CN215029017U - Kit for extracting trace uromodulin - Google Patents

Abstract

The utility model discloses a kit for trace uromodulin draws. The kit comprises a centrifugal filtration sleeve; the centrifugal filter sleeve comprises a centrifugal column and a collecting pipe; the centrifugal column is a pipe body with openings at two ends, and a cover matched with the centrifugal column is arranged at the opening at the top of the centrifugal column; the collecting pipe is a pipe body with a pipe orifice at one end; the outer diameter of the centrifugal column is smaller than the inner diameter of the pipe orifice of the collecting pipe, the whole pipe body of the centrifugal column is nested in the pipe orifice of the collecting pipe in a matching mode, the centrifugal column cannot fall to the bottom of the collecting pipe, and the centrifugal column is detachably connected with the collecting pipe; the bottom end of the centrifugal column is provided with a filter layer for filtering uromodulin. Adopt the utility model discloses the kit detects and need not to collect a large amount of urine specimens and carry out the protein purification, can directly use a small amount of urine specimens directly to extract, uses this kit to detect consuming time fewly, and the cost is low.

Description

Kit for extracting trace uromodulin

Technical Field

The utility model relates to a kit for trace uromodulin draws belongs to the biotechnology field.

Background

Many studies have shown that uromodulin has a protective effect in kidney injury and that the level of uromodulin may be a biomarker predictive of kidney injury. Uromodulin is rich in N-sugars, the N-sugar content accounting for 30% of the protein molecular weight. Glycosylation is an important post-translational modification of uromodulin. Under disease conditions, dysglycosylation of uromodulin may precede changes in protein levels. Glycosylation is an important post-translational modification and may be more sensitive than protein levels alone to predict disease. Glycosylation is a key component of immunomodulatory activity of uromodulin, and detection of glycosylated antigens may become an important marker for predicting renal injury in future clinical diagnosis.

In the detection of glycosylation, regardless of the detection method using mass spectrometry or lectin chip, it is necessary to extract and purify uromodulin without destroying its protein activity and glycosyl structure. The existing extraction method of the uromodulin adopts diatomite mixing and a funnel and filter paper filtration purification method, so that the uromodulin is wasted more, and therefore, the traditional method has the disadvantages of large urine volume, long time consumption, reduced secretion amount of the uromodulin after kidney injury, larger required urine volume, long clinical urine retention time, time consumption for extraction and reduced diagnosis timeliness and practicability.

SUMMERY OF THE UTILITY MODEL

The utility model aims at providing a kit for trace uromodulin draws.

The utility model provides a kit for extracting trace uromodulin, which comprises a centrifugal filtering sleeve;

the centrifugal filter sleeve comprises a centrifugal column and a collecting pipe; the centrifugal column is a pipe body with openings at two ends, and a cover matched with the centrifugal column is arranged at the opening at the top of the centrifugal column; the collecting pipe is a pipe body with a pipe orifice at one end;

the outer diameter of the centrifugal column is smaller than the inner diameter of the pipe orifice of the collecting pipe, the whole pipe body of the centrifugal column is nested in the pipe orifice of the collecting pipe in a matching mode, the centrifugal column cannot fall to the bottom of the collecting pipe, and the centrifugal column is detachably connected with the collecting pipe;

the bottom end of the centrifugal column is provided with a filter layer for filtering uromodulin, and the filter layer and the centrifugal column are arranged integrally and cannot be detached.

In the above kit for microalbumin extraction, the ratio of the volume of the collection tube to the volume of the centrifugal column may be 2: 1.

in the kit for extracting the micro uromodulin, the filter layer is whatman filter paper.

In the kit for extracting the trace uromodulin, the bottom of the tube body of the collecting tube is arranged in a round bottom manner.

In the kit for extracting the uromodulin, the kit for extracting the uromodulin also comprises 3 g of diatomite, 3 high-speed centrifuge tubes and/or 1 dialysis bag.

In the above kit for extracting uromodulin in a trace amount, the kit for extracting uromodulin in a trace amount further includes a box;

place in the box centrifugal filtration sleeve pipe, diatomaceous earth the high-speed centrifuge tube and/or the dialysis bag, just diatomaceous earth holds through wrapping bag or packing carton.

The method for extracting uromodulin by using the kit for extracting trace uromodulin in the utility model comprises the following steps:

1) mixing 2g of diatomite with deionized water to obtain a mixed solution;

2) pouring the mixed solution into the centrifugal column through the top opening of the centrifugal column, then centrifuging the centrifugal filter sleeve, and discarding the liquid in the collection tube;

3) adding 5ml of urine sample into the centrifugal column in the step 2), centrifuging the centrifugal filter sleeve, and discarding liquid in the collecting pipe;

4) adding 0.02M PBS solution into the centrifugal column in the step 3) for centrifugation, then centrifuging the centrifugal filter sleeve, and discarding the liquid in the collection tube;

5) taking out the diatomite in the centrifugal column in the step 4), mixing the diatomite with ultrapure water, stirring the mixture on a magnetic stirrer in a cold storage at 4 ℃ for 15-20min, then loading the mixed solution into a high-speed centrifugal tube, and centrifuging;

6) taking out the high-speed centrifuge tube in the step 5) to suck the supernatant, and discarding the precipitate;

7) adding 0.1M PB buffer solution (phosphate buffer solution) and NaCl into the supernatant, wherein the ion concentration of the PB buffer solution is 0.025mol/L and the ion concentration of sodium is 0.14 mol/L;

8) mixing 1g of diatomite with deionized water, and pouring the mixture into a new centrifugal column of the centrifugal filter sleeve for centrifugation;

9) pouring the solution collected in the step 7) into the centrifugal column in the step 8) for centrifugation, discarding the liquid in the collection tube, and repeating the steps 7) -9) until the liquids in the step 7) are centrifuged;

the utility model has the advantages of it is following:

the kit of the utility model is adopted for detection, a large amount of urine samples do not need to be collected for protein purification, and a small amount of urine samples can be directly used for extraction; the nested arrangement of the centrifugal column and the collecting tube ensures that the detection using the kit has less time consumption and low cost, is convenient for clinical development, and strives for precious time for clinical early diagnosis and treatment.

Drawings

Fig. 1 is a schematic structural diagram of a centrifugal filtration sleeve in the kit for extracting uromodulin in minute quantities.

The individual labels in the figure are as follows:

1, a cover; 2, centrifugal column; 3 collecting pipes and 4 filtering layers.

FIG. 2 shows the purity of uromodulin obtained by separating the kit for extracting trace uromodulin according to the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, but the present invention is not limited to the following embodiments.

In the following examples, 0.1M phosphate buffer was composed of 2.9g of Na₂HPO₄.12H₂O，0.3g NaH₂PO₄.2H₂O, 100ml deionized water.

The 0.02M PBS solution is prepared from the following components in percentage by weight: 8g NaCl, 5.8g Na₂HPO₄·12H₂O，0.6g NaH₂PO₄·2H₂O, dd water 1000ml dialysis bag treatment: 2% NaHCO₃(pH 8.0) 20g NaHCO₃，0.372g EDTA-Na₂，dd water 1000ml；1M EDTA-Na₂:0.372g EDTA-Na₂1000ml of deionized water, pH 8.0.

Examples 1,

As shown in figure 1, the utility model discloses a kit for trace uromodulin extraction, including the centrifugal filtration sleeve pipe.

The centrifugal filter sleeve comprises a centrifugal column 2 and a collecting pipe 3; the centrifugal column 2 is a pipe body with openings at two ends, and the top opening of the centrifugal column is provided with a cover 1 matched with the centrifugal column; the collecting pipe 3 is a pipe body with a pipe orifice at one end; the outer diameter of the centrifugal column 2 is smaller than the inner diameter of the pipe orifice of the collecting pipe 3, the whole pipe body of the centrifugal column 2 is nested in the pipe orifice of the collecting pipe 3 in a matching mode, the centrifugal column 2 cannot fall to the bottom of the collecting pipe 3, the centrifugal column 2 is detachably connected with the collecting pipe 3, and the ratio of the volume of the collecting pipe 3 to the volume of the centrifugal column 2 is 2: 1. The bottom end of the centrifugal column 2 is provided with a filter layer 4 (specifically, whatman filter paper) for filtering uromodulin. The filter layer 4 and the centrifugal column 2 are arranged integrally and are not detachable.

Further, the bottom of the tube body of the collecting tube 3 is arranged in a round bottom mode.

Further, the kit for extracting the microalbumin also comprises 3 g of diatomite, 3 high-speed centrifuge tubes and/or 1 dialysis bag.

Further, the kit for extracting the micro uromodulin also comprises a box;

centrifugal filter sleeve pipe, diatomaceous earth, high-speed centrifuge tube and/or dialysis bag are placed to the box in, and diatomaceous earth holds through wrapping bag or packing carton.

The method for extracting uromodulin by using the kit for extracting trace uromodulin in the utility model comprises the following experimental steps:

1. 5ml of human urine sample is left for standby;

2. uniformly mixing 2g of diatomite and 10ml of deionized water in a glass beaker to obtain a mixed solution;

3. pouring the mixed solution into a centrifugal column gently through an opening at the top of the centrifugal column at 2500pm for 2min, discarding the liquid in a collecting pipe, and not changing the pipe;

4. adding 5ml of human urine sample into the centrifugal column in the step 3, centrifuging at 2500rpm for 5min, centrifuging the centrifugal filter sleeve, and removing liquid in the collection tube;

5. continuously adding 10ml of 0.02M PBS into the centrifugal column in the step 4, centrifuging for 2min, centrifuging the centrifugal filter sleeve, and discarding the liquid in the collecting pipe;

6. taking out the diatomite and mixing with 30ml of ultrapure water uniformly, stirring for 30min on a magnetic stirrer at 4 ℃, filling the mixed solution into a 50ml high-speed centrifugal tube, and balancing; centrifuging at 20000g for 20 min;

7. after the centrifugation is finished, gently taking out the high-speed centrifuge tube to absorb the supernatant, and discarding the precipitate;

8. adding 0.1M PB buffer (phosphate buffer) and NaCl to the supernatant to make the ion concentration of PB buffer 0.025mol/L and the sodium ion concentration 0.14mol/L

9. Mixing 1g of diatomaceous earth with deionized water, pouring into a new centrifugal column of a centrifugal filter sleeve, centrifuging at 2500pm, and centrifuging for 2 min.

10. Pouring the solution collected in the step 8) into the centrifugal column in the step 9) for centrifugation at 2500rpm for 2min, discarding the liquid in the collection tube, and repeating the steps 8) to 10) until the liquids in the step 7) are centrifuged;

11. mixing the diatomite and the ultrapure water in the step 9), stirring for 15-20min on a magnetic stirrer in a cold storage at 4 ℃, then loading the mixed solution into a high-speed centrifugal tube, centrifuging and balancing; 20000g for 20 min.

12. Gently taking out the centrifuge tube in the step 10), sucking the supernatant liquid and discarding the precipitate.

13. And (3) putting the supernatant obtained in the step (11) into a new high-speed centrifuge tube, centrifuging at 20000g for 10min, and removing precipitates.

14. The liquid in step 12 is subpackaged into dialysis bags and dialyzed in deionized water at 4 degrees for 5 hours or at 4 degrees overnight.

16. Filtering the liquid in the dialysis bag in the step 13) with a 0.02 μm filter to remove impurities, and collecting the filtrate.

17. And (3) carrying out ultrafiltration concentration on the filtrate, concentrating the protein by using an ultrafiltration tube with the flow cut-off of 30000, centrifuging at 3000rpm for 5-10 min, observing the concentration state, and adjusting the centrifugation time.

As can be seen from FIG. 2, the molecular weight of uromodulin is about 95kDa, and the Coomassie blue staining result shows that the uromodulin purity (first 4 protein samples) extracted by the kit of the present invention is similar to that extracted by the conventional method (5 th protein sample).

Claims (6)

1. A kit for extracting trace uromodulin is characterized in that: the kit comprises a centrifugal filtration sleeve;

the centrifugal filter sleeve comprises a centrifugal column and a collecting pipe; the centrifugal column is a pipe body with openings at two ends, and a cover matched with the centrifugal column is arranged at the opening at the top of the centrifugal column; the collecting pipe is a pipe body with a pipe orifice at one end;

the outer diameter of the centrifugal column is smaller than the inner diameter of the pipe orifice of the collecting pipe, the whole pipe body of the centrifugal column is nested in the pipe orifice of the collecting pipe in a matching mode, the centrifugal column cannot fall to the bottom of the collecting pipe, and the centrifugal column is detachably connected with the collecting pipe;

the bottom end of the centrifugal column is provided with a filter layer for filtering uromodulin.

2. The kit for microalbuminuria extraction according to claim 1, wherein: the ratio of the volume of the collection tube to the volume of the spin column is 2: 1.

3. the kit for microalbuminuria extraction according to claim 1, wherein: the filter layer is whatman filter paper.

4. The kit for microalbuminuria extraction according to claim 1, wherein: the bottom of the tube body of the collecting tube is arranged at a round bottom.

5. The kit for microalbuminuria extraction according to claim 1, wherein: the kit for extracting the microalbumin also comprises 3 g of diatomite, 3 high-speed centrifuge tubes and/or 1 dialysis bag.

6. The kit for microalbumin extraction according to claim 4, wherein: the kit for extracting the trace uromodulin also comprises a box;

place in the box centrifugal filtration sleeve pipe, diatomaceous earth the high-speed centrifuge tube and/or the dialysis bag, just diatomaceous earth holds through wrapping bag or packing carton.

Images