Preparation and application of novel diagnostic kit for evaluating autophagy function of mitochondrial network

Abstract

The invention discloses a novel diagnostic kit for evaluating the autophagy function of a mitochondrial network, which is used for detecting the content of autophagy related proteins NADH, ROMO1, PINK1 and TOM40 of four mitochondrial networks of a human body sample by utilizing an enzyme-linked immunosorbent assay (ELISA) and comprehensively judging the functional state of the mitochondrial of the human body. According to the kit, a microplate reader is used for measuring the absorbance (OD value) of a sample, and the concentration of the sample is calculated through a standard curve. Results show that the kit can accurately detect the content of four proteins, comprehensively judge the mitochondrial network autophagy ability of a human body sample and understand the mitochondrial function state. The kit for diagnosing the mitochondrial function of the double-antibody sandwich method combined with four proteins can conveniently and accurately evaluate the autophagy ability of the mitochondrial network in a human body specimen. The invention discloses preparation of a diagnostic kit for evaluating the autophagy ability of a mitochondrial network. The diagnostic kit can be used for evaluating cell mitochondrial states in cell mitochondrial functions, related disease diagnosis and treatment indexes and scientific research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to a kit that can be used for evaluating the autophagy function of human mitochondrial network. Background Art

[0002] Mitochondrial function is regulated by multiple mitochondrial network autophagy-related proteins. Although there are relatively detailed studies on these proteins in the cell mitochondrial network autophagy, there is no diagnostic kit for evaluating the mitochondrial network function. In particular, the combination of NADH dehydrogenase, ROMO1, PINK1 and TOM40 can more accurately reflect the evaluation of the cell mitochondrial network autophagy. This kit detects the content of four mitochondrial network autophagy-related proteins in human samples by the double antibody sandwich method in the ELISA method. Including NADH dehydrogenase, ROMO1, PINK1 and TOM40. By detecting the OD value corresponding to each sample plate well, and then calculating the concentration result of the corresponding protein from the standard curve, the mitochondrial function status of the sample can be judged. If the content of TOM40, NADH, PINK1 and ROMO1 proteins is within the normal range, it means that the mitochondrial function is good; if the content of TOM40, NADH and PINK1 proteins is lower than the lower or very low range, and the content of ROMO1 protein is higher than the normal range, the mitochondrial function is poor or very poor.

[0003] Complex I (NADH dehydrogenase): Mitochondrial genetic defects can lead to defects in complex I, II, III, IV or V, which can lead to mitochondrial energy metabolism disorders, often damaging organs or tissues with vigorous energy metabolism such as the central nervous system, muscles, liver, and kidneys. Among them, complex I-related mitochondrial diseases (mitochondrial diseases, MDs) are the most common, which is related to the structure of complex I itself. Complex I is the largest complex, so the subunit composition is the most complex, and the possibility of mutation also increases accordingly, mainly pathogenic mutations in genes related to complex I subunits and specific assembly factors encoded by mtDNA or nDNA. When NADH dehydrogenase function is impaired, it will lead to an increase in the NADH / NAD+ ratio, which will inhibit metabolic reactions that depend on NAD+, such as glycolysis, and affect mitochondrial function. Severe defects can cause fatal diseases in newborns, and can also manifest as neurodegenerative diseases in adults, such as Alzheimer's disease and Parkinson's disease, mainly due to point mutations in different subunits. Common clinical phenotypes include: Leigh syndrome, Leigh-like syndrome, Leber hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic acidosis and stroke-like neonatal cardiomyopathy, severe neonatal lactic acidosis, leukoencephalopathy, nonspecific encephalopathy, etc. Increased NADH expression can enhance mitochondrial energy metabolism and mitochondrial function. Its protein content is within the normal range or slightly higher, indicating that the mitochondrial function is good, otherwise it indicates that the mitochondrial function is poor.

[0004] Reactive oxygen species modulator 1 (Romo1) is a novel 79-amino acid transmembrane protein located on mitochondria that can induce intracellular ROS production. Its mechanism of action is that Romo1 can mediate tumor necrosis factor-α (TNF-α) signal transduction to induce mitochondria to produce intracellular reactive oxygen species. Studies have shown that a variety of ROMO1-related cancer cell lines increase ROMO1-induced ROS, leading to resistance to 5-FU. In addition to other pathways, ROMO1 can also affect MMPs through ROMO1-induced ROS, and this important mitochondrial protein may be involved in metastasis and cell invasion in other organs. Chung et al. studied liver cancer tissue samples and liver cancer cell lines. Compared with healthy adjuvant tissue, the ROMO1 gene was increased in cancer tissue, and the increase in ROMO1 gene expression in hepatocellular carcinoma cell lines was similar to that in normal cell lines. Related studies have shown that the ROMO1 protein level in cancer tissue was compared with healthy individuals, and ROMO1 was found to be significantly increased in cancer samples. When ROMO1 content is relatively low, the mitochondria produce less reactive oxygen species, which can reduce autophagy caused by reactive oxygen species damage to mitochondria and maintain mitochondrial function. On the contrary, when ROMO1 content is higher than normal, it may lead to increased ROS levels, causing mitochondrial autophagy to affect mitochondrial function and poor mitochondrial function.

[0005] The PINK1 / Parkin signaling pathway is the most typical indicator of mitophagy, which is involved in the maintenance of mitochondrial membrane potential, the ubiquitination degradation of the mitochondrial membrane protein Mfn2, and the selective degradation of mitochondrial structure. Under normal circumstances, the expression level of PINK1 is extremely low and cannot be detected. Under specific mitochondrial stress conditions, only when the endogenous PINK1 expression level increases to a certain extent can it be detected by protein immunoblotting or microscopy, and it is mainly located in the outer membrane of mitochondria. When mitochondrial function is disordered, the expression level of PINK1 protein in the cell is significantly reduced, the mitochondrial potential is reduced, and the mitochondrial function is reduced. Conversely, when the PINK1 content is at or slightly above the normal range, the mitochondrial function is in good condition.

[0006] Translocase of outer mitochondrial membrane,

[0007] TOM40 is a major member of the mitochondrial outer membrane complex, which is a protein channel of the mitochondrial outer membrane and is responsible for most mitochondrial precursor proteins passing through the mitochondrial outer membrane. The mitochondrial outer membrane complex is composed of channel proteins (TOM40) and receptor proteins (TOM20, TOM22, TOM70) and small mitochondrial outer membrane proteins (TOM5, TOM6, TOM7). Except for TOM40, the remaining proteins are fixed on the mitochondrial outer membrane by a single transmembrane segment. TOM40 is an outer membrane channel necessary for maintaining mitochondrial function and is responsible for transporting precursor proteins synthesized in the cytoplasm into mitochondria. TOM40 plays an important role in the mitochondrial fusion process and affects the morphology and distribution of mitochondria. The dynamic changes of mitochondria, such as fission and fusion, are necessary to maintain their normal function. TOM40 plays an important role in maintaining the function of mitochondria. It can selectively recognize and import proteins, and can also transport other molecules, including metabolites, inorganic ions and nucleic acids. It is an essential conduction channel for protein import into mitochondria. Studies have shown that when TOM40 expression increases, the overall function and activity of mitochondria increase. Therefore, when TOM40 content is within the normal range or increased, mitochondrial function is good. Otherwise, it is poor. Summary of the invention

[0008] 1. Purpose of the invention

[0009] The purpose of the present invention is to disclose a method for constructing and using a kit for evaluating the autophagy function of mitochondria network, and to provide a reference for evaluating the functional state of mitochondria in human cells and for diagnosing and treating diseases related to mitochondrial function in scientific research.

[0010] (II) Technical solution

[0011] In order to solve the above problems, the present invention proposes a construction method and application of a mitochondrial function evaluation kit, in which four specific antibodies against the target antigen are fixed in a 96-well microplate as a solid phase carrier. Then, standard substances or samples to be tested are added to these microwells respectively to bind the target antigen to the fixed antibodies. Subsequently, biotinylated antibodies against the same antigen are added to form a sandwich structure. After washing away the unbound biotinylated antibodies, avidin labeled with horseradish peroxidase (HRP) is added. After thorough washing again, OPD substrate is added for color development reaction. Under the catalytic action of peroxidase, OPD is converted into orange-red, and the reaction is terminated by acid. The depth of the color is proportional to the concentration of the target antigen in the sample. The absorbance (OD value) is measured at a wavelength of 450 nanometers using an enzyme reader, and the antigen concentration in the sample is determined by a standard curve.

[0012] The following steps are involved:

[0013] S1. In the present invention, an ELISA plate coated with four protein antibodies, TOM40, NADH, PINK1 and ROMO1, was first prepared: the antibody solution was coated to the corresponding position of the ELISA plate at room temperature, and after the coating was completed, it was dried and sealed and stored at -20 degrees Celsius. Other reagents were stored separately according to their storage conditions.

[0014] S2. In the present invention, after the kit is balanced to room temperature, the centrifuged human sample is added to the microplate for reaction and color development. First, the average optical density (OD) value of each standard and test sample is calculated, and then the OD value of the blank well is subtracted to obtain the corrected OD value. Next, on a double logarithmic coordinate paper, a standard curve of a logistic function containing four parameters is drawn with concentration as the horizontal axis and OD value as the vertical axis, and the data of the blank group is excluded during the drawing process. If the OD value of a sample exceeds the upper limit of the standard curve, the sample needs to be appropriately diluted and re-measured, and the dilution multiple should be considered when calculating the final concentration. In this way, the content of the target protein in the sample can be accurately obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the arrangement of four proteins in a 96-well plate of the kit in Example 1.

[0016] Figure 2 This is a curve diagram of the NADH standard in Example 2.

[0017] Figure 3 This is a curve diagram of the ROMO1 standard product in Example 2.

[0018] Figure 4 This is a curve diagram of the PINK1 standard product in Example 2.

[0019] Figure 5 This is a curve diagram of the TOM40 standard product in Example 2.

[0020] Figure 6 This is a bar graph showing the contents of four proteins detected by the kit in Example 2. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the embodiments.

[0022] Example 1: Construction of a kit for evaluating the autophagy function of mitochondrial network

[0023] 1 Kit Materials

[0024] 1.1 Required self-provided test equipment

[0025] (1) 37℃ constant temperature box. (2) High-precision pipette and pipette tip: 0.5-10 uL, 5-50 uL, 20-200 uL, 200-1000 uL. (3) Microplate reader (450 nm). (4) Distilled water or deionized water.

[0026] 1.2 Kit Composition

[0027]

[0028] 2 Experimental methods

[0029] 2.1 Notes

[0030] (1) Strictly follow the specified time and temperature conditions for incubation to ensure the accuracy of the results. All reagents must be returned to room temperature (20-25°C) before use. After use, the reagents should be refrigerated immediately.

[0031] (2) Improper plate washing may lead to inaccurate results. Make sure that the liquid in the wells is as dry as possible each time. Avoid letting the microwells dry during the incubation process.

[0032] (3) During operation, remove any residual liquid and fingerprints on the bottom of the plate, otherwise it will affect the OD reading.

[0033] (4) During storage and incubation, avoid direct exposure to strong light.

[0034] (5) No reagent should be exposed to bleach or its strong fumes. Bleach components will destroy the biological activity of the reagents in the kit.

[0035] (9) Recombinant proteins from sources other than the kit may not match the antibodies in this kit and thus cannot be recognized.

[0036] 2.2 Preparation of ELISA plates

[0037] First, we prepared a 96-well ELISA plate that can simultaneously detect TOM40, NADH, PINK1 and ROMO1, four proteins. The positions of each protein are shown in Figure 1 .

[0038] (1) Dilute and dissolve the four coating antibodies with a highly absorbent ELISA coating buffer.

[0039] (2) Prepare the ELISA plate, add 100 μl of the coating antibody working solution to each well, cover with the membrane, and then refrigerate at 2-8 degrees Celsius overnight.

[0040] (3) Shake off the liquid in the wells without washing. Then add 200 μl of ready-to-use ELISA blocking solution to each well, cover the membrane, and incubate at 37 degrees Celsius for 1 hour.

[0041] (4) Shake off the liquid in the wells again without washing, and then dry the ELISA plate at 37 degrees Celsius for 30 minutes. After drying, seal it with a desiccant and store it at -20 degrees Celsius for up to 6 months.

[0042] Example 2: Detection of mitochondrial network autophagy function evaluation kit

[0043] 1. Sample pretreatment

[0044] (1) Serum: Collect the whole blood sample in a serum separator tube and let it stand at room temperature for 2 hours or at 4°C overnight. Then centrifuge at 1000×g for 20 minutes and take the supernatant for use, or store the supernatant at -20°C or -80°C, but avoid multiple freeze-thaw cycles.

[0045] (2) Plasma: Use EDTA or heparin as an anticoagulant when collecting blood samples. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes after blood collection. Take the supernatant for testing, or store the supernatant at -20°C or -80°C. Also avoid multiple freezing and thawing.

[0046] (3) Tissue homogenization: Wash tissue samples with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (because red blood cell lysis will affect the measurement results). After weighing, mince the tissue and add PBS containing protease inhibitors to a glass homogenizer at a weight-to-volume ratio of 1:9 (for example, 1 gram of tissue corresponds to 9 ml of PBS, the specific ratio can be adjusted and recorded according to experimental requirements), and grind thoroughly on ice. To further disrupt cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge at 5000×g for 5 to 10 minutes and take the supernatant for detection.

[0047] (4) Cell culture supernatant: After centrifugation at 1000 × g for 20 minutes, take the supernatant for detection, or store the supernatant at -20°C or -80°C to avoid repeated freezing and thawing.

[0048] (5) Other biological samples: Centrifuge at 1000×g for 20 minutes and collect the supernatant for testing.

[0049] (6) Sample appearance: Ensure that the sample is clear and transparent, and remove suspended matter by centrifugation.

[0050] (7) Sample storage: If the sample is used within one week, it can be stored at 4°C; if it needs to be stored for a long time, it should be divided into single-use portions and frozen at -20°C (for use within one month) or -80°C (for use within six months), and multiple freeze-thaw cycles should be avoided. Hemolyzed samples may affect the final test results, so it is not recommended to use hemolyzed samples for this test.

[0051] 2 Kit pretreatment

[0052] (1) Please take out the reagent kit from the refrigerator 10 minutes in advance and equilibrate it to room temperature.

[0053] (2) Preparation of standard gradient working solution: Add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved, then gently mix (concentration is 10ng / mL), and then dilute according to the following concentrations: 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL, 0.312ng / mL, 0.156ng / mL, 0ng / mL. Multiple dilution method: Take 7 EP tubes, add 500μL of universal diluent to each tube, draw 500μL from the 10ng / mL standard working solution into the first EP tube and mix to make a 5ng / mL standard working solution, and follow this step to draw and mix in sequence. The last tube is directly used as a blank well, and there is no need to draw liquid from the second to last tube.

[0054] (3) Preparation of biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to 1× working concentration with universal diluent (e.g. 10 μL concentrated solution + 990 μL universal diluent) and use on the same day.

[0055] (4) Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to 1× working concentration with universal diluent (e.g. 10 μL concentrated solution + 990 μL universal diluent) and use on the same day.

[0056] (5) Preparation of 1× washing solution: Take 10 ml of 20× washing solution and add it to 190 ml of distilled water (the concentrated washing solution taken out from the refrigerator may have crystals, which is a normal phenomenon. It can be left at room temperature and shaken evenly. Wait until the crystals are completely dissolved before preparing it).

[0057] 3 Operation method

[0058] (1) After equilibration at room temperature for 10 minutes, remove the required strips from the aluminum foil bag. Seal the unused strips in a ziplock bag and return them to 4°C for storage.

[0059] (2) Sample addition: Add 100 μl of sample or standard of different concentrations to the corresponding wells, and add 100 μL of universal diluent to the blank control well. Cover with the sealing film and incubate at 37°C for 1 hour. It is recommended to dilute the sample to be tested at least 1 times with universal diluent before adding it to the ELISA plate for testing to reduce the error of the matrix effect on the test results. When calculating the sample concentration, it is necessary to multiply the corresponding dilution factor. It is recommended to set up duplicate wells for all samples and standards to be tested during the test process.

[0060] (3) Add biotinylated antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of biotinylated antibody working solution directly to each well, cover with sealing film, and incubate at 37°C for 1 hour.

[0061] (4) Washing: Discard the liquid, add 300 μL of 1x washing solution to each well, let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 3 times (you can also use a plate washer to wash the plate).

[0062] (5) Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film, and incubate at 37°C for 30 minutes.

[0063] (6) Washing the plate: Discard the liquid again and wash the plate according to the method in step 4 for a total of 5 times.

[0064] (7) Add substrate: Add 90 μL of TMB substrate to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0065] (8) Add stop solution: Take out the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the optical density of each well at a wavelength of 450 nm.

[0066] 4 Calculation of sample protein content

[0067] (1) Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, draw the standard curve of the four-parameter logistic function on the double logarithmic coordinate paper (remove the value of the blank group when drawing the graph). The corresponding standard curves of NADH, ROMO1, PINK1 and TOM40 proteins are shown in Figure 2-Figure 5 .

[0068] (2) If the sample OD value is higher than the upper limit of the standard curve, it should be appropriately diluted and re-measured and multiplied by the corresponding dilution factor when calculating the sample concentration.

[0069] (3) Typical data of the table of concentrations corresponding to the OD values ​​of each protein:

[0070] Table 1 Typical data of NADH

[0071] Normal range: The NADH dehydrogenase concentration in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the expression of NADH dehydrogenase in the serum of healthy volunteers was 0.06-0.08 ng / ml. NADH dehydrogenase is a key enzyme in the mitochondrial electron transport chain and is essential for maintaining the normal function of mitochondria. When the content of NADH dehydrogenase is low, properly increasing the content can significantly increase the synthesis of intracellular NAD+, thereby enhancing the metabolic capacity and biocatalytic efficiency of cells. Therefore, when the NADH content is at or slightly higher than the normal concentration, it means that the mitochondrial function is good. Otherwise, the mitochondrial function is poor.

[0072] Table 2 ROMO1 typical data

[0073] Normal range: The concentration of Romo1 protein in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the expression of ROMO1 protein in the serum of healthy volunteers was 0.056-0.094 ng / ml. High concentrations of ROMO1 may lead to abnormalities in mitochondrial morphology and function, such as mitochondrial fragmentation and loss of cristae, which in turn affect mitochondrial respiration and energy production. Therefore, if the ROMO1 content is at or below the normal concentration, it means that the mitochondrial function is good, otherwise the mitochondrial function is poor.

[0074] Table 3 PINK1 typical data

[0075] Normal range: The PINK1 protein concentration in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the PINK1 protein expression in the serum of healthy volunteers was 0.22-0.3 ng / ml. Low PINK1 content may weaken the autophagy process and affect the balance of mitochondrial quality and quantity. Therefore, PINK1 content at or slightly above the normal concentration indicates good mitochondrial function, and vice versa.

[0076] Table 4 TOM40 typical data

[0077] Normal range: The concentration of TOM40 protein in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the expression of TOM40 protein in the serum of healthy volunteers was 0.36-0.54 ng / ml. TOM40 is a key protein that mediates mitochondrial outer membrane fusion. Its high content can promote the transport of mitochondrial metabolites and enhance the overall function and activity of mitochondria. Therefore, when the TOM40 content is at or slightly higher than the normal concentration, it means that the mitochondrial function is good. Otherwise, the mitochondrial function is poor.

[0078] In summary, if Figure 6 As shown, black represents that the contents of the four proteins are within the normal concentration range, gray represents a lower concentration range, and light gray represents an extremely low concentration range. If the contents of TOM40, NADH, PINK1 and ROMO1 proteins are within the normal range, it means that the mitochondrial function is good; if the contents of TOM40, NADH and PINK1 proteins are lower than the lower or extremely low range, and the content of ROMO1 protein is higher than the normal range, then the mitochondrial function is poor or extremely poor.

Claims

1. A novel diagnostic kit for evaluating the autophagy function of the mitochondrial network.

2. A novel diagnostic kit for evaluating the autophagy function of mitochondrial network according to claim 1, characterized in that The ELISA method was used to determine the contents of four mitochondrial network autophagy-related proteins, NADH, ROMO1, PINK1 and TOM40, in human samples to determine the mitochondrial network autophagy function of the samples.

3. The NADH, ROMO1, PINK1 and TOM40 proteins according to claim 2, characterized in that The ELISA kit is composed of a 96-well ELISA plate (12 columns and 8 rows). From left to right, every 3 columns are the standard and sample positions of TOM40, NADH, PINK1 and ROMO1, respectively. The first 2 columns are standards and the last column is a sample.

4. A novel diagnostic kit for evaluating the autophagy function of mitochondrial network according to claim 1, characterized in that It can be used for in vitro detection of mitochondrial autophagy function in the human body, including tissue specimens, cell culture supernatant, blood, peripheral blood mononuclear cells (PBMC) and urine. It can be applied to a wide range of samples, with simple sample pretreatment steps. After cell disruption and centrifugation, the supernatant is collected for standby use.

5. The application of a novel diagnostic kit for evaluating the autophagy function of mitochondrial network as claimed in claim 1 in evaluating the functional status of mitochondria in human cells, including the application in evaluating mitochondrial function in the diagnosis and treatment of diseases related to mitochondrial function in cells.

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