulating the FOS / AP-1 pathway. However, the relationship between SIRT3 and the development, severity, and prognosis of pulmonary hypertension is still unclear. Summary of the invention

[0005] In the process of exploring SIRT3 and the occurrence, development and treatment of pulmonary hypertension, our research group found that there is a positive correlation between the serum SIRT3 level of patients with pulmonary hypertension and the severity of pulmonary hypertension, and the serum SIRT3 level is positively correlated with pulmonary arterial hypertension (PAH) diseases such as idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH), suggesting that serum SIRT3 level can be used as a biomarker for the diagnosis of pulmonary hypertension. There is no report on this research at home and abroad. Based on this research finding, the present invention includes the following technical solutions.

[0006] The first aspect of the present invention provides the use of silent information regulator 3 (SIRT3) in the preparation of a pulmonary arterial hypertension detection reagent.

[0007] In one embodiment, the pulmonary hypertension refers to idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH).

[0008] Preferably, in the above application, the SIRT3 refers to the SIRT3 protein in serum.

[0009] A second aspect of the present invention provides a pulmonary arterial hypertension detection kit for implementing the above application, wherein the detection target is silent information regulator 3 (SIRT3) in a biological sample.

[0010] Preferably, the above detection method is to measure the SIRT3 protein content in the biological sample.

[0011] In a preferred embodiment, the above-mentioned pulmonary arterial hypertension detection kit can use the antibody-antigen affinity binding principle to determine the antigen SIRT3 content in the biological sample. In this case, the kit at least includes:

[0012] (1) an antibody capable of binding to SIRT3, which may also be referred to herein as a SIRT3 first antibody or a SIRT3 primary antibody; and

[0013] (2) A standard sample consisting of a solution containing a known amount of SIRT3, wherein SIRT3 can be a natural SIRT3 protein extracted from humans or mammals or a recombinant SIRT3 protein produced by fermentation of genetically engineered bacteria.

[0014] In this case, the kit can detect SIRT3 by latex immunoturbidimetry using a SIRT3 antibody, or chemiluminescence method such as magnetic particle chemiluminescence method, or radioimmunoassay (RIA) using a radioactive element labeled SIRT3 antibody. That is, the kit can be a latex immunoturbidimetry kit, a magnetic particle chemiluminescence kit, or a radioimmunoassay kit, or an immunochromatographic test strip.

[0015] Optionally, the above-mentioned pulmonary hypertension detection kit may further include: (3) when SIRT3 binds to the antibody defined in (1), a labeled antibody that can bind to SIRT3, which may also be referred to as SIRT3 secondary antibody herein, and the label on the secondary antibody can catalyze or activate chemiluminescent compounds or fluorescent dyes, thereby quickly converting colorless substrates into colored products, or causing light changes, or converting non-fluorescent fluorescent dyes into strong fluorescent products, for example, the label is an enzyme (such as horseradish peroxidase or alkaline phosphatase); or the label on the second antibody is a chemiluminescent compound or fluorescent dye that can be catalyzed or activated to develop color or emit light, such as acridinium ester.

[0016] That is, the kit contains two different SIRT3 antibodies (1) and (3), one of which is a capture antibody for capturing antigen SIRT3, also known as the "first antibody" or "primary antibody", i.e., the antibody defined in item (1) above; the other is a detection antibody for detecting captured antigen SIRT3, also known as the "binding antibody" or "second antibody" or "secondary antibody", i.e., the antibody defined in item (3) above. These two antibodies successively bind to different antigenic epitopes (antigenic determinants) of the antigen SIRT3 protein to form a first antibody-antigen SIRT3 conjugate, and then the first antibody-antigen SIRT3 conjugate is combined with the second antibody to produce a "sandwich" structure of the first antibody-antigen-second antibody to implement the "double antibody sandwich method" to detect the antigen SIRT3 protein.

[0017] In this case, the pulmonary arterial hypertension detection kit is a double antibody sandwich kit, for example, selected from the following groups: A. enzyme-linked immunosorbent assay (ELISA) kit; B. magnetic particle chemiluminescence kit; C. immunochromatographic test strip.

[0018] The first antibody and the second antibody may be monoclonal antibodies or polyclonal antibodies. For example, the first antibody is a monoclonal antibody and the second antibody is a polyclonal antibody.

[0019] Optionally, the above-mentioned SIRT3 first antibody can be a first antibody labeled with quantum dots or fluorescent microspheres, and the above-mentioned SIRT3 first antibody can also be labeled with colloidal gold. Quantum dots are nanoparticles composed of semiconductor materials such as cadmium selenide CdSe. When quantum dots are excited by an ordinary light, they can release fluorescence that is tens of times stronger than ordinary fluorescein, and have the characteristics of a wide excitation spectrum and a narrow emission spectrum. The emission spectrum can also be adjusted according to the different semiconductor materials and the different sizes of nanoparticles, and the emission light has high stability and will not be quenched. It is precisely because of these advantages of quantum dot fluorescence that antibodies labeled with quantum dot nanoparticles can increase the luminescence intensity by hundreds of times compared to antibodies labeled with ordinary fluorescein, thereby making the signal sensitivity of the reagent reach the level of chemiluminescence.

[0020] Preferably, when the above kit is an ELISA kit, it further comprises:

[0021] (4) An initiator solution, used to initiate a chemical reaction between the marker on the SIRT3 secondary antibody and the chemiluminescent compound or color developing substrate or fluorescent dye that is a reaction target of the marker.

[0022] When the kit is an ELISA kit, preferably the SIRT3 primary antibody or the solid phase carrier for fixing the SIRT3 primary antibody, such as a multi-well plate or magnetic particles, is connected with a chemiluminescent compound or a fluorescent dye, thereby making the immunoassay method simpler and the quantification more accurate.

[0023] Preferably, the biological sample can be selected from the following group: whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretions, tissue fluid, body fluid, urine, such as peripheral venous blood.

[0024] Preferably, any of the above kits also includes instructions for use.

[0025] The instructions may be written on bottles, test tubes and the like, boards, or on a separate piece of paper, or on the outside or inside of a container, such as a paper with an operation demonstration video APP download window such as a QR code. The instructions may also be in multimedia form, such as a CD, U disk, network disk, etc.

[0026] Optionally, the instructions may also include a standard comparison table for distinguishing whether a patient has pulmonary hypertension, wherein the standard comparison table records a cutoff value or a cutoff value of serum SIRT3 concentration (SIRT3 serum level) for distinguishing pulmonary hypertension.

[0027] For example, the cutoff value for pulmonary arterial hypertension (PAH) can be 3.6 ng / ml. Above this cutoff value, PAH can be basically diagnosed, and below this cutoff value, PAH can be basically excluded. The cutoff value for idiopathic pulmonary arterial hypertension (IPAH) can be 4.1 ng / ml. Above this cutoff value, IPAH can be basically diagnosed, and below this cutoff value, IPAH can be basically excluded. The cutoff value for chronic thromboembolic pulmonary hypertension (CTEPH) can be 3.6 ng / ml, which is the same as the cutoff value for pulmonary arterial hypertension (PAH). Above this cutoff value, CTEPH can be basically diagnosed, and below this cutoff value, CTEPH can be basically excluded.

[0028] The present invention discovered for the first time that silent information regulator 3 (SIRT3) has a significant indicative effect on the occurrence and development of pulmonary hypertension and can be used as a biomarker for the disease. Therefore, the detection of SIRT3 can be used to evaluate patients' myocardial hypertrophy and cardiac function. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The statistical bar graph of serum SIRT3 content in people with pulmonary arterial hypertension (PAH, IPAH, CTEPH) and the normal group (Control) is shown.

[0030] Figure 2 The receiver operating curve (ROC curve) of serum SIRT3 for diagnosing different types of pulmonary arterial hypertension (PAH, IPAH, CTEPH) is shown. DETAILED DESCRIPTION

[0031] SIRT3 is also known as silent information regulator 2-related enzyme 3 (silent mating type information regulation 2 homolog-3). It is known that SIRT3 is mainly located in mitochondria and widely distributed in mitochondria-rich tissues and organs such as the kidney, brain, heart and liver. It can deacetylate histones and non-histones and play an important role in regulating cell metabolism, cell cycle, apoptosis and cell lifespan. SIRT3 regulates the biological activity of related target proteins by deacetylation, and plays a protective role in resisting oxidative stress response, improving endothelial cell function and many other cardiovascular diseases.

[0032] The present invention reveals the relationship between serum SIRT3 levels and pulmonary hypertension, proving that SIRT3 can be used as a biomarker for pulmonary hypertension in the diagnosis and prognosis of pulmonary hypertension. It is expected that the determination of serum SIRT3 protein content can assist in the diagnosis of pulmonary hypertension, and further assist in the diagnosis of idiopathic pulmonary hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH), thereby greatly facilitating the diagnosis of pulmonary hypertension.

[0033] In view of the use of SIRT3 as a biomarker for pulmonary hypertension, it is obvious to those skilled in the art that various types of reagent products such as kits can be developed as auxiliary diagnostic tools for pulmonary hypertension. For example, the kit forms that can be developed include but are not limited to: latex immunoturbidimetry kits containing only one SIRT3 antibody (monoclonal antibody or polyclonal antibody, preferably monoclonal antibody), chemiluminescence kits such as magnetic microparticle chemiluminescence kits, radioimmunoassay kits; enzyme-linked immunosorbent assay (ELISA) kits containing two different SIRT3 antibodies (monoclonal antibody and / or polyclonal antibody), magnetic microparticle chemiluminescence kits, immunochromatographic test strips, etc.

[0034] The SIRT3 used in the preparation of pulmonary arterial hypertension detection reagent can be a natural SIRT3 protein extracted from humans or mammals, or it can be a recombinant human SIRT3 protein produced by fermentation of genetically engineered bacteria. Preferably, the amino acid sequence of the SIRT3 protein is consistent with that of the human SIRT3 protein and maintains the same antigen recognition epitope / antigenic determinant cluster.

[0035] SIRT3 monoclonal antibodies and SIRT3 polyclonal antibodies can be commercial antibodies purchased from commercial channels, or they can be prepared using conventional technical means, such as using SIRT3, preferably human SIRT3, as an antigen. Typically, monoclonal antibodies can be prepared by immunizing animals with SIRT3 protein, and the steps include: after the protein SIRT3 is used as an immunogen and fully mixed with an equal volume of Freund's complete adjuvant, the animal, such as a mouse, is immunized subcutaneously at multiple points; after a few weeks, the serum titer is measured, and mice with good immune responsiveness are selected for multiple booster immunizations, and then spleen cells are taken and fused with myeloma cells using PEG mediation according to conventional methods, and selected and cultured with HAT conditioned medium; after fusion, CO is placed in the culture medium. 2After 9-11 days of culture at 37°C in an incubator, a large cell clone appeared in the well; screening was started by indirect ELISA on the 11th day; the wells with positive initial screening were cloned 4 times by limiting dilution method to make the screened cells multiply in large numbers, and then the cells were expanded, frozen, and ascites was prepared; the mice were treated with pristane, and hybridoma cells were inoculated into the abdominal cavity one week later, and ascites was collected 10 days later. The titer of the monoclonal antibody was determined by indirect ELISA method, and the monoclonal antibody with the required titer was obtained; after ammonium sulfate precipitation, the monoclonal antibody was purified by Protein G affinity purification, and the antibody was packaged and freeze-dried and stored at low temperature.

[0036] Similarly, polyclonal antibodies can be prepared by immunizing animals with SIRT3 protein, and the steps include: selecting rabbits as immunized animals; in basic immunization, mixing SIRT3 protein as an immunogen with an equal volume of Freund's complete adjuvant - after fully emulsifying, injecting it subcutaneously at multiple points on the back of the rabbit; boosting immunization once every 4 weeks, injecting it subcutaneously at multiple points on the back after the antigen is fully emulsified with incomplete Freund's adjuvant; bleeding from the carotid artery on the 10th day after the last boosting immunization, and separating the serum; determining the titer of the prepared polyclonal antibody by indirect ELISA, obtaining polyclonal antibodies with antibody titers that meet the requirements, cannulating the carotid artery to collect blood, and separating the serum; after ammonium sulfate precipitation, Protein G affinity purification is performed to separate and purify the monoclonal antibody, and the antibody is packaged and freeze-dried and stored at low temperature.

[0037] The use of SIRT3 monoclonal antibodies and SIRT3 polyclonal antibodies to detect antigenic SIRT3 proteins in biological samples is based on immunological techniques. There are many detection methods for immunological techniques known in the field of clinical medicine. In general, the level (content) of SIRT3 in a biological sample can be determined using a method selected from the following group: protein blotting; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; immunofluorescence assay; mass spectrometry; fluorescence activated cell sorting (FACS); immunoelectrophoresis assay. In some embodiments, the antibody or its antigen-binding portion is detectably labeled or produces a detectable signal.

[0038] In some embodiments, antibody reagent can be detectably labeled. In some embodiments, antibody reagent can be attached to a solid support (e.g., bound to a solid support). In some embodiments, a solid support can include particles (including but not limited to agarose or latex beads or particles or magnetic particles), beads, nanoparticles, polymers, substrates, slides, coverslips, plates, culture dishes, holes, films and / or gratings. Solid supports can include many different materials, including but not limited to polymers, plastics, resins, polysaccharides, materials based on silicon or silicon dioxide, carbon, metals, inorganic glass and films.

[0039] In one embodiment, the test, method and / or system as described herein may include an ELISA. In an exemplary embodiment, the first antibody reagent may be immobilized on a solid support (typically a polystyrene microtiter plate). The solid support may be contacted with a sample obtained from a subject, and the antibody reagent will bind ("capture") its specific antigen (SIRT3). The solid support may then be contacted with a second labeled antibody reagent (e.g., a detection antibody reagent). The detection antibody reagent may, for example, contain a detectable signal, be covalently linked to an enzyme, or itself may be linked to an enzyme by bioconjugation for secondary antibody detection. The presence of the signal indicates that both the first antibody reagent and the second "detection" antibody reagent immobilized on the support are bound to the antigen, i.e., the presence of the signal indicates the presence of the SIRT3 molecule. Between each step, the plate is typically washed with a mild detergent solution to remove any non-specifically bound proteins or antibodies. After the final washing step, the plate is developed by adding an enzyme substrate to produce a visible signal that indicates the content of SIRT3 in the sample. Older ELISAs use chromogenic substrates, but newer tests use fluorescent substrates with higher sensitivity. There are other different formats of ELISA, which are well known to those skilled in the art.

[0040] In one embodiment, the assays, systems, and methods described herein may include a lateral flow immunoassay test (LFIA), also known as an immunochromatographic assay, or a strip test, to measure or determine the level of SIRT3 in a sample. LFIA is a simple device designed to detect the presence (or absence) of SIRT3 in a sample. There are currently many LFIA tests used for medical diagnostics or for home testing, point-of-care testing, or laboratory use. The LFIA test is a form of immunoassay in which the test sample flows along a solid matrix by capillary action. After the sample is applied to the test strip, it encounters a colored antibody reagent mixed with the sample, and if it binds to a portion of the sample, it passes through a line or area on the substrate that has been pretreated with a second antibody reagent. Depending on the level of SIRT3 present in the sample, the colored antibody reagent may bind to the test line or area. LFIA is essentially an immunoassay that is suitable for operation along a single axis to accommodate a test strip format or a test paper format. Strip tests are widely used and can be easily modified by those skilled in the art to detect a large number of antigens from fluid samples (e.g., urine, blood, water samples, etc.). The strip test is also called the dip stick test, and the name comes from the literal action of "dipping" the test strip into the fluid sample to be tested. The LFIA strip test is easy to use, requires minimal training, and can be easily used on-site as part of a point-of-care test (POCT) diagnostic. The LFIA test can be operated as a competitive or sandwich test. Sandwich LFIA is similar to sandwich ELISA. The sample first encounters colored particles labeled with an antibody reagent specific to the target (e.g., a SIRT3-specific antibody reagent). The test line will also contain an antibody reagent (e.g., a SIRT3-specific antibody reagent). The test line will appear as a colored strip in a positive sample. In some embodiments, the lateral flow immunoassay can be a double antibody sandwich test, a competitive test, a quantitative test, or a variant thereof. There are many variations of lateral flow technology. Multiple capture areas can also be applied to create multiple tests.

[0041] A typical test strip consists of the following components: (1) a sample application area, including an absorbent pad (i.e., matrix or material) to which the test sample is applied; (2) a conjugate or reagent pad - which contains one or more antibody reagents specific for the target, which may be coupled to colored particles (usually colloidal gold particles or latex microspheres); (3) a test result area containing a reaction membrane - typically a hydrophobic nitrocellulose or cellulose acetate membrane to which the antibody reagent is immobilized, which serves as a capture zone or test line (there may also be a control zone, which contains antibodies specific for the antibody reagent coupled to the particles or microspheres); and (4) an optional wick or waste reservoir - another absorbent pad designed to draw the sample through the reaction membrane by capillary action and collect it. The components of the strip are usually fixed to an inert backing material and can be provided in the form of a simple dipstick or in a plastic housing with a sample port and reaction window showing the capture zone and control zone. Although not absolutely necessary, most tests will include a second line containing antibodies that can absorb free latex / gold to confirm that the test is performed correctly.

[0042] In some embodiments, one or more antibody reagents described herein may comprise a detectable label and / or comprise the ability to produce a detectable signal (e.g., by catalyzing a reaction that converts a compound into a detectable product). Detectable labels may include, for example, light absorbing dyes, fluorescent dyes, or radioactive labels. Detectable labels, methods of detecting them, and methods of incorporating them into antibody reagents are well known in the art.

[0043] In some embodiments, detectable label can include the mark that can be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, electromagnetic, radiochemistry or chemical means (such as fluorescence, chemiluminescence or chemiluminescence, or any other suitable means).The detectable label used in the methods described herein can be a primary label (wherein the label includes a part that can be directly detected or produces a part that can be directly detected) or a secondary label (wherein the detectable label is combined with another part to produce a detectable signal, for example, as is common in the immunolabeling using two antibodies and three antibodies). Detectable label can be connected to antibody reagents by covalent or non-covalent means. Alternatively, detectable label can be connected, for example, by direct labeling via ligand-receptor binding to arrange to realize the molecule or other such specific recognition molecules that are bonded to antibody reagents. Detectable label can include but is not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates and enzymes.

[0044] In other embodiments, the detection antibody is labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected based on fluorescence. In some embodiments, the detectable label can be a fluorescent dye molecule or fluorophore, including but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluorescamine, Cy3TM, Cy5TM, allophycocyanin, Texas Red, champagne chlorophyll (peridenin chlorophyll), cyanines, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and OregonGreenTM, rhodamine and its derivatives such as Texas Red and tetrarhodamine isothiocyanate (TRITC), biotin, phycoerythrin, AMCA, CyDyesTM, 6-carboxymethylfluorescein (known commonly as abbreviated FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N, N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-hydroxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, such as Cy3, Cy5 and Cy7 dyes; coumarins, such as umbelliferone; benzylamine dyes, such as Hoechst33258; phenanthridinium dyes, such as Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, such as Cy3, Cy5 and other cyanine dyes; BODIPY dyes and quinoline dyes.

[0045] In some embodiments, the detectable label in the radioimmunoassay (RIA) can be a radioactive label, including but not limited to 3H, 125I, 35S, 14C, 32 P and 33 P.

[0046] In some embodiments, the detectable label can be an enzyme, including but not limited to horseradish peroxidase and alkaline phosphatase. Enzyme labels can produce, for example, chemiluminescent signals, color signals, or fluorescent signals. Enzymes contemplated for detectably labeled antibody reagents include but are not limited to: malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0047] In some embodiments, the detectable label is a chemiluminescent label, including but not limited to lucigenin, luminol, fluorescein, isoluminol, acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0048] In some embodiments, the detectable label can be a spectrally colorimetric label, including but not limited to colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, and latex) beads.

[0049] In some embodiments, the antibody can also be labeled with a detectable label, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS or biotin. Other detection systems can also be used, such as the biotin-streptavidin system. In this system, antibodies with immunoreactivity (i.e., specificity) to the biomarker of interest are biotinylated. Streptavidin-peroxidase conjugates and chromogenic substrates are used to determine the amount of biotinylated antibodies bound to the biomarker. This streptavidin-peroxidase detection kit is commercially available.

[0050] Antibody reagents can also use fluorescent emitting metals such as 152 Eu or other metals of the lanthanide series can be detectably labeled. These metals can be attached to antibody reagents using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0051] As described herein, the tests and methods may involve determining whether a subject has an increased level of SIRT3 relative to a reference level. In some embodiments, the reference level of SIRT3 may be the level of SIRT3 in a healthy subject who does not suffer from or is not diagnosed as suffering from (pulmonary hypertension, such as hypertensive myocardial hypertrophy). In some embodiments, the reference level may be the level in a sample of similar cell type, sample type, sample treatment, and / or the level obtained from an object of similar age, gender, and other demographic parameters to the object whose amount of SIRT3 is to be determined. In some embodiments, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source, and contain the same composition, such as the same number and type of cells and / or the same type of sample material. Therefore, in some embodiments, the level of increased SIRT3 may vary with changes in demographic factors such as age, gender, genotype, environmental factors, and individual medical history. In some embodiments, the reference level may include the level of SIRT3 in a sample of the same type taken from an object that does not show any signs or symptoms of (e.g., hypertensive myocardial hypertrophy). In some embodiments, the reference level of SIRT3 may be the expression level of SIRT3 in a prior sample obtained from an object. This allows direct analysis of any level changes in that individual.

[0052] In some embodiments, if the level of SIRT3 is at least 1.25 times, e.g., at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times or more of the reference level, then the level of SIRT3 may be increased relative to the reference level. In some embodiments, the expression level of SIRT3 may be normalized relative to a reference value.

[0053] As used herein, the term "biological sample", "specimen", "sample" or "test sample" refers to a sample collected or separated from an organism, such as a urine sample from a subject. Exemplary biological samples include, but are not limited to: biological fluid samples; serum; plasma; urine; saliva; and / or tissue samples, etc. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pretreated (or preprocessed) biological samples. In some embodiments, the test sample may include cells from a subject. As used herein, the term "biological fluid" refers to any fluid obtained from a biological source, including but not limited to blood, urine, and body secretions.

[0054] The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g., isolated at a previous time point and isolated by the same person or another person). In addition, the test sample can be a freshly collected or previously collected sample.

[0055] In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase "untreated test sample" refers to a test sample that has not undergone any prior sample pretreatment except dilution and / or suspension in a solution. Exemplary methods for treating test samples include, but are not limited to, centrifugation, filtration, ultrasonic treatment, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, such as a frozen tissue. Before using the methods, tests, and systems described herein, the frozen sample can be thawed. After thawing, the frozen sample can be centrifuged before the methods, tests, and systems described herein are handled. In some embodiments, the test sample is a clarified test sample, for example, prepared by centrifugation and collecting the supernatant containing the clarified test sample. In some embodiments, the test sample can be a pre-processed test sample, for example, a supernatant or filtrate produced by a process selected from centrifugation, filtration, thawing, purification, and any combination thereof. In some embodiments, the test sample can be treated with chemical and / or biological reagents. During processing, chemical and / or biological reagents can be used to protect and / or maintain the stability of the sample, including biomolecules (e.g., nucleic acids and proteins) therein. An exemplary agent is a protease inhibitor, which is often used to protect or maintain the stability of proteins during processing. The skilled artisan is well aware of methods and processes suitable for pre-processing of biological samples required for determining SIRT3 levels as described herein.

[0056] In some embodiments, the methods, assays, and systems described herein may further comprise the step of obtaining a test sample from a subject. In some embodiments, the subject may be a human subject.

[0057] By using the method, test and system of the present invention to detect the SIRT3 protein content in biological samples to diagnose pulmonary arterial hypertension (PAH) or further subdivide IPAH and CTEPH, it is obvious that great convenience is provided for the diagnosis of pulmonary arterial hypertension. Compared with right heart catheter detection, the advantages of the present invention include: 1. Through a simple blood test of SIRT3 content, it is possible to know whether the patient has pulmonary arterial hypertension and understand the degree of development of pulmonary arterial hypertension. 2. Less trauma, easy to implement, and high patient acceptance. 3. It is capable of monitoring the pressure changes of pulmonary arterial hypertension multiple times in a short period of time. Compared with echocardiography estimation, the advantages of the present invention are also reflected in the low skill requirements for detection personnel. Even if there is no cardiac color Doppler ultrasound machine in grassroots hospitals, or the level of ultrasound physicians is limited, PAH, IPAH and CTEPH diagnosis can be carried out.

[0058] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] In the embodiments of the present invention, if there is no specific description on the experimental operation temperature, the temperature generally refers to room temperature (10-40° C.).

[0060] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, are all percentages by weight.

[0061] Example 1: Grouping of test subjects and method for detecting protein SIRT3 content

[0062] 1. Population recruitment: Normal volunteers were recruited from the Department of Cardiology, Zhongshan Hospital Affiliated to Fudan University. Patients with pulmonary hypertension were recruited from the Pulmonary Vascular Disease Subspecialty, Department of Cardiology, Zhongshan Hospital Affiliated to Fudan University. Both groups of people underwent physical examination, blood biochemical examination, blood index examination, and echocardiography.

[0063] The recruited pulmonary hypertension patients were divided into groups: 40 patients without pulmonary hypertension and 80 patients with pulmonary hypertension (including 30 patients with idiopathic pulmonary hypertension and 27 patients with thromboembolic pulmonary hypertension). All three groups underwent physical examination, blood biochemical examination, and echocardiography.

[0064] The clinical information and data of the two groups are shown in Table 1.

[0065] 2. Cardiac ultrasound examination: Experienced cardiologists from Zhongshan Hospital of Fudan University performed echocardiography on the individuals to assess the severity of pulmonary hypertension. Hemodynamic parameters were assessed, including pulmonary artery diastolic pressure (PADP), pulmonary artery systolic pressure (PASP) and mean pulmonary artery pressure (mPAP), mean right ventricular pressure, mean right atrial pressure, and pulmonary vascular resistance. The results are shown in Table 1.

[0066] Table 1. Comparison of clinical baseline data and serum SIRT3 levels between patients with pulmonary hypertension and controls

[0067]

[0068] 3. Detection of serum SIRT3 content: Human serum SIRT3 content was detected by ELISA detection kit provided by R&D Company in London, UK. The ELISA experiment for detecting human SIRT3 content includes the following steps:

[0069] (1) Before the experiment, dissolve the serum sample and all reagents in the ELISA kit at room temperature.

[0070] (2) Dilute the standard and serum samples 10-fold.

[0071] (3) Prepare a 96-well ELISA plate (purchased from ThermoFisher), add 10 μl of standard or serum sample to the microplate pre-coated with SIRT3-specific antibody, and incubate at 37°C for 60 minutes.

[0072] (4) Add avidin-horseradish peroxidase conjugate (incubate at 37°C for 30 minutes).

[0073] (5) Add tetramethylbenzidine (incubate at 37°C for 20 minutes) and 2 SO 4 Terminate the reaction.

[0074] (6) The color change was measured spectrophotometrically at 450 nm.

[0075] (7) Using the values ​​and concentrations of the standard, a curve is created to obtain a calculation formula. The sample values ​​are inserted into the formula to obtain the ELISA sample concentration. The ELISA sample concentration is multiplied by 10 to obtain the SIRT3 concentration in the original serum.

[0076] The results are shown in Table 1 and Figure 1 shown.

[0077] Example 2: Data Analysis and Statistical Methods

[0078] 1. Pearson correlation analysis

[0079] Pearson is a statistical analysis method, which is performed by Graphpad software (version 6.01) in the following examples. The software has many statistical methods. Enter the data into the Graphpad software, click the 'Analyze' button, select Pearson statistics, and the calculation results can be generated: P value and correlation coefficient r value. The results of the Pearson correlation analysis of serum SIRT3 content and measured pulmonary artery pressure are shown in Table 2.

[0080] Table 2. Correlation between serum SIRT3 content and measured pulmonary artery pressure

[0081]

[0082] 2. ROC curve analysis of the diagnostic effect of serum SIRT3 on pulmonary hypertension. In the following examples, STATA (version 15.1) was used. The data were imported into STATA analysis software, and the graph-ROC curve was clicked to make a receiver operating characteristic (ROC) curve analysis to calculate the sensitivity, specificity and area under the curve (AUC). The results are shown in Table 3 and Figure 2 shown.

[0083] Table 3. Diagnostic effect of serum SIRT3 content on different types of pulmonary artery pressure

[0084] variable Sensitivity Specificity Cutoff value Area under the curve 95% Confidence Interval P SIRT3-PAH 85.0％ 80.0％ 3.6 (ng / ml) 0.894 (0.839,0.950) <0.001 SIRT3-IPAH 72.5％ 80.0％ 4.1 (ng / ml) 0.835 (0.742,0.928) <0.001 SIRT3-CTEPTH 85.0％ 88.9％ 3.6 (ng / ml) 0.921 (0.851,992) <0.001

[0085] As can be seen from Table 3, the cutoff value of pulmonary arterial hypertension (PAH) is 3.6 ng / ml, of which the cutoff value of idiopathic pulmonary arterial hypertension (IPAH) is 4.1 ng / ml, and the cutoff value of chronic thromboembolic pulmonary arterial hypertension (CTEPH) is 3.6 ng / ml, which is the same as the cutoff of pulmonary arterial hypertension (PAH).

[0086] 3. Table 1, Figure 2 The analysis was done by 1-way ANOVA test; the analysis of Table 2 was done by Pearson correlation analysis; Figure 2 The drawing was completed by Graphpad software (version 6.01); the data analysis in Table 3 was completed by STATA (version 15.1).

[0087] Results and Discussion

[0088] 1. Table 1 and Figure 1 The clinical data of two groups of people are shown in the figure, including 40 cases of non-pulmonary hypertension and 80 cases of pulmonary hypertension. The results showed that the serum SIRT3 content of patients with pulmonary hypertension increased significantly: through enzyme-linked immunosorbent assay (ELISA), it was found that the pulmonary artery pressure in the pulmonary hypertension, idiopathic pulmonary hypertension and thromboembolic pulmonary hypertension groups was higher than that in the normal group.

[0089] 2. The data in Table 2 show that serum SIRT3 content is significantly positively correlated with pulmonary artery systolic pressure, diastolic pressure and mean pressure (P<0.001), that is, with the increase of pulmonary artery pressure (that is, with the increase of pulmonary artery systolic pressure, diastolic pressure and mean pressure), serum SIRT3 content gradually increases. In addition, serum SIRT3 content is not significantly correlated with age and gender.

[0090] 3. Figure 2 The ROC curve analysis in Table 3 showed that serum SIRT3 had a good diagnostic efficacy for patients with pulmonary hypertension (including idiopathic pulmonary hypertension and thromboembolic pulmonary hypertension).

[0091] In summary, through the analysis of population samples, we have confirmed for the first time the correlation between serum SIRT3 content and pulmonary hypertension pressure, which will help develop a new non-invasive diagnostic method for pulmonary hypertension. Given that the current gold standard for diagnosing pulmonary hypertension is still invasive right heart catheterization, if serum SIRT3 content can be detected and calibrated, it will provide a new non-invasive method for diagnosing pulmonary hypertension.

[0092] The description of the above embodiments is to facilitate those skilled in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. According to the disclosure of the present invention, improvements and modifications made without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Application of SIRT3 in the preparation of pulmonary arterial hypertension detection reagents.

2. The use according to claim 1, characterized in that: The pulmonary hypertension refers to idiopathic pulmonary hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH).

3. The use according to claim 1, characterized in that: The SIRT3 refers to the SIRT3 protein in serum.

4. A pulmonary hypertension detection kit for implementing the use as claimed in claim 1, characterized in that: The detection target is SIRT3 in biological samples.

5. The pulmonary hypertension detection kit according to claim 4, characterized in that: The detection method is to measure the SIRT3 protein content in biological samples.

6. The pulmonary hypertension detection kit according to claim 5, characterized in that: The antibody-antigen affinity binding principle is used to determine the antigen SIRT3 content in biological samples, including: (1) an antibody capable of binding to SIRT3; and (2) A standard sample consisting of a solution containing a known amount of SIRT3, wherein SIRT3 is SIRT3 protein extracted from humans or mammals or a recombinant SIRT3 protein produced by fermentation of genetically engineered bacteria.

7. The pulmonary hypertension detection kit according to claim 8, characterized in that: It is a latex immunoturbidimetry kit, a magnetic particle chemiluminescence kit, a radioimmunoassay kit, or an immunochromatographic test strip.

8. The pulmonary hypertension detection kit according to claim 6, characterized in that: Also includes: (3) A labeled antibody capable of binding to SIRT3 when SIRT3 binds to the antibody defined in (1).

9. The pulmonary hypertension detection kit according to claim 6, characterized in that: The double antibody sandwich kit is selected from the following group: A. Enzyme-linked immunosorbent assay kit; B. Magnetic particle chemiluminescence kit; C. Immunochromatographic test strips.

10. The pulmonary hypertension detection kit according to claim 3, characterized in that: The biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretions, tissue fluid, body fluid, urine, and more preferably serum.

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