Application of RANTES in preparation of product for diagnosing or evaluating pelvic organ prolapse and product

Abstract

The invention discloses application of RANTES in preparation of a product for diagnosing or evaluating pelvic organ prolapse and the product, belongs to the technical field of biological medicine, and particularly relates to detection of the concentration content of RANTES in a blood sample on the molecular level by taking the blood sample of a subject as a detection object. A large number of experiments prove that the concentration content of RANTES molecules in blood can be used as a detection index for diagnosing or evaluating pelvic organ prolapse, and early intervention and subsequent treatment of pelvic organ prolapse are facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to the application of RANTES in the preparation of products for diagnosing or evaluating pelvic organ prolapse and the products. Background Art

[0002] Regulated upon activation normal T cell expressed and secreted (RANTES) is a member of the CC subfamily of chemotactic cytokines. It can chemotactic T cells, eosinophils and basophils, and plays an active role in recruiting leukocytes to inflammatory sites. In addition to being a typical chemotactic cytokine, some new biological characteristics of RANTES have gradually been discovered in recent years. Studies have shown that the level of plasma RANTES can be regarded as a marker for the early stages of Alzheimer's disease. In patients with Alzheimer's disease, the higher the level of RANTES, the more obvious the inflammatory response and the worse the insulin metabolism function (see "Normal T cell expression and secretion (RANTES) levels in the peripheral blood of patients with Alzheimer's disease are regulated after activation" "Chinese Journal of Neural Regeneration Research (English Edition) 2021 Issue 4, 775-779). In addition, RANTES can also be used as a highly expressed cytokine and combined with tumor markers in colorectal diagnosis and treatment, which has a better diagnostic effect on colorectal cancer than the detection of carcinoembryonic antigen (CEA) alone (see "Application of combined detection of RANTES and tumor markers in the diagnosis and treatment of colorectal cancer" "International Journal of Laboratory Medicine" | Issue S01, 2023, 4-10).

[0003] Pelvic organ prolapse (POP) is a type of pelvic floor support tissue weakness caused by various reasons, which causes the pelvic organs to descend and shift, leading to abnormal organ position and function. The main symptom is the prolapse of vulvar masses, accompanied or not accompanied by abnormal urination and defecation, vulvar bleeding, inflammation, etc., which affect the patient's quality of life to varying degrees. Known diagnostic markers for POP include UNC45A (Unc-45 myosin chaperone protein), HSP10 (heat shock protein 10), Utrophin (Duchenne muscular dystrophy-related protein) and ZC3H8 (CCCH type zinc finger protein) (see Chinese patents CN111856038A, Chinese patents CN111596069A, Chinese patents CN111596068A and Chinese patents CN111596067A).

[0004] Currently, there is no report documenting the use of RANTES in the diagnosis and evaluation of pelvic organ prolapse. Summary of the invention

[0005] The purpose of the present invention is to provide an application of RANTES in the preparation of a product for diagnosing or evaluating pelvic organ prolapse. For the first time, RANTES is used as a molecular marker to efficiently, sensitively and accurately realize the diagnosis or evaluation of pelvic organ prolapse. At the same time, the present invention also provides a product for diagnosing or evaluating pelvic organ prolapse, providing a new detection basis and standard for the existing clinical screening of pelvic organ prolapse.

[0006] The present invention is achieved through the following technical solution: application of RANTES in the preparation of a product for diagnosing or evaluating pelvic organ prolapse, wherein the product uses a blood sample of a subject as a detection object and detects the concentration of RANTES in the blood sample at the molecular level.

[0007] The blood sample includes whole blood, serum or plasma.

[0008] When the concentration of RANTES in the blood sample is higher than 33 ng / mL, the subject is diagnosed or assessed as having pelvic organ prolapse; when the concentration of RANTES in the blood sample is lower than 33 ng / mL, the subject is diagnosed or assessed as not having pelvic organ prolapse.

[0009] The detection methods include immunofluorescence analysis, ELISA, biochip method and electrochemiluminescence method.

[0010] The products include kits, detection reagents or other medically feasible products.

[0011] The present invention also provides a product for diagnosing or evaluating pelvic organ prolapse as used in the above application.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention is based on the fact that the content of RANTES molecules in the blood of patients with pelvic organ prolapse is significantly higher than that in healthy people. A large number of experiments have shown that the concentration of RANTES molecules in the blood can be used as a detection indicator for diagnosing or evaluating pelvic organ prolapse, which is beneficial for early intervention and subsequent treatment of pelvic organ prolapse.

[0013] (2) The present invention is the first to use the RANTES molecule as a detection indicator for diagnosing or evaluating whether a subject suffers from pelvic organ prolapse. Its content can be detected in the blood, and the test results can be obtained using known detection methods such as immunofluorescence analysis, ELISA, biochip method, electrochemiluminescence method, etc., which can achieve high-sensitivity and high-precision rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is the A450 value-RANTES concentration standard curve involved in Example 2. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0016] Example 1: Preparation of a diagnostic or assessment kit for pelvic organ prolapse for detecting RANTES molecules Reagent preparation: carbonate buffer at pH 9; carbonate buffer at pH 9.6, 1 mol / L; anhydrous methanol; bovine serum albumin solution; phosphate buffer at pH 7.2, 0.05 mol / L; dimethylformamide; biotinyl-N-hydroxysuccinimide ester; avidin-horseradish peroxidase; 3',3',5,5'-tetramethylbenzidine; RANTES protein standard; sample diluent.

[0017] Instrument preparation: constant temperature box; magnetic stirrer; precision pipette; microplate reader capable of measuring absorbance at 450 nm.

[0018] The specific preparation steps are as follows: (1) Prepare a multiwell plate coated with mouse anti-human RANTES monoclonal antibody (purchased from abcam, catalog number ab52562).

[0019] The mouse anti-human RANTES monoclonal antibody is diluted with a carbonate buffer at pH=9 to a diluent with a concentration of 10 μg / mL, anhydrous methanol is added to the antibody diluent, and the amount of anhydrous methanol added is limited to 3 mL / 100 mL, and then the mouse anti-human RANTES monoclonal antibody diluent containing methanol is added to each well of a 96-well plate at an amount of 100 μL / well, and coated at 4°C for 12 hours; after the coating time expires, a bovine serum albumin solution with a concentration of 1g / 100mL is added to each well of the multi-well plate at an amount of 120 μL / well, and a blocking reaction is carried out at 37°C for 1 hour; after the blocking reaction is completed, the multi-well plate is washed with a phosphate buffer at pH=7.2 and a concentration of 0.05 mol / L, and when the unreacted substances on the multi-well plate are removed, the multi-well plate coated with the mouse anti-human RANTES monoclonal antibody is obtained, and then the multi-well plate is placed in a refrigerator at 4°C for storage for standby use.

[0020] (2) Prepare biotin-labeled rabbit anti-human RANTES polyclonal antibody (purchased from abcam, catalog number ab271272) (Biotin-RANTES Ab) detection solution.

[0021] N,N-dimethylformamide was used as solvent and biotinyl-N-hydroxysuccinimide ester was used as solute to prepare an acyl-N-hydroxysuccinimide ester solution with a concentration of 50 μg / μL. A carbonate buffer solution with a pH of 9.6 and a concentration of 1 mol / L was used as solvent and rabbit anti-human RANTES polyclonal antibody was used as solute to prepare a rabbit anti-human RANTES polyclonal antibody solution with a concentration of 24 mg / mL. The acyl-N-hydroxysuccinimide ester solution and the rabbit anti-human RANTES polyclonal antibody solution were weighed according to the mass ratio of acyl-N-hydroxysuccinimide ester to rabbit anti-human RANTES polyclonal antibody = 1:7 to form a mixed solution. The mixed solution of acyl-N-hydroxysuccinimide ester solution and rabbit anti-human RANTES polyclonal antibody was reacted at room temperature for 4 hours under stirring (magnetic stirrer, stirring speed 200 rpm) to obtain biotin-labeled rabbit anti-human RANTES monoclonal antibody (Biotin-RANTES Ab); after the reaction is completed, the reaction solution containing biotin-labeled rabbit anti-human RANTES polyclonal antibody (Biotin-RANTES Ab) is placed in a dialysis bag and dialyzed with a phosphate buffer having a pH of 9.2 and a concentration of 0.05 mol / L at 4°C for 12 hours, during which the dialysate is replaced 3 times; subsequently, bovine serum albumin is added to the dialyzed reaction solution, and the amount of bovine serum albumin added is limited to a concentration of 4 g / 100 mL.

[0022] The dialyzed reaction solution containing biotin-labeled rabbit anti-human RANTES polyclonal antibody (Biotin-RANTESAb) and bovine serum albumin prepared in the above process is measured with glycerol in a volume ratio of 1:1, and mixed evenly at room temperature to form a biotin-labeled rabbit anti-human RANTES polyclonal antibody (Biotin-RANTES Ab) detection solution, which is stored at -20°C.

[0023] (3) Each kit is equipped with 12 mL of avidin-horseradish peroxidase, 12 mL of chromogenic substrate 3',3',5,5'-tetramethylbenzidine, 1 mL of RANTES protein standard (RANTES concentration is 50 ng / mL) and 30 mL of sample diluent. The sample diluent is prepared by adding bovine serum albumin to 0.02 mol / L Tris buffer (TBS). The amount of bovine serum albumin added is limited to its concentration of 2 g / 100 mL. The volume ratio of avidin-horseradish peroxidase to biotin-labeled rabbit anti-human RANTES polyclonal antibody detection solution is 1:1.

[0024] Example 2: Detection of RANTES concentration in a sample to be tested This example is a process of using the diagnosing or evaluating kit for pelvic organ prolapse prepared in Example 1 to detect the RANTES concentration of a sample to be tested.

[0025] Source of samples to be tested: Women in the outpatient, inpatient and physical examination center of West China Second Hospital of Sichuan University were selected as experimental subjects. According to the existing diagnostic methods, the experimental subjects were divided into a pelvic floor prolapse group and a healthy control group, and plasma samples from the two groups of women were collected as test samples, totaling 72 samples, of which samples 1-36 were taken from the healthy control group, and samples 37-72 were taken from the pelvic floor prolapse group.

[0026] Inclusion criteria for the samples to be tested: 40 to 80 years old, excluding women with malignant tumors, fibroids, pelvic masses and mental disorders, women diagnosed with POP were included in the pelvic floor prolapse group; 40 to 80 years old, no abnormal liver and kidney function biochemistry, no malignant tumors shown by patient self-report, imaging examinations and pathological examinations, no abnormal tumor markers, no pelvic masses, no mental disorders, no pelvic floor dysfunction or medical history, women diagnosed without POP were included in the normal control group.

[0027] The specific detection methods are as follows: 1. Samples to be tested (1) Preparation of standard samples The RANTES protein standard provided in the kit described in Example 1 was set as the standard sample with the highest RANTES concentration, named standard sample 0, whose RANTES concentration was 50 ng / mL, and then the RANTES protein standard was diluted with the sample diluent provided in the kit described in Example 1 to standards with RANTES concentrations of 2000 pg / mL, 666.7 pg / mL, 222.2 pg / mL, 74.07 pg / mL, 24.69 pg / mL, 8.23 ​​pg / mL, and 2.74 pg / mL, respectively, and named them standard sample 1, standard sample 2, standard sample 3, standard sample 4, standard sample 5, standard sample 6, and standard sample 7, respectively, for a total of 7 standards.

[0028] (2) Sample Female outpatients and inpatients of West China Second Hospital of Sichuan University were selected as experimental subjects. According to the existing diagnostic method, the experimental subjects were divided into a pelvic floor prolapse group and a healthy control group. Anticoagulated blood samples of the two groups of women were collected as specimens, totaling 72 specimens, of which specimens 1-36 were taken from the healthy control group, and specimens 37-72 were taken from the pelvic floor prolapse group.

[0029] (3) Blank control The sample diluent provided in the kit described in Example 1 is a blank control.

[0030] 2. Sample testing (1) Place all reagents and samples at room temperature (18-25°C) before testing.

[0031] (2) Add the standard sample, blank control, and sample prepared in step 1 to the corresponding wells of the 96-well plate coated with mouse anti-human RANTES monoclonal antibody. Set up duplicate wells for each standard sample, blank control, and sample. The amount of sample added to each well is 100 μL. Cover the 96-well plate with a film and place the 96-well plate on a shaker and incubate at room temperature for 2.5 hours.

[0032] (3) After the incubation time is up, aspirate all the liquid in the wells and set the washing program on the automatic plate washer to wash each well four times with 300 μL / well washing solution (phosphate buffer with a concentration of 0.02 mol / L or Tris buffer with a concentration of 0.02 mol / L). The washing solution should stay in the well for 10 to 15 seconds each time. After washing, turn the 96-well plate over so that the wells face downward and tap them gently on absorbent filter paper to completely remove the remaining washing solution in each well.

[0033] (4) Add 100 μL of biotinylated antibody (Biotin-RANTES Ab) detection solution to each well of the 96-well plate, then cover the 96-well plate with a film and place the 96-well plate on a shaker and incubate at room temperature for 1 hour. After the incubation time is over, aspirate the liquid in each well and wash the 96-well plate according to the method in step (3).

[0034] (5) Add 100 μL of avidin-horseradish peroxidase to each well of the 96-well plate, then cover the 96-well plate with a film and place the 96-well plate on a shaker and incubate at room temperature for 45 minutes. After the incubation time is over, aspirate the liquid in each well and wash the 96-well plate according to the method in step (3).

[0035] (6) Add 100 μL of 3',3',5,5'-tetramethylbenzidine to each well of the 96-well plate and incubate the plate in the dark for 30 min at room temperature. After the incubation time is over, add 50 μL of stop solution to each well. The stop solution is an aqueous solution of sulfuric acid and sodium sulfite with a concentration of 2 mol / L and 0.1 mol / L, respectively.

[0036] (7) Place the 96-well plate in an ELISA reader and measure the A450 value (optical density or absorbance value) of each well. The results are shown in Tables 1 to 3 below. The A450 value of the blank control is 0.060.

[0037] Table 1: A450 values ​​of standard samples Table 2: A450 values ​​of samples 1-36 Table 3: A450 values ​​of samples 37-72 (8) Based on the A450 value of each standard sample and the RANTES concentration in each standard sample, a standard curve of A450 value-RANTES concentration was prepared (see Figure 1 ), and the calculation formula y = 0.0011x + 0.1139 was obtained, where y is the A450 value of the sample being tested and x is the RANTES content in the sample being tested.

[0038] (9) Based on the A450 value of each sample, the concentration of RANTES in each sample was calculated using the formula y = ((max / (x-min)-1)^(-1 / Hillslope))*EC50 (see Table 6). The calculation results are shown in Tables 4 and 5 below.

[0039] Table 4: Calculation results of samples 1-36 (RANTES concentration unit: ng / mL) Table 5: Calculation results of samples 37-72 (RANTES concentration unit: ng / mL) Table 6: Formula and parameters of the four-parameter method The average RANTES concentration of samples 1-36 in Table 4 was calculated to be 17.21±19.27 ng / mL. The average RANTES concentration of samples 37-72 in Table 5 was calculated to be 81.12±55.62 ng / mL. From the calculation results, it can be seen that the difference between the average RANTES concentration in samples 1-36 taken from the healthy control group and the average RANTES concentration in samples 37-72 taken from the pelvic floor prolapse group is statistically significant.

[0040] The detection limit is set according to the ROC curve calculation result of the sample. Therefore, the RANTES detection limit set in the kit of the present invention is 33 ng / mL, that is, a RANTES concentration in the test sample higher than 33 ng / mL is judged as positive (i.e., suffering from female pelvic organ prolapse), and a RANTES concentration lower than 33 ng / mL is judged as negative (i.e., not suffering from female pelvic organ prolapse).

[0041] Example 3: Specificity and sensitivity test This example is a specificity and sensitivity test for diagnosing or evaluating pelvic organ prolapse using the kit described in Example 1.

[0042] (1) Specificity Source of samples to be tested: Women in the outpatient, inpatient and physical examination center of West China Second Hospital of Sichuan University were selected as experimental subjects. According to the existing diagnostic methods, the experimental subjects were divided into a pelvic floor prolapse group and a healthy control group, and plasma samples from the two groups of women were collected as test samples, totaling 72 samples, of which samples 1-36 were taken from the healthy control group, and samples 37-72 were taken from the pelvic floor prolapse group.

[0043] The inclusion criteria and detection methods of the samples to be tested are the same as those in Example 2.

[0044] The test results are shown in Table 7 below.

[0045] Table 7: Detection results of the samples to be tested The specificity was determined based on the ROC curve, and the specificity of the test kit for the test sample was calculated to be 86.11%.

[0046] (2) Sensitivity Source of samples to be tested: Women in the outpatient, inpatient and physical examination center of West China Second Hospital of Sichuan University were selected as experimental subjects. According to the existing diagnostic methods, the experimental subjects were divided into a pelvic floor prolapse group and a healthy control group, and plasma samples from the two groups of women were collected as test samples, totaling 72 samples, of which samples 1-36 were taken from the healthy control group, and samples 37-72 were taken from the pelvic floor prolapse group.

[0047] The inclusion criteria and detection methods of the samples to be tested are the same as those in Example 2.

[0048] The sensitivity was determined by the ROC curve, and the sensitivity of the test kit of the present invention to the test sample was calculated to be 80.56%.

[0049] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of RANTES in the preparation of a product for diagnosing or evaluating pelvic organ prolapse, characterized in that: The product uses a blood sample of a subject as a detection object and detects the concentration of RANTES in the blood sample at the molecular level.

2. The use according to claim 1, characterized in that: The blood sample includes whole blood, serum or plasma.

3. The use according to claim 1, characterized in that: When the concentration of RANTES in the blood sample is higher than 33 ng / mL, the subject is diagnosed or assessed as having pelvic organ prolapse; when the concentration of RANTES in the blood sample is lower than 33 ng / mL, the subject is diagnosed or assessed as not having pelvic organ prolapse.

4. The use according to claim 1, characterized in that: The detection methods include immunofluorescence analysis, ELISA, biochip method and electrochemiluminescence method.

5. The use according to claim 1, characterized in that: The products include test kits, detection reagents or other medically feasible products.

6. A product for diagnosing or evaluating pelvic organ prolapse for use as claimed in any one of claims 1 to 5.

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