Application and detection method of kidney injury biomarker protein

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a kidney injury biomarker protein detection method which comprises the following specific steps: S1, acquiring a urine sample to be detected, and centrifuging to obtain supernate; s2, adding 1 [mu] g / ml of PBS of a goat anti-rabbit antibody into an ELISA reaction plate, and then carrying out sealed incubation on the ELISA reaction plate; drying the reaction plate; washing with PBS (Phosphate Buffer Solution) (PBST) containing 0.2% of Twain-20, beating the reaction plate to dry, and repeating for two times to obtain a coated ELISA (Enzyme-Linked Immunosorbent Assay) plate; s3, respectively dissolving DKK3 and beta2-MG protein standard substances with PBS, respectively diluting, and drawing standard curves for the protein standard solutions with different concentrations; s4, respectively and sequentially adding 50 microliters of protein standard solutions or detection urine samples with different concentrations, 100 microliters of HRP labeled antibodies, 100 microliters of TMB developing solutions and 50 microliters of stop solutions into the coated ELISA plate holes; s5, within 15 minutes after the reaction is terminated, putting the reaction plate into a microplate reader, and reading an OD450 value; and S6, drawing a standard curve, and calculating the concentrations of DKK3 and beta2-MG proteins according to the standard curve.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application and detection method of a kidney injury biomarker protein. Background Art

[0002] In recent years, the harm of chronic kidney disease has increased significantly worldwide. According to the "2019 Global Health Estimates Report" released by the World Health Organization in December 2020, kidney disease has become one of the "top ten causes of death in the world." It is predicted that by 2040, the number of years of life lost due to chronic kidney disease will rise to fifth place worldwide. Kidney disease has a high incidence rate, poor treatment effect, is difficult to detect in the early stages of damage, and can cause irreversible damage to the kidneys. Therefore, the key to prevention and control lies in finding sensitive biomarkers.

[0003] DKK3 (Dickkopf-3) is a secretory protein produced by the renal tubules, which can specifically reflect the damage of the renal tubules. In different types of kidney diseases, the level of urinary DKK3 protein is significantly increased, making DKK3 a potential biomarker of kidney damage. Studies have found that the level of DKK3 protein in the urine of patients with chronic kidney disease is significantly higher than that of normal people.

[0004] In the prior art, β2-MG protein is a sensitive indicator of glomerular filtration function. When various primary or secondary glomerular lesions affect the glomerular filtration function, the β2-MG protein index in urine will increase. In long-term observations, it was found that β2-MG protein, as a marker of renal tubular damage, can only reflect the disorder of renal tubular reabsorption function, and cannot directly reflect the damage of the renal tubules.

[0005] To this end, the present invention provides an application and detection method of a renal injury biomarker protein. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art and solve at least one problem raised in the background technology, an application and detection method of a renal injury biomarker protein is proposed.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for detecting a renal injury biomarker protein according to the present invention, the specific steps of the detection method are as follows:

[0008] S1: Obtain the urine sample to be tested and centrifuge it to obtain the supernatant;

[0009] S2: Add 1 μg / ml goat anti-rabbit antibody in PBS to the ELISA reaction plate and seal it for incubation; pat the reaction plate dry; wash with PBS containing 0.2% Twain-20 (PBST), pat the reaction plate dry, repeat twice to obtain a coated ELISA plate;

[0010] S3: Dissolve the DKK3 and β2-MG protein standards in PBS, dilute them, and draw standard curves for protein standard solutions of different concentrations;

[0011] S4: Add 50 μL of protein standard solution or urine sample of different concentrations, 100 μL of HRP-labeled antibody, 100 μL of TMB colorimetric solution and 50 μL of stop solution to the coated ELISA plate wells respectively;

[0012] S5: Within 15 minutes after stopping the reaction, place the reaction plate in a microplate reader and read the OD 450 value;

[0013] S6: Draw a standard curve, and calculate the concentrations of DKK3 and β2-MG proteins based on the standard curve.

[0014] Preferably, the specific formula of the reagent is: carbonate buffer of goat anti-rabbit DKK3 antibody, carbonate buffer of goat anti-rabbit β2-MG antibody, 1% BSA blocking buffer, standard, HRP labeled secondary antibody solution, washing solution, TMB color developing solution, and stop solution.

[0015] Preferably, the specific operation steps in S1 are:

[0016] S1-1: Take 3 mL of urine;

[0017] S1-2: centrifuged at 3000 rpm for 5 minutes;

[0018] S1-3: Take the supernatant as the urine sample for testing.

[0019] Preferably, the sealed incubation in S2 is sealed incubation at a temperature of 4° C. for 24 hours.

[0020] Preferably, the specific dilution concentration in S3 is:

[0021] DKK3 protein: 3.125, 6.25, 12.5, 25, 50, 100 ng / mL;

[0022] β2-MG protein: 2.5, 5, 10, 20, 40, 80 ng / mL.

[0023] The beneficial effects of the present invention are as follows:

[0024] The application and detection method of a renal injury biomarker protein described in the present invention, urinary DKK3 protein is a protein secreted by renal tubular epithelial cells, can be excreted from the body with urine, has higher sensitivity and specificity than the existing classic renal injury biomarker β2-MG protein, DKK3 protein in urine will increase significantly in the early stage of renal tubular injury, and the non-invasive method is easily accepted by the testers, which is conducive to the promotion of the concept of early screening for renal tubular injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings.

[0026] Figure 1 is a flow chart of the method in the present invention;

[0027] Figure 2 is a line graph of cadmium levels in renal tissue (A) and urine (B) of rats with cadmium exposure-mediated renal injury in the present invention;

[0028] Figure 3 It is a bar graph of the levels of early sensitive markers of renal tubular injury DKK3 (A) and β2-MG (B) in urine of rats with cadmium exposure-mediated renal injury in the present invention;

[0029] Figure 4 It is a bar graph of the levels of DKK3 (A) and β2-MG (B), early sensitive markers of renal tubular injury, in urine of occupational population in the present invention;

[0030] Figure 5 It is a data table of the baseline of occupational cadmium exposure population in the present invention; DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Specific examples are given below.

[0033] like Figure 1 As shown, a method for detecting a renal injury biomarker protein according to an embodiment of the present invention has the following specific steps:

[0034] S1: Obtain the urine sample to be tested and centrifuge it to obtain the supernatant;

[0035] S2: Add 1 μg / ml goat anti-rabbit antibody in PBS to the ELISA reaction plate and seal it for incubation; pat the reaction plate dry; wash with PBS containing 0.2% Twain-20 (PBST), pat the reaction plate dry, repeat twice to obtain a coated ELISA plate;

[0036] S3: Dissolve the DKK3 and β2-MG protein standards in PBS, dilute them, and draw standard curves for protein standard solutions of different concentrations;

[0037] S4: Add 50 μL of protein standard solution or urine sample of different concentrations, 100 μL of HRP-labeled antibody, 100 μL of TMB colorimetric solution and 50 μL of stop solution to the coated ELISA plate wells respectively;

[0038] S5: Within 15 minutes after stopping the reaction, place the reaction plate in a microplate reader and read the OD 450 value;

[0039] S6: Draw a standard curve, and calculate the concentrations of DKK3 and β2-MG proteins based on the standard curve.

[0040] Further, such as Figure 1 As shown, the specific formula of the reagent is: carbonate buffer of goat anti-rabbit DKK3 antibody, carbonate buffer of goat anti-rabbit β2-MG antibody, 1% BSA blocking buffer, standard, HRP labeled secondary antibody solution, washing solution, TMB color developing solution, and stop solution.

[0041] Further, such as Figure 1 As shown, the specific operation steps in S1 are:

[0042] S1-1: Take 3 mL of urine;

[0043] S1-2: centrifuged at 3000 rpm for 5 minutes;

[0044] S1-3: Take the supernatant as the urine sample for testing.

[0045] Further, such as Figure 1 As shown, the sealed incubation in S2 is sealed incubation at a temperature of 4° C. for 24 hours.

[0046] Further, such as Figure 1 As shown, the specific dilution concentration in S3 is:

[0047] DKK3 protein: 3.125, 6.25, 12.5, 25, 50, 100 ng / mL;

[0048] β2-MG protein: 2.5, 5, 10, 20, 40, 80 ng / mL.

[0049] Embodiment 1

[0050] Preparation of test subjects and cadmium poisoning: Rats were randomly divided into 4 groups and gavaged with cadmium chloride solution at 0, 1, 3, and 9 mg / kg per day for 90 consecutive days.

[0051] Sample collection: After the cadmium exposure was completed and before the rats were killed, the 24-hour urine of each group of rats was collected using a metabolic cage, and the kidneys of the rats were collected when they were killed.

[0052] The above urine and kidney tissue were tested for cadmium concentration, and the steps were as follows:

[0053] 1. Place 1g tissue sample, 1ml blood sample and 1ml urine sample in 5ml concentrated nitric acid respectively, and use 65% HNO3 to digest the kidney tissue and urine sample by microwave digestion.

[0054] 2. Use ultraCLAVE to digest for 1 hour and 40 minutes, and use deionized water to make up to the volume to obtain the sample solution to be tested.

[0055] 3. Prepare standard curves of different cadmium concentration gradients and use ICP-MS method to detect the concentration of cadmium in kidney tissue and urine samples.

[0056] like Figure 2 As shown, as the cadmium dose increased, the levels of urinary cadmium and renal cadmium increased, indicating that the animal model of cadmium-mediated renal tubular injury was successfully established.

[0057] The above urine supernatant was subjected to ELISA test, the steps are as follows:

[0058] 1. Add carbonate buffer (PH 9.6) containing 2ug / ml goat anti-rabbit antibody to the ELISA reaction plate, seal and incubate overnight at 4℃;

[0059] 2. Take out the reaction plate, turn the ELISA plate upside down on absorbent paper, pat dry, wash the plate wells with PBS (PH 7.4), repeat three times, add 200 μL 1% BSA blocking buffer to each well, cover the plate with sticky plastic, and incubate at room temperature in the dark for 2 h;

[0060] 3. Discard the blocking buffer and wash the plate twice with PBS to obtain a coated ELISA plate;

[0061] 4. Room temperature balance and obtaining supernatant: Take the urine sample out of the -80℃ refrigerator and thaw it at 4℃. After it is completely thawed, centrifuge it at 1000g for 15 minutes to collect the supernatant;

[0062] 5. Set up blank wells, standard wells and sample wells, and mark them on the plate;

[0063] 6. Add standard: add 50 μl of standard to the standard wells in sequence, with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng / ml, respectively. Set up at least three parallel wells for each concentration.

[0064] 7. Add urine sample: add 50 μl of urine supernatant to be tested into the sample wells in sequence, and set up three parallel wells for each sample;

[0065] 8. Add antibody-HRP binding solution: add 100 μl of horseradish peroxidase (HRP)-labeled rabbit anti-DKK3 antibody to each well;

[0066] 9. Incubation: Seal the reaction wells with a sealing film and incubate in a 37°C water bath in the dark for 60 minutes;

[0067] 10. Wash the plate: discard the contents in the wells, turn the ELISA plate upside down on absorbent paper, pat dry, fill each well with washing solution (PBS containing 0.2% Tween 20), let it stand for 1-2 minutes, discard the washing solution, turn the ELISA plate upside down on absorbent paper, pat dry, and repeat this 5 times;

[0068] 11. Color development: Add 100 μl of TMB color development solution to each well, shake to mix, and incubate at room temperature in the dark for 15 minutes;

[0069] 12. Stop: Add 50 μl of stop solution to each well and shake to mix;

[0070] 13. Detection: After 10 minutes, place the ELISA plate in an ELISA reader and measure the OD value of each well at a wavelength of 450 nm;

[0071] 14. Draw the standard curve: In the Excel worksheet, use the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis to draw the linear regression curve of the standard. Calculate the sample concentration: Calculate the concentration value of each sample according to the curve equation obtained above;

[0072] 15. The detection of urine β2-MG protein is the same as that of urine DKK3.

[0073] like Figure 3 As shown in the data, there was no statistical difference in urinary β2-MG protein between the treatment group and the control group, while there was a statistical difference in DKK3 between the high-dose group (9 mg / kg / d) and the control group (P<0.01), indicating that urinary DKK3 protein tended to increase when renal tubular damage occurred, suggesting that increased urinary DKK3 protein may have an early warning effect on renal tubular damage caused by cadmium exposure.

[0074] Embodiment 2

[0075] Collection of urine samples from the population: Investigators distributed clean and sterile 50ml conical bottles on site and asked the subjects to collect mid-morning urine, about 30ml / person. After collection, the samples were fully mixed, packaged and stored in a -80℃ refrigerator. They were thawed at 4℃ before urine testing. ICP-MS was used to detect the level of cadmium in urine, and ELISA was used to detect the levels of DKK3 and β2-MG proteins in urine.

[0076] This study included 384 workers as research subjects, and the included population had to meet the following conditions: ① complete basic information, complete biological sample collection, and engaged in cadmium exposure for more than half a year; ② no history of occupational diseases, suspected occupational diseases, occupational contraindications, heavy metal poisoning in the past year, etc.; ③ no kidney-related diseases.

[0077] The relevant data of the included population include Figure 5 As shown: A total of 384 people were included, with a median age of 44 (42, 46) and a median length of service of 24 (21.5, 25). Among them, 58.9% were male, 66.7% were smokers, and 81.2% were drinkers. The levels of urinary cadmium, urinary DKK3, and urinary β2-MG protein were 4.46 (3.03, 6.45) μg / gCr, 380.55 (232.19, 648.04) pg / gCr, and 62.96 (40.43, 102.52) μg / gCr, respectively. Figure 5 As shown, there was no statistical difference in urinary β2-MG protein between the Q1 group and the Q3 group, while there was a statistical difference in DKK3 between the Q1 and Q3 groups (P<0.01), indicating that the DKK3 level increased with the increase of urinary cadmium level, further suggesting that urinary DKK3 protein is a biomarker for renal tubular damage.

[0078] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for detecting a renal injury biomarker protein, characterized in that: The specific steps of this detection method are as follows: S1: Obtain the urine sample to be tested and centrifuge it to obtain the supernatant; S2: Add 1 μg / ml goat anti-rabbit antibody in PBS to the ELISA reaction plate and seal it for incubation; pat the reaction plate dry; wash with PBS containing 0.2% Twain-20 (PBST), pat the reaction plate dry, repeat twice to obtain a coated ELISA plate; S3: Dissolve the DKK3 and β2-MG protein standards in PBS, dilute them, and draw standard curves for protein standard solutions of different concentrations; S4: Add 50 μL of protein standard solution or urine sample of different concentrations, 100 μL of HRP-labeled antibody, 100 μL of TMB colorimetric solution and 50 μL of stop solution to the coated ELISA plate wells respectively; S5: Within 15 minutes after stopping the reaction, place the reaction plate in a microplate reader and read the OD 450 value; S6: Draw a standard curve, and calculate the concentrations of DKK3 and β2-MG proteins based on the standard curve.

2. A kidney injury biomarker protein ELISA detection reagent according to claim 1, characterized in that: The specific formula of the reagent is: carbonate buffer of goat anti-rabbit DKK3 antibody, carbonate buffer of goat anti-rabbit β2-MG antibody, 1% BSA blocking buffer, standard substance, HRP labeled secondary antibody solution, washing solution, TMB color developing solution, and stop solution.

3. A method for detecting a renal injury biomarker protein according to claim 1, characterized in that: The specific operation steps in S1 are: S1-1: Take 3 mL of urine; S1-2: centrifuged at 3000 rpm for 5 minutes; S1-3: Take the supernatant as the urine sample for testing.

4. A method for detecting a renal injury biomarker protein according to claim 1, characterized in that: The sealed incubation in S2 is sealed incubation at a temperature of 4° C. for 24 hours.

5. A method for detecting a renal injury biomarker protein according to claim 1, characterized in that: The specific dilution concentration in S3 is: DKK3 protein: 3.125, 6.25, 12.5, 25, 50, 100 ng / mL; β2-MG protein: 2.5, 5, 10, 20, 40, 80ng / mL.

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