WO2023056924A1 - Uromodulin sda antigen glycosylation detection kit and use thereof in prediction of early kidney injury - Google Patents

Abstract

Disclosed are a uromodulin Sda antigen glycosylation detection kit and the use thereof in prediction of early kidney injury. Provided in the present invention is the use of a Sda carbohydrate antigen on uromodulin as a marker in any one of the following scenarios: screening or auxiliary diagnosis of kidney injury; prediction of the risk of developing kidney injury; and distinguishing of patients with kidney injury from a healthy control. Also provided in the present invention is an enzyme linked immunosorbent assay kit for detecting the content of the Sda carbohydrate antigen on uromodulin in urine. By means of the present invention, it is researched and confirmed that the use of the Sda carbohydrate antigen on uromodulin as a marker of kidney injury is beneficial for the continuous improvement of detection methods, promotes the early diagnosis of kidney injury, and establishes a biological marker of kidney injury with better sensitivity and specificity. The detection method of the present invention greatly simplifies the process, the detection scheme requires simple and easy-to-obtain equipment and reagents, and the detection method is less time-consuming and low-cost and facilitates clinical development.

Description

Uromodulin Sda Antigen Glycosylation Detection Kit and Its Application in Predicting Early Kidney Injury technical field

The invention relates to the field of biomedicine, in particular to a kit for detecting glycosylation of uromodulin Sda antigen and its application in predicting early kidney damage.

Background technique

Numerous studies have shown that uromodulin has a protective role in kidney injury. The level of uromodulin may become a biomarker for predicting renal injury. Uromodulin is rich in N-glycans, which account for 30% of the molecular weight of the protein. Glycosylation is an important post-translational modification of uromodulin. In disease states, abnormal glycosylation of uromodulin can precede changes in protein levels. Changes in glycosylation levels may predict disease more sensitively than changes in protein levels alone. There is a special Sda antigen in the N-glycomodification of uromodulin. Sda antigen is an erythrocyte antigen, which is widely distributed on Caucasian erythrocytes, and Sda can be detected in saliva, urine and milk. The Sda antigen contained in uromodulin is located at the polyantennary N-sugar terminus. Its structural composition is GalNAcβ(1,4)[Neu5Acα(2,3)]Galβ(1,4)GlcNAcβ(1,3)Gal. The rate-limiting enzyme for the synthesis of Sda antigen is β(1,4) acetamidogalactosyltransferase 2 (beta-1,4-N-acetyl-galactosaminyltransferase2, B4GALNT2), which is expressed in gastrointestinal cells and renal outer medulla (consisting of the thick ascending limb of the loop of Henle expressing uromodulin) expression.

Many functions of uromodulin are affected by glycosylation. The anti-calculus ability of uromodulin depends on the integrity of the sugar chain of uromodulin, especially the presence of sialic acid in it. Hallson et al. found that the ability of uromodulin to inhibit the formation of calcium oxalate and calcium phosphate crystals was lost when uromodulin was deglycosylated or desialic acid treated. The ability of uromodulin to resist urinary tract infection is also mediated by the N-glycans of uromodulin.

Cavallone et al found that the high mannose of uromodulin can bind to the type I pili of Escherichia coli (E.coli) and protect the urinary system from E. coli infection. Serafini et al. summarized previous studies and found that sialic acid on uromodulin Sda antigen can combine with S pili of Escherichia coli to protect the kidney from bacterial infection. The sialic acid level of uromodulin decreases in patients with interstitial cystitis, but the level of uromodulin does not change significantly. The cytoprotective effect of asialo uromodulin decreases, which may be involved in the pathogenesis of interstitial cystitis.

The technical means currently used for detection of uromodulin glycosylation include mass spectrometry detection, liquid chromatography detection, lectin chip detection and nuclear magnetic resonance detection. Among them, matrix-assisted laser desorption ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS) is often the first choice for N-glycan analysis due to its advantages of providing rich glycoform information. However, mass spectrometry relies on molecular weight for N-glycan analysis. chain prediction, and the diversity of N-glycan chains leads to insufficient accuracy of mass spectrometry results. In addition, mass spectrometry detection requires a large amount of protein samples and takes a long time, which is not convenient for clinical detection and promotion. Lectin chip technology is developed in recent years for New technology for glycosylation detection. Lectin is a protein that can specifically recognize and bind sugar. Lectin chip is a high-throughput experimental method. It pre-coats multiple lectins on the same glass plate at the same time , add the analytes that are pre-labeled with antibodies or dyes to the lectin chip board, and the signal results can be obtained by using the specific recognition of lectins on sugars. Since the report in 2005, lectin chips have been widely used in cancer, Bacteria, fungi, stem cells and diabetes and other related research. As a high-throughput new technology, it is expensive and more suitable for scientific research rather than clinical detection. In addition, the existing mass spectrometry detection and lectin chip detection technology are both Uromodulin needs to be separated and purified to exclude other protein interference, and urine specimens cannot be used directly for detection. Uromodulin purification requires the collection of a large amount of urine for protein extraction, which increases the complexity of the test.

invention disclosure

The purpose of the present invention is to provide a kit for detecting glycosylation of uromodulin Sda antigen and its application in predicting early kidney injury.

In the first aspect, the present invention claims to protect the use of the Sda sugar antigen on uromodulin as a marker in any of the following:

(A1) Preparation of products for screening or auxiliary diagnosis of kidney injury, or screening or auxiliary diagnosis of kidney injury;

(A2) Prepare a product for predicting the risk of kidney injury, or predict the risk of kidney injury;

(A3) Preparation of a product for distinguishing renally impaired patients from healthy controls, or distinguishing renally impaired patients from healthy controls.

In the second aspect, the present invention claims the application of the substance used to detect the Sda sugar antigen on uromodulin in any of the following:

(A1) Preparation of products for screening or auxiliary diagnosis of kidney injury, or screening or auxiliary diagnosis of kidney injury;

(A2) Prepare a product for predicting the risk of kidney injury, or predict the risk of kidney injury;

(A3) Preparation of a product for distinguishing renally impaired patients from healthy controls, or distinguishing renally impaired patients from healthy controls.

Further, the detection of the Sda sugar antigen on uromodulin is to detect the content of the Sda sugar antigen on uromodulin in the urine sample.

Further, the substance for detecting the Sda sugar antigen on uromodulin is a substance capable of specifically binding to the Sda sugar antigen on uromodulin.

In a specific embodiment of the present invention, the substance used to detect the Sda sugar antigen on uromodulin is DBA lectin or the enzyme-linked immunosorbent assay kit described in the sixth aspect below.

In the third aspect, the present invention claims a kit having the following functions (B1) and/or (B2) and/or (B3), which contains substances for detecting the Sda sugar antigen on uromodulin.

(B1) Screening or auxiliary diagnosis of kidney injury;

(B2) Predict the risk of kidney injury;

(B3) Distinguish between patients with renal impairment and healthy controls.

Wherein, the substance used for detecting the Sda sugar antigen on uromodulin may be a substance capable of specifically binding to the Sda sugar antigen on uromodulin.

In a specific embodiment of the present invention, the substance for detecting the Sda sugar antigen on uromodulin is specifically DBA lectin or the enzyme-linked immunosorbent assay kit described in the sixth aspect below.

In a fourth aspect, the present invention claims to protect a system having the following functions (B1) and/or (B2) and/or (B3):

(B1) Screening or auxiliary diagnosis of kidney injury;

(B2) Predict the risk of kidney injury;

(B3) Distinguish between patients with renal impairment and healthy controls.

The system includes:

(a1) Reagents and/or instruments (such as a microplate reader) for detecting the Sda sugar antigen on uromodulin.

(a2) device;

The device includes a data receiving module, a data storage module, a data comparison module and a judgment module;

The data receiving module is configured to receive the content value of the Sda sugar antigen on uromodulin in the subject's urine sample;

The data storage module is configured to store a judgment threshold;

The data comparison module is configured to receive the content value of the Sda sugar antigen on uromodulin in the subject's urine sample sent by the data receiving module, and call the judgment threshold from the data storage module Compared with the content value of the Sda sugar antigen on uromodulin in the subject's urine sample;

The judging module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to a predetermined judgment condition, and judge that the subject who meets the predetermined judgment condition is or is a candidate for a kidney injury patient , determining that the subject who does not meet the predetermined determination condition is or is a candidate for a non-kidney-impaired patient (such as a healthy control).

Wherein, the predetermined judgment condition may be: if the content value of the Sda sugar antigen on uromodulin in the urine sample of the subject is less than the judgment threshold, then the subject is or is a candidate for a kidney injury patient ; otherwise, the subject is or is a candidate for a non-renal impaired patient (eg, a healthy control).

The judgment threshold is the content value of Sda sugar antigen on uromodulin in the urine sample of non-kidney-impaired patients (such as healthy controls).

Further, the instrument can be a microplate reader. The reagent may include healthy human uromodulin as a standard.

In the fifth aspect, the present invention claims the application of the aforementioned kit or the aforementioned system in any of the following:

(A1) Preparation of products for screening or auxiliary diagnosis of kidney injury, or screening or auxiliary diagnosis of kidney injury;

(A2) Prepare a product for predicting the risk of kidney injury, or predict the risk of kidney injury;

(A3) Preparation of a product for distinguishing renally impaired patients from healthy controls, or distinguishing renally impaired patients from healthy controls.

In the above aspects, the kidney injury can specifically be acute kidney injury (AKI). Can be defined according to the 2012 KDIGO guidelines.

In the above aspects, the healthy controls all refer to healthy adults with normal renal function in physical examination, no basic kidney disease, no urinary system infection, and age > 18 years old.

In the sixth aspect, the present invention claims an enzyme-linked immunosorbent assay kit for detecting the content of Sda sugar antigen on uromodulin in urine.

The enzyme-linked immunoimmunoassay kit for detecting the content of Sda sugar antigen on uromodulin in urine as claimed in the present invention includes standard antigens, capture objects and detection objects. The standard antigen is healthy human uromodulin; the capture antibody is anti-uromodulin antibody; and the detection substance is biotin-labeled lectin capable of recognizing Sda.

Further, the anti-uromodulin antibody may be an anti-uromodulin polyclonal antibody. Of course, it may also be an anti-uromodulin monoclonal antibody having an equivalent effect.

In a specific embodiment of the present invention, the anti-uromodulin antibody is specifically the sheep anti-human uromodulin polyclonal antibody produced by Meridian Company with the product number K90071C-1.

Further, the lectin capable of recognizing Sda may be DBA (Vectorlabs, B-1035-5). Of course, other lectins capable of recognizing Sda with equivalent effects may also be used.

Furthermore, the ELISA kit may also contain horseradish peroxidase-labeled avidin and/or sample diluent.

Wherein, the avidin is a substance having avidin binding properties. Specifically, the substance with binding properties to avidin may be streptavidin, avidin, vitellavidin or avidin-like.

In a specific embodiment of the present invention, the avidin is specifically streptavidin.

Wherein, the sample diluent is a buffer.

In a specific embodiment of the present invention, the buffer is 0.01M PBS buffer at pH 7.4.

Of course, the ELISA kit may also contain at least one of the following: enzyme label plate, plating buffer, washing solution, blocking solution, color developing solution and stop solution.

In a specific embodiment of the invention, the plating buffer is a carbonate buffer at pH 9.6.

In a specific embodiment of the present invention, the washing solution is a phosphate buffer solution (ie 0.1% PBST) with a pH of 7.4 containing only 0.1% by volume of Tween 20.

In a specific embodiment of the present invention, the blocking solution is a phosphate buffered saline solution (i.e. 1% BSA) with a pH of 7.4 containing only 10 g/L BSA.

In a specific embodiment of the present invention, the color developing solution is tetramethylbenzidine (TMB).

In a specific embodiment of the present invention, the stop solution is 1M H2SO4.

In the seventh aspect, the present invention claims the application of the aforementioned ELISA kit in detecting the content of Sda sugar antigen on uromodulin in urine.

In said application, urine is used as the sample to be tested, and the test is performed after being diluted with buffer solution.

In a specific embodiment of the present invention, the buffer is 0.01M PBS buffer at pH 7.4.

In the above application, the urine sample cannot be diluted with ultrapure water, and the ultrapure water dilution detection system cannot detect it.

In the application, different concentrations of the standard antigen corresponding to the detected Sda antigen content can be used as a standard curve (the standard antigen is uromodulin from healthy humans).

In the eighth aspect, the present invention claims to protect the application of the ELISA kit described above in the preparation of products for the auxiliary diagnosis of diseases related to the abnormal expression of the Sda sugar antigen on uromodulin, or the ELISA described above Application of the kit in auxiliary diagnosis of diseases related to abnormal expression of Sda sugar antigen on uromodulin.

Description of drawings

Fig. 1 is a schematic diagram of the detection principle of the enzyme-linked immunosorbent assay kit for detecting the content of Sda sugar antigen on uromodulin in urine according to the present invention.

Fig. 2 is the standard curve of the ELISA kit for detecting the content of Sda sugar antigen on uromodulin in urine according to the present invention. Vertical axis: Y (measured OD450 value); horizontal axis: X (Sda sugar antigen concentration U/μl).

Figure 3 shows the expression levels of DBA in AKI patients (N=10 cases) and healthy controls (N=10 cases). A is the detection result of lectin chip; B is the detection result of lectin-ELISA (the combination of uromodulin and biotin-labeled DBA).

Fig. 4 is the detection of Sda antigen concentration on uromodulin in samples from AKI patients (N=17 cases) and healthy controls (N=17 cases) by lectin-ELISA (uromodulin combined with biotin-labeled DBA).

The best way to practice the invention

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Example 1 Preparation and use of an enzyme-linked immunosorbent assay kit for detecting the content of Sda sugar antigen on uromodulin in urine

One. Preparation of an enzyme-linked immunosorbent assay kit for detecting the content of Sda sugar antigen on uromodulin (UMOD) in urine

The ELISA kit for detecting the content of Sda sugar antigen on uromodulin in urine provided by the present invention includes: standard antigen, capture object, detection object, and enzyme plate, plating buffer, washing solution, blocking solution , horseradish peroxidase-labeled streptavidin (HRP-SA), chromogenic solution and stop solution, etc. Wherein, the standard antigen is healthy human uromodulin (healthy human uromodulin contains normal content of Sda glycosylation); the capture object is an anti-uromodulin antibody (meridian company uromodulin polyclonal antibody K90071C-1 ), the detection substance is a biotin-labeled lectin DBA that can recognize Sda. The schematic diagram of the detection is shown in Figure 1.

(1) Conventional equipment and reagents

1. ELISA plate (96-well enzyme plate), Thermo Company.

2. Plating buffer: carbonate buffer pH 9.6.

3. Washing solution: Phosphate buffered saline (i.e. PBST buffer) with a pH of 7.4 containing only 0.1% by volume of Tween 20, referred to as 0.1% PBST.

4. Blocking solution: Phosphate buffer solution with a pH of 7.4 containing only 10g/L BSA, referred to as 1% BSA.

5. Streptavidin labeled with horseradish peroxidase (Vectorlabs SA-5014-1).

6. Chromogenic substrate: Tetramethylbenzidine (TMB).

7. Stop solution: 1M H ₂ SO ₄ .

8. Sample diluent: 0.01M PBS buffer with pH 7.4.

(2) Standard antigen (healthy human uromodulin) sample preparation and purity testing

Healthy human uromodulin sample preparation:

1) Take 24h urine from healthy people, prepare buffer solution 0.02M PBS, 0.1M PB.

2) Mix 20g of diatomaceous earth with 24h of urine, stir at 4°C for 20min, add to a Buchner funnel covered with 18.5cm Whatman filter paper, and suck with a vacuum pump.

3) After filtering, rinse the diatomaceous earth with 800ml PBS.

4) Take out the diatomite after pumping water, mix it with 150ml of ultrapure water, and place it in a refrigerator at 4°C for 30 minutes.

5) Dispense the mixture into 50ml high-speed centrifuge tubes, balance the balance and centrifuge at high speed at 20000g, 20min, 4°C.

6) After centrifugation, take out the centrifuge tube, save the supernatant, and discard the precipitate (gently, diatomaceous earth is easy to float). Add 0.1M PB and NaCl to the supernatant to a concentration of 0.025M/0.14M (usually 46ml of PB and 1.5g of NaCl). The mixture was stirred at 4 °C for 20 min.

7) Add the mixed solution to a 7cm Buchner funnel covered with Whatman filter paper and filter, rinse with 200ml PBS.

8) Drain the diatomite again, mix the diatomite with 50ml of ultrapure water, place in a 4°C refrigerator and stir for 30min.

9) Put the mixed solution into a 50ml high-speed centrifuge tube, balance the balance and centrifuge at a high speed at 20000g for 20min at 4°C.

10) After centrifugation, put the supernatant into a dialysis bag (cut-off flow rate: 30kD), the volume of which should not exceed 2/3, seal both ends, place in 5L of deionized water and dialyze overnight at 4°C.

11) On the second day, the dialyzed liquid was taken out, and the liquid was filtered with a 0.2 μM filter to remove impurities.

12) Concentrate by ultrafiltration, use a 30kD ultrafiltration tube (Millipore) to concentrate the protein. 3000rpm, 4°C, observe the concentrated state, adjust the centrifugation time.

13) After concentration, use nanodrop and BCA kit to detect protein concentration.

14) Coomassie brilliant blue/silver staining to detect protein purity.

15) The specimens were subpackaged, freeze-dried and stored in a -80°C refrigerator.

Coomassie brilliant blue test purity :

1) Prepare 10% SDS-PAGE gel and electrophoresis solution.

2) Sample preparation 2-4μg protein sample, add water and 5×loading buffer to make an appropriate volume. The reduced sample needs to be boiled for 10 minutes, and the non-reduced sample does not need to be boiled.

3) Sample loading, electrophoresis, constant voltage 80V, after the sample reaches the separation gel layer, adjust the voltage to 120V.

4) Take out the gel after electrophoresis, and cut off the stacking gel on the upper layer and the bottom loading.

5) Coomassie brilliant blue staining, 30min-1h.

6) For decolorization, change the decolorization solution every 20-30 minutes, and then decolorize overnight at 4°C. Take a picture and save it.

(3) Detection substance (biotin-labeled lectin DBA capable of recognizing Sda)

DBA is a commercial reagent from Vectorlabs B1035-5.

2. How to use the enzyme-linked immunosorbent assay kit for detecting the content of Sda sugar antigen on uromodulin in urine

1. Use standard antigen to make standard curve

(1) Prepare a serial concentration of uromodulin (containing Sda sugar antigen) standard solution: the concentration of the standard antigen (healthy human uromodulin) prepared in step 1 is 2000pg/μl, 1000pg/μl, 500pg/μl, 250pg/μl, 100pg/μl, 50pg/μl, 10pg/μl, and use the diluent as a blank well (0pg/μl).

(2) After diluting the uromodulin polyclonal antibody of Meridian Company (Cat. No. K90071C-1 sheep anti-human uromodulin polyclonal antibody) with pH9.6 bicarbonate buffer 1:4000 (volume ratio), pre-coated overnight at 4°C on the detection plate. The experimental system is 100 μl.

(3) Block with 1% BSA and incubate at 37°C for 60 min.

(4) Wash the plate with 0.1% PBST, 300 μl/well, wash three times, and pat dry with absorbent paper; load the Sda sugar antigen standard solution on uromodulin with a series of concentrations prepared in step (1), at 37°C, Incubate for 60min.

(5) Wash the plate with 0.1% PBST, 300 μl/well, wash three times, pat dry with absorbent paper; dilute biotin-labeled DBA (Vectorlabs, B-1035-5) at 1:2000 (volume ratio), load the sample, 100 μl/well , 37°C, 60min.

(6) Wash the plate with 0.1% PBST, 300 μl/well, wash three times, pat dry with absorbent paper; dilute horseradish peroxidase-labeled streptavidin at 1:2000 (volume ratio), load the sample, 100 μl/well, 37°C, 50min.

(7) Wash the plate with 0.1% PBST, 300 μl/well, wash three times, and pat dry with absorbent paper; TMB chromogenic solution develops color at room temperature for 10-20 minutes, and stops with 1M H2SO4 sulfuric acid.

(8) Read at 450nm on a microplate reader.

(9) Among the above-mentioned standards of various concentrations, the Sda concentration on uromodulin corresponds to 2000U/μl, 1000U/μl, 500U/μl, 250U/μl, 100U/μl, 50U/μl, 10U/μl, 0U/μl (the Sda content carried by every 1pg of uromodulin in the standard is judged as 1U). Take the above-mentioned value as the abscissa and the corresponding OD450 as the ordinate respectively, and plot to obtain a standard curve (as shown in Figure 2 and Table 1).

Table 1. Calculation equation of uromodulin Sda sugar antigen concentration, four-parameter Logistic curve fitting

2. Determination of the content of Sda sugar antigen on uromodulin in the urine to be tested

Replace the standard antigen (normally glycosylated uromodulin) in step 1 with the human urine sample to be tested, dilute it 10 times (volume) with 0.01M pH 7.4 PBS buffer for detection (note that urine samples cannot be used Ultrapure water dilution, the ultrapure water dilution detection system can not be detected), the obtained OD450 value is substituted into the standard curve obtained in step 1, thereby obtaining the content of the Sda sugar antigen on the uromodulin in the human urine sample to be tested.

Example 2. Discovery and application of the Sda sugar antigen on uromodulin as a kidney injury marker

1. Case inclusion

1. Population inclusion criteria

Acute Kidney Injury (AKI) patients: 10 cases, in line with the increase in serum creatinine ≥ 0.3 mg/dL (26.5 μmol/L) within 48 hours or urine output < 0.5 mL/kg/h for 6 hours.

Healthy Controls (CK): 10 healthy adults aged >18 years old with normal renal function, no underlying renal disease, and no urinary system infection.

2. Population exclusion criteria

(1) History of renal disease before operation

(2) Urinary tract infection

(3) The operation is kidney surgery.

3. Basic information of subjects

All 10 patients with acute kidney injury were postoperative (non-renal surgery), 5 males and 5 females, with an average age of 57±4 years, meeting the criteria for acute kidney injury.

The 10 healthy controls were laboratory workers with normal renal function in physical examination, normal renal function in physical examination, no basic kidney disease, and no urinary system infection.

2. Sample collection and processing

For different subsequent detection methods, the amount of urine collected is different, as follows:

1. The lectin-ELISA method of lectin chip and biotin-directly labeled uromodulin requires a urine volume of at least 1000 ml, and the sample is processed immediately after the urine is retained to extract and purify uromodulin.

Uromodulin extraction method:

1) Mix 20g of diatomaceous earth with urine, stir at 4°C for 20min, add it into a Buchner funnel covered with 18.5cm Whatman filter paper, and suck it with a vacuum pump.

2) After filtering, rinse the diatomaceous earth with 800ml PBS.

3) Take out the diatomite after pumping water, mix it with 150ml of ultrapure water, and place it in a 4°C freezer for 30 minutes.

4) Dispense the mixture into 50ml high-speed centrifuge tubes, balance the balance and centrifuge at high speed at 20000g for 20min at 4°C.

5) After centrifugation, take out the centrifuge tube, keep the supernatant, and discard the precipitate (gently, diatomaceous earth is easy to float). Add 0.1M PB and NaCl to the supernatant to a concentration of 0.025M/0.14M (usually 46ml of PB and 1.5g of NaCl). The mixture was stirred at 4 °C for 20 min.

6) Add the mixed solution to a 7cm Buchner funnel covered with Whatman filter paper to filter, rinse with 200ml PBS.

7) Drain the diatomaceous earth again, mix the diatomite with 50ml of ultrapure water, place in a 4°C refrigerator and stir for 30min.

8) Put the mixed solution into a 50ml high-speed centrifuge tube, balance the balance and centrifuge at a high speed at 20000g for 20min at 4°C.

9) After centrifugation, put the supernatant into a dialysis bag (cut-off flow: 30kD), the volume of which should not exceed 2/3, seal the two ends, place in 5L deionized water and dialyze overnight at 4°C.

10) On the second day, the dialyzed liquid was taken out, and the liquid was filtered with a 0.2 μM filter to remove impurities.

11) Concentrate by ultrafiltration, using a 30kD ultrafiltration tube (Millipore) to concentrate the protein. 3000rpm, 4°C, observe the concentrated state, adjust the centrifugation time.

12) After concentration, use nanodrop and BCA kit to detect protein concentration.

13) Coomassie brilliant blue/silver staining to detect protein purity.

15) The specimens were subpackaged, freeze-dried and stored in a -80°C refrigerator.

Coomassie brilliant blue test purity:

1) Prepare 10% SDS-PAGE gel and electrophoresis solution.

2) Sample preparation 2-4μg protein sample, add water and 5×loading buffer to make an appropriate volume. The reduced sample needs to be boiled for 10 minutes, and the non-reduced sample does not need to be boiled.

3) Sample loading, electrophoresis, constant voltage 80V, after the sample reaches the separation gel layer, adjust the voltage to 120V.

4) Take out the gel after electrophoresis, and cut off the stacking gel on the upper layer and the bottom loading.

5) Coomassie brilliant blue staining, 30min-1h.

6) For decolorization, change the decolorization solution every 20-30 minutes, and then decolorize overnight at 4°C. Take a picture and save it.

2. Use biotin-labeled DBA to directly detect the content of Sda antigen on uromodulin: 5 ml of urine needs to be collected, centrifuged at 2500 rpm for 20 minutes, and the supernatant is separated. Take one volume of the supernatant and add 9 volumes of 0.01MPBS Buffer, directly use the diluted sample for detection, and the supernatant can also be stored in a freezer at -80°C for future detection. After the frozen sample is dissolved, use 0.01MPBS buffer directly, (according to sample: buffer 1:9 volume ratio) dilution.

3. Sample testing

1. Use commercial lectin chip kit (GA-Lectin-70, Raybiotech, USA) to detect the expression level of Sda carbohydrate antigen in AKI patients and healthy controls

details as follows:

1) Material preparation: commercial lectin chip kit (GA-Lectin-70, Raybiotech, USA), uromodulin dry powder.

2) Sample preparation: resuspend 30 μg of lyophilized uromodulin in ultrapure water. Samples were labeled with biotin and then dialyzed overnight at 4°C.

3) Chip detection: the chip was equilibrated to room temperature, placed in a drying oven to dry for 2 hours, and then sealed with 100 μl diluent for 30 minutes on the glass slide. Diluted samples were added to the test wells, and the arrays were incubated overnight at 4°C. Pour samples from each well and wash wells and slides with 1× Wash Buffer I with gentle shaking at room temperature. Subsequently, 80 μl Cy3-streptavidin was added to each well. Array plates were incubated for 1 h at room temperature in the dark. Decant the Cy3-streptavidin, then wash the wells and slides. The slides were dried by centrifugation at 1,000 rpm for 3 min. Finally, slides were scanned with an Agilent SureScan Dx Microarray Scanner chip scanner at 532nm, 100% Power. The chip contains 70 lectins to detect 8 specific carbohydrates, including mannose (Man), fucose (Fuc), sialic acid, galactose (Gal), glucose (Glc), lactose (Lac), N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc).

2. Using lectin-ELISA method (uromodulin and biotin-labeled DBA) to detect the expression level of Sda sugar antigen in AKI patients and healthy controls

See embodiment 1 for details.

3. Using lectin-ELISA method (biotin directly labeled uromodulin) to detect the expression level of Sda sugar antigen in AKI patients and healthy controls

1) Diatomaceous earth method to purify uromodulin.

2) The purified uromodulin was labeled with a biotin kit (Thermo 21435).

3) The unbiotinylated DBA lectin was diluted 1:2000 (volume ratio) with bicarbonate buffer solution of pH 9.6, pre-coated on the detection plate, and kept overnight at 4°C. The experimental system is 100 μl.

4) Block with 1% BSA, incubate at 37°C for 60min

5) Wash the plate with 0.1% PBST, 300 μl/well, wash three times, and pat dry with absorbent paper; dilute biotin-labeled uromodulin with 0.01MPBS to a concentration of 1 μg/ml, load the sample, and incubate at 37°C for 60 minutes.

6) Wash plate with 0.1% PBST, 300 μl/well, wash three times, pat dry with absorbent paper; dilute horseradish peroxidase-labeled streptavidin at 1:2000 (volume ratio), load sample, 100 μl/well, 37 ℃, 50min.

7) Wash the plate with 0.1% PBST, 300 μl/well, three times, and pat dry with absorbent paper; TMB chromogenic solution develops color at room temperature for 10-20 minutes, and stops with 1M H ₂ SO ₄ sulfuric acid.

8) Read at 450nm on a microplate reader.

4. Data processing

Each sample was repeated twice, and the mean value was taken. The t-test was used for the healthy group and the AKI group, and P<0.05 was considered statistically significant.

5. Results and Analysis

The results of the lectin chip showed that the expression level of the Sda sugar antigen detected by the lectin DBA was most significantly different between AKI patients and healthy controls, P=0.002 (A in FIG. 3 ).

The present invention uses the lectin-ELISA method to re-detect the recognition of DBA lectin on the specific glycoform of uromodulin, respectively uses biotin to directly label uromodulin for detection, and uromodulin and biomarker for DBA detection. Through preliminary experiments, it was found that the direct detection of biotin-labeled uromodulin was unsuccessful for DBA detection, and this method could not be used (the experimental system was complex, time-consuming, and the detection results were not effective). Uromodulin was combined with biotin-labeled DBA, and then horseradish peroxidase-labeled avidin was used to detect the binding strength. The results showed that the expression of DBA was significantly decreased in AKI patients, P=0.02 (B in FIG. 3 ).

Further, increasing the number of cases, using the lectin-ELISA method (uromodulin and biotin-labeled DBA) for patients with acute kidney injury AKI (N=17 cases, including 10 cases described in step 1), normal controls (N =17 cases, including the 10 cases described in step 1) The concentration of Sda antigen on uromodulin was detected. AKI patients (54.9+/-43.9U/ml) were significantly lower than normal controls (169.6+/-111.5U/ml), P=0.026. See Figure 4 for details.

industrial application

The determination of the Sda sugar antigen on the uromodulin of the present invention as a marker of renal injury is helpful to the continuous improvement of detection methods, promotes the early diagnosis of renal injury, and establishes biological markers of renal injury with better sensitivity and specificity. In addition, compared with the existing technical solutions, the enzyme-linked immunosorbent assay method for the Sda sugar antigen content on uromodulin in urine provided by the present invention does not need to collect a large number of urine samples for protein purification, and can directly use a small amount of urine samples to directly detection, which greatly simplifies the detection process. The detection scheme requires that the equipment and reagents are simple and easy to obtain, and the use of the detection method is less time-consuming, low-cost, and convenient for clinical development. The expression level of Sda antigen changes when the kidney is injured, and the rapid and convenient detection method buys precious time for early clinical diagnosis and treatment.

Claims (21)

1. The use of Sda sugar antigen on uromodulin as a marker in any of the following:

   (A1) Preparation of products for screening or auxiliary diagnosis of kidney injury, or screening or auxiliary diagnosis of kidney injury;

   (A2) Prepare a product for predicting the risk of kidney injury, or predict the risk of kidney injury;

   (A3) Preparation of a product for distinguishing renally impaired patients from healthy controls, or distinguishing renally impaired patients from healthy controls.
2. Application of the substance for detecting the Sda sugar antigen on uromodulin in any of the following:

   (A1) Preparation of products for screening or auxiliary diagnosis of kidney injury, or screening or auxiliary diagnosis of kidney injury;

   (A2) Prepare a product for predicting the risk of kidney injury, or predict the risk of kidney injury;

   (A3) Preparation of a product for distinguishing renally impaired patients from healthy controls, or distinguishing renally impaired patients from healthy controls.
3. The application according to claim 2, characterized in that: the detection of the Sda sugar antigen on uromodulin is to detect the content of the Sda sugar antigen on uromodulin in the urine sample.
4. The application according to claim 2, characterized in that: the substance used to detect the Sda sugar antigen on uromodulin is a substance capable of specifically binding to the Sda sugar antigen on uromodulin.
5. The application according to claim 4, characterized in that: the substance for detecting the Sda sugar antigen on uromodulin is the lectin DBA or the kit according to any one of claims 12-17.
6. A kit with the functions shown in (B1) and/or (B2) and/or (B3) below, containing substances for detecting the Sda sugar antigen on uromodulin;

   (B1) Screening or auxiliary diagnosis of kidney injury;

   (B2) Predict the risk of kidney injury;

   (B3) Distinguish between patients with renal impairment and healthy controls.
7. A system with the following functions (B1) and/or (B2) and/or (B3):

   (B1) Screening or auxiliary diagnosis of kidney injury;

   (B2) Predict the risk of kidney injury;

   (B3) Distinguish between patients with renal impairment and healthy controls;

   The system includes:

   (a1) reagents and/or instruments for detecting the Sda sugar antigen on uromodulin;

   (a2) device;

   The device includes a data receiving module, a data storage module, a data comparison module and a judgment module;

   The data receiving module is configured to receive the content value of the Sda sugar antigen on uromodulin in the subject's urine sample;

   The data storage module is configured to store a judgment threshold;

   The data comparison module is configured to receive the content value of the Sda sugar antigen on uromodulin in the subject's urine sample sent by the data receiving module, and call the judgment threshold from the data storage module Compared with the content value of the Sda sugar antigen on uromodulin in the subject's urine sample;

   The judging module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to a predetermined judgment condition, and judge that the subject who meets the predetermined judgment condition is or is a candidate for a kidney injury patient , determining that the subject who does not meet the predetermined determination condition is or is a candidate for a non-kidney-impaired patient.
8. The system according to claim 7, characterized in that: the predetermined judgment condition is: if the content value of the Sda sugar antigen on uromodulin in the subject's urine sample is less than the judgment threshold, then the The subject is or is a candidate for a renal impairment patient; otherwise, the subject is or is a candidate for a non-renal impairment patient.
9. The system according to claim 7 or 8, characterized in that: the judgment threshold is the content value of the Sda sugar antigen on uromodulin in the urine sample of the non-kidney-impaired patient.
10. The application of the kit according to claim 6 or the system according to any one of claims 7-9 in any of the following:

    (A1) Preparation of products for screening or auxiliary diagnosis of kidney injury, or screening or auxiliary diagnosis of kidney injury;

    (A2) Prepare a product for predicting the risk of kidney injury, or predict the risk of kidney injury;

    (A3) Preparation of a product for distinguishing renally impaired patients from healthy controls, or distinguishing renally impaired patients from healthy controls.
11. The application or kit or system according to any one of claims 1-10, characterized in that: the kidney injury is acute kidney injury.
12. An enzyme-linked immunosorbent assay kit for detecting the content of Sda sugar antigen on uromodulin in urine, comprising standard antigens, capture objects and detection objects; the standard antigen is healthy human uromodulin; the capture antibody It is an antibody against uromodulin, and the detection substance is a biotin-labeled lectin capable of recognizing Sda.
13. The ELISA kit according to claim 12, characterized in that: the anti-uromodulin antibody is an anti-uromodulin polyclonal antibody.
14. The ELISA kit according to claim 12, characterized in that: the lectin capable of recognizing Sda is DBA.
15. The ELISA kit according to any one of claims 12-14, characterized in that: the ELISA kit also contains horseradish peroxidase-labeled avidin and/or sample diluent.
16. According to the ELISA kit according to any one of claims 12-15, it is characterized in that: the ELISA kit also contains at least one of the following: ELISA plate, plating buffer, washing solution, Blocking solution, chromogenic solution and stop solution.
17. ELISA kit according to claim 16, characterized in that

    The sample diluent is 0.01M PBS buffer at pH 7.4; and/or

    The plating buffer is a carbonate buffer at pH 9.6; and/or

    The washing solution is a phosphate buffer solution with a pH of 7.4 containing only 0.1% by volume of Tween 20; and/or

    The blocking solution is a phosphate buffer solution with a pH of 7.4 containing only 10g/L BSA; and/or

    The chromogenic solution is tetramethylbenzidine; and/or

    The stop solution is 1M H ₂ SO ₄ .
18. The application of the ELISA kit according to any one of claims 12-17 in detecting the content of Sda sugar antigen on uromodulin in urine.
19. The application according to claim 18, characterized in that: in the application, urine is used as the sample to be tested, and the test is performed after being diluted with a buffer;
20. The application according to claim 19, characterized in that: the buffer is PBS buffer.
21. The application of the enzyme-linked immunoassay kit according to any one of claims 12-17 in the following: preparation of products for auxiliary diagnosis of diseases related to the abnormal expression of Sda sugar antigen on uromodulin; or, auxiliary diagnosis and uromodulin Diseases related to the abnormal expression of Sda sugar antigen on the protein.

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