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

Abstract

The invention discloses preparation and application of a novel diagnostic kit for a mitochondrial network regeneration function evaluation test, and in the invention, combined detection of related proteins DRP1, MFN2, OPA1 and TIM23 is carried out by utilizing four mitochondrial networks, so that the mitochondrial regeneration function state of sample cells is judged. The kit disclosed by the invention is used for detecting samples such as human blood, urine, cell cultures and tissues by utilizing an ELISA (Enzyme-Linked Immunosorbent Assay) method. Results show that the kit can accurately detect the content of four proteins so as to judge the function of sample mitochondria. The mitochondrial network regeneration capacity function evaluation kit based on a double-antibody sandwich method in an ELISA method can conveniently and accurately evaluate the mitochondrial network regeneration capacity in a human body specimen. The invention discloses preparation and application of the novel diagnostic kit for evaluating the regeneration function of the mitochondrial network. The diagnostic kit plays an auxiliary role in mitochondrial detection in scientific research and disease diagnosis and treatment. The method is applied to mitochondrial function evaluation in all diseases with abnormal cell mitochondrial functions.

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 regeneration function of human mitochondrial network. Background Art

[0002] Mitochondrial function is regulated by multiple network regeneration proteins. Although there are relatively detailed studies on these proteins in the cell mitochondrial network regeneration, there is no diagnostic kit for evaluating the mitochondrial network function. In particular, the combination of the four proteins DRPI, MFN2, OPA1 and TIM23 can more accurately reflect the evaluation of the cell mitochondrial network regeneration ability. This kit detects four mitochondrial network regeneration-related proteins in the sample by the double antibody sandwich method. Including mitochondrial fission protein DRPI, fusion proteins MFN2 and OPA1, inner membrane translocase TIM23. By detecting the OD value corresponding to each sample well, and then calculating the concentration result of each protein from the standard curve, the mitochondrial network regeneration function of the sample can be judged. If the content of the four proteins is at a normal concentration, it means that mitochondrial fusion and fission are in a good balance, then the mitochondrial network in the sample is in a vigorous renewal state, so the mitochondrial function is relatively good.

[0003] The main executor of mitochondrial fission is dynamin-related protein 1 (Drp1). Mitochondrial fission increases the number of mitochondria, reconstructs the mitochondrial network, and enhances mitochondrial function. Drp1 is mainly distributed in the cytoplasm and mediates the fission of organelles by translocation to the mitochondrial surface. Drp1 is a core protein that regulates mitochondrial fission and is also a GTP hydrolase. Drp1 is mainly distributed in the cytoplasm. Once it responds to signals in the cell that promote mitochondrial fission, Drp1 is recruited to the mitochondrial surface by its receptor protein located on the mitochondria and polymerizes into a ring structure, using the energy of GTP hydrolysis to split the mitochondria into two. Drp1 assembles on the mitochondrial surface to form helical oligomers, and undergoes self-interaction-mediated guanosine triphosphate (GTP) hydrolysis and subsequent membrane contraction, resulting in the rupture of the inner and outer mitochondrial membranes. The researchers found that in the early stage of ischemic injury, the morphology of mitochondria changed, while mitochondrial dysfunction occurred in the late stage. Drp1 can inhibit mitochondrial autophagy by upregulating the expression of mito-Clec16a, while mito-Clec16a can inhibit the recruitment of mito-Parkin and inhibit the formation of autophagosomes in vascular tissue after ischemic injury. Therefore, when the content of Drp1 is at a normal concentration or slightly higher, it can be beneficial to mitochondrial fission, inhibit autophagy, reconstruct mitochondrial network regeneration, and enhance mitochondrial function.

[0004] Mitochondrial fusion protein 2 (Mfn2) is a transmembrane motor protein on the outer membrane of mitochondria. It not only regulates mitochondrial fusion, but also plays an important role in cellular energy metabolism, apoptosis, cell proliferation, mitochondrial endoplasmic reticulum connection, endoplasmic reticulum stress and mitochondrial autophagy. Studies have found that abnormal expression or functional loss of Mfn2 can lead to mitochondrial dysfunction, which in turn causes a variety of liver diseases, such as metabolic associated fatty liver disease (MAFLD), viral hepatitis, liver fibrosis, HCC and acute-on-chronic liver failure (ACLF). Sebastián et al. found that the expression level of Mfn2 was positively correlated with the insulin signaling pathway in the liver and muscle, and individuals with low Mfn2 expression levels were more prone to insulin resistance. The lack of Mfn2 also increases the concentration of hydrogen peroxide, causing mitochondrial dysfunction and endoplasmic reticulum stress in the liver and muscle. The stress response caused by this inflammatory factor interacts with the insulin resistance and type 2 diabetes caused by obesity, which will cause a more serious closed vicious cycle. In addition to MFN2, OPA1 regulates mitochondrial inner membrane fusion, MFN1 is a mediator of mitochondrial fusion, and DRP1 is responsible for mitochondrial fission. In fibroblasts, the absence of MFN2 leads to a decrease in mitochondrial membrane potential and glucose oxidation capacity. Therefore, normal or slightly elevated MFN2 expression is conducive to the reconstruction of the mitochondrial network and good mitochondrial function. On the contrary, low levels may lead to continuous mitochondrial fragmentation and affect the normal function of mitochondria.

[0005] Mitochondrial dynamics have shown early on that OPA1 is directly involved in inner membrane fusion, mitochondrial DNA maintenance, and cristae formation. Mitochondrial cristae are bioenergetic chambers with curved edges formed by the inner membrane, connected to the inner membrane through cristae junctions. The dynamic changes of cristae and cristae junctions under different physiological conditions are called cristae remodeling. The effects of cristae remodeling on mitochondrial phospholipid metabolism and protein transport are important links in the development and progression of neurodegenerative diseases. The absence of OPA1 not only leads to mitochondrial fragmentation, which is obviously related to its fusion activity, but also reduces cristae biogenesis, because the IMM becomes vesicular, as observed by EM. Purified short Mgm1 (s-Mgm1) and short OPA1 (s-OPA1) are able to interact with IMM-like liposomes, induce local membrane bending, and deform them into tubular structures. The tubularization of these DLPs on the membrane is consistent with the formation of IMM protrusions between cristae. Among the mitochondrial fusion proteins, Mfn1 and Mfn2 are associated with the outer mitochondrial membrane (OMM), while OPA1 is the only inner mitochondrial membrane (IMM) protein found to coordinate fusion events. OPA1 has multiple isoforms (8 different splice variants), which are mainly divided into long (L-) and short (S-) isoforms. Several reports have shown different roles played by the long and short isoforms. L-OPA1 plays an important role in elongation / fusion activity, while the short isoform plays an important role in mitochondrial energetics and cristae maintenance. L-OPA1 is degraded to S-OPA1 by two major proteases such as OMA1 and YME1L, and their activities are increased during mitochondrial dysfunction and fragmentation. OPA1 plays an important role in mitochondrial fusion, energetics / OXPHOS, cristae / mt-DNA maintenance, etc. Normal or slightly elevated OPA1 expression is beneficial to remodeling the mitochondrial network and enhancing mitochondrial function.

[0006] The TIM23 complex (presequence translocase of the inner mitochondrial membrane) is the main transport and assembly complex of the mitochondrial inner membrane. In the process of functioning, it can exist in two different states: free state and bound state with the PAM complex (presequence translocase-associated motor) (TIM23-PAM complex). The TIM23 complex is mainly composed of four subunits, including Tim23, Tim50, Tim17 and Tim21. Tim17 is homologous to Tim23, but only a short fragment of its N-terminus is exposed in the membrane gap. Tim17 and Tim23 constitute the TIM23 complex integration transmembrane center, which plays an important role in the opening of the TIM23 complex pore. Normal or slightly elevated TIM23 expression is conducive to the transport of mitochondrial proteins and metabolites, maintaining the normal function of mitochondria, and is of great significance for the regeneration function of the mitochondrial network. Summary of the invention

[0007] 1. Purpose of the invention

[0008] The purpose of the present invention is to disclose a method for constructing and using a kit for evaluating mitochondrial network regeneration function. The present invention plays an auxiliary role in mitochondrial detection in scientific research and disease diagnosis and treatment, and evaluates mitochondrial function in all diseases with abnormal mitochondrial function in cells.

[0009] (II) Technical solution

[0010] In order to solve the above problems, the present invention proposes a construction method and application of a mitochondrial network regeneration function evaluation kit, wherein four antibodies specific to the antigen to be tested are coated in a 96-well microplate to prepare a solid phase carrier, and standard products or samples are added to the microwells respectively, wherein the antigen to be tested is bound to the antibody connected to the solid phase carrier, and then a biotinylated antibody to the antigen to be tested is added to form a sandwich, and the unbound biotinylated antibody is washed, and HRP-labeled avidin is added, and after thorough washing again, OPD substrate is added for color development. OPD is converted into orange-red under the catalysis of peroxidase, and the reaction is terminated with acid. The depth of the color is positively correlated with the antigen to be tested in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme marker, and the sample concentration is calculated by the standard curve. If the content of mitochondrial-related proteins is within the normal range, it means that the mitochondrial function is good and the network regeneration ability is strong, and the opposite is true if it is lower than the normal concentration.

[0011] The following steps are involved:

[0012] S1. In the present invention, we first prepare an ELISA plate coated with four protein antibodies, DRP1, MFN2, OPA1 and TIM23. First, at room temperature, four antibody solutions are coated on the ELISA plate corresponding to each protein position, and after coating, the ELISA plate is sealed with a desiccant and stored at -20 degrees Celsius. Other reagents are stored separately according to their storage conditions.

[0013] S2. The present invention measures the OD values ​​of the standard and the sample at a wavelength of 450nm by using an ELISA plate, and the average OD value minus the OD value of the blank well is used as the correction value. With concentration as the horizontal coordinate and OD value as the vertical coordinate, a standard curve of the four-parameter logistic function is drawn on a double logarithmic coordinate paper (minus the value of the blank group); 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. Finally, the target protein content in the sample is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the arrangement of four proteins in a 96-well plate of the kit in Example 1.

[0015] Figure 2 Schematic diagram of the preparation of the standard gradient working solution of the kit in Example 2.

[0016] Figure 3 This is a curve diagram of the DRP1 standard in Example 2.

[0017] Figure 4 This is a curve diagram of the MFN2 standard product in Example 2.

[0018] Figure 5 This is a curve diagram of the OPA1 standard in Example 2.

[0019] Figure 6 This is a curve diagram of the TIM23 standard product in Example 2.

[0020] Figure 7 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 mitochondrial network regeneration function

[0023] 1 Kit Materials

[0024] 1.1 Required self-provided test equipment

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

[0026] 1.2 Kit Composition

[0027]

[0028] 2. Preparation of ELISA plates

[0029] In the present invention, we prepared a 96-well ELISA plate that can simultaneously detect four proteins, DRP1, MFN2, OPA1 and TIM23. The positions of each protein are shown in Figure 1 .

[0030] (1) First, use a highly absorbent ELISA coating buffer to fully dilute and dissolve the four antibodies.

[0031] (2) Prepare the ELISA plate and inject 100 μl of the coated antibody solution into each well, with each three columns corresponding to one protein. Then seal the plate with a membrane and refrigerate it at 2-8 degrees Celsius overnight.

[0032] (3) Shake off the liquid in the wells without washing. Then, add 200 μl of ready-to-use ELISA blocking solution to each well, seal the membrane, and incubate at 37 degrees Celsius for 1 hour.

[0033] (4) Shake off the liquid in the wells again without washing. Next, dry the ELISA plate at 37 degrees Celsius for 30 minutes. After drying, add desiccant and seal it, then store it at -20 degrees Celsius.

[0034] Note: Incubate at the specified time and temperature, ensure that the reagents are used at room temperature of 20-25°C, and refrigerate in time; wash the plate correctly to avoid liquid residue and drying in the wells to avoid affecting the results; remove liquid and fingerprints on the bottom of the plate to avoid affecting the OD value reading; avoid direct sunlight on the reagents and ELISA plates; avoid contact between the reagents and bleach to avoid destroying the biological activity.

[0035] Example 2: Mitochondrial network regeneration function evaluation kit detection

[0036] 1. Sample processing and requirements

[0037] (1) Serum: Place the whole blood sample collected in a serum separation tube at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes and take the supernatant. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing.

[0038] (2) Plasma: Collect the specimen using EDTA or heparin as an anticoagulant and centrifuge it at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be tested or stored at -20°C or -80°C. However, repeated freezing and thawing should be avoided.

[0039] (3) Tissue homogenization: Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results), weigh and mince the tissue. Add the minced tissue and the corresponding volume of PBS (generally at a weight-to-volume ratio of 1:9, for example, 1g of tissue sample corresponds to 9mL of PBS. The specific volume can be adjusted appropriately according to experimental needs and recorded. It is recommended to add protease inhibitors to PBS) into a glass homogenizer and grind thoroughly on ice. In order to further lyse tissue cells, the homogenate can be ultrasonically disrupted or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and take the supernatant for detection.

[0040] (4) Cell culture supernatant: Centrifuge at 1000×g for 20 minutes and take the supernatant for testing, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing.

[0041] (5) Other biological specimens: Centrifuge at 1000×g for 20 minutes and remove the supernatant for testing.

[0042] (6) Sample appearance: The sample should be clear and transparent, and suspended matter should be removed by centrifugation.

[0043] (7) Sample preservation: If the sample is to be tested within 1 week after collection, it can be stored at 4°C. If it cannot be tested in time, please divide it into aliquots according to the amount for single use and freeze it at -20°C (for testing within 1 month) or -80°C (for testing within 6 months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test results, so hemolyzed samples are not suitable for this test.

[0044] 2. Preparation of the test kit before testing

[0045] (1) Take out the kit from the refrigerator 10 minutes in advance and equilibrate to room temperature.

[0046] (2) Preparation of standard gradient working solution: Add 1 mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved and then gently mix (concentration is 10 ng / mL), then dilute according to the following concentrations: 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.312 ng / mL, 0.156 ng / mL, 0 ng / mL. Serial dilution method: Take 7 EP tubes, add 500 μL of universal diluent to each tube, draw 500 μL from the 10 ng / mL standard working solution into the first EP tube and mix to make a 5 ng / 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. For details, please refer to the following steps. Figure 2 .

[0047] (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.

[0048] (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 concentrate + 990 μL universal diluent) and use on the same day.

[0049] (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).

[0050] 3. Detection operation steps

[0051] (1) Take out the required strips from the sealed bag after equilibration at room temperature for 10 minutes.

[0052] (2) Sample addition: Add 100 μl of samples and standards of different concentrations to the corresponding wells, and add 100 μL of universal diluent to the blank wells. Cover with the sealing film and incubate at 37°C for 1 hour. (Recommendation: Dilute the sample to be tested with universal diluent at least 1 times before adding it to the ELISA plate for testing. This will reduce the influence of matrix effect on the error of the test results. When calculating the sample concentration, multiply it by the corresponding dilution factor. It is recommended to set up duplicate wells for all samples and standards to be tested).

[0053] (3) Add biotinylated antibody: Remove the ELISA plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well, cover with sealing film, and incubate at 37°C for 1 hour.

[0054] (4) Washing the plate: discard the liquid, add 300 μL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this process 3 times (you can also use a plate washer to wash the plate).

[0055] (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.

[0056] (6) Washing the plate: Discard the liquid and wash the plate according to the method in step 4 for 5 times.

[0057] (7) Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0058] (8) Add stop solution: Take out the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm.

[0059] 4 Result calculation

[0060] (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 DRPI, MFN2, OPA1 and TIM23 proteins are shown in Figure 3-Figure 6 .

[0061] (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 the corresponding dilution factor should be multiplied when calculating the sample concentration.

[0062] (3) Typical data of the table of concentrations corresponding to the OD values ​​of four proteins:

[0063] Table 1 DRP1 typical data

[0064]

[0065] Normal range: The concentration of DRP1 protein in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the expression of DRP1 protein in the serum of healthy volunteers was 0.92-2.16 ng / ml. DRP1 is involved in the fission of mitochondria. Low content may affect the maintenance of mitochondrial morphology and structure, leading to excessive fusion of mitochondria and affecting their normal function. The protein content is within the normal range or slightly higher, indicating that the mitochondrial function is good and the network regeneration ability is strong.

[0066] Table 2 MFN2 typical data

[0067]

[0068] Normal range: The Mfn2 protein concentration in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the Mfn2 protein expression in the serum of healthy volunteers was 0.032-0.166 ng / ml. Mfn2 plays a key role in the fusion process of mitochondria. Its low content may cause continuous fragmentation of mitochondria and affect the normal function of mitochondria. The protein content is within the normal range or slightly higher, indicating that the mitochondrial function is good and the network regeneration ability is strong.

[0069] Table 3 OPA1 typical data

[0070]

[0071] Normal range: The concentration of OPA1 protein in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the expression of OPA1 protein in the serum of healthy volunteers was 0.92-2.16 ng / ml. OPA1 is a key protein mediating mitochondrial inner membrane fusion. Its low content may inhibit the mitochondrial inner membrane fusion process and affect the dynamic network structure of mitochondria. The protein content is within the normal range or slightly higher, indicating that the mitochondrial function is good and the network regeneration ability is strong.

[0072] Table 4 TIM23 typical data

[0073]

[0074] Normal range: The concentration of TIM23 protein in the serum of 40 healthy volunteers was detected by enzyme-linked immunosorbent assay (EUSA), and it was found that the expression of TIM23 protein in the serum of healthy volunteers was 0.22-0.3 ng / ml. TIM23-mediated protein input is a core pathway for mitochondrial biogenesis. Low TIM23 content may affect the normal biogenesis and function of mitochondria, and thus affect the energy supply and metabolism of mitochondria. Protein content within the normal range or slightly higher indicates good mitochondrial function and strong network regeneration ability.

[0075] Combining the above results, Figure 7 As shown, gray represents protein content in the normal range, black represents in the lower range, and light gray represents in the extremely low range. The protein content of DRP1, MFN2, OPA1 and TIM23 is in the normal range, indicating that the mitochondrial function is good and the network regeneration ability is strong; in the lower range, it indicates that the mitochondrial function is poor and the network regeneration ability is poor; in the extremely low range, it indicates that the mitochondrial function is extremely poor and the network regeneration ability is extremely poor.

Claims

1. A novel diagnostic kit for evaluating the regeneration function of mitochondrial network in cells.

2. A novel diagnostic kit for evaluating the regeneration function of mitochondrial network of cells as claimed in claim 1, characterized in that The ELISA method was used to determine the contents of four mitochondrial network regeneration-related proteins, DRPI, MFN2, OPA1 and TIM23, in the samples to comprehensively judge the mitochondrial network regeneration capacity of the samples.

3. A novel diagnostic kit for evaluating the regeneration function of mitochondrial network of cells as claimed in claim 1, characterized in that The standard curve was obtained by the double antibody sandwich method in the ELISA method under the ELISA instrument, and the concentrations of DRPI, MFN2, OPA1 and TIM23 in the test samples were calculated.

4. The DRPI, MFN2, OPA1 and TIM23 proteins of claim 2, characterized in that The ELISA kit enzyme labeling plate is composed of 96-well plates with 12 columns and 8 rows. From left to right, every 3 columns are DRPI, MFN2, OPA1 and TIM23 standard and sample positions, the first 2 columns are standards, and the last column is a sample.

5. The novel diagnostic kit for evaluating the regeneration function of mitochondrial network of cells as described in claim 1 can be used for in vitro detection of mitochondrial function evaluation of cells in human blood, peripheral blood mononuclear cells (PBMC), urine, cell culture supernatant and tissue specimens.

6. Use of a novel diagnostic kit for evaluating the regeneration function of mitochondrial network in claim 1 in the evaluation of mitochondrial function in the diagnosis and treatment of diseases related to mitochondrial function in the human body.

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