Reagent and kit for detecting HJURP-S-S-PRDX1 disulfide bond compound and application of reagent and kit

Abstract

The invention discloses a reagent for detecting an HJURP-S-S-PRDX1 disulfide bond complex. The reagent comprises a capture antibody, a detection antibody and an enzyme substrate, the capture antibody is an anti-HJURP specific antibody, and the detection antibody is a horse radish peroxidase labeled anti-PRDX1 specific antibody; the enzyme substrate is a catalytic substrate of horse radish peroxidase. The kit prepared by utilizing the reagent can be used for detecting the content of the HJURP-S-S-PRDX1 disulfide bond complex in PCa tissues and predicting the sensitivity of PCa patients to ferroptosis inducers, a more accurate and reliable basis is provided for treatment of PCa, and meanwhile, the kit is more economical, simpler and more efficient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a reagent, a kit, and their applications for detecting the HJURP-S-S-PRDX1 disulfide bond complex. Background Art

[0002] Inducing ferroptosis, as a promising treatment strategy for prostate cancer (PCa), shows good application prospects whether used alone or in combination with hormone therapy. Therefore, it is crucial to clarify the mechanism of regulating ferroptosis in PCa cells.

[0003] It is pointed out in the article "HJURP inhibits sensitivity to ferroptosis inducers in prostate cancer cells by enhancing the peroxidase activity of PRDX1" that HJURP forms a disulfide bond complex HJURP-S-S-PRDX1 with PRDX1 through Cys327 and Cys457. This disulfide bond binding can enhance the antioxidant ability of PRDX1, thereby leading to a decrease in the ROS level and ultimately inhibiting the sensitivity of PCa cells to ferroptosis inducers. Therefore, clarifying the content of HJURP-S-S-PRDX1 in PCa tissues helps predict the sensitivity of PCa cells to ferroptosis inducers, providing a new direction and basis for the precise treatment of tumors.

[0004] Disulfide bonds can be used as diagnostic markers for the HJURP-S-S-PRDX1 complex. The common existing techniques for detecting disulfide bonds are as follows:

[0005] (1) Spectroscopic methods

[0006] Infrared spectroscopy: Disulfide bonds absorb infrared light at specific wavelengths. By analyzing the corresponding characteristic absorption peaks in the infrared spectrum, the presence of disulfide bonds can be judged, but it is difficult to accurately determine their positions.

[0007] Ultraviolet absorption spectroscopy: Disulfide bonds have specific absorption characteristics in the ultraviolet region. By measuring the ultraviolet absorption spectrum of a sample, the number of disulfide bonds can be quantitatively calculated according to the absorption intensity, etc., but it is difficult to accurately determine their positions.

[0008] Nuclear magnetic resonance (NMR): By measuring the nuclear magnetic properties of atomic nuclei in a magnetic field, detailed information about the molecular structure is provided, and the presence and position of disulfide bonds can be determined, but the requirements for samples are high and the experimental cost is high.

[0009] (2) Mass spectrometry

[0010] The protein is enzymatically digested or chemically hydrolyzed to obtain peptide segments containing disulfide bonds, which are analyzed by a mass spectrometer. According to the mass difference of the peptide segments before and after the disulfide bonds are broken, the position and number of disulfide bonds are determined. It has high sensitivity, high resolution and accuracy, and can provide detailed structural information, but the equipment is expensive, the operation is complex, and the sample pretreatment is cumbersome.

[0011] (3) Chemical method

[0012] Disulfide bond reduction and reductive electrophoresis: Incubate the protein with a reducing agent (such as dithiothreitol) to break the disulfide bonds, and then analyze by reductive electrophoresis. The disulfide bond situation is judged according to the change of the electrophoresis band. It is suitable for the detection of endogenous disulfide bonds, but the operation is cumbersome.

[0013] Ethyl thiocyanate reduction method: Oxidize the disulfide bonds in the protein to pseudo-disulfide bonds with ethyl thiocyanate, add a reducing agent to reduce, and calculate the disulfide bond content by measuring the oxidation amount of the reducing agent. However, when detecting protein complexes, it may lead to incomplete formation of pseudo-disulfide bonds or calculation deviation of the reducing agent consumption, affecting the quantitative accuracy.

[0014] Dithioimide staining method: Dithioimide specifically reacts with disulfide bonds to form a sulfur blue staining product, and the absorbance is measured by ultraviolet spectroscopy or colorimetry to calculate the disulfide bond content. However, when measuring protein complexes, the steric hindrance of the protein may prevent the dye molecule from binding to the internal disulfide bonds, resulting in false negatives or low signal intensity.

[0015] (4) Other methods

[0016] X-ray crystallography: Perform X-ray diffraction on protein crystals, and obtain the three-dimensional structure of the protein by analyzing the diffraction data. The position and number of disulfide bonds can be determined, but high-quality protein crystals need to be obtained and the data processing is complex.

[0017] Atomic force microscopy: Can directly observe disulfide bonds at the nanoscale and provide information on the position and number, but requires complex instruments and professional techniques.

[0018] Biosensor technology: Utilize the specific interaction between biosensors and disulfide bonds to generate signal responses, with the advantages of high sensitivity, high selectivity and real-time monitoring, but requires complex instruments.

[0019] The existing methods for detecting disulfide bonds have problems such as inaccurate detection, the need for complex instruments and high costs. Therefore, it is of great significance to provide an economical, simple and efficient method for detecting the content of the HJURP-S-S-PRDX1 disulfide bond complex in PCa tissues, so as to predict the sensitivity of PCa patients to ferroptosis inducers and provide a more accurate and reliable basis for the treatment of PCa. Summary of the invention

[0020] The object of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and to provide a reagent for detecting the HJURP-S-S-PRDX1 disulfide bond complex.

[0021] The second object of the present invention is to provide the application of the above reagent in the preparation of a kit for detecting the HJURP-S-S-PRDX1 disulfide bond complex.

[0022] The third object of the present invention is to provide a kit for detecting the HJURP-S-S-PRDX1 disulfide bond complex.

[0023] The fourth object of the present invention is to provide a method for using the above kit.

[0024] The fifth object of the present invention is to provide the application of the above kit in detecting the HJURP-S-S-PRDX1 disulfide bond complex in prostate cancer tissues.

[0025] The above objects of the present invention are achieved by the following technical solutions:

[0026] The present invention provides a reagent for detecting the HJURP-S-S-PRDX1 disulfide bond complex, and the reagent includes a capture antibody, a detection antibody and an enzyme substrate; the capture antibody is an anti-HJURP specific antibody, the detection antibody is a horseradish peroxidase-labeled anti-PRDX1 specific antibody; the enzyme substrate is a catalytic substrate of horseradish peroxidase.

[0027] Inducing ferroptosis, as a promising treatment strategy for prostate cancer (PCa), shows good application prospects whether used alone or in combination with hormone therapy. Therefore, it is crucial to clarify the mechanism regulating ferroptosis in PCa cells. Existing studies have shown that HJURP forms a disulfide bond complex HJURP-S-S-PRDX1 with PRDX1 through Cys327 and Cys457. This disulfide bond binding can enhance the antioxidant capacity of PRDX1, thereby leading to a decrease in ROS levels and ultimately inhibiting the sensitivity of PCa cells to ferroptosis inducers. Therefore, clarifying the content of HJURP-S-S-PRDX1 in PCa tissues helps predict the sensitivity of PCa cells to ferroptosis inducers. Accordingly, the present invention provides a detection reagent that utilizes a pair of specific antibodies against HJURP and PRDX1 respectively. One antibody is coated on a solid-phase carrier as a capture antibody, and the other antibody is labeled with an enzyme (such as horseradish peroxidase HRP) as a detection antibody. When a sample containing the HJURP-S-S-PRDX1 complex is added, the complex will be bound by the capture antibody, and then the enzyme-labeled detection antibody is added to form a "capture antibody-HJURP-S-S-PRDX1 complex-detection antibody" sandwich structure. After adding the enzyme substrate, HRP catalyzes the substrate to undergo a color reaction, and the color depth is proportional to the content of the HJURP-S-S-PRDX1 disulfide bond complex in the sample. The absorbance is measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the content of the complex in the sample is calculated according to the standard curve. This kit aims to detect the content of the HJURP-S-S-PRDX1 disulfide bond complex in PCa tissues, predict the sensitivity of PCa cells to ferroptosis, and provide a new direction and basis for the precision treatment of tumors.

[0028] Furthermore, the reagent further includes a termination solution, a sample diluent, and a washing buffer.

[0029] Furthermore, the sample diluent includes a buffer, a protein stabilizer, and a preservative. The sample diluent is used to dilute blood samples to ensure that the complexes in the samples are within a suitable detection concentration range and to maintain the stability of the samples.

[0030] Even further, the buffer is PBS, the protein stabilizer is bovine serum albumin BSA, and the preservative is sodium azide.

[0031] Furthermore, the washing buffer includes a PBS buffer containing a surfactant. It is used to wash the microplate to remove unbound substances and reduce non-specific signals.

[0032] Even further, the surfactant is Tween-20.

[0033] Furthermore, the enzyme substrate is 3,3',5,5'-tetramethylbenzidine. 3,3',5,5'-tetramethylbenzidine will undergo a color reaction under the catalysis of HRP.

[0034] Furthermore, the stop solution is a sulfuric acid solution. It is used to terminate the enzymatic reaction and stop the color reaction at a certain stage for accurate determination of absorbance.

[0035] Even further, the stop solution is a 2M sulfuric acid solution.

[0036] The present invention provides the application of the above reagents in the preparation of a kit for detecting the HJURP-S-S-PRDX1 disulfide complex.

[0037] The present invention also provides a kit for detecting the HJURP-S-S-PRDX1 disulfide complex, and the kit contains the above reagents.

[0038] Furthermore, the kit further includes a solid-phase carrier; the solid-phase carrier is coated with an anti-HJURP specific antibody.

[0039] Even further, the solid-phase carrier is a microplate.

[0040] Preferably, the microplate is an 8×12 well removable strip.

[0041] Furthermore, the kit also includes a sealing film and standards. The sealing film is used to seal the microplate during incubation to prevent evaporation and contamination of the solution.

[0042] Furthermore, the standards are HJURP-S-S-PRDX1 disulfide complex standards with concentration gradients of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL respectively.

[0043] Furthermore, the kit should be stored at 2-8°C and avoid direct sunlight. The enzyme conjugate and substrate solution need to be stored in the dark, and the validity period is generally 12-18 months. Within the validity period, the performance indicators of the kit should meet the quality control requirements.

[0044] The present invention also provides a method for using the above kit, including the following steps:

[0045] S1. Sample treatment: Dilute the sample with a diluent;

[0046] S2. Sample addition: Set blank wells, standard wells, and sample wells respectively, add the corresponding samples and seal the plate, and incubate at 35-40°C for 1-2 hours;

[0047] S3. Washing: Discard the liquid in the wells and wash 4-6 times with a washing buffer;

[0048] S4. Add detection antibody: Add the detection antibody, mix well, seal the plate, and incubate at 35 - 40 °C for 30 - 60 minutes; discard the liquid in the wells and wash.

[0049] S5. Color development: Add the enzyme substrate, mix well, and incubate at 35 - 40 °C in the dark for 15 - 20 minutes.

[0050] S6. Terminate the reaction and measure absorbance: Add the termination solution, mix well; measure the absorbance of each well.

[0051] Furthermore, the sample in step S1 is a sample treated with a lysis buffer containing protease inhibitor, phosphatase inhibitor, and phenylmethylsulfonyl fluoride and methyl methanesulfonate. The research results of the present invention show that the test samples using this kit need to be specially treated to preserve disulfide bonds and inhibit the formation of new disulfide bonds of thiol groups. Specifically, immediately after sampling, the sample is placed in a cryotube and quickly frozen in liquid nitrogen to avoid air oxidation of the thiol groups (-SH) of HJURP and PRDX1, which may cause errors in the experimental results. Add an appropriate amount of cell lysis buffer (lysis buffer containing 0.1% protease inhibitor, 1% phosphatase inhibitor, and 1% phenylmethylsulfonyl fluoride), and shake on a rocker at room temperature for 10 minutes. After lysis, centrifuge at high speed and take the supernatant. Transfer the supernatant to a new EP tube. Add Methyl methanethiosulfonate (MMTS, final concentration 20 mM) to the supernatant to further block the thiol groups.

[0052] Furthermore, the lysis buffer contains 0.1% protease inhibitor, 1% phosphatase inhibitor, and 1% phenylmethylsulfonyl fluoride.

[0053] Specifically, the present invention provides a method for specifically detecting the HJURP - S - S - PRDX1 disulfide bond complex, including the following steps:

[0054] S1. Preparation: Take the kit out of the refrigerator and equilibrate it to room temperature (20 - 25 °C). At the same time, prepare the required experimental equipment, such as pipettes, microplate readers, etc.

[0055] S2. Sample treatment: Collect venous blood, treat it with an anticoagulant (such as EDTA), centrifuge at 3000 rpm for 10 minutes to separate the plasma. Dilute the plasma with the sample diluent in an appropriate ratio (such as 1:10).

[0056] S3. Sample addition: Set blank wells (only add sample diluent), standard wells (add 100 μL of standard products with different concentrations to each well), and sample wells (add 100 μL of the diluted sample to each well) respectively, gently shake and mix well, seal the plate with a sealing film, and incubate at 37 °C for 1 - 2 hours.

[0057] S4. Washing: Discard the liquid in the wells, fill each well with washing buffer, let it stand for 30 seconds and then spin dry, repeat washing 5 times and pat dry.

[0058] S5. Add detection antibody: Add 100 μL of detection antibody to each well, gently shake to mix, seal the plate, and incubate at 37°C for 30-60 minutes.

[0059] S6. Wash again: same as step 4.

[0060] S7. Color development: Add 90 μL of enzyme substrate solution to each well, gently shake to mix, and incubate at 37°C in the dark for 15-20 minutes. At this time, the solution will gradually develop color.

[0061] S8. Stop reaction: Add 50 μL of stop solution to each well and shake gently to mix. The blue color will immediately turn into yellow.

[0062] S9. Determine absorbance: Measure the absorbance (OD value) of each well at a wavelength of 450 nm on a microplate reader.

[0063] S10. Calculation of results

[0064] (1) Draw a standard curve with the concentration of the standard as the horizontal axis and the corresponding average OD value as the vertical axis. Professional data analysis software (such as Origin) can be used for curve fitting, generally using a four-parameter equation.

[0065] (2) According to the OD value of the sample, the concentration of the HJURP-SS-PRDX1 disulfide complex in the corresponding sample is obtained from the standard curve. If the sample is diluted, the obtained concentration needs to be multiplied by the dilution factor to obtain the actual complex content of the sample.

[0066] The results of the present invention show that the kit can be used to detect the content of HJURP-SS-PRDX1 disulfide bond complex in PCa tissues, predict the sensitivity of PCa cells to ferroptosis, and provide a new direction for the precision treatment of tumors.

[0067] Therefore, the present invention also provides the use of the above kit in detecting the HJURP-SS-PRDX1 disulfide bond complex in prostate cancer tissue.

[0068] The present invention also provides the use of the above-mentioned kit in preparing a product for detecting the HJURP-SS-PRDX1 disulfide bond complex in prostate cancer tissue.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention provides a reagent for detecting the HJURP-S-S-PRDX1 disulfide complex. The reagent includes a capture antibody, a detection antibody, and an enzyme substrate; the capture antibody is a specific antibody against HJURP, and the detection antibody is a horseradish peroxidase-labeled specific antibody against PRDX1; the enzyme substrate is a catalytic substrate of horseradish peroxidase. The kit prepared using this reagent can be used to detect the content of the HJURP-S-S-PRDX1 disulfide complex in PCa tissues, predict the sensitivity of PCa patients to ferroptosis inducers, and provide a more accurate and reliable basis for the treatment of PCa. At the same time, this kit is also more economical, simple, and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 It is an intermediate in which JURP forms a disulfide bond with PRDX1 through Cys327 and Cys457.

[0072] Figure 2 It is for detecting the HJURP-S-S-PRDX1 disulfide complex in prostate cancer cells by the kit of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0073] The present invention will be further described below in conjunction with the drawings in the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0074] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0075] Example 1 Detection of the HJURP-S-S-PRDX1 Disulfide Complex in Prostate Cancer Cells

[0076] According to the article "HJURP inhibits sensitivity to ferroptosis inducers in prostate cancer cells by enhancing the peroxidase activity of PRDX1", HJURP is an oncogene upregulated in PCa cells and plays a role in tumor proliferation. HJURP forms a disulfide bond complex HJURP-S-S-PRDX1 with PRDX1 through Cys327 and Cys457 residues. This disulfide bond binding promotes the redox cycle of PRDX1 and inhibits its overoxidation. As a result, HJURP enhances the peroxidase activity of PRDX1, thereby reducing the ROS level and further inhibiting lipid peroxidation induced by ferroptosis inducers. These findings reveal the potential of HJURP / PRDX1 as a novel therapeutic target and biomarker for ferroptosis in PCa patients.

[0077] To directly demonstrate the existence of disulfide bond binding between PRDX1 and HJURP, samples were subjected to CO-IP and non-reducing gel western blotting, which confirmed the formation of a disulfide bond-linked complex (HJURP-S-S-PRDX1) between HJURP and PRDX1 in prostate cancer cells ( Figure 1 A-D). In addition, endogenous HJURP in C4-2 cells treated with 10 μM H2O2 for 2 minutes was processed, and HJURP-S-S-X with a molecular weight greater than 75 KDa was analyzed by LC-MS / MS. Consistent with the above results, many unique PRDX1 peptides were found in the HJURP disulfide bond-linked conjugate ( Figure 1 E), further confirming the formation of a disulfide bond-linked intermediate between HJURP and PRDX1 in prostate cancer cells.

[0078] To determine the cysteines in HJURP that are involved in the formation of the HJURP-S-S-PRDX1 intermediate, HJURP contains 14 cysteines, and each residue was mutated separately. The results showed that mutations of Cys327, Cys348, and Cys457 significantly reduced the level of HJURP S-S-X, while the mutation of Cys646 only reduced the level of HJURP-SS-X peptides with a mass greater than 310 KDa ( Figure 1 F). However, only the mutations of Cys327 and Cys457 inhibited the formation of the PRDX1-S-S-HJURP conjugate ( Figure 1F). In view of these results, mutations were simultaneously introduced at both Cys327 and Cys457 sites, which completely inhibited the formation of disulfide bonds between HJURP and PRDX1, eliminating the formation of the PRDX1-S-S-HJURP conjugate. Figure 1 F). The above results indicate that in prostate cancer cells, PRDX1 forms disulfide bonds with cysteine 327 and cysteine 457 of HJURP.

[0079] Therefore, it is of great significance that the present invention provides a kit capable of detecting the content of the HJURP-S-S-PRDX1 disulfide bond complex in PCa tissues, predicting the sensitivity of PCa cells to ferroptosis, and providing a new direction and basis for the precision treatment of tumors.

[0080] Example 2 Preparation of the kit

[0081] I. Detection principle

[0082] The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method is adopted. Using a pair of specific antibodies against HJURP and PRDX1 respectively, one antibody is coated on the microplate as the capture antibody, and the other antibody is labeled with an enzyme (such as horseradish peroxidase HRP) as the detection antibody. When a sample containing the HJURP-PRDX1 disulfide bond complex is added to the microplate, the complex will be bound by the capture antibody, and then the detection antibody labeled with the enzyme is added to form a sandwich structure of "capture antibody-HJUR-S-S-PRDX1 complex-detection antibody". After adding the enzyme substrate, the enzyme catalyzes the substrate to undergo a color reaction, and the color intensity is proportional to the content of the HJURP-S-S-PRDX1 complex in the sample. The absorbance is measured by an enzyme-linked immunosorbent assay reader, and the content of the complex in the sample is calculated according to the standard curve.

[0083] II. Components of the kit

[0084] 1. Microplate: Coated with anti-HJURP specific antibody (capture antibody), 8×12 well removable strip.

[0085] 2. Standard: A series of HJURP-S-S-PRDX1 complex standards with known concentrations, used for drawing the standard curve. For example, the concentration gradient can be 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL.

[0086] 3. Enzyme conjugate (detection antibody): Anti-PRDX1 specific antibody labeled with horseradish peroxidase (HRP).

[0087] 4. Sample diluent: Used to dilute blood samples, ensure that the complexes in the samples are within an appropriate detection concentration range, and maintain the stability of the samples. The components may include buffers (such as PBS), protein stabilizers (such as bovine serum albumin BSA), preservatives (such as sodium azide), etc.

[0088] 5. Washing buffer: Used to wash microplates, remove unbound substances, and reduce non-specific signals. Generally, it is a PBS buffer containing surfactants such as Tween-20.

[0089] 6. Substrate solution (enzyme substrate): Commonly used such as tetramethylbenzidine (TMB) substrate solution, which will undergo a color reaction under the catalysis of HRP.

[0090] 7. Stop solution: Generally, it is a 2M sulfuric acid solution, used to terminate the enzymatic reaction, stop the color reaction at a certain stage, so as to accurately measure the absorbance.

[0091] 8. Sealing film: Used to seal microplates during incubation to prevent solution evaporation and contamination.

[0092] 9. Instruction manual: Details information such as the composition of the kit, storage conditions, operation steps, result calculation, precautions, etc.

[0093] III. Storage Conditions

[0094] The kit should be stored at 2 - 8°C, avoiding direct sunlight. Different components should be stored according to the requirements of the instruction manual. For example, the enzyme conjugate and substrate solution need to be stored in the dark. The validity period is generally 12 - 18 months. Within the validity period, the performance indicators of the kit should meet the quality control requirements.

[0095] Example 3 Operation Steps of the Kit

[0096] 1. Preparation: Take the kit out of the refrigerator and equilibrate it to room temperature (20 - 25°C). At the same time, prepare the required experimental equipment, such as pipettes, microplate readers, etc.

[0097] 2. Sample treatment: Collect venous blood, treat it with an anticoagulant (such as EDTA), centrifuge at 3000 rpm for 10 minutes to separate the plasma. Dilute the plasma with the sample diluent at an appropriate ratio (such as 1:10).

[0098] 3. Sample addition: Set up blank wells (only add sample diluent), standard wells (add 100 μL of standards with different concentrations to each well), and sample wells (add 100 μL of diluted samples to each well) respectively. Gently mix well, seal the plate with a sealing film, and incubate at 37°C for 1 - 2 hours.

[0099] 4. Washing: Discard the liquid in the wells, fill each well with the washing buffer, let it stand for 30 seconds and then shake off the liquid. Repeat the washing 5 times and pat dry.

[0100] 5. Add enzyme conjugate (detection antibody): Add 100 μL of enzyme conjugate to each well, gently shake to mix, seal the plate, and incubate at 37°C for 30-60 minutes.

[0101] 6. Wash again: Same as step 4.

[0102] 7. Color development: Add 90 μL of enzyme substrate solution to each well, gently shake to mix, and incubate at 37°C in the dark for 15-20 minutes. At this time, the solution will gradually develop color.

[0103] 8. Stop reaction: Add 50 μL of stop solution to each well and gently shake to mix. The blue color will immediately turn into yellow.

[0104] 9. Determine absorbance: Measure the absorbance (OD value) of each well at a wavelength of 450 nm on an ELISA reader.

[0105] 10. Calculation of results

[0106] (1) Draw a standard curve with the concentration of the standard as the horizontal axis and the corresponding average OD value as the vertical axis. Professional data analysis software (such as Origin) can be used for curve fitting, generally using a four-parameter equation.

[0107] (2) According to the OD value of the sample, the concentration of the HJURP-SS-PRDX1 complex in the corresponding sample is obtained from the standard curve. If the sample is diluted, the concentration obtained needs to be multiplied by the dilution factor to obtain the actual complex content of the sample.

[0108] 11. Quality Control

[0109] (1) Internal control: In each test, three internal control samples with high, medium and low concentration levels are set, and the content of HJURP-SS-PRDX1 disulfide complex is known and stable. The accuracy and repeatability of this experiment are judged by analyzing the test results of the internal control samples. If the test results of the internal control samples are out of the expected range, the results of this experiment are invalid and need to be retested.

[0110] (2) Precision test: Use the same batch of kits to test the same sample multiple times (e.g. 20 times) and calculate the intra-batch precision, expressed as the coefficient of variation (CV). Generally, the intra-batch CV is required to be ≤10%. At the same time, use multiple batches of kits to test the same sample and calculate the inter-batch precision, which is required to be ≤15%.

[0111] (3) Linear range verification: Use a sample of the HJURP-S-S-PRDX1 complex with a known concentration and perform the detection according to the operation steps of the kit to verify the linear relationship between the detection result and the theoretical concentration. Generally, it is required that within the linear range claimed by the kit, the correlation coefficient r ≥ 0.99.

[0112] (4) Specificity verification: Select other protein complexes with a structure similar to the HJURP-S-S-PRDX1 disulfide bond complex or the individual HJURP and PRDX1 proteins and perform the detection according to the operation steps of the kit. There should be no obvious cross-reaction, that is, the detection result should be close to the blank control.

[0113] Example 4 Detection of the Kit

[0114] Under sterile conditions, sample from the surgically resected or biopsied prostate cancer tissue (the same sample as in Example 1), avoid using necrotic tissue, and select the area with concentrated tumor cells and good viability. Immediately after sampling, place the sample into a cryotube and quickly freeze it in liquid nitrogen to avoid air oxidation of the sulfhydryl groups (-SH) of HJURP and PRDX1, which may cause errors in the experimental results. Mash the sample and add tissue digestive solution (such as 0.5% collagenase Type2 and 0.05% trypsin) to digest for 20 - 30 minutes, pass through a 70um sieve, and collect prostate cancer cells. Add an appropriate amount of cell lysis buffer (lysis buffer containing 0.1% protease inhibitor, 1% phosphatase inhibitor, and 1% phenylmethylsulfonyl fluoride), and shake on a rocker at room temperature for 10 minutes. After lysis, centrifuge with a high-speed centrifuge and take the supernatant. Transfer the supernatant to a new EP tube. Add Methylmethanethiosulfonate (MMTS, final concentration 20 mM) to the supernatant to further block the sulfhydryl groups, and boil the sample at 95 °C for 10 minutes to disrupt the tertiary structure of the protein. Then use the kit in Example 2 and detect the content of the HJURP-S-S-PRDX1 disulfide bond complex in the sample according to the usage method in Example 3.

[0115] The results are as Figure 2 shown. The content of the HJURP-S-S-PRDX1 disulfide bond complex in the tissues of 5 prostate cancer patients was detected. Three replicate wells were set for each patient. The detection results using the kit in Example 2 were the same as those in Example 1. It shows that the kit of the present invention can be used to detect the HJURP-S-S-PRDX1 disulfide bond complex in PCa tissues, and can also be used to detect the content of the HJURP-S-S-PRDX1 disulfide bond complex, predict the sensitivity of PCa cells to ferroptosis, and provide a new direction for the precision treatment of tumors.

Claims

1. A reagent for detecting the HJURP-S-S-PRDX1 disulfide bond complex, characterized in that, The reagent includes a capture antibody, a detection antibody, and an enzyme substrate; the capture antibody is a specific antibody against HJURP, and the detection antibody is a horseradish peroxidase-labeled specific antibody against PRDX1; the enzyme substrate is a catalytic substrate of horseradish peroxidase.

2. The reagent according to claim 1, wherein The reagent further includes a stop solution, a sample diluent, and a washing buffer.

3. Use of the reagent according to any one of claims 1 to 2 in the preparation of a kit for detecting the HJURP-S-S-PRDX1 disulfide bond complex.

4. A kit for detecting the HJURP-S-S-PRDX1 disulfide bond complex, characterized in that, The kit contains the reagent according to any one of claims 1 to 2.

5. The kit according to claim 4, characterized in that, The kit further includes a solid-phase carrier; the solid-phase carrier is coated with a specific antibody against HJURP.

6. The kit according to claim 4, wherein The kit also includes a sealing plate film and a standard product.

7. The kit according to claim 6, characterized in that, The standard product is a standard product of the HJURP-S-S-PRDX1 disulfide bond complex with concentration gradients of 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL respectively.

8. The method of using the kit according to any one of claims 4 to 7, characterized in that The usage method includes the following steps: S1. Sample treatment: Dilute the sample with a diluent. S2. Sample addition: Respectively set blank wells, standard product wells, and sample wells, add the corresponding samples, seal the plate, and incubate at 35-40 °C for 1-2 hours. S3. Washing: Discard the liquid in the wells and wash 4-6 times with the washing buffer. S4. Addition of enzyme conjugate: Add the enzyme conjugate, mix well, seal the plate, and incubate at 35-40 °C for 30-60 minutes; discard the liquid in the wells and wash. S5. Color development: Add the substrate, mix well, and incubate in the dark at 35-40 °C for 15-20 minutes. S6. Terminate the reaction and measure the absorbance: Add the stop solution, mix well; measure the absorbance of each well.

9. The use method according to claim 8, wherein The sample in step S1 is a sample treated with a lysis buffer containing protease inhibitor, phosphatase inhibitor, and phenylmethylsulfonyl fluoride and methyl methanesulfonate.

10. Use of the kit according to any one of claims 4 to 7 in the detection of the HJURP-S-S-PRDX1 disulfide bond complex in prostate cancer tissues.

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