Mitochondrial cristae integrity protein detection kit and preparation method and application thereof

Abstract

This invention discloses a mitochondrial cristae integrity protein detection kit, its preparation method, and its applications, belonging to the field of protein detection technology. Addressing the problems of cumbersome operation, difficult quantification, and insufficient accuracy in existing mitochondrial cristae detection technologies, this invention provides a protein detection kit targeting TMEM65 and MIC60 together. The kit includes specific antibodies, chromogenic solutions, blocking solutions, washing solutions, standards, positive controls, and negative controls, simultaneously detecting the expression levels of TMEM65 and MIC60 proteins in samples to jointly determine mitochondrial cristae integrity. This kit features high specificity, high sensitivity, simple operation, low cost, and high-throughput detection capabilities, making it suitable for tumor metabolism research, basic research on mitochondrial diseases, drug screening, and cell function assessment, with broad research and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a mitochondrial cristae integrity protein detection kit, its preparation method, and its application. Background Technology

[0002] Mitochondria are core organelles in eukaryotic cells responsible for energy metabolism, fatty acid oxidation, and cell signaling regulation. The highly folded inner membrane of mitochondria forms the cristae, a crucial structural basis for oxidative phosphorylation, electron transport chain assembly, and efficient ATP synthesis. The morphology, number, and structural integrity of mitochondrial cristae directly determine mitochondrial homeostasis and cell fate. Structural abnormalities are often accompanied by a series of pathophysiological changes, including mitochondrial functional impairment, metabolic reprogramming, enhanced oxidative stress, and apoptosis, and are closely related to the development of various major diseases such as tumors, neurodegenerative diseases, metabolic syndrome, and cardiovascular diseases. In the field of tumor research, mitochondrial cristae structural disorder has been proven to participate in tumor cell metabolic remodeling, invasion and metastasis, and drug resistance formation. Particularly in digestive system tumors such as gastric cancer, abnormally elevated mitochondrial fatty acid oxidation and cristae structural changes jointly drive malignant tumor progression. Therefore, establishing a stable, specific, sensitive, and scalable method for detecting mitochondrial cristae integrity has extremely important scientific value and application prospects for elucidating disease pathogenesis, conducting early clinical diagnosis, performing high-throughput drug screening, and carrying out basic research related to mitochondria.

[0003] Currently, methods for detecting and evaluating mitochondrial cristae integrity are mainly divided into three categories, but all have significant technical shortcomings. The first category is direct structural observation methods, represented by transmission electron microscopy. This method can directly display ultrastructural changes such as morphology, density, breakage, and expansion of mitochondrial cristae and is considered the "gold standard" in the industry. However, this method requires complex sample pretreatment procedures, including fixation, dehydration, embedding, and ultrathin sectioning. It relies on high-value electron microscopy equipment, resulting in high detection costs, extremely low throughput, and long processing times. Furthermore, the results are highly dependent on the operator's subjective interpretation, making it difficult to achieve standardized and quantitative analysis, and unsuitable for large-scale clinical sample testing and high-throughput drug screening scenarios. The second category is indirect functional detection methods, represented by mitochondrial membrane potential, ATP content, and oxygen consumption rate. These methods infer structural damage by reflecting the overall functional state of mitochondria. While relatively simple to operate, they lack structural specificity, cannot distinguish cristae structural damage types, are insensitive to early and subtle structural changes, and are easily affected by cellular metabolic state, external stimuli, and signaling pathways. The detection results deviate significantly from the actual cristae structural state, making accurate evaluation difficult. The third category is molecular detection methods using a single protein as a marker. Among them, MIC60 (Mitofilin), as a core component of the MICOS complex, is crucial for maintaining the stability of crest junction sites, and its decreased expression often indicates crest structure damage. However, in practical applications, single MIC60 markers are easily affected by cellular stress, post-transcriptional regulation, protein degradation, and multiple signaling pathways, resulting in insufficient specificity, poor stability, and a high risk of false positives or false negatives, thus failing to meet the needs of accurate detection.

[0004] TMEM65 is a newly discovered transmembrane protein located on the inner mitochondrial membrane. It possesses typical mitochondrial targeting sequences and transmembrane domains, participating in mitochondrial structure maintenance, energy metabolism, and fatty acid oxidation regulation. Existing research indicates that TMEM65 is abnormally highly expressed in various tumors, including gastric cancer, and is closely related to tumor invasion, metastasis, and poor patient prognosis. However, current published research focuses only on the signal regulation mechanism of TMEM65 in tumor development and progression, and has not yet been developed as a marker for mitochondrial cristae structure, nor has a detection system combining TMEM65 and MIC60 been established. In summary, existing technologies have significant shortcomings in terms of detection cost, ease of operation, structural specificity, quantitative accuracy, and high-throughput applicability. The industry urgently needs a protein detection technology that is structurally specific, provides reliable results, is simple to operate, low-cost, and quantifiable to compensate for the deficiencies in current methods for evaluating mitochondrial cristae integrity.

[0005] Against this backdrop, this invention proposes a mitochondrial cristae integrity protein detection kit using TMEM65 and MIC60 as dual-protein joint markers. By simultaneously detecting the expression levels of the two key proteins, it achieves accurate evaluation of cristae structure, effectively overcoming the shortcomings of single markers such as instability, difficulty in quantification by electron microscopy, and non-specificity of functional detection, providing a new tool for mitochondrial-related research and disease diagnosis and treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a mitochondrial cristae integrity protein detection kit with high specificity, high sensitivity, simple operation, quantification, and high throughput, as well as its preparation method and application, to solve the problems of existing detection technologies being cumbersome to operate, difficult to quantify, insufficient inaccuracy, high cost, and unable to be applied on a large scale.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A kit for detecting mitochondrial cristae integrity proteins is characterized in that: the kit includes a primary antibody working solution and a secondary antibody working solution, the primary antibody working solution includes a TMEM65 protein-specific antibody and a MIC60 protein-specific antibody, and the secondary antibody working solution includes horseradish peroxidase-labeled goat anti-rabbit IgG.

[0008] Preferably, the primary antibody working solution includes TBST buffer, which comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20.

[0009] Preferably, the volume ratio of TMEM65 specific antibody to TBST buffer is 1:200~1000.

[0010] Preferably, the volume ratio of MIC60 specific antibody to TBST buffer is 1:200~1000.

[0011] Preferably, the working solution of the secondary antibody contains TBST buffer, which includes 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl and 0.05-0.2% Tween-20.

[0012] Preferably, the volume ratio of horseradish peroxidase-labeled goat anti-rabbit IgG to TBST buffer is 1:1000~3000.

[0013] Preferably, the kit includes an antigen retrieval solution, which is a citrate antigen retrieval solution with a pH of 5.9-6.1 and a concentration of 0.005-0.02 mol / L.

[0014] Preferably, the kit includes a blocking solution, which is a normal goat serum blocking solution.

[0015] Preferably, the primary antibody working solution includes 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and the final concentration of the 4-hydroxy-2,5-dimethyl-3(2H)-furanone primary antibody working solution is 6~9 μmol / L.

[0016] At a suitable concentration in the primary antibody working solution described in this invention, 4-hydroxy-2,5-dimethyl-3(2H)-furanone can significantly increase the absorbance value of antibody-antigen binding, reduce the coefficient of variation of detection results, and improve the detection sensitivity and repeatability of the kit. 4-hydroxy-2,5-dimethyl-3(2H)-furanone may improve the spatial conformation of antigen epitopes and reduce steric hindrance in antigen-antibody binding, thereby enhancing the recognition efficiency and binding affinity of TMEM65 and MIC60 specific antibodies for target proteins. Simultaneously, it may stabilize the antibody spatial structure and reduce non-specific hydrophobic binding, thereby improving the signal intensity and uniformity of the immune response and providing a stable and efficient antigen-antibody binding environment for immunohistochemistry and immunoassay.

[0017] More preferably, the secondary antibody working solution includes 2-methylthio-4-amino-6-hydroxypyrimidine, and the final concentration of 2-methylthio-4-amino-6-hydroxypyrimidine in the secondary antibody working solution is 1~4 μmol / L.

[0018] At an appropriate concentration in the secondary antibody working solution described in this invention, 2-methylthio-4-amino-6-hydroxypyrimidine can further enhance the detection signal intensity, reduce background interference and staining fluctuations, and decrease the coefficient of variation of detection results. This synergistic effect makes positive staining more uniform and quantitative results more reliable, significantly optimizing the overall stability and accuracy of the detection system. This component may act on the binding interface between the secondary and primary antibodies, enhancing the stability of the immune complex, improving the signal output efficiency of the enzyme-labeled secondary antibody, and strengthening the intensity and consistency of the colorimetric reaction. Simultaneously, it may reduce systematic errors in the immune reaction, achieving overall signal enhancement, thus adapting to the needs of large-sample, high-throughput standardized detection.

[0019] This invention also discloses a method for preparing the reagent kit of claim 1, characterized by comprising the following steps: Rabbit monoclonal antibodies targeting conserved antigenic epitopes in the exposed mitochondrial membrane region of human TMEM65 protein and conserved antigenic epitopes of human MIC60 protein were selected as primary antibodies, and horseradish peroxidase-labeled goat anti-rabbit IgG was selected as secondary antibodies. The primary antibody and secondary antibody were diluted with TBST buffer containing 5% bovine serum albumin at pH 7.4 to prepare working solutions for the primary antibody and secondary antibody, respectively. Antigen retrieval solution, blocking solution, washing solution, colorimetric solution, counterstaining solution, and mounting medium were prepared and aliquoted to obtain the mitochondrial cristae integrity protein detection kit.

[0020] This invention also discloses an in vitro method for detecting mitochondrial cristae integrity, comprising: The paraffin sections of the sample to be tested were dewaxed to water, and antigen retrieval was performed under high temperature and high pressure with citrate buffer to block endogenous peroxidase. After washing with TBST buffer, block with blocking buffer, incubate with primary antibody working solution at 4°C, wash again, and then incubate with secondary antibody working solution. DAB developing solution, hematoxylin counterstaining, differentiation, blue reversion, dehydration and clearing, mounting; A semi-quantitative scoring method was used, with scores based on the proportion of positive cells and staining intensity. The ratio of TMEM65 to MIC60 scores was calculated simultaneously to determine the integrity status of mitochondrial cristae.

[0021] The present invention also discloses the application of a mitochondrial cristae integrity protein detection kit, which is used for the detection of mitochondrial cristae integrity in isolated tissue and cell samples.

[0022] Preferably, the kit is used for tumor metabolism research, basic research on mitochondrial diseases, high-throughput drug screening, and cell function assessment.

[0023] This invention also provides a method for preparing a mitochondrial cristae integrity protein detection kit and a method for detecting protein expression levels, as detailed below: Step 1: Preparation of the Mitochondrial Creat Integrity Protein Detection Kit: The kit includes primary antibody working solution, secondary antibody working solution, antigen retrieval solution, blocking solution, washing solution, chromogenic solution, counterstaining solution, and mounting medium. Rabbit monoclonal antibodies targeting conserved antigenic epitopes in the exposed mitochondrial intermembrane region of human TMEM65 protein and rabbit monoclonal antibodies targeting conserved antigenic epitopes of human MIC60 protein are used as primary antibodies, and horseradish peroxidase-labeled goat anti-rabbit IgG is used as the secondary antibody. The primary antibody is diluted with TBST buffer (pH 7.4) containing 5% bovine serum albumin to prepare the primary antibody working solution, and the secondary antibody is diluted with TBST buffer (pH 7.4) containing 5% bovine serum albumin to prepare the secondary antibody working solution. After aliquoting, the mitochondrial cristae integrity protein detection kit is obtained.

[0024] Preferably, the antigen retrieval solution is a citrate antigen retrieval solution with a pH of 5.9-6.1 and a concentration of 0.005-0.02 mol / L.

[0025] Preferably, the blocking solution is normal goat serum blocking solution.

[0026] Preferably, the washing solution is a TBST buffer solution with a pH of 7.3 to 7.5.

[0027] Preferably, the colorimetric solution is a DAB colorimetric solution.

[0028] Preferably, the counterstaining solution is a hematoxylin counterstaining solution.

[0029] Preferably, the sealing medium is a neutral resin sealing medium.

[0030] Preferably, the TBST buffer comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20.

[0031] Preferably, the volume ratio of TMEM65 specific antibody to TBST buffer is 1:200~1000.

[0032] Preferably, the volume ratio of MIC60 specific antibody to TBST buffer is 1:200~1000.

[0033] Preferably, the volume ratio of the secondary antibody to TBST buffer is 1:1000~3000.

[0034] Preferably, the primary antibody working solution includes 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and the final concentration of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in the primary antibody working solution is 6~9 μmol / L.

[0035] More preferably, the secondary antibody working solution includes 2-methylthio-4-amino-6-hydroxypyrimidine, and the final concentration of 2-methylthio-4-amino-6-hydroxypyrimidine in the secondary antibody working solution is 1~4 μmol / L.

[0036] Step 2: Detecting protein expression levels: Take paraffin sections of the tissue to be tested, dewax to water using routine methods, and perform antigen retrieval using citrate buffer under high temperature and high pressure for 1-3 minutes, then allow to cool naturally to room temperature; add 2-4% H2O2 solution and incubate at room temperature in the dark for 5-15 minutes to block endogenous peroxidase; wash the sections 2-4 times with TBST buffer, 3-8 minutes each time; add normal goat serum blocking solution and block at 36-38℃ for 20-40 minutes; discard the blocking solution, add primary antibody working solution, and incubate in a humidified chamber at 0-5℃. 10-15 h; wash 2-4 times with TBST buffer, 3-10 min each time, add secondary antibody working solution, incubate at 36-38℃ for 20-40 min; after thorough washing with TBST buffer, add DAB chromogenic solution and develop at room temperature for 3-5 min, rinse with tap water to stop the development; counterstain with hematoxylin for 0.5-2 min, differentiate with hydrochloric acid alcohol, turn blue with ammonia, undergo gradient dehydration and clearing, and mount with neutral resin; observe under a microscope, the appearance of brownish-yellow granules in the cytoplasm is used to determine the positive expression of TMEM65 and MIC60 proteins.

[0037] Preferably, a semi-quantitative scoring method is used, which scores the sample based on the proportion of positive cells and the staining intensity, and simultaneously calculates the ratio of the two protein scores. The integrity status of the mitochondrial crest structure is determined by referring to the criteria for judging the integrity of mitochondrial crests.

[0038] This invention, employing a dual-protein co-targeting approach of TMEM65 and MIC60, combined with an optimized antibody system and specific synergistic components, offers the following advantages: It establishes for the first time a dual-marker detection system targeting the core regulatory axis of mitochondrial cristae structure. TMEM65 and MIC60 exhibit direct protein-protein interactions and jointly maintain the integrity of mitochondrial cristae; their combined detection accurately reflects the cristae structural state, overcoming the shortcomings of single-marker detection, such as high false positive / false negative rates, difficulty in quantification under electron microscopy, and non-specificity in functional detection. The kit exhibits high specificity and sensitivity; the synergistic components significantly enhance antigen-antibody binding affinity and detection repeatability, with low background interference and good staining uniformity. It is simple to operate, low-cost, requires no high-end equipment, and is compatible with various types of samples, including paraffin sections and cell samples. It enables high-throughput, standardized detection, suitable for tumor metabolism research, basic research on mitochondrial diseases, drug screening, and cell function assessment. Furthermore, it can achieve stratification of gastric cancer metastasis risk, with significantly superior detection efficiency compared to traditional methods, possessing both scientific research value and clinical translational potential. Therefore, this invention provides a mitochondrial cristae integrity protein detection kit and its preparation and application technology that is highly targeted, stable, easy to operate, widely applicable, and possesses both high specificity and mitochondrial structure assessment value. Attached Figure Description

[0039] Figure 1 This diagram illustrates the differences in TMEM65 protein expression in different gastric cancer cells.

[0040] Figure 2 This diagram illustrates the differences in protein expression of TMEM65 and PPARA between TMEM65-KO cells and the control group.

[0041] Figure 3 This is a schematic diagram illustrating the location of TMEM65 expression as detected by cell fluorescence.

[0042] Figure 4 This is a schematic diagram of TMEM65 protein immunohistochemical staining in cancerous and adjacent tissues of a gastric cancer patient.

[0043] Figure 5 A schematic diagram illustrating the effect of immunoprecipitation on protein precipitation in HGC27 cells.

[0044] Figure 6 A schematic diagram illustrating the effect of immunoprecipitation on protein precipitation in 293T cells.

[0045] Figure 7 This is a schematic diagram of target protein overlap and intersection analysis.

[0046] Figure 8 This diagram illustrates the binding of endogenous TMEM65 and MIC60, MIC19 and CS in HGC27 / GCIY cells.

[0047] Figure 9 This is a schematic diagram of the combination of TMEM65 and MIC60 in the TurboID input and IP (Strep) samples.

[0048] Figure 10 This diagram illustrates the differences in the expression of TMEM65 and MIC60 in different cells.

[0049] Figure 11 A schematic diagram illustrating the differences in migration ability of cells expressing different amounts of TMEM65 and MIC60.

[0050] Figure 12 This diagram illustrates the differences in expression of TMEM65, MIC60, and PPARA in different cells.

[0051] Figure 13 This is a schematic diagram illustrating the morphology of mitochondrial cristae and the accumulation of intracellular lipid droplets in cells from different treatment groups observed by cryo-transmission electron microscopy.

[0052] Figure 14 This is a schematic diagram illustrating the morphology of mitochondrial cristae in different tissue samples observed by cryo-transmission electron microscopy. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0055] This embodiment provides a method for preparing a mitochondrial crest integrity protein detection kit and a method for detecting protein expression levels, as detailed below.

[0056] Step 1: Preparation of the Mitochondrial Creat Integrity Protein Detection Kit: The kit includes primary antibody working solution, secondary antibody working solution, antigen retrieval solution, blocking solution, washing solution, chromogenic solution, counterstaining solution, and mounting medium. Rabbit monoclonal antibodies targeting conserved antigenic epitopes in the exposed mitochondrial intermembrane region of human TMEM65 protein and rabbit monoclonal antibodies targeting conserved antigenic epitopes of human MIC60 protein are used as primary antibodies, and horseradish peroxidase-labeled goat anti-rabbit IgG is used as the secondary antibody. The primary antibody is diluted with TBST buffer containing 5% bovine serum albumin at pH 7.4 to prepare the primary antibody working solution, and the secondary antibody is diluted with TBST buffer containing 5% bovine serum albumin at pH 7.4 to prepare the secondary antibody working solution. The antigen retrieval solution was 0.01 mol / L pH 6.0 citrate antigen retrieval solution; the blocking solution was normal goat serum blocking solution; the washing solution was pH 7.4 TBST buffer; the chromogenic solution was DAB chromogenic solution; the counterstaining solution was hematoxylin counterstaining solution; and the mounting medium was neutral resin mounting medium. All commercially available qualified reagents were used. After aliquoting, the mitochondrial cristae integrity protein detection kit was obtained. The TBST buffer contained 20 mmol / L Tris-HCl, 150 mmol / L NaCl, and 0.1% Tween-20; the volume ratio of TMEM65 specific antibody to TBST buffer was 1:500, the volume ratio of MIC60 specific antibody to TBST buffer was 1:500, and the volume ratio of secondary antibody to TBST buffer was 1:2000.

[0057] Step 2: Detecting Protein Expression Levels: Take paraffin sections of the tissue to be tested, dewax to water using standard procedures, and perform antigen retrieval using citrate buffer under high temperature and high pressure for 2 minutes, followed by natural cooling to room temperature. Add 3% H2O2 solution and incubate at room temperature in the dark for 10 minutes to block endogenous peroxidase. Wash the sections three times with TBST buffer, 5 minutes each time. Add normal goat serum blocking solution and block at 37°C for 30 minutes. Discard the blocking solution, add primary antibody working solution, and incubate in a humidified chamber at 4°C for 12 hours. Wash three times with TBST buffer, 5 minutes each time, add secondary antibody working solution, and incubate at 37°C. Incubate at a constant temperature for 30 min; after thorough washing with TBST buffer, add DAB chromogenic solution and develop at room temperature for 4 min, then rinse with tap water to stop the development; counterstain with hematoxylin for 1 min, differentiate with hydrochloric acid alcohol, return to blue with ammonia, undergo gradient dehydration and clearing treatment, and mount with neutral resin; observe under a microscope, and determine positive expression of TMEM65 and MIC60 proteins based on the appearance of brownish-yellow granules in the cytoplasm. Use a semi-quantitative scoring method, score according to the proportion of positive cells and staining intensity, and simultaneously calculate the ratio of the two protein scores. Refer to the criteria for judging the integrity of mitochondrial cristae to clarify the structural integrity status of the mitochondrial cristae in the sample.

[0058] Example 2: The only difference between this example and Example 1 is in the preparation steps of the mitochondrial cristae integrity protein detection kit. In Example 2, 4-hydroxy-2,5-dimethyl-3(2H)-furanone is added to the primary antibody working solution. The final concentration of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in the primary antibody working solution is 6 μmol / L.

[0059] Example 3: The only difference between this example and Example 2 is that in the preparation steps of the mitochondrial cristae integrity protein detection kit, the final concentration of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in the primary antibody working solution in Example 3 was adjusted from 6 μmol / L to 9 μmol / L.

[0060] Example 4: The only difference between this example and Example 3 is in the preparation steps of the mitochondrial cristae integrity protein detection kit. In Example 4, 2-methylthio-4-amino-6-hydroxypyrimidine is added to the secondary antibody working solution. The final concentration of 2-methylthio-4-amino-6-hydroxypyrimidine in the secondary antibody working solution is 2 μmol / L.

[0061] Example 5: The only difference between this example and Example 4 is that in the preparation steps of the mitochondrial cristae integrity protein detection kit, the final concentration of 2-methylthio-4-amino-6-hydroxypyrimidine in the secondary antibody working solution in Example 5 is adjusted from 2 μmol / L to 3.5 μmol / L.

[0062] Comparative Example 1: The only difference between this comparative example and Example 2 is that in the preparation steps of the mitochondrial cristae integrity protein detection kit, the final concentration of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in the primary antibody working solution of Comparative Example 1 was adjusted from 6 μmol / L to 12 μmol / L.

[0063] Comparative Example 2: The only difference between this comparative example and Example 3 is that in the preparation steps of the mitochondrial crest integrity protein detection kit, 4-hydroxy-2,5-dimethyl-3(2H)-furanone was replaced with acetone, and the final concentration of acetone in the primary antibody working solution was 9 μmol / L.

[0064] Comparative Example 3: The only difference between this comparative example and Example 5 is that in the preparation steps of the mitochondrial cristae integrity protein detection kit, 2-methylthio-4-amino-6-hydroxypyrimidine is replaced with urea, and the final concentration of urea in the secondary antibody working solution is 3.5 μmol / L.

[0065] Example 1: Expression and subcellular localization analysis of TMEM65 and MIC60 proteins in gastric cancer tissues and cells.

[0066] Test samples: human normal gastric epithelial cells GES1, gastric cancer cell lines HGC27, GCIY, AGS, and NUGC3; cancer tissues and paired adjacent normal tissues from 112 patients with gastric adenocarcinoma; and 433 gastric cancer tissue samples with complete clinical information.

[0067] Test methods: Western blot was used to detect the expression of TMEM65 and MIC60 (MIC60) proteins in GES1 and gastric cancer cells; subcellular localization of the proteins was observed by immunofluorescence double labeling assay, and co-localization was verified by mitochondrial markers; the expression of TMEM65 and MIC60 in tissue microarrays was detected by immunohistochemistry (IHC), and the positive rate and staining intensity were statistically analyzed by semi-quantitative scoring method to analyze the correlation between their expression and their association with clinical characteristics.

[0068] Differences in TMEM65 protein expression in different gastric cancer cells, such as Figure 1 As shown, the differences in protein expression of TMEM65 and PPARA between TMEM65-KO cells and the control group are as follows: Figure 2 As shown in the figure, Western blot results indicated that the expression levels of TMEM65 and MIC60 in gastric cancer cells were significantly higher than those in GES1 normal cells, with the highest expression levels observed in HGC27 and NUGC3 cells. The expression location of TMEM65 was detected by cell fluorescence as shown in the figure. Figure 3 As shown, DAPI labeled the cell nucleus, and FITC and PE labeled the expression of TMEM65 and mitochondrial marker proteins, respectively. Immunofluorescence and mitochondrial component separation experiments confirmed that TMEM65 and MIC60 are both located in the inner mitochondrial membrane and exhibit significant co-localization, making them core proteins for maintaining mitochondrial cristae structure. Immunohistochemical staining of TMEM65 protein in cancerous and adjacent tissues from gastric cancer patients using the DAB method was performed. Figure 4 As shown, immunohistochemistry revealed that the positive rates and staining intensity of TMEM65 and MIC60 in the tumor region of gastric cancer tissue were significantly higher than those in adjacent normal tissue. These results clarified the mitochondrial localization of TMEM65 and MIC60 and their gastric cancer-specific high expression characteristics, providing tissue and cellular level evidence for protein detection kits.

[0069] Experimental Example 2: Verification of the interaction between TMEM65 and MIC60 proteins and their regulation of mitochondrial cristae structure.

[0070] Test samples: HGC27 and GCIY cells with TMEM65 knockout, MIC60 knockdown, and TMEM65 overexpression, and mouse subcutaneous xenografts of gastric cancer.

[0071] Test methods: Immunoprecipitation was performed in HGC27 and 293T cells, and the protein precipitation effect was verified by Western blot. The immunoprecipitated samples were sent for mass spectrometry analysis. Target proteins with fold change > 2 in both groups were subjected to overlap analysis, and the proteins were sorted according to the detected unique peptides. The direct interaction between TMEM65 and MIC60 was verified by endogenous co-immunoprecipitation (Co-IP) and TurboID mass spectrometry analysis. The regulation of MIC60 protein stability by TMEM65 was detected by Western blot. The number, morphology, and structural integrity of mitochondrial cristae in gene-modified cells and mouse xenograft tissues were observed by cryo-transmission electron microscopy.

[0072] Immunoprecipitation was performed in HGC27 cells to verify the protein precipitation effect. Figure 5 As shown, the protein precipitation effect was verified by immunoprecipitation in 293T cells. Figure 6 As shown, the target protein overlap and intersection analysis is as follows: Figure 7 As shown, the binding of endogenous TMEM65 and MIC60, MIC19 and CS in HGC27 / GCIY cells is as follows: Figure 8 As shown, the combination of TMEM65 and MIC60 in the TurboID input and IP (Strep) samples is as follows. Figure 9 As shown, green arrows indicate protein locations; immunoprecipitation and mass spectrometry confirmed a direct and stable endogenous protein interaction between TMEM65 and MIC60, with TMEM65 stabilizing MIC60 protein levels by inhibiting its ubiquitination and degradation; differences in TMEM65 and MIC60 expression in different cells are shown below. Figure 10 As shown, the differences in migration ability of cells expressing different amounts of TMEM65 and MIC60 are as follows: Figure 11 As shown, the expression differences of TMEM65, MIC60, and PPARA in different cells are as follows: Figure 12 As shown in the figure, Western blot results indicate that MIC60 protein levels are significantly reduced after TMEM65 knockout, TMEM65 overexpression can upregulate MIC60 and PPARα, while MIC60 knockdown does not affect TMEM65 expression.

[0073] Cryo-transmission electron microscopy observation of mitochondrial cristae morphology in cells of different treatment groups, such as Figure 13 As shown, intracellular lipid droplet accumulation was detected by cell fluorescence, with DAPI labeling the cell nucleus and Lipid-TOX labeling the lipid droplets at 400X (left). Cryo-transmission electron microscopy was used to observe the morphology of mitochondrial cristae in cells from different treatment groups at bar=500 nm (right). Cryo-transmission electron microscopy was also used to observe the morphology of mitochondrial cristae in different tissue samples. Figure 14As shown, cryo-transmission electron microscopy revealed that a decrease in TMEM65 or MIC60 leads to a reduction in the number and structural damage of mitochondrial cristae. When both are co-expressed normally, the mitochondrial cristae structure remains intact. The results in mouse xenograft tissue were consistent with those in in vitro cells. These results confirm, from the perspectives of protein interaction and ultrastructure, that the TMEM65 / MIC60 axis is a key functional axis for regulating the integrity of mitochondrial cristae.

[0074] Experimental Example 3: Antigen-antibody binding affinity test of mitochondrial crest integrity protein detection kit.

[0075] Test samples: Mitochondrial cristae integrity protein detection kits prepared for each example and comparative example.

[0076] Test method: The binding affinity between the specific antibody in the kit and the TMEM65 antigen was determined by enzyme-linked immunosorbent assay (ELISA). The purified antigen was coated onto the ELISA plate, and the primary antibody working solution of each example and comparative kit was added for incubation. After washing, the corresponding secondary antibody working solution was added, and the absorbance value at 450 nm was measured after color development. The average absorbance value reflects the binding affinity.

[0077] The results of the antigen-antibody binding affinity test of the mitochondrial cristae integrity protein assay kit are shown in Table 1.

[0078] Table 1. Results of antigen-antibody binding affinity test of the mitochondrial cristae integrity protein detection kit.

[0079] Example 1 uses a basic immunohistochemical detection system with a specific monoclonal antibody targeting a conserved epitope. The antigen-antibody binding ability is stable and reliable, and the absorbance is within the standard effective range for ELISA, meeting the needs of clinical and research sample detection. Example 2 adds an appropriate concentration of 4-hydroxy-2,5-dimethyl-3(2H)-furanone to the primary antibody working solution. By improving the spatial conformation of the antigen epitope and reducing binding steric hindrance, the binding affinity between the antibody and the antigen is significantly enhanced, and the absorbance is significantly higher than in Example 1. Example 3 further optimizes the concentration of this enhancer, making the antigen-antibody binding interface more stable and continuously improving the affinity. Example 4 adds 2-methylthio-4-amino-6-hydroxypyrimidine to the secondary antibody working solution based on the primary antibody enhancement. Enhanced immune complex stability and improved enzyme-labeled signal output efficiency resulted in reasonable signal amplification, with absorbance slightly higher than in Example 3, fully conforming to the conventional immunological principle that the primary antibody determines specificity and binding capacity, while the secondary antibody is only responsible for signal amplification. Example 5 employed the optimal ratio of dual synergistic components, achieving optimal antigen-antibody binding efficiency and detection signal intensity. Comparative Example 1, due to synergist concentration exceeding the appropriate range, could not effectively improve antigen-antibody binding efficiency, and the affinity enhancement effect was limited. Comparative Example 2 replaced the synergistic component with acetone, losing the antigen epitope optimization effect, and the antibody binding capacity decreased significantly. Comparative Example 3 replaced the signal optimization component with urea, failing to achieve effective signal amplification and improved binding stability, and the detection performance was significantly inferior to Examples 4 and 5.

[0080] Example 4: Repeatability evaluation of the mitochondrial crest integrity protein detection kit.

[0081] Test samples: Mitochondrial cristae integrity protein detection kits prepared in Examples 1-5 and Comparative Examples 1-3.

[0082] Test method: Paraffin sections of the same gastric cancer tissue were selected as the unified test samples. Five independent repeated immunohistochemical tests were performed using the kits of each example and comparative example. The same experimental conditions and operating procedures were strictly followed. ImageProPlus software was used to perform quantitative analysis of the average optical density (IOD) of the positive stained areas and calculate the coefficient of variation (CV) of the TMEM65 protein detection results.

[0083] The repeatability evaluation results of the mitochondrial cristae integrity protein detection kit are shown in Table 2.

[0084] Table 2. Repeatability evaluation results of the mitochondrial cristae integrity protein detection kit.

[0085] Example 1 demonstrates a basic immunohistochemical detection system. Multiple independent repeat tests showed good consistency, with the coefficient of variation within an acceptable range, meeting the stable testing requirements for routine clinical samples and basic research. Example 2 adds an appropriate concentration of 4-hydroxy-2,5-dimethyl-3(2H)-furanone to the primary antibody working solution. This stabilizes antigen-antibody binding efficiency, reduces staining fluctuations and non-specific binding, significantly lowering the coefficient of variation compared to Example 1 and noticeably improving repeatability. Example 3 further optimizes the concentration of the synergistic component, improving staining uniformity and result stability, further reducing the coefficient of variation. Example 4 adds 2-methylthio-4-amino-2-hydroxy-2-dimethyl ... 6-Hydroxypyrimidine further improves detection repeatability by optimizing signal output uniformity and reducing systematic errors. Example 5 uses the optimal ratio of dual synergistic components, achieving the best staining uniformity, signal stability, and result consistency, with highly consistent results across multiple tests. Comparative Example 1, due to the synergist concentration exceeding the suitable range, could not fully stabilize the detection system, resulting in a higher coefficient of variation than Examples 2 and 3. In Comparative Example 2, replacing the synergistic component with acetone decreased antigen-antibody binding stability, increased detection error, and significantly reduced repeatability. In Comparative Example 3, replacing the signal optimization component with urea only slightly reduced non-specific binding and could not achieve efficient and stable results, with a higher coefficient of variation than Examples 4 and 5, but slightly better than Example 1.

[0086] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0087] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A kit for detecting mitochondrial cristae integrity proteins, characterized in that: The kit includes a primary antibody working solution and a secondary antibody working solution. The primary antibody working solution includes a TMEM65 protein-specific antibody and a MIC60 protein-specific antibody, and the secondary antibody working solution includes horseradish peroxidase-labeled goat anti-rabbit IgG.

2. The mitochondrial cristae integrity protein detection kit according to claim 1, characterized in that: The primary antibody working solution includes TBST buffer, which comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20; the volume ratio of the TMEM65 specific antibody to the TBST buffer is 1:200-1000, and the volume ratio of the MIC60 specific antibody to the TBST buffer is 1:200-1000.

3. The mitochondrial cristae integrity protein detection kit according to claim 1, characterized in that: The secondary antibody working solution contains TBST buffer, which includes 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl and 0.05-0.2% Tween-20; the volume ratio of horseradish peroxidase-labeled goat anti-rabbit IgG to TBST buffer is 1:1000-3000.

4. The mitochondrial cristae integrity protein detection kit according to claim 1, characterized in that: The kit includes an antigen retrieval solution, which is a 0.005~0.02mol / L citrate antigen retrieval solution with a pH of 5.9~6.

1.

5. The mitochondrial cristae integrity protein detection kit according to claim 1, characterized in that: The kit includes a blocking solution, which is a normal goat serum blocking solution.

6. The mitochondrial cristae integrity protein detection kit according to claim 1, characterized in that: The primary antibody working solution includes 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and the final concentration of the 4-hydroxy-2,5-dimethyl-3(2H)-furanone primary antibody working solution is 6~9 μmol / L.

7. A method for preparing the kit according to any one of claims 1 to 6, characterized in that: Includes the following steps, Rabbit monoclonal antibodies targeting conserved antigenic epitopes in the exposed mitochondrial membrane region of human TMEM65 protein and conserved antigenic epitopes of human MIC60 protein were selected as primary antibodies, and horseradish peroxidase-labeled goat anti-rabbit IgG was selected as secondary antibodies. The primary antibody and secondary antibody were diluted with TBST buffer containing 5% bovine serum albumin at pH 7.4 to prepare working solutions for the primary antibody and secondary antibody, respectively. Antigen retrieval solution, blocking solution, washing solution, colorimetric solution, counterstaining solution, and mounting medium were prepared and aliquoted to obtain the mitochondrial cristae integrity protein detection kit.

8. A method for detecting the integrity of mitochondrial cristae in vitro, characterized in that: Immunohistochemical detection of ex vivo samples using the kit described in any one of claims 1 to 6 includes: The paraffin sections of the sample to be tested were dewaxed to water, and antigen retrieval was performed under high temperature and high pressure with citrate buffer to block endogenous peroxidase. After washing with TBST buffer, block with blocking buffer, incubate with primary antibody working solution at 4°C, wash again, and then incubate with secondary antibody working solution. DAB developing solution, hematoxylin counterstaining, differentiation, blue reversion, dehydration and clearing, mounting; A semi-quantitative scoring method was used, with scores based on the proportion of positive cells and staining intensity. The ratio of TMEM65 to MIC60 scores was calculated simultaneously to determine the integrity status of mitochondrial cristae.

9. The application of the mitochondrial cristae integrity protein detection kit according to any one of claims 1 to 7, characterized in that: The kit is used to detect the integrity of mitochondrial cristae in ex vivo tissue and cell samples.

10. The application according to claim 9, characterized in that: The kit is used for tumor metabolism research, basic research on mitochondrial diseases, high-throughput drug screening, and cell function assessment.

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