Novel biomarker detection method for kidney disease diagnosis

Abstract

The invention relates to the technical field of biomedicine detection, and discloses a novel biomarker detection method for kidney disease diagnosis, which comprises the following steps: acquiring a biological sample of a subject, pre-treating the collected biological sample, enriching novel biomarkers in a sample treatment object by using a specific affinity reagent, and detecting the novel biomarkers in the sample treatment object. Carrying out quantitative detection on the enriched novel biomarkers by adopting a detection technology based on mass spectrum to obtain expression quantity data of the novel biomarkers, comparing the detected expression quantity data of the novel biomarkers with a preset normal reference value range, and judging whether the subject has the risk of kidney diseases or not. Through combined detection of multiple novel biomarkers, the accuracy and reliability of kidney disease diagnosis are remarkably improved, the kidney injury condition is reflected from different angles, mutual complementation is achieved, the kidney health state is evaluated more comprehensively, and misdiagnosis and missed diagnosis are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to a novel biomarker detection method for diagnosing kidney diseases. Background Art

[0002] As an important excretory and endocrine organ in the human body, the kidney is responsible for filtering blood, maintaining water and electrolyte balance, regulating blood pressure and other key physiological functions. In recent years, with the aging of the population and the increase in the incidence of chronic diseases such as diabetes and hypertension, the prevalence of kidney disease has also been increasing year by year. According to relevant statistics, the global incidence of chronic kidney disease has reached 10%-15%, which means that one in every 7-10 people may suffer from kidney disease to varying degrees. In China, the number of patients with chronic kidney disease has exceeded 100 million, and the awareness rate is only about 12.5%. Most patients have progressed to the middle and late stages of the disease when they are discovered, and have missed the best time for treatment.

[0003] There are many types of kidney diseases, including glomerulonephritis, nephrotic syndrome, chronic kidney disease, diabetic nephropathy, hypertensive nephropathy, etc. Different types of kidney diseases have complex pathogenesis and diverse clinical manifestations. Early symptoms are often atypical and easily overlooked. Moreover, the kidneys have a strong compensatory function. In the early stages of the disease, even if some renal units are damaged, the remaining renal units can still maintain normal physiological functions, making it difficult to detect subtle kidney lesions through routine examinations. This not only increases the difficulty of early diagnosis of kidney disease, but also places higher demands on clinical diagnostic technology.

[0004] In the existing kidney disease diagnosis technology, commonly used diagnostic indicators such as blood creatinine and urea nitrogen often only show obvious changes when kidney damage has developed to a certain extent, making it difficult to achieve early diagnosis of kidney disease. In addition, the traditional single marker detection method has relatively low accuracy and specificity, which can easily lead to misdiagnosis or missed diagnosis. For different types of kidney diseases, there is a lack of a comprehensive, accurate and early screening diagnostic method. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a new biomarker detection method for kidney disease diagnosis, which solves the problems of accuracy and timeliness defects in kidney disease diagnosis and detection in the prior art.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: A new biomarker detection method for diagnosing kidney disease, comprising the following steps:

[0007] S1. Sample collection: obtaining a biological sample from a subject, wherein the biological sample is one or more of blood, urine or tissue fluid;

[0008] S2. Sample pretreatment: pretreatment of the collected biological samples, including one or more operations of centrifugation, filtration, protein precipitation, and nucleic acid extraction, to obtain sample processing materials suitable for detection;

[0009] S3. Biomarker enrichment: using specific affinity reagents to enrich novel biomarkers in the sample processing material, wherein the novel biomarkers are neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 and urinary β2-microglobulin, and the specific affinity reagents are antibodies against the novel biomarkers;

[0010] S4. Detection: Use mass spectrometry-based detection technology to quantitatively detect the enriched new biomarkers and obtain the expression data of the new biomarkers;

[0011] S5. Result analysis: Compare the expression data of the new biomarker detected with the preset normal reference value range. If the expression level of the new biomarker exceeds the normal reference value range, it is judged that the subject is at risk of kidney disease.

[0012] Preferably, in the sample collection step, the collection volume of the blood sample is 3-4 mL, the collection volume of the urine sample is 15-25 mL, and the collection volume of the tissue fluid sample is 1-1.5 mL.

[0013] Preferably, in the sample pretreatment step, the conditions for the centrifugation operation are: rotation speed 5000-7000r / min, time 8-12 minutes, the filter membrane pore size used in the filtration operation is 0.22μm, the precipitant used for protein precipitation is acetonitrile, the volume ratio of the precipitant to the sample is 2:1, and the nucleic acid extraction uses a commercial nucleic acid extraction kit.

[0014] Preferably, in the biomarker enrichment step, the specific affinity reagent and the sample treatment material are incubated at 25° C. for 60 minutes, and the concentration of the specific affinity reagent is 5 μg / mL.

[0015] Preferably, the mass spectrometry-based detection technology is a liquid chromatography-mass spectrometry technology, the chromatographic column of the liquid chromatography-mass spectrometry technology is a C18 reverse phase chromatographic column, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution containing 0.1% formic acid, and the gradient elution program is: 0-5 minutes, 8%-20% B; 5-15 minutes, 20%-40% B; 15-20 minutes, 40%-90% B; 20-25 minutes, 90% B; 25-30 minutes, 90%-8% B.

[0016] Preferably, the preparation method of the neutrophil gelatinase-associated lipocalin is:

[0017] Gene cloning: The gene fragment encoding neutrophil gelatinase-associated lipocalin was cloned from a human cDNA library;

[0018] Vector construction: insert the cloned gene fragment into the expression vector to construct a recombinant expression vector;

[0019] Transformation and expression: The recombinant expression vector was transformed into Escherichia coli, and the expression of neutrophil gelatinase-associated lipocalin gene was induced by inducer;

[0020] Protein purification: The expressed Escherichia coli was disrupted and the neutrophil gelatinase-associated lipocalin was purified by ion exchange chromatography and other methods.

[0021] Preferably, the preparation method of kidney injury molecule-1 is:

[0022] Gene acquisition: Total RNA was extracted from normal human kidney tissue, and cDNA encoding kidney injury molecule-1 was obtained by reverse transcription-PCR technology;

[0023] Expression vector construction: insert the obtained cDNA into a suitable eukaryotic expression vector;

[0024] Cell transfection: transfect the constructed expression vector into mammalian cell lines;

[0025] Protein extraction and purification: The transfected cells were cultured, the cell culture supernatant was collected, and the kidney injury molecule-1 protein was extracted and purified by immunoaffinity chromatography.

[0026] Preferably, the preparation method of urinary β2-microglobulin is:

[0027] Gene synthesis: artificially synthesize the gene encoding β2-microglobulin based on the known β2-microglobulin gene sequence;

[0028] Vector ligation: connect the synthesized gene to the prokaryotic expression vector;

[0029] Induced expression: The ligated vector was transformed into Escherichia coli, and the expression of β2-microglobulin was induced using IPTG inducer;

[0030] Separation and purification: The induced expression E. coli was lysed, and β2-microglobulin was separated and purified by gel filtration chromatography and ion exchange chromatography.

[0031] Preferably, the kidney disease is one or more of glomerulonephritis, nephrotic syndrome, chronic kidney disease, diabetic nephropathy, and hypertensive nephropathy.

[0032] The present invention provides a novel biomarker detection method for diagnosing kidney disease, which has the following beneficial effects:

[0033] 1. Compared with the traditional single marker detection method, the present invention significantly improves the accuracy and reliability of kidney disease diagnosis by jointly detecting three new biomarkers, namely neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 and urinary β2-microglobulin. Multiple biomarkers reflect kidney damage from different angles and complement each other, which can more comprehensively evaluate the health status of the kidneys, reduce the occurrence of misdiagnosis and missed diagnosis, and achieve early detection and intervention of the disease, thereby buying precious treatment time for patients and improving prognosis.

[0034] 2. During the sample pretreatment process, the present invention determines the specific conditions of operations such as centrifugation, filtration, protein precipitation, and detailed parameters for biomarker enrichment, so that the entire detection process is standardized and normalized, ensuring the stability and repeatability of the test results. In addition, this method requires less samples, reducing the pain and burden on patients. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0036] Embodiment 1:

[0037] The embodiment of the present invention provides a novel biomarker detection method for kidney disease diagnosis, which is used for detecting glomerulonephritis patients, and comprises the following steps:

[0038] S1. Sample collection

[0039] Twenty patients with clinical diagnosis of glomerulonephritis and 20 healthy volunteers matched in age and gender were selected as controls. 3.5 mL of venous blood was collected from each subject using a vacuum blood collection tube; 20 mL of the first midstream urine in the morning was collected; 1.2 mL of tissue fluid was collected from the kidneys of the subject using a sterile syringe under ultrasound guidance. The operation process strictly followed the principle of sterility to ensure that the sample was not contaminated;

[0040] S2. Sample pretreatment

[0041] Blood sample: Place the collected blood sample in a centrifuge and centrifuge at 6000r / min for 10 minutes to separate blood cells from plasma. Carefully aspirate the upper plasma and transfer it to a new centrifuge tube. Add 2 times the volume of acetonitrile and gently invert to mix to precipitate the protein. Centrifuge again at 10000r / min for 15 minutes and take the supernatant, which is the blood sample processed after preliminary treatment.

[0042] Urine sample: First, filter the urine sample through a 0.22 μm filter membrane to remove cell debris, impurities, etc. in the urine. Then, follow the blood sample protein precipitation procedure to add acetonitrile to precipitate the protein. The centrifugation conditions are the same as those after the blood sample precipitation. Take the supernatant as the urine sample treatment material;

[0043] Interstitial fluid samples: Centrifuge the interstitial fluid samples at 5000r / min for 8 minutes to remove possible cells and tissue debris. Then, use the same protein precipitation method as blood samples, add acetonitrile to precipitate proteins, and take the supernatant after centrifugation as the interstitial fluid sample processing material. If it is necessary to extract nucleic acids from the samples, a commercial nucleic acid extraction kit can be used to extract according to the operating steps in the kit manual;

[0044] S3. Biomarker Enrichment

[0045] The blood, urine and tissue fluid sample treatments obtained above were transferred to corresponding reaction tubes respectively, and specific affinity reagents (antibodies) for neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KI M-1) and urinary β2-microglobulin (β2-MG) were added to each reaction tube, with an antibody concentration of 5 μg / mL, and the reaction tubes were placed on a constant temperature shaker at 25° C. and incubated for 60 minutes to allow the antibodies to fully bind to the corresponding biomarkers in the sample treatments, thereby achieving enrichment of the biomarkers;

[0046] S4. Detection

[0047] Liquid chromatography-mass spectrometry (LC-MS) was used to quantitatively detect the enriched biomarkers;

[0048] Chromatographic conditions: A C18 reverse phase column was selected, the column temperature was set to 35°C, mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the gradient elution program was as follows: 0-5 minutes, the proportion of phase B increased linearly from 8% to 20%, the polarity of the mobile phase gradually decreased, and the elution ability gradually increased, which helped to elute some relatively strong polar compounds from the chromatographic column first. As the proportion of phase B increased, the elution ability gradually increased, so that substances with slightly weaker polarity also began to have the opportunity to be eluted; 5-15 minutes, the proportion of phase B increased linearly from 20% to 40%, the polarity of the mobile phase further decreased, and the elution ability was further enhanced, which could elute more medium polar compounds from the chromatographic column, realizing the separation of compounds with different polarities. Separation; 15-20 minutes, the proportion of phase B increases linearly from 40% to 90%. At this time, the polarity of the mobile phase is greatly reduced, and the elution ability is greatly enhanced, so that some compounds with weak polarity and strong interaction with the stationary phase of the chromatographic column are eluted, ensuring that all components in the sample can be effectively separated; 20-25 minutes, the proportion of phase B is maintained at 90%, ensuring that compounds with very weak polarity have enough time to be completely eluted out of the chromatographic column, and at the same time, the chromatographic column is also rinsed to thoroughly wash out impurities that may remain in the column; 25-30 minutes, the proportion of phase B decreases linearly from 90% to 8%, the flow rate is set to 0.3mL / min, and the polarity of the mobile phase gradually returns to the initial state, preparing for the next sample analysis and allowing the chromatographic column to return to conditions suitable for the next injection and separation;

[0049] Mass spectrometry conditions: Electrospray ionization source (ESI) was used, positive ion mode detection, ion source spray voltage was 3.5 kV, capillary temperature was 320 °C, sheath gas flow rate was 35 arb, auxiliary gas flow rate was 10 arb, NGAL, KIM-1 and β2-MG were scanned in multiple reaction monitoring (MRM) mode, and quantitative analysis was performed based on the characteristic ion pairs of each biomarker;

[0050] S5. Results Analysis

[0051] The expression data of NGAL, KIM-1 and β2-MG in glomerulonephritis patients and healthy controls were collated. The normal reference value range was obtained by testing biological samples of 100 healthy people and calculating them using statistical methods (mean ± standard deviation). The analysis showed that the expression levels of NGAL, KIM-1 and β2-MG in the glomerulonephritis patient group were significantly higher than those in the healthy control group, and the expression levels of most patients exceeded the normal reference value range. The receiver operating characteristic curve analysis showed that the sensitivity of this detection method for diagnosing glomerulonephritis was 85% and the specificity was 90%.

[0052] Embodiment 2:

[0053] The present invention provides a novel biomarker detection method for diagnosing kidney disease, which is used to detect diabetic nephropathy patients, and includes the following steps:

[0054] S1. Sample collection

[0055] Thirty patients diagnosed with diabetic nephropathy and 30 healthy controls were selected. 4 mL of blood sample, 25 mL of urine sample and 1.5 mL of renal puncture tissue fluid were obtained under strict aseptic operation;

[0056] S2. Sample pretreatment

[0057] The sample pretreatment steps are basically the same as those in Example 1. Depending on the sample type, the sample is processed according to the corresponding operating conditions of centrifugation, filtration, protein precipitation and nucleic acid extraction;

[0058] S3. Biomarker Enrichment

[0059] Also at 25°C, the sample treatments were incubated with specific affinity reagents (antibodies) at a concentration of 5 μg / mL for 60 min to enrich NGAL, KIM-1, and β2-MG;

[0060] S4. Detection

[0061] The enriched biomarkers were quantitatively detected using the same liquid chromatography-mass spectrometry technology and detection conditions as in Example 1;

[0062] S5. Results Analysis

[0063] The test data of diabetic nephropathy patients and healthy controls were compared and analyzed. The results showed that the expression of NGAL, KIM-1 and β2-MG in diabetic nephropathy patients was significantly higher than that in healthy controls, and was related to the course of diabetes and blood sugar control level. With the extension of diabetes course and poor blood sugar control, the expression of biomarkers increased more significantly. Taking the normal reference value range as the judgment standard, the accuracy of this detection method for diabetic nephropathy diagnosis reached 88%.

[0064] Embodiment three:

[0065] The present invention provides a novel biomarker detection method for diagnosing kidney disease, which is used to detect patients with hypertensive nephropathy, and includes the following steps:

[0066] S1. Sample collection

[0067] 25 hypertensive nephropathy patients and 25 healthy volunteers were included. 3 mL of venous blood and 15 mL of urine were collected, and a small amount of renal tissue fluid (1 mL) was obtained under the operation of a professional doctor;

[0068] S2. Sample pretreatment

[0069] According to the prescribed sample pretreatment conditions, blood, urine and tissue fluid samples were centrifuged, filtered, protein precipitated and other operations were performed in sequence. If nucleic acid extraction was involved, commercial kits were used;

[0070] S3. Biomarker Enrichment

[0071] The sample treatment was incubated with specific affinity antibodies at a concentration of 5 μg / mL for 60 minutes at 25°C to complete the enrichment of biomarkers;

[0072] S4. Detection

[0073] Liquid chromatography-mass spectrometry was used to quantitatively detect the enriched biomarkers by setting the same chromatographic column, mobile phase and gradient elution procedure as in the first two embodiments, and the mass spectrometry conditions were also kept consistent;

[0074] S5. Results Analysis

[0075] Analysis of the test data showed that the expression levels of NGAL, KIM-1 and β2-MG in patients with hypertensive nephropathy were significantly higher than those in the healthy control group, and were closely related to the course of hypertension and blood pressure control. In patients with poor long-term blood pressure control, the expression levels of biomarkers increased more significantly. By comparing with the normal reference value range, the sensitivity of this detection method for the diagnosis of hypertensive nephropathy was 82%, and the specificity was 88%.

[0076] Through the above examples, blood, urine and tissue fluid samples were collected from patients with glomerulonephritis, diabetic nephropathy, hypertensive nephropathy and healthy controls, and the samples were pretreated according to specific requirements. The biomarkers were enriched by incubation at 25°C and 5μg / mL concentration for 60 minutes using specific affinity reagents, and the same liquid chromatography-mass spectrometry technology was used for detection. The results showed that the expression levels of NGAL, KIM-1 and β2-MG in patients with the three diseases were significantly higher than those in the healthy control group, and were closely associated with disease-related factors. The detection method has a high diagnostic efficiency for different kidney diseases.

[0077] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A novel biomarker detection method for diagnosing kidney disease, characterized in that: The following steps are involved: S1. Sample collection: obtaining a biological sample from a subject, wherein the biological sample is one or more of blood, urine or tissue fluid; S2. Sample pretreatment: pretreatment of the collected biological samples, including one or more operations of centrifugation, filtration, protein precipitation, and nucleic acid extraction, to obtain sample processing materials suitable for detection; S3. Biomarker enrichment: using specific affinity reagents to enrich novel biomarkers in the sample processing material, wherein the novel biomarkers are neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 and urinary β2-microglobulin, and the specific affinity reagents are antibodies against the novel biomarkers; S4. Detection: Use mass spectrometry-based detection technology to quantitatively detect the enriched new biomarkers and obtain the expression data of the new biomarkers; S5. Result analysis: Compare the expression data of the new biomarker detected with the preset normal reference value range. If the expression level of the new biomarker exceeds the normal reference value range, it is judged that the subject is at risk of kidney disease.

2. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: In the sample collection step, the collection volume of the blood sample is 3-4 mL, the collection volume of the urine sample is 15-25 mL, and the collection volume of the tissue fluid sample is 1-1.5 mL.

3. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: In the sample pretreatment step, the conditions for the centrifugation operation are: rotation speed 5000-7000r / min, time 8-12 minutes, the filter membrane pore size used in the filtration operation is 0.22μm, the precipitant used for protein precipitation is acetonitrile, the volume ratio of the precipitant to the sample is 2:1, and the nucleic acid extraction uses a commercial nucleic acid extraction kit.

4. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: In the biomarker enrichment step, the specific affinity reagent and the sample treatment material are incubated at 25° C. for 60 minutes, and the concentration of the specific affinity reagent is 5 μg / mL.

5. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: The mass spectrometry-based detection technology is a liquid chromatography-mass spectrometry technology, the chromatographic column of the liquid chromatography-mass spectrometry technology is a C18 reverse phase chromatographic column, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution containing 0.1% formic acid, and the gradient elution program is: 0-5 minutes, 8%-20% B; 5-15 minutes, 20%-40% B; 15-20 minutes, 40%-90% B; 20-25 minutes, 90% B; 25-30 minutes, 90%-8% B.

6. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: The preparation method of the neutrophil gelatinase-associated lipocalin is as follows: Gene cloning: The gene fragment encoding neutrophil gelatinase-associated lipocalin was cloned from a human cDNA library; Vector construction: insert the cloned gene fragment into the expression vector to construct a recombinant expression vector; Transformation and expression: The recombinant expression vector was transformed into Escherichia coli, and the expression of neutrophil gelatinase-associated lipocalin gene was induced by inducer; Protein purification: The expressed Escherichia coli was disrupted and the neutrophil gelatinase-associated lipocalin was purified by ion exchange chromatography and other methods.

7. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: The preparation method of the kidney injury molecule-1 is: Gene acquisition: Total RNA was extracted from normal human kidney tissue, and cDNA encoding kidney injury molecule-1 was obtained by reverse transcription-PCR technology; Expression vector construction: insert the obtained cDNA into a suitable eukaryotic expression vector; Cell transfection: transfect the constructed expression vector into mammalian cell lines; Protein extraction and purification: The transfected cells were cultured, the cell culture supernatant was collected, and the kidney injury molecule-1 protein was extracted and purified by immunoaffinity chromatography.

8. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: The preparation method of the urinary β2-microglobulin is: Gene synthesis: artificially synthesize the gene encoding β2-microglobulin based on the known β2-microglobulin gene sequence; Vector ligation: connect the synthesized gene to the prokaryotic expression vector; Induced expression: The ligated vector was transformed into Escherichia coli, and the expression of β2-microglobulin was induced using IPTG inducer; Separation and purification: The induced expression E. coli was lysed, and β2-microglobulin was separated and purified by gel filtration chromatography and ion exchange chromatography.

9. A novel biomarker detection method for kidney disease diagnosis according to claim 1, characterized in that: The kidney disease is one or more of glomerulonephritis, nephrotic syndrome, chronic kidney disease, diabetic nephropathy, and hypertensive nephropathy.